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Sample records for dictyostelium contractile vacuoles

  1. New insights into roles of acidocalcisomes and contractile vacuole complex in osmoregulation in protists.

    Science.gov (United States)

    Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

    2013-01-01

    While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. © 2013, Elsevier Inc. All Rights Reserved.

  2. New Insights into the Roles of Acidocalcisomes and the Contractile Vacuole Complex in Osmoregulation in Protists

    Science.gov (United States)

    Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

    2013-01-01

    While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. PMID:23890380

  3. Subcellular localization of ammonium transporters in Dictyostelium discoideum

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    Davis Carter T

    2008-12-01

    Full Text Available Abstract Background With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. Results Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. Conclusion Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not

  4. Loss of Cln3 Function in the Social Amoeba Dictyostelium discoideum Causes Pleiotropic Effects That Are Rescued by Human CLN3

    Science.gov (United States)

    Huber, Robert J.

    2014-01-01

    The neuronal ceroid lipofuscinoses (NCL) are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL) is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3− cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3− cells was precocious and cln3− slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3− cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3− cells, strongly supports the use of this new model for JNCL research. PMID:25330233

  5. Loss of Cln3 function in the social amoeba Dictyostelium discoideum causes pleiotropic effects that are rescued by human CLN3.

    Directory of Open Access Journals (Sweden)

    Robert J Huber

    Full Text Available The neuronal ceroid lipofuscinoses (NCL are a group of inherited, severe neurodegenerative disorders also known as Batten disease. Juvenile NCL (JNCL is caused by recessive loss-of-function mutations in CLN3, which encodes a transmembrane protein that regulates endocytic pathway trafficking, though its primary function is not yet known. The social amoeba Dictyostelium discoideum is increasingly utilized for neurological disease research and is particularly suited for investigation of protein function in trafficking. Therefore, here we establish new overexpression and knockout Dictyostelium cell lines for JNCL research. Dictyostelium Cln3 fused to GFP localized to the contractile vacuole system and to compartments of the endocytic pathway. cln3- cells displayed increased rates of proliferation and an associated reduction in the extracellular levels and cleavage of the autocrine proliferation repressor, AprA. Mid- and late development of cln3- cells was precocious and cln3- slugs displayed increased migration. Expression of either Dictyostelium Cln3 or human CLN3 in cln3- cells suppressed the precocious development and aberrant slug migration, which were also suppressed by calcium chelation. Taken together, our results show that Cln3 is a pleiotropic protein that negatively regulates proliferation and development in Dictyostelium. This new model system, which allows for the study of Cln3 function in both single cells and a multicellular organism, together with the observation that expression of human CLN3 restores abnormalities in Dictyostelium cln3- cells, strongly supports the use of this new model for JNCL research.

  6. The vacuole within

    Science.gov (United States)

    Ellis, Kathryn; Hoffman, Brenton D.; Bagnat, Michel

    2013-01-01

    The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuoles results in a shortened embryo along the anterior-posterior axis. Interestingly, we discovered that notochord vacuoles are also essential for proper spine morphogenesis and that vacuole defects lead to scoliosis of the spine. Here we discuss the cellular organization of the notochord and how key features of its architecture allow the notochord to function in embryonic axis elongation and spine formation. PMID:23887209

  7. Loss of Cln3 impacts protein secretion in the social amoeba Dictyostelium.

    Science.gov (United States)

    Huber, Robert J

    2017-07-01

    Neuronal ceroid lipofuscinosis (NCL), also referred to as Batten disease, is the most common form of childhood neurodegeneration. Mutations in CLN3 cause the most prevalent subtype of the disease, which manifests during early childhood and is currently untreatable. The precise function of the CLN3 protein is still not known, which has inhibited the development of targeted therapies. In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. The results of the present study indicate that this phenotype may be at least partly due to aberrant protein secretion in cln3 - cells. It is well-established that Cln3 localizes primarily to the contractile vacuole (CV) system in Dictyostelium, and to a lesser extent, compartments of the endocytic pathway. Intriguingly, the CV system has been linked to the secretion of proteins that do not contain a signal peptide for secretion (i.e., unconventional protein secretion). Proteins that do contain a signal peptide are secreted via a conventional mechanism involving the endoplasmic reticulum, transport through the Golgi, and secretion via vesicle release. In this study, Cln3 was observed to co-localize with the Golgi marker wheat germ agglutinin suggesting that Cln3 participates in both secretion mechanisms. Chimeras of wild-type (WT) and cln3 - cells displayed delayed streaming and aggregation, and interestingly, cln3 - cells starved in conditioned media (CM) harvested from starving WT cells showed near normal timing of streaming and aggregation suggesting aberrant protein secretion in Cln3-deficient cells. Based on these observations, LC-MS/MS was used to reveal the protein content of CM from starved cells (mass spectrometry data are available via ProteomeXchange with identifier PXD004897). A total of 450 proteins were detected in WT and cln3 - CM, of which 3 were absent in cln3 - CM. Moreover, 12 proteins that were present in

  8. Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection

    Science.gov (United States)

    Swart, A. Leoni; Harrison, Christopher F.; Eichinger, Ludwig; Steinert, Michael; Hilbi, Hubert

    2018-01-01

    Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of “effector” proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors. PMID:29552544

  9. Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection

    Directory of Open Access Journals (Sweden)

    A. Leoni Swart

    2018-03-01

    Full Text Available Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV. LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of “effector” proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

  10. Calcium Signals from the Vacuole

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    Gerald Schönknecht

    2013-10-01

    Full Text Available The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.

  11. Phospholipase Cδ regulates germination of Dictyostelium spores

    NARCIS (Netherlands)

    Dijken, Peter van; Haastert, Peter J.M. van

    2001-01-01

    Background: Many eukaryotes, including plants and fungi make spores that resist severe environmental stress. The micro-organism Dictyostelium contains a single phospholipase C gene (PLC); deletion of the gene has no effect on growth, cell movement and differentiation. In this report we show that PLC

  12. Cell vacuolation caused by Vibrio cholerae hemolysin.

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    Figueroa-Arredondo, P; Heuser, J E; Akopyants, N S; Morisaki, J H; Giono-Cerezo, S; Enríquez-Rincón, F; Berg, D E

    2001-03-01

    Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (approximately 1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (approximately 16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

  13. Fractalkine depresses cardiomyocyte contractility.

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    David Taube

    Full Text Available Our laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E2 EP4 receptor sub-type (EP4 KO exhibit reduced cardiac function. Gene array on left ventricles (LV showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE2 and contributes to depressed contractility via alterations in intracellular calcium.Fractalkine was measured in LV of 28-32 week old male EP4 KO and wild type controls (WT by ELISA and the effect of PGE2 on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso stimulation.LV fractalkine was increased in EP4 KO mice but surprisingly, PGE2 regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca(2+ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery.Fractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium.

  14. Cell Vacuolation Caused by Vibrio cholerae Hemolysin

    Science.gov (United States)

    Figueroa-Arredondo, Paula; Heuser, John E.; Akopyants, Natalia S.; Morisaki, J. Hiroshi; Giono-Cerezo, Silvia; Enríquez-Rincón, Fernando; Berg, Douglas E.

    2001-01-01

    Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (∼1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (∼16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses. PMID:11179335

  15. Retrotransposon Domestication and Control in Dictyostelium discoideum

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    Marek Malicki

    2017-10-01

    Full Text Available Transposable elements, identified in all eukaryotes, are mobile genetic units that can change their genomic position. Transposons usually employ an excision and reintegration mechanism, by which they change position, but not copy number. In contrast, retrotransposons amplify via RNA intermediates, increasing their genomic copy number. Hence, they represent a particular threat to the structural and informational integrity of the invaded genome. The social amoeba Dictyostelium discoideum, model organism of the evolutionary Amoebozoa supergroup, features a haploid, gene-dense genome that offers limited space for damage-free transposition. Several of its contemporary retrotransposons display intrinsic integration preferences, for example by inserting next to transfer RNA genes or other retroelements. Likely, any retrotransposons that invaded the genome of the amoeba in a non-directed manner were lost during evolution, as this would result in decreased fitness of the organism. Thus, the positional preference of the Dictyostelium retroelements might represent a domestication of the selfish elements. Likewise, the reduced danger of such domesticated transposable elements led to their accumulation, and they represent about 10% of the current genome of D. discoideum. To prevent the uncontrolled spreading of retrotransposons, the amoeba employs control mechanisms including RNA interference and heterochromatization. Here, we review TRE5-A, DIRS-1 and Skipper-1, as representatives of the three retrotransposon classes in D. discoideum, which make up 5.7% of the Dictyostelium genome. We compile open questions with respect to their mobility and cellular regulation, and suggest strategies, how these questions might be addressed experimentally.

  16. Evidence for nucleolar subcompartments in Dictyostelium

    International Nuclear Information System (INIS)

    Catalano, Andrew; O’Day, Danton H.

    2015-01-01

    Highlights: • Two nucleolar subcompartments (NoSC1, NoSC2) were found in Dictyostelium. • Specific nucleolar proteins localize to different nucleolar subcompartments. • Specific proteins exit NoSC1 and NoSC2 differently upon Actinomycin D treatment. • KRKR appears to function as an NoSC2 nucleolar subcompartment localization signal. - Abstract: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during

  17. Evidence for nucleolar subcompartments in Dictyostelium

    Energy Technology Data Exchange (ETDEWEB)

    Catalano, Andrew, E-mail: acatalano@ccny.cuny.edu [Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario L5L 1C6 (Canada); O’Day, Danton H., E-mail: danton.oday@utoronto.ca [Department of Biology, University of Toronto at Mississauga, 3359 Mississauga Rd. N., Mississauga, Ontario L5L 1C6 (Canada); Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario M5S 3G5 (Canada)

    2015-01-24

    Highlights: • Two nucleolar subcompartments (NoSC1, NoSC2) were found in Dictyostelium. • Specific nucleolar proteins localize to different nucleolar subcompartments. • Specific proteins exit NoSC1 and NoSC2 differently upon Actinomycin D treatment. • KRKR appears to function as an NoSC2 nucleolar subcompartment localization signal. - Abstract: The nucleolus is a multifunctional nuclear compartment usually consisting of two to three subcompartments which represent stages of ribosomal biogenesis. It is linked to several human diseases including viral infections, cancer, and neurodegeneration. Dictyostelium is a model eukaryote for the study of fundamental biological processes as well as several human diseases however comparatively little is known about its nucleolus. Unlike most nucleoli it does not possess visible subcompartments at the ultrastructural level. Several recently identified nucleolar proteins in Dictyostelium leave the nucleolus after treatment with the rDNA transcription inhibitor actinomycin-D (AM-D). Different proteins exit in different ways, suggesting that previously unidentified nucleolar subcompartments may exist. The identification of nucleolar subcompartments would help to better understand the nucleolus in this model eukaryote. Here, we show that Dictyostelium nucleolar proteins nucleomorphin isoform NumA1 and Bud31 localize throughout the entire nucleolus while calcium-binding protein 4a localizes to only a portion, representing nucleolar subcompartment 1 (NoSC1). SWI/SNF complex member Snf12 localizes to a smaller area within NoSC1 representing a second nucleolar subcompartment, NoSC2. The nuclear/nucleolar localization signal KRKR from Snf12 localized GFP to NoSC2, and thus also appears to function as a nucleolar subcompartment localization signal. FhkA localizes to the nucleolar periphery displaying a similar pattern to that of Hsp32. Similarities between the redistribution patterns of Dictyostelium nucleolar proteins during

  18. Regulation of TORC2 complex in Dictyostelium discoideum

    NARCIS (Netherlands)

    Khanna, Ankita

    2016-01-01

    Dictyostelium is an amoeba that lives in the soil where it feeds on bacteria. During scarcity of food, Dictyostelium cells undergo a highly regulated developmental process in which the cells aggregate by chemotaxing towards pulsatile emission of extracellular cAMP from a signaling center; the cells

  19. Arachidonic acid is a chemoattractant for Dictyostelium discoideum ...

    Indian Academy of Sciences (India)

    SEARCHU

    binding proteins and calmodulin-dependent phosphorylation linked to calmodulin-dependent chemotaxis to folic and cAMP in Dictyostelium; Cell Signal 13 575–584. Gerisch G and Hess B 1974 Cyclic-AMP-controlled oscillations in suspended Dictyostelium cells: Their relation to morphogenetic cell interactions; Proc. Natl.

  20. Initiation and elimination of vacuoles in microencapsulated shells

    International Nuclear Information System (INIS)

    Du Kai; You Dan

    2000-01-01

    Two mechanisms of vacuole formation in microencapsulated micro-shells wall are introduced. It is verified that phase separation of trace amount of water in the organic solvent is the most possible course of vacuole formation

  1. Raman Microspectroscopy of the Yeast Vacuoles

    Czech Academy of Sciences Publication Activity Database

    Bednárová, Lucie; Palacký, J.; Bauerová, Václava; Hrušková-Heidingsfeldová, Olga; Pichová, Iva; Mojzeš, P.

    2012-01-01

    Roč. 27, 5-6 (2012), s. 503-507 ISSN 0712-4813 R&D Projects: GA ČR GAP208/10/0376; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Raman microspectroscopy * living cell * yeast * vacuole * chemical composition * polyphospate * Candida albicans Subject RIV: CE - Biochemistry Impact factor: 0.530, year: 2012

  2. Analysis of Rheb in the cellular slime mold Dictyostelium discoideum

    Indian Academy of Sciences (India)

    2014-01-27

    Jan 27, 2014 ... lyse the caspase-independent cell death mechanism. It is a ..... we observed exclusive expression in the prespore region. (figure 5A). There were ..... disc formation in Dictyostelium discoideum is an early event in culmination.

  3. Dispatch. Dictyostelium chemotaxis: fascism through the back door?

    Science.gov (United States)

    Insall, Robert

    2003-04-29

    Aggregating Dictyostelium cells secrete cyclic AMP to attract their neighbours by chemotaxis. It has now been shown that adenylyl cyclase is enriched in the rear of cells, and this localisation is required for normal aggregation.

  4. Isolation of Protein Storage Vacuoles and Their Membranes.

    Science.gov (United States)

    Shimada, Tomoo; Hara-Nishimura, Ikuko

    2017-01-01

    Protein-storage vacuoles (PSVs) are specialized vacuoles that sequester large amounts of storage proteins. During seed development, PSVs are formed de novo and/or from preexisting lytic vacuoles. Seed PSVs can be subdivided into four distinct compartments: membrane, globoid, matrix, and crystalloid. In this chapter, we introduce easy methods for isolation of PSVs and their membranes from pumpkin seeds. These methods facilitate the identification and characterization of PSV components.

  5. The vacuole within: How cellular organization dictates notochord function

    OpenAIRE

    Ellis, Kathryn; Hoffman, Brenton D.; Bagnat, Michel

    2013-01-01

    The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuole...

  6. Structural comparison of contractile nanomachines

    Directory of Open Access Journals (Sweden)

    Sebastian Kube

    2015-05-01

    Full Text Available Contractile molecular machines are a common feature among bacteriophages and prokaryotes. Due to their stability and the large size, contractile-tailed bacteriophages are traditionally investigated by electron microscopic methods. Complemented by crystallographic studies, a molecular model of contraction for the T4 phage was developed. Lately, also related contractile structures like the Photorhabdus virulence cassette-like particles, the R-Type pyocins and the contractile tubule of the bacterial Type VI secretion system have been analyzed by cryo electron microscopy. Photorhabdus virulence cassette particles and R-Type pyocins are toxin complexes reminiscent of bacteriophage tails that are secreted by bacteria to kill their insect host or competing bacteria. In contrast, the Type VI secretion system is an intracellular apparatus for injection of effector proteins into bacterial and eukaryotic cells. Although it shares homology with other contractile systems, the Type VI secretion system is additionally equipped with a recycling function, which makes it suitable for multiple rounds of action. Starting from the 3D reconstructions, we compare these molecular machines structurally and functionally to their viral counterparts and summarize the current knowledge on their respective mode of action.

  7. Arachidonic acid is a chemoattractant for Dictyostelium discoideum

    Indian Academy of Sciences (India)

    Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells ... Arachidonic acid; chemotaxis; fatty acids; iplA ... Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of ...

  8. Diffusion-Assisted Aggregation and Synchronization in Dictyostelium discoideum

    Science.gov (United States)

    Nagano, Seido

    1998-05-01

    In biological pattern formation, chemotaxis and cell adhesion are essential. However, we lack quantitative data and a theory to understand their coordination. The cellular dynamics theory presented can clarify how Dictyostelium discoideum amoebae use diffusible cyclic adenosine 3',5'-monophosphate, and coordinate chemotaxis and cell adhesion during aggregation.

  9. Biochemistry and genetics of inositol phosphate metabolism in Dictyostelium

    NARCIS (Netherlands)

    vanHaastert, PJM; van Dijken, P.

    1997-01-01

    Biochemical and genetic data on the metabolism of inositol phosphates in the microorganism Dictyostelium are combined in a scheme composed of in five subroutes. The first subroute is the inositol cycle as found in other organisms:inositol is incorporated into phospholipids that are hydrolysed by PLC

  10. Characterization of two unusual guanylyl cyclases from Dictyostelium

    NARCIS (Netherlands)

    Roelofs, Jeroen; Haastert, Peter J.M. van

    2002-01-01

    Guanylyl cyclase A (GCA) and soluble guanylyl cyclase (sGC) encode GCs in Dictyostelium and have a topology similar to 12-transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell

  11. The genome of the social amoeba Dictyostelium discoideum

    DEFF Research Database (Denmark)

    Eichinger, L; Pachebat, J A; Glöckner, G

    2005-01-01

    The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins,...

  12. Calcium regulates the expression of a Dictyostelium discoideum ...

    Indian Academy of Sciences (India)

    In a screen for calcium-regulated gene expression during growth and development of Dictyostelium discoideum we have identified an asparaginyl tRNA synthetase (ddAsnRS) gene, the second tRNA synthetase gene identified in this organism. The ddAsnRS gene shows many unique features. One, it is repressed by ...

  13. Involvement of Sib Proteins in the Regulation of Cellular Adhesion in Dictyostelium discoideum▿ †

    OpenAIRE

    Cornillon, Sophie; Froquet, Romain; Cosson, Pierre

    2008-01-01

    Molecular mechanisms ensuring cellular adhesion have been studied in detail in Dictyostelium amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in Dictyostelium, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a Dictyostelium population. In order to understand the mechani...

  14. Pumping up the volume - vacuole biogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Krüger, Falco; Schumacher, Karin

    2017-07-08

    Plant architecture follows the need to collect CO 2, solar energy, water and mineral nutrients via large surface areas. It is by the presence of a central vacuole that fills much of the cell volume that plants manage to grow at low metabolic cost. In addition vacuoles buffer the fluctuating supply of essential nutrients and help to detoxify the cytosol when plants are challenged by harmful molecules. Despite their large size and multiple important functions, our knowledge of vacuole biogenesis and the machinery underlying their amazing dynamics is still fragmentary. In this review, we try to reconcile past and present models for vacuole biogenesis with the current knowledge of multiple parallel vacuolar trafficking pathways and the molecular machineries driving membrane fusion and organelle shape. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Lydie Michaillat

    Full Text Available The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

  16. A secreted protein is an endogenous chemorepellant in Dictyostelium discoideum

    OpenAIRE

    Phillips, Jonathan E.; Gomer, Richard H.

    2012-01-01

    Chemorepellants may play multiple roles in physiological and pathological processes. However, few endogenous chemorepellants have been identified, and how they function is unclear. We found that the autocrine signal AprA, which is produced by growing Dictyostelium discoideum cells and inhibits their proliferation, also functions as a chemorepellant. Wild-type cells at the edge of a colony show directed movement outward from the colony, whereas cells lacking AprA do not. Cells show directed mo...

  17. Chemotaxis of Dictyostelium discoideum: collective oscillation of cellular contacts.

    Directory of Open Access Journals (Sweden)

    Edith Schäfer

    Full Text Available Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.

  18. Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts

    Science.gov (United States)

    Schäfer, Edith; Tarantola, Marco; Polo, Elena; Westendorf, Christian; Oikawa, Noriko; Bodenschatz, Eberhard; Geil, Burkhard; Janshoff, Andreas

    2013-01-01

    Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells. PMID:23349816

  19. Identification and recombinant expression of anandamide hydrolyzing enzyme from Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Neelamegan Dhamodharan

    2012-06-01

    Full Text Available Abstract Background Anandamide (Arachidonoyl ethanolamide is a potent bioactive lipid studied extensively in humans, which regulates several neurobehavioral processes including pain, feeding and memory. Bioactivity is terminated when hydrolyzed into free arachidonic acid and ethanolamine by the enzyme fatty acid amide hydrolase (FAAH. In this study we report the identification of a FAAH homolog from Dictyostelium discoideum and its function to hydrolyze anandamide. Results A putative FAAH DNA sequence coding for a conserved amidase signature motif was identified in the Dictyostelium genome database and the corresponding cDNA was isolated and expressed as an epitope tagged fusion protein in either E.coli or Dictyostelium. Wild type Dictyostelium cells express FAAH throughout their development life cycle and the protein was found to be predominantly membrane associated. Production of recombinant HIS tagged FAAH protein was not supported in E.coli host, but homologous Dictyostelium host was able to produce the same successfully. Recombinant FAAH protein isolated from Dictyostelium was shown to hydrolyze anandamide and related synthetic fatty acid amide substrates. Conclusions This study describes the first identification and characterisation of an anandamide hydrolyzing enzyme from Dictyostelium discoideum, suggesting the potential of Dictyostelium as a simple eukaryotic model system for studying mechanisms of action of any FAAH inhibitors as drug targets.

  20. Naringenin is a novel inhibitor of Dictyostelium cell proliferation and cell migration

    International Nuclear Information System (INIS)

    Russ, Misty; Martinez, Raquel; Ali, Hind; Steimle, Paul A.

    2006-01-01

    Naringenin is a flavanone compound that alters critical cellular processes such as cell multiplication, glucose uptake, and mitochondrial activity. In this study, we used the social amoeba, Dictyostelium discoideum, as a model system for examining the cellular processes and signaling pathways affected by naringenin. We found that naringenin inhibited Dictyostelium cell division in a dose-dependent manner (IC 5 ∼ 20 μM). Assays of Dictyostelium chemotaxis and multicellular development revealed that naringenin possesses a previously unrecognized ability to suppress amoeboid cell motility. We also found that naringenin, which is known to inhibit phosphatidylinositol 3-kinase activity, had no apparent effect on phosphatidylinositol 3,4,5-trisphosphate synthesis in live Dictyostelium cells; suggesting that this compound suppresses cell growth and migration via alternative signaling pathways. In another context, the discoveries described here highlight the value of using the Dictyostelium model system for identifying and characterizing the mechanisms by which naringenin, and related compounds, exert their effects on eukaryotic cells

  1. Hydrolytic enzymes in the central vacuole of plant cells

    International Nuclear Information System (INIS)

    Boller, T.; Kende, H.

    1979-01-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast: (a) purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation; (b) hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation; and (c) vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β-N-acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. None of the vacuolar enzymes investigated ws found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter

  2. The relationship between vacuolation and initiation of PCD in rice (Oryza sativa) aleurone cells

    Science.gov (United States)

    Zheng, Yan; Zhang, Heting; Deng, Xiaojiang; Liu, Jing; Chen, Huiping

    2017-01-01

    Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.

  3. Pleiotropic Actions of Helicobacter pylori Vacuolating Cytotoxin, VacA

    OpenAIRE

    Isomoto, Hajime; Moss, Joel; Hirayama, Toshiya

    2010-01-01

    Helicobacter pylori produces a vacuolating cytotoxin, VacA, and most virulent H. pylori strains secrete VacA. VacA binds to two types of receptor-like protein tyrosine phosphatase (RPTP), RPTPα and RPTPβ, on the surface of host cells. VacA bound to RPTPβ, relocates and concentrates in lipid rafts in the plasma membrane. VacA causes vacuolization, membrane anion-selective channel and pore formation, and disruption of endosomal and lysosomal activity in host cells. Secreted VacA is processed in...

  4. Modulatory effects of taurine on jejunal contractility

    Directory of Open Access Journals (Sweden)

    Q.Y. Yao

    2014-12-01

    Full Text Available Taurine (2-aminoethanesulfonic acid is widely distributed in animal tissues and has diverse pharmacological effects. However, the role of taurine in modulating smooth muscle contractility is still controversial. We propose that taurine (5-80 mM can exert bidirectional modulation on the contractility of isolated rat jejunal segments. Different low and high contractile states were induced in isolated jejunal segments of rats to observe the effects of taurine and the associated mechanisms. Taurine induced stimulatory effects on the contractility of isolated rat jejunal segments at 3 different low contractile states, and inhibitory effects at 3 different high contractile states. Bidirectional modulation was not observed in the presence of verapamil or tetrodotoxin, suggesting that taurine-induced bidirectional modulation is Ca2+ dependent and requires the presence of the enteric nervous system. The stimulatory effects of taurine on the contractility of isolated jejunal segments was blocked by atropine but not by diphenhydramine or by cimetidine, suggesting that muscarinic-linked activation was involved in the stimulatory effects when isolated jejunal segments were in a low contractile state. The inhibitory effects of taurine on the contractility of isolated jejunal segments were blocked by propranolol and L-NG-nitroarginine but not by phentolamine, suggesting that adrenergic β receptors and a nitric oxide relaxing mechanism were involved when isolated jejunal segments were in high contractile states. No bidirectional effects of taurine on myosin phosphorylation were observed. The contractile states of jejunal segments determine taurine-induced stimulatory or inhibitory effects, which are associated with muscarinic receptors and adrenergic β receptors, and a nitric oxide associated relaxing mechanism.

  5. Modulatory effects of taurine on jejunal contractility

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Q.Y.; Chen, D.P.; Ye, D.M.; Diao, Y.P.; Lin, Y. [Dalian Medical University, Dalian, Liaoning (China)

    2014-10-14

    Taurine (2-aminoethanesulfonic acid) is widely distributed in animal tissues and has diverse pharmacological effects. However, the role of taurine in modulating smooth muscle contractility is still controversial. We propose that taurine (5-80 mM) can exert bidirectional modulation on the contractility of isolated rat jejunal segments. Different low and high contractile states were induced in isolated jejunal segments of rats to observe the effects of taurine and the associated mechanisms. Taurine induced stimulatory effects on the contractility of isolated rat jejunal segments at 3 different low contractile states, and inhibitory effects at 3 different high contractile states. Bidirectional modulation was not observed in the presence of verapamil or tetrodotoxin, suggesting that taurine-induced bidirectional modulation is Ca{sup 2+} dependent and requires the presence of the enteric nervous system. The stimulatory effects of taurine on the contractility of isolated jejunal segments was blocked by atropine but not by diphenhydramine or by cimetidine, suggesting that muscarinic-linked activation was involved in the stimulatory effects when isolated jejunal segments were in a low contractile state. The inhibitory effects of taurine on the contractility of isolated jejunal segments were blocked by propranolol and L-NG-nitroarginine but not by phentolamine, suggesting that adrenergic β receptors and a nitric oxide relaxing mechanism were involved when isolated jejunal segments were in high contractile states. No bidirectional effects of taurine on myosin phosphorylation were observed. The contractile states of jejunal segments determine taurine-induced stimulatory or inhibitory effects, which are associated with muscarinic receptors and adrenergic β receptors, and a nitric oxide associated relaxing mechanism.

  6. Modulatory effects of taurine on jejunal contractility

    International Nuclear Information System (INIS)

    Yao, Q.Y.; Chen, D.P.; Ye, D.M.; Diao, Y.P.; Lin, Y.

    2014-01-01

    Taurine (2-aminoethanesulfonic acid) is widely distributed in animal tissues and has diverse pharmacological effects. However, the role of taurine in modulating smooth muscle contractility is still controversial. We propose that taurine (5-80 mM) can exert bidirectional modulation on the contractility of isolated rat jejunal segments. Different low and high contractile states were induced in isolated jejunal segments of rats to observe the effects of taurine and the associated mechanisms. Taurine induced stimulatory effects on the contractility of isolated rat jejunal segments at 3 different low contractile states, and inhibitory effects at 3 different high contractile states. Bidirectional modulation was not observed in the presence of verapamil or tetrodotoxin, suggesting that taurine-induced bidirectional modulation is Ca 2+ dependent and requires the presence of the enteric nervous system. The stimulatory effects of taurine on the contractility of isolated jejunal segments was blocked by atropine but not by diphenhydramine or by cimetidine, suggesting that muscarinic-linked activation was involved in the stimulatory effects when isolated jejunal segments were in a low contractile state. The inhibitory effects of taurine on the contractility of isolated jejunal segments were blocked by propranolol and L-NG-nitroarginine but not by phentolamine, suggesting that adrenergic β receptors and a nitric oxide relaxing mechanism were involved when isolated jejunal segments were in high contractile states. No bidirectional effects of taurine on myosin phosphorylation were observed. The contractile states of jejunal segments determine taurine-induced stimulatory or inhibitory effects, which are associated with muscarinic receptors and adrenergic β receptors, and a nitric oxide associated relaxing mechanism

  7. Modulatory effects of taurine on jejunal contractility

    Science.gov (United States)

    Yao, Q.Y.; Chen, D.P.; Ye, D.M.; Diao, Y.P.; Lin, Y.

    2014-01-01

    Taurine (2-aminoethanesulfonic acid) is widely distributed in animal tissues and has diverse pharmacological effects. However, the role of taurine in modulating smooth muscle contractility is still controversial. We propose that taurine (5-80 mM) can exert bidirectional modulation on the contractility of isolated rat jejunal segments. Different low and high contractile states were induced in isolated jejunal segments of rats to observe the effects of taurine and the associated mechanisms. Taurine induced stimulatory effects on the contractility of isolated rat jejunal segments at 3 different low contractile states, and inhibitory effects at 3 different high contractile states. Bidirectional modulation was not observed in the presence of verapamil or tetrodotoxin, suggesting that taurine-induced bidirectional modulation is Ca2+ dependent and requires the presence of the enteric nervous system. The stimulatory effects of taurine on the contractility of isolated jejunal segments was blocked by atropine but not by diphenhydramine or by cimetidine, suggesting that muscarinic-linked activation was involved in the stimulatory effects when isolated jejunal segments were in a low contractile state. The inhibitory effects of taurine on the contractility of isolated jejunal segments were blocked by propranolol and L-NG-nitroarginine but not by phentolamine, suggesting that adrenergic β receptors and a nitric oxide relaxing mechanism were involved when isolated jejunal segments were in high contractile states. No bidirectional effects of taurine on myosin phosphorylation were observed. The contractile states of jejunal segments determine taurine-induced stimulatory or inhibitory effects, which are associated with muscarinic receptors and adrenergic β receptors, and a nitric oxide associated relaxing mechanism. PMID:25387674

  8. Compensatory Hypertrophy of Skeletal Muscle: Contractile Characteristics

    Science.gov (United States)

    Ianuzzo, C. D.; Chen, V.

    1977-01-01

    Describes an experiment using rats that demonstrates contractile characteristics of normal and hypertrophied muscle. Compensatory hypertrophy of the plantaris muscle is induced by surgical removal of the synergistic gastrocnemium muscle. Includes methods for determination of contractile properties of normal and hypertrophied muscle and…

  9. A secreted protein is an endogenous chemorepellant in Dictyostelium discoideum.

    Science.gov (United States)

    Phillips, Jonathan E; Gomer, Richard H

    2012-07-03

    Chemorepellants may play multiple roles in physiological and pathological processes. However, few endogenous chemorepellants have been identified, and how they function is unclear. We found that the autocrine signal AprA, which is produced by growing Dictyostelium discoideum cells and inhibits their proliferation, also functions as a chemorepellant. Wild-type cells at the edge of a colony show directed movement outward from the colony, whereas cells lacking AprA do not. Cells show directed movement away from a source of recombinant AprA and dialyzed conditioned media from wild-type cells, but not dialyzed conditioned media from aprA(-) cells. The secreted protein CfaD, the G protein Gα8, and the kinase QkgA are necessary for the chemorepellant activity of AprA as well as its proliferation-inhibiting activity, whereas the putative transcription factor BzpN is dispensable for the chemorepellant activity of AprA but necessary for inhibition of proliferation. Phospholipase C and PI3 kinases 1 and 2, which are necessary for the activity of at least one other chemorepellant in Dictyostelium, are not necessary for recombinant AprA chemorepellant activity. Starved cells are not repelled by recombinant AprA, suggesting that aggregation-phase cells are not sensitive to the chemorepellant effect. Cell tracking indicates that AprA affects the directional bias of cell movement, but not cell velocity or the persistence of cell movement. Together, our data indicate that the endogenous signal AprA acts as an autocrine chemorepellant for Dictyostelium cells.

  10. Targets downstream of Cdk8 in Dictyostelium development

    Directory of Open Access Journals (Sweden)

    Skelton Jason

    2011-01-01

    Full Text Available Abstract Background Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. Results Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2 with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD and one transcriptionally (short chain dehydrogenase/reductase (SDR1. Conclusions This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

  11. Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

    International Nuclear Information System (INIS)

    Hara-Nishimura, I.; Nishimura, M.

    1987-01-01

    The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulse-labeled with [ 35 S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, γ and δ. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg 2+ , and Cu 2+ , but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles

  12. Rimmed vacuoles in Becker muscular dystrophy have similar features with inclusion myopathies.

    Science.gov (United States)

    Momma, Kazunari; Noguchi, Satoru; Malicdan, May Christine V; Hayashi, Yukiko K; Minami, Narihiro; Kamakura, Keiko; Nonaka, Ikuya; Nishino, Ichizo

    2012-01-01

    Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45-48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.

  13. Rimmed vacuoles in Becker muscular dystrophy have similar features with inclusion myopathies.

    Directory of Open Access Journals (Sweden)

    Kazunari Momma

    Full Text Available Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45-48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.

  14. A secreted factor represses cell proliferation in Dictyostelium

    OpenAIRE

    Brock, Debra A.; Gomer, Richard H.

    2005-01-01

    Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that i...

  15. External stimulation strength controls actin response dynamics in Dictyostelium cells

    Science.gov (United States)

    Hsu, Hsin-Fang; Westendorf, Christian; Tarantola, Marco; Zykov, Vladimir; Bodenschatz, Eberhard; Beta, Carsten

    2015-03-01

    Self-sustained oscillation and the resonance frequency of the cytoskeletal actin polymerization/depolymerization have recently been observed in Dictyostelium, a model system for studying chemotaxis. Here we report that the resonance frequency is not constant but rather varies with the strength of external stimuli. To understand the underlying mechanism, we analyzed the polymerization and depolymerization time at different levels of external stimulation. We found that polymerization time is independent of external stimuli but the depolymerization time is prolonged as the stimulation increases. These observations can be successfully reproduced in the frame work of our time delayed differential equation model.

  16. Hydrolytic enzymes in the central vacuole of plant cells.

    Science.gov (United States)

    Boller, T; Kende, H

    1979-06-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.THE INTRACELLULAR ACTIVITIES OF THE FOLLOWING ACID HYDROLASES WERE PRIMARILY LOCALIZED IN THE VACUOLE OF TOBACCO CELLS: alpha-mannosidase, beta-N-acetylglucosaminidase, beta-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar

  17. A secreted factor represses cell proliferation in Dictyostelium.

    Science.gov (United States)

    Brock, Debra A; Gomer, Richard H

    2005-10-01

    Many cells appear to secrete factors called chalones that limit their proliferation, but in most cases the factors have not been identified. We found that growing Dictyostelium cells secrete a 60 kDa protein called AprA for autocrine proliferation repressor. AprA has similarity to putative bacterial proteins of unknown function. Compared with wild-type cells, aprA-null cells proliferate faster, while AprA overexpressing cells proliferate slower. Growing wild-type cells secrete a factor that inhibits the proliferation of wild-type and aprA- cells; this activity is not secreted by aprA- cells. AprA purified by immunoprecipitation also slows the proliferation of wild-type and aprA- cells. Compared with wild type, there is a higher percentage of multinucleate cells in the aprA- population, and when starved, aprA- cells form abnormal structures that contain fewer spores. AprA may thus decrease the number of multinucleate cells and increase spore production. Together, the data suggest that AprA functions as part of a Dictyostelium chalone.

  18. Cheating by exploitation of developmental prestalk patterning in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Anupama Khare

    2010-02-01

    Full Text Available The cooperative developmental system of the social amoeba Dictyostelium discoideum is susceptible to exploitation by cheaters-strains that make more than their fair share of spores in chimerae. Laboratory screens in Dictyostelium have shown that the genetic potential for facultative cheating is high, and field surveys have shown that cheaters are abundant in nature, but the cheating mechanisms are largely unknown. Here we describe cheater C (chtC, a strong facultative cheater mutant that cheats by affecting prestalk differentiation. The chtC gene is developmentally regulated and its mRNA becomes stalk-enriched at the end of development. chtC mutants are defective in maintaining the prestalk cell fate as some of their prestalk cells transdifferentiate into prespore cells, but that defect does not affect gross developmental morphology or sporulation efficiency. In chimerae between wild-type and chtC mutant cells, the wild-type cells preferentially give rise to prestalk cells, and the chtC mutants increase their representation in the spore mass. Mixing chtC mutants with other cell-type proportioning mutants revealed that the cheating is directly related to the prestalk-differentiation propensity of the victim. These findings illustrate that a cheater can victimize cooperative strains by exploiting an established developmental pathway.

  19. Cheating by Exploitation of Developmental Prestalk Patterning in Dictyostelium discoideum

    Science.gov (United States)

    Khare, Anupama; Shaulsky, Gad

    2010-01-01

    The cooperative developmental system of the social amoeba Dictyostelium discoideum is susceptible to exploitation by cheaters—strains that make more than their fair share of spores in chimerae. Laboratory screens in Dictyostelium have shown that the genetic potential for facultative cheating is high, and field surveys have shown that cheaters are abundant in nature, but the cheating mechanisms are largely unknown. Here we describe cheater C (chtC), a strong facultative cheater mutant that cheats by affecting prestalk differentiation. The chtC gene is developmentally regulated and its mRNA becomes stalk-enriched at the end of development. chtC mutants are defective in maintaining the prestalk cell fate as some of their prestalk cells transdifferentiate into prespore cells, but that defect does not affect gross developmental morphology or sporulation efficiency. In chimerae between wild-type and chtC mutant cells, the wild-type cells preferentially give rise to prestalk cells, and the chtC mutants increase their representation in the spore mass. Mixing chtC mutants with other cell-type proportioning mutants revealed that the cheating is directly related to the prestalk-differentiation propensity of the victim. These findings illustrate that a cheater can victimize cooperative strains by exploiting an established developmental pathway. PMID:20195510

  20. Evidence for a Messenger Function of Cyclic GMP During Phosphodiesterase Induction in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Pasveer, Frank J.; Meer, Rob C. van der; Heijden, Paul R. van der; Walsum, Hans van; Konijn, Theo M.

    1982-01-01

    Chemotactic stimulation of vegetative or aggregative Dictyostelium discoideum cells induced a transient elevation of cyclic GMP levels. The addition of chemoattractants to postvegetative cells by pulsing induced phosphodiesterase activity. The following lines of evidence suggest a messenger function

  1. Shigella subverts the host recycling compartment to rupture its vacuole.

    Science.gov (United States)

    Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer L; Danckaert, Anne; Enninga, Jost

    2014-10-08

    Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Characteristics of weak base-induced vacuoles formed around individual acidic organelles.

    Science.gov (United States)

    Hiruma, Hiromi; Kawakami, Tadashi

    2011-01-01

    We have previously found that the weak base 4-aminopyridine induces Brownian motion of acidic organelles around which vacuoles are formed, causing organelle traffic disorder in neurons. Our present study investigated the characteristics of vacuoles induced by weak bases (NH(4)Cl, aminopyridines, and chloroquine) using mouse cells. Individual vacuoles included acidic organelles identified by fluorescent protein expression. Mitochondria and actin filaments were extruded outside the vacuoles, composing the vacuole rim. Staining with amine-reactive fluorescence showed no protein/amino acid content in vacuoles. Thus, serous vacuolar contents are probably partitioned by viscous cytosol, other organelles, and cytoskeletons, but not membrane. The weak base (chloroquine) was immunochemically detected in intravacuolar organelles, but not in vacuoles. Early vacuolization was reversible, but long-term vacuolization caused cell death. The vacuolization and cell death were blocked by the vacuolar H(+)-ATPase inhibitor and Cl--free medium. Staining with LysoTracker or LysoSensor indicated that intravacuolar organelles were strongly acidic and vacuoles were slightly acidic. This suggests that vacuolization is caused by accumulation of weak base and H(+) in acidic organelles, driven by vacuolar H(+)-ATPase associated with Cl(-) entering, and probably by subsequent extrusion of H(+) and water from organelles to the surrounding cytoplasm.

  3. Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation

    Science.gov (United States)

    Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

    2017-01-01

    Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration. PMID:28915712

  4. Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

    International Nuclear Information System (INIS)

    Lombardi, M.L.; Knecht, D.A.; Lee, J.

    2008-01-01

    The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca 2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca 2+ ] i ) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca 2+ ] i . We observed small, brief, Ca 2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd 3+ ) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca 2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells

  5. Control of cyclin C levels during development of Dictyostelium.

    Directory of Open Access Journals (Sweden)

    David M Greene

    2010-05-01

    Full Text Available Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores. Cells in which the gene encoding Cdk8 has been deleted fail to enter aggregates due to a failure of early gene expression.We have monitored the expression levels of cyclin C protein during development and find levels decrease after the multicellular mound is formed. This decrease is triggered by extracellular cAMP that, in turn, is working in part through an increase in intracellular cAMP. The loss of cyclin C is coincident with a reduction in the association of Cdk8 with a high molecular weight complex in the nucleus. Overexpression of cyclin C and Cdk8 lead to an increased rate of early development, consistent with the levels being rate limiting.Overall these results show that both cyclin C and Cdk8 are regulated during development in response to extracellular signals and the levels of these proteins are important in controlling the timing of developmental processes. These findings have important implications for the role of these proteins in controlling development, suggesting that they are targets for developmental signals to regulate gene expression.

  6. Neuronal vacuolation and spinocerebellar degeneration associated with altered neurotransmission

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    Aggeliki Giannakopoulou

    2017-06-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species, such as the domestic dog (Canis lupus familiaris, and many are caused by mutations in the same genes as corresponding human conditions. In the present study, we report an inherited neurodegenerative condition, termed ‘neuronal vacuolation and spinocerebellar degeneration’ (NVSD which affects neonatal or young dogs, mainly Rottweilers, which recently has been linked with the homozygosity for the RAB3GAP1:c.743delC allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM, a severe developmental disorder characterized predominantly by abnormalities of the nervous system including axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 proteins, respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, autophagy, axonal transport and synaptic transmission. The present study attempts to carry out a detailed histopathological examination of NVSD disease, extending from peripheral nerves to lower brain structures focusing on the neurotransmitter alterations noted in the cerebellum, the major structure affected. NVSD dogs presented with progressive cerebellar ataxia and some clinical manifestations that recapitulate the WARBM phenotype. Neuropathological examination revealed dystrophic axons, neurodegeneration and intracellular vacuolization in specific nuclei. In the cerebellum, severe vacuolation of cerebellar nuclei neurons, atrophy of Purkinje cells, and diminishing of GABAergic and glutamatergic fibres constitute the most striking lesions. The balance of evidence suggests that the neuropathological lesions are a reaction to the altered neurotransmission. The canine phenotype could serve as a model to delineate the disease-causing pathological mechanisms in RAB3GAP1 mutation.

  7. Thioploca spp: filamentous sulfur bacteria with nitrate vacuoles

    DEFF Research Database (Denmark)

    Jørgensen, BB; Gallardo, VA

    1999-01-01

    communities of large Thioploca species live along the Pacific coast of South America and in other upwelling areas of high organic matter sedimentation with bottom waters poor in oxygen and rich in nitrate. Each cell of these thioplocas harbors a large liquid vacuole which is used as a storage for nitrate...... with a concentration of lip to 506 mM. The nitrate is used as an electron acceptor for sulfide oxidation and the bacteria may grow autotrophically or mixotrophically using acetate or other organic molecules as carbon source. The filaments stretch up into the overlying seawater, from which they take up nitrate...

  8. Purification and proteomics of pathogen-modified vacuoles and membranes

    Directory of Open Access Journals (Sweden)

    Jo-Ana eHerweg

    2015-06-01

    Full Text Available Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e. the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

  9. A Dictyostelium chalone uses G proteins to regulate proliferation

    Directory of Open Access Journals (Sweden)

    Hanson Nana E

    2009-07-01

    Full Text Available Abstract Background Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. Results We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA. Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus, whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. Conclusion This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  10. A Dictyostelium chalone uses G proteins to regulate proliferation.

    Science.gov (United States)

    Bakthavatsalam, Deenadayalan; Choe, Jonathan M; Hanson, Nana E; Gomer, Richard H

    2009-07-27

    Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  11. Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

    Science.gov (United States)

    van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

    2015-04-01

    Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  12. Evolutionarily conserved sites in yeast tropomyosin function in cell polarity, transport and contractile ring formation

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    Susanne Cranz-Mileva

    2015-08-01

    Full Text Available Tropomyosin is a coiled-coil protein that binds and regulates actin filaments. The tropomyosin gene in Schizosaccharomyces pombe, cdc8, is required for formation of actin cables, contractile rings, and polar localization of actin patches. The roles of conserved residues were investigated in gene replacement mutants. The work validates an evolution-based approach to identify tropomyosin functions in living cells and sites of potential interactions with other proteins. A cdc8 mutant with near-normal actin affinity affects patch polarization and vacuole fusion, possibly by affecting Myo52p, a class V myosin, function. The presence of labile residual cell attachments suggests a delay in completion of cell division and redistribution of cell patches following cytokinesis. Another mutant with a mild phenotype is synthetic negative with GFP-fimbrin, inferring involvement of the mutated tropomyosin sites in interaction between the two proteins. Proteins that assemble in the contractile ring region before actin do so in a mutant cdc8 strain that cannot assemble condensed actin rings, yet some cells can divide. Of general significance, LifeAct-GFP negatively affects the actin cytoskeleton, indicating caution in its use as a biomarker for actin filaments.

  13. Identification and characterization of peptide: N- glycanase from Dictyostelium discoideum

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    Gosain Anuradha

    2012-06-01

    Full Text Available Abstract Background Peptide: N- glycanase (PNGase enzyme cleaves oligosaccharides from the misfolded glycoproteins and prepares them for degradation. This enzyme plays a role in the endoplasmic reticulum associated degradation (ERAD pathway in yeast and mice but its biological importance and role in multicellular development remain largely unknown. Results In this study, the PNGase from the cellular slime mold, Dictyostelium discoideum (DdPNGase was identified based on the presence of a common TG (transglutaminase core domain and its sequence homology with the known PNGases. The domain architecture and the sequence comparison validated the presence of probable functional domains in DdPNGase. The tertiary structure matched with the mouse PNGase. Here we show that DdPNGase is an essential protein, required for aggregation during multicellular development and a knockout strain of it results in small sized aggregates, all of which did not form fruiting bodies. The in situ hybridization and RT-PCR results show higher level of expression during the aggregate stage. The expression gets restricted to the prestalk region during later developmental stages. DdPNGase is a functional peptide:N-glycanase enzyme possessing deglycosylation activity, but does not possess any significant transamidation activity. Conclusions We have identified and characterized a novel PNGase from D. discoideum and confirmed its deglycosylation activity. The results emphasize the importance of PNGase in aggregation during multicellular development of this organism.

  14. Crystallization and preliminary characterization of dihydropteridine reductase from Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Chen, Cong; Seo, Kyung Hye; Kim, Hye Lim; Zhuang, Ningning; Park, Young Shik; Lee, Kon Ho

    2008-01-01

    The dihydropteridine reductase from D. discoideum has been crystallized. Diffraction data were collected from a rectangular-shaped crystal to 2.16 Å resolution. Dihydropteridine reductase from Dictyostelium discoideum (dicDHPR) can produce d-threo-BH 4 [6R-(1′R,2′R)-5,6,7,8-tetrahydrobiopterin], a stereoisomer of l-erythro-BH 4 , in the last step of tetrahydrobiopterin (BH 4 ) recycling. In this reaction, DHPR uses NADH as a cofactor to reduce quinonoid dihydrobiopterin back to BH 4 . To date, the enzyme has been purified to homogeneity from many sources. In this report, the dicDHPR–NAD complex has been crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as a precipitant. Rectangular-shaped crystals were obtained. Crystals grew to maximum dimensions of 0.4 × 0.6 × 0.1 mm. The crystal belonged to space group P2 1 , with unit-cell parameters a = 49.81, b = 129.90, c = 78.76 Å, β = 100.00°, and contained four molecules in the asymmetric unit, forming two closely interacting dicDHPR–NAD dimers. Diffraction data were collected to 2.16 Å resolution using synchrotron radiation. The crystal structure has been determined using the molecular-replacement method

  15. The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants.

    Science.gov (United States)

    Kringstad, R; Kenyon, W H; Black, C C

    1980-09-01

    A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.The yield of vacuoles is approximately 44%. The major vacuole layer is 15 hours when left in Ficoll; however, dispersion into media of various osmotic concentrations resulted in decreased stability. Addition of mercaptobenzothiazole, CaCl(2), MgCl(2), bovine serum albumin, ethylenediaminetetraacetic acid, polyethylene glycol 600, and KH(2)PO(4) to the vacuole isolation media did not increase the stability of the isolated vacuoles.THIS TECHNIQUE WITH ONLY SLIGHT MODIFICATIONS HAS BEEN USED TO ISOLATE LEAF CELL VACUOLES FROM THE FOLLOWING CRASSULACEAN ACID METABOLISM PLANTS: pineapple, Kalanchoë fedtschenkoi, and Echeveria elegans. Spinach leaves also were used successfully.

  16. pH measurement of tubular vacuoles of an arbuscular mycorrhizal fungus, Gigaspora margarita.

    Science.gov (United States)

    Funamoto, Rintaro; Saito, Katsuharu; Oyaizu, Hiroshi; Aono, Toshihiro; Saito, Masanori

    2015-01-01

    Arbuscular mycorrhizal fungi play an important role in phosphate supply to the host plants. The fungal hyphae contain tubular vacuoles where phosphate compounds such as polyphosphate are accumulated. Despite their importance for the phosphate storage, little is known about the physiological properties of the tubular vacuoles in arbuscular mycorrhizal fungi. As an indicator of the physiological state in vacuoles, we measured pH of tubular vacuoles in living hyphae of arbuscular mycorrhizal fungus Gigaspora margarita using ratio image analysis with pH-dependent fluorescent probe, 6-carboxyfluorescein. Fluorescent images of the fine tubular vacuoles were obtained using a laser scanning confocal microscope, which enabled calculation of vacuolar pH with high spatial resolution. The tubular vacuoles showed mean pH of 5.6 and a pH range of 5.1-6.3. These results suggest that the tubular vacuoles of arbuscular mycorrhizal fungi have a mildly acidic pH just like vacuoles of other fungal species including yeast and ectomycorrhizal fungi.

  17. A new social gene in Dictyostelium discoideum, chtB

    Directory of Open Access Journals (Sweden)

    Santorelli Lorenzo A

    2013-01-01

    Full Text Available Abstract Background Competitive social interactions are ubiquitous in nature, but their genetic basis is difficult to determine. Much can be learned from single gene knockouts in a eukaryote microbe. The mutants can be competed with the parent to discern the social impact of that specific gene. Dictyostelium discoideum is a social amoeba that exhibits cooperative behavior in the construction of a multicellular fruiting body. It is a good model organism to study the genetic basis of cooperation since it has a sequenced genome and it is amenable to genetic manipulation. When two strains of D. discoideum are mixed, a cheater strain can exploit its social partner by differentiating more spore than its fair share relative to stalk cells. Cheater strains can be generated in the lab or found in the wild and genetic analyses have shown that cheating behavior can be achieved through many pathways. Results We have characterized the knockout mutant chtB, which was isolated from a screen for cheater mutants that were also able to form normal fruiting bodies on their own. When mixed in equal proportions with parental strain cells, chtB mutants contributed almost 60% of the total number of spores. To do so, chtB cells inhibit wild type cells from becoming spores, as indicated by counts and by the wild type cells’ reduced expression of the prespore gene, cotB. We found no obvious fitness costs (morphology, doubling time in liquid medium, spore production, and germination efficiency associated with the cheating ability of the chtB knockout. Conclusions In this study we describe a new gene in D. discoideum, chtB, which when knocked out inhibits the parental strain from producing spores. Moreover, under lab conditions, we did not detect any fitness costs associated with this behavior.

  18. BTG interacts with retinoblastoma to control cell fate in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Daniele Conte

    Full Text Available BACKGROUND: In the genesis of many tissues, a phase of cell proliferation is followed by cell cycle exit and terminal differentiation. The latter two processes overlap: genes involved in the cessation of growth may also be important in triggering differentiation. Though conceptually distinct, they are often causally related and functional interactions between the cell cycle machinery and cell fate control networks are fundamental to coordinate growth and differentiation. A switch from proliferation to differentiation may also be important in the life cycle of single-celled organisms, and genes which arose as regulators of microbial differentiation may be conserved in higher organisms. Studies in microorganisms may thus contribute to understanding the molecular links between cell cycle machinery and the determination of cell fate choice networks. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that in the amoebozoan D. discoideum, an ortholog of the metazoan antiproliferative gene btg controls cell fate, and that this function is dependent on the presence of a second tumor suppressor ortholog, the retinoblastoma-like gene product. Specifically, we find that btg-overexpressing cells preferentially adopt a stalk cell (and, more particularly, an Anterior-Like Cell fate. No btg-dependent preference for ALC fate is observed in cells in which the retinoblastoma-like gene has been genetically inactivated. Dictyostelium btg is the only example of non-metazoan member of the BTG family characterized so far, suggesting that a genetic interaction between btg and Rb predated the divergence between dictyostelids and metazoa. CONCLUSIONS/SIGNIFICANCE: While the requirement for retinoblastoma function for BTG antiproliferative activity in metazoans is known, an interaction of these genes in the control of cell fate has not been previously documented. Involvement of a single pathway in the control of mutually exclusive processes may have relevant implication in the

  19. Ras proteins have multiple functions in vegetative cells of Dictyostelium.

    Science.gov (United States)

    Bolourani, Parvin; Spiegelman, George; Weeks, Gerald

    2010-11-01

    During the aggregation of Dictyostelium cells, signaling through RasG is more important in regulating cyclic AMP (cAMP) chemotaxis, whereas signaling through RasC is more important in regulating the cAMP relay. However, RasC is capable of substituting for RasG for chemotaxis, since rasG⁻ cells are only partially deficient in chemotaxis, whereas rasC⁻/rasG⁻ cells are totally incapable of chemotaxis. In this study we have examined the possible functional overlap between RasG and RasC in vegetative cells by comparing the vegetative cell properties of rasG⁻, rasC⁻, and rasC⁻/rasG⁻ cells. In addition, since RasD, a protein not normally found in vegetative cells, is expressed in vegetative rasG⁻ and rasC⁻/rasG⁻ cells and appears to partially compensate for the absence of RasG, we have also examined the possible functional overlap between RasG and RasD by comparing the properties of rasG⁻ and rasC⁻/rasG⁻ cells with those of the mutant cells expressing higher levels of RasD. The results of these two lines of investigation show that RasD is capable of totally substituting for RasG for cytokinesis and growth in suspension, whereas RasC is without effect. In contrast, for chemotaxis to folate, RasC is capable of partially substituting for RasG, but RasD is totally without effect. Finally, neither RasC nor RasD is able to substitute for the role that RasG plays in regulating actin distribution and random motility. These specificity studies therefore delineate three distinct and none-overlapping functions for RasG in vegetative cells.

  20. Molecular markers for granulovacuolar degeneration are present in rimmed vacuoles.

    Directory of Open Access Journals (Sweden)

    Masahiro Nakamori

    Full Text Available BACKGROUND: Rimmed vacuoles (RVs are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM and distal myopathy with RVs (DMRV. Granulovacuolar degeneration (GVD bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers. METHODS: Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1 tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK], (2 lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1, and (3 other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43] in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization. RESULTS: GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs. CONCLUSIONS: These results suggest that RVs of muscle

  1. Actomyosin contractility rotates the cell nucleus.

    Science.gov (United States)

    Kumar, Abhishek; Maitra, Ananyo; Sumit, Madhuresh; Ramaswamy, Sriram; Shivashankar, G V

    2014-01-21

    The cell nucleus functions amidst active cytoskeletal filaments, but its response to their contractile stresses is largely unexplored. We study the dynamics of the nuclei of single fibroblasts, with cell migration suppressed by plating onto micro-fabricated patterns. We find the nucleus undergoes noisy but coherent rotational motion. We account for this observation through a hydrodynamic approach, treating the nucleus as a highly viscous inclusion residing in a less viscous fluid of orientable filaments endowed with active stresses. Lowering actin contractility selectively by introducing blebbistatin at low concentrations drastically reduced the speed and coherence of the angular motion of the nucleus. Time-lapse imaging of actin revealed a correlated hydrodynamic flow around the nucleus, with profile and magnitude consistent with the results of our theoretical approach. Coherent intracellular flows and consequent nuclear rotation thus appear to be an intrinsic property of cells.

  2. Spontaneous actin dynamics in contractile rings

    Science.gov (United States)

    Kruse, Karsten; Wollrab, Viktoria; Thiagarajan, Raghavan; Wald, Anne; Riveline, Daniel

    Networks of polymerizing actin filaments are known to be capable to self-organize into a variety of structures. For example, spontaneous actin polymerization waves have been observed in living cells in a number of circumstances, notably, in crawling neutrophils and slime molds. During later stages of cell division, they can also spontaneously form a contractile ring that will eventually cleave the cell into two daughter cells. We present a framework for describing networks of polymerizing actin filaments, where assembly is regulated by various proteins. It can also include the effects of molecular motors. We show that the molecular processes driven by these proteins can generate various structures that have been observed in contractile rings of fission yeast and mammalian cells. We discuss a possible functional role of each of these patterns. The work was supported by Agence Nationale de la Recherche, France, (ANR-10-LABX-0030-INRT) and by Deutsche Forschungsgemeinschaft through SFB1027.

  3. Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

    Science.gov (United States)

    Li, Dong Xiao; Hu, Hai Yan; Li, Gan; Ru, Zhen Gang; Tian, Hui Qiao

    2017-09-01

    Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca 2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

  4. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  5. Phototaxis during the slug stage of Dictyostelium discoideum: a model study

    NARCIS (Netherlands)

    Marée, A.F.M.; Panfilov, A.V.; Hogeweg, P.

    1999-01-01

    During the slug stage, the cellular slime mould Dictyostelium discoideum moves towards light sources. We have modelled this phototactic behaviour using a hybrid cellular automata/partial differential equation model. In our model, individual amoebae are not able to measure the direction from which

  6. Overexpression of the cAMP Receptor 1 in Growing Dictyostelium Cells

    NARCIS (Netherlands)

    Johnson, Ronald L.; Vaughan, Roxanne A.; Caterina, Michael J.; Haastert, Peter J.M. van; Devreotes, Peter N.

    1991-01-01

    cAR1, the cAMP receptor expressed normally during the early aggregation stage of the Dictyostelium developmental program, has been expressed during the growth stage, when only low amounts of endogenous receptors are present. Transformants expressing cAR1 have 7-40 times over growth stage and

  7. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.W.G.; Haastert, van P.J.M.

    2004-01-01

    The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

  8. Sensitization of Dictyostelium chemotaxis by phosphoinositide-3-kinase-mediated self-organizing signalling patches.

    NARCIS (Netherlands)

    Postma, M.; Roelofs, J.; Goedhart, J.; Loovers, H.M.; Visser, A.J.; van Haastert, P.J.

    2004-01-01

    The leading edge of Dictyostelium cells in chemoattractant gradients can be visualized using green fluorescent protein (GFP) tagged to the pleckstrin-homology (PH) domain of cytosolic regulator of adenylyl cyclase (CRAC), which presumable binds phosphatidylinositol-(3,4,5)triphosphate

  9. Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van; Kesbeke, Fanja

    1983-01-01

    Chemoattractants induce a transient accumulation of cGMP levels in Dictyostelium. Intracellular cGMP levels reach a peak at 10 s and prestimulated cGMP levels are recovered at about 30 s. Intracellular and extracellular cGMP levels were detected simultaneously after stimulation of D. lacteum cells

  10. Normal chemotaxis in Dictyostelium discoideum cells with a depolarized plasma membrane potential

    NARCIS (Netherlands)

    Duijn, Bert van; Vogelzang, Sake A.; Ypey, Dirk L.; Molen, Loek G. van der; Haastert, Peter J.M. van

    1990-01-01

    We examined a possible role for the plasma membrane potential in signal transduction during cyclic AMP-induced chemotaxis in the cellular slime mold Dictyostelium discoideum. Chemotaxis, cyclic GMP and cyclic AMP responses in cells with a depolarized membrane potential were measured. Cells can be

  11. Transient Kinetics of a cGMP-dependent cGMP-specific Phosphodiesterase from Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van

    1984-01-01

    Chemotactic stimulation of Dictyostelium discoideum cells induces a fast transient increase of cGMP levels which reach a peak at 10 s. Prestimulation levels are recovered in ~30 s, which is achieved mainly by the action of a guanosine 3',5'-monophosphate cGMP-specific phosphodiesterase. This enzyme

  12. Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

    NARCIS (Netherlands)

    Engel, Ruchira; Hink, Mark A.; Bosgraaf, Leonard; Haastert, Peter J.M. van; Visser, Antonie J.W.G.

    2004-01-01

    The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

  13. Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

    NARCIS (Netherlands)

    Ruchira, A.; Hink, M.A.; Bosgraaf, L.; Haastert, van P.J.M.; Visser, A.J.W.G.

    2004-01-01

    The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

  14. Vacuolating encephalitis in mice infected by human coronavirus OC43

    International Nuclear Information System (INIS)

    Jacomy, Helene; Talbot, Pierre J.

    2003-01-01

    Involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. Human respiratory coronaviruses (HCoV) can infect neural cells, persist in human brain, and activate myelin-reactive T cells. As a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of HCoV-OC43 through the development of an experimental animal model. Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. This acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. Damage to the CNS was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. Although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals

  15. Nano rare-earth oxides induced size-dependent vacuolization: an independent pathway from autophagy.

    Science.gov (United States)

    Zhang, Ying; Yu, Chenguang; Huang, Guanyi; Wang, Changli; Wen, Longping

    2010-09-07

    Four rare earth oxides have been shown to induce autophagy. Interestingly, we often noticed plentiful vacuolization, which was not always involved in this autophagic process. In this study, we investigated three other rare-earth elements, including Yttrium (Y), Ytterbium (Yb), and Lanthanum (La). Autophagic effect could be induced by all of them but only Y(2)O(3) and Yb(2)O(3) could cause massive vacuolization. Y(2)O(3) and Yb(2)O(3) treated by sonication or centrifugation to reduce particle size were used to test vacuolization level in HeLa cell lines. The results showed that rare earth oxides-induced vacuolization is size-dependent and differs from autophagic pathway. To further clarify the characteristics of this autophagic process, we used MEF Atg-5 (autophagy associated gene 5) knockout cell line, and the result showed that the autophagic process induced by rare earth oxides is Atg-5-dependent and the observed vacuolization was independent from autophagy. Similar results could also be observed in our tests on 3-methyladenine(3-MA), a well-known autophagy inhibitor. In conclusion, for the first time, we clarified the relationship between massive vacuolization and autophagic process induced by rare earth oxides and pointed out the size effect of rare earth oxides on the formation of vacuoles, which give clues to further investigation on the mechanisms underlying their biological effects.

  16. Cell stiffness, contractile stress and the role of extracellular matrix

    International Nuclear Information System (INIS)

    An, Steven S.; Kim, Jina; Ahn, Kwangmi; Trepat, Xavier; Drake, Kenneth J.; Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne; Fredberg, Jeffrey J.; Biswal, Shyam

    2009-01-01

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  17. Cell stiffness, contractile stress and the role of extracellular matrix

    Energy Technology Data Exchange (ETDEWEB)

    An, Steven S., E-mail: san@jhsph.edu [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Kim, Jina [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Ahn, Kwangmi [Division of Biostatistics, Penn State College of Medicine, Hershey, PA 17033 (United States); Trepat, Xavier [CIBER, Enfermedades Respiratorias, 07110 Bunyola (Spain); Drake, Kenneth J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Kumar, Sarvesh; Ling, Guoyu; Purington, Carolyn; Rangasamy, Tirumalai; Kensler, Thomas W.; Mitzner, Wayne [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Fredberg, Jeffrey J. [Division of Molecular and Integrative Physiological Sciences, Harvard School of Public Health, Boston, MA 02115 (United States); Biswal, Shyam [Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room E-7616, Baltimore, MD 21205 (United States); Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, MD 21205 (United States)

    2009-05-15

    Here we have assessed the effects of extracellular matrix (ECM) composition and rigidity on mechanical properties of the human airway smooth muscle (ASM) cell. Cell stiffness and contractile stress showed appreciable changes from the most relaxed state to the most contracted state: we refer to the maximal range of these changes as the cell contractile scope. The contractile scope was least when the cell was adherent upon collagen V, followed by collagen IV, laminin, and collagen I, and greatest for fibronectin. Regardless of ECM composition, upon adherence to increasingly rigid substrates, the ASM cell positively regulated expression of antioxidant genes in the glutathione pathway and heme oxygenase, and disruption of a redox-sensitive transcription factor, nuclear erythroid 2 p45-related factor (Nrf2), culminated in greater contractile scope. These findings provide biophysical evidence that ECM differentially modulates muscle contractility and, for the first time, demonstrate a link between muscle contractility and Nrf2-directed responses.

  18. Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

    Directory of Open Access Journals (Sweden)

    Coreen Johnson

    Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

  19. Cadmium translocation by contractile roots differs from that in regular, non-contractile roots.

    Science.gov (United States)

    Lux, Alexander; Lackovič, Andrej; Van Staden, Johannes; Lišková, Desana; Kohanová, Jana; Martinka, Michal

    2015-06-01

    Contractile roots are known and studied mainly in connection with the process of shrinkage of their basal parts, which acts to pull the shoot of the plant deeper into the ground. Previous studies have shown that the specific structure of these roots results in more intensive water uptake at the base, which is in contrast to regular root types. The purpose of this study was to find out whether the basal parts of contractile roots are also more active in translocation of cadmium to the shoot. Plants of the South African ornamental species Tritonia gladiolaris were cultivated in vitro for 2 months, at which point they possessed well-developed contractile roots. They were then transferred to Petri dishes with horizontally separated compartments of agar containing 50 µmol Cd(NO3)2 in the region of the root base or the root apex. Seedlings of 4-d-old maize (Zea mays) plants, which do not possess contractile roots, were also transferred to similar Petri dishes. The concentrations of Cd in the leaves of the plants were compared after 10 d of cultivation. Anatomical analyses of Tritonia roots were performed using appropriately stained freehand cross-sections. The process of contraction required specific anatomical adaptation of the root base in Tritonia, with less lignified and less suberized tissues in comparison with the subapical part of the root. These unusual developmental characteristics were accompanied by more intensive translocation of Cd ions from the basal part of contractile roots to the leaves than from the apical-subapical root parts. The opposite effects were seen in the non-contractile roots of maize, with higher uptake and transport by the apical parts of the root and lower uptake and transport by the basal part. The specific characteristics of contractile roots may have a significant impact on the uptake of ions, including toxic metals from the soil surface layers. This may be important for plant nutrition, for example in the uptake of nutrients from

  20. A Rab-centric perspective of bacterial pathogen-occupied vacuoles.

    Science.gov (United States)

    Sherwood, Racquel Kim; Roy, Craig R

    2013-09-11

    The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges, including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provide insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    International Nuclear Information System (INIS)

    Polatnick, J.; Wool, S.H.

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [ 3 H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

  2. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    Energy Technology Data Exchange (ETDEWEB)

    Polatnick, J.; Wool, S.H. (United States Department of Agriculture, Science and Education, Greenport, New York (USA). Agricultural Research, Plum Island Animal Disease Center)

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated (/sup 3/H) uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

  3. Oculopharyngeal Weakness, Hypophrenia, Deafness, and Impaired Vision: A Novel Autosomal Dominant Myopathy with Rimmed Vacuoles

    Directory of Open Access Journals (Sweden)

    Ting Chen

    2016-01-01

    Conclusions: We reported a novel autosomal dominant myopathy with rimmed vacuoles characterized by dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision, but the causative gene has not been found and needs further study.

  4. THE TONOPLAST TRANSPORT SYSTEMS OF PLANT VACUOLES AND THEIR POTENTIAL APPLICATION IN BIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    S. V. Isayenkov

    2013-06-01

    Full Text Available The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

  5. Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

    Science.gov (United States)

    Ellis, Kathryn; Bagwell, Jennifer; Bagnat, Michel

    2013-03-04

    The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

  6. Characterization of the anion sensitive ATPase in intact vacuoles of Kalanchoe diagremontiana

    Energy Technology Data Exchange (ETDEWEB)

    Kobza, J.; Uribe, E.G.

    1986-04-01

    A method for the isolation of intact vacuoles from K. daigremontiana was developed which produced high yields of relatively pure vacuoles as determined by marker enzyme contamination. Upon isolation, the vacuoles were stabilized by the inclusion of 5% (w/v) ficoll. Enzyme activity was insensitive to vanadate and azide but was strongly inhibited by DCCD. Enzyme activity was strictly dependent on the inclusion of Mg/sup 2 +/ and was stimulated by anions as depicted by the series, NO/sub 3//sup -/ < Br/sup -/ < SO/sub 4//sup -/ < HCO/sub 3//sup -/ < Cl/sup -/. It was found that in intact vacuoles the ATPase activity was stimulated by phosphate to a level equivalent to that found with the chloride. The enzyme exhibited Michaelis-Menten kinetics with a Km for Mg-ATP complex of 0.51 mM.

  7. Contractility Dispersion in Long QT Syndrome

    Directory of Open Access Journals (Sweden)

    MH Nikoo

    2009-09-01

    Full Text Available Background: Previous studies, using M mode echocardiography, provided unexpected evidence of a mechanical alteration in patients with long QT syndrome. The aim of this study was to evaluate entire left ventricular (LV wall motion characteristics in patients with long QT syndrome using tissue Doppler imaging. Methods: We enrolled 17 patients with congenital long QT syndrome [11 female and 6 male], aged 21 to 45 years. 10 subjects without cardiac disease were also selected as a control group. Two-dimensional tissue Doppler imaging (TDI recording of the LV was obtained from the basal and mid-segments from apical four-chamber, two-chamber, and long-axis views. ‘Myocardial Contraction Duration’ [MCD] was defined as the time from start of R wave on ECG to end of S wave on TDI. MCD was measured in the six LV wall positions: septal, anteroseptal, lateral, inferior, posterior and anterior positions.Results: LV contractility dispersion was significantly greater in long QT syndrome patients compared to control group [0.051 ± 0.011 vs. 0.016 ± 0.06; P < 0.001]. Conclusion: Our study evaluated left ventricular dispersion of contractility duration in patients with long QT syndrome. This mechanical dispersion may be a reflection of the inhomogeneity of repolarisation in the long QT syndrome.

  8. Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells

    International Nuclear Information System (INIS)

    Canut, H.; Alibert, G.; Carrasco, A.; Boudet, A.M.

    1986-01-01

    In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([ 14 C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([ 14 C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole

  9. Vacuolating Cytotoxin of Helicobacter pylori Plays a Role during Colonization in a Mouse Model of Infection

    OpenAIRE

    Salama, Nina R.; Otto, Glen; Tompkins, Lucy; Falkow, Stanley

    2001-01-01

    Helicobacter pylori, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. H. pylori strains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gast...

  10. The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

    DEFF Research Database (Denmark)

    Takeda, Kozue; Cabrera, Margarita; Rohde, Jan

    2008-01-01

    At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate...

  11. The Dictyostelium discoideum cellulose synthase: Structure/function analysis and identification of interacting proteins

    Energy Technology Data Exchange (ETDEWEB)

    Richard L. Blanton

    2004-02-19

    OAK-B135 The major accomplishments of this project were: (1) the initial characterization of dcsA, the gene for the putative catalytic subunit of cellulose synthase in the cellular slime mold Dictyostelium discoideum; (2) the detection of a developmentally regulated event (unidentified, but perhaps a protein modification or association with a protein partner) that is required for cellulose synthase activity (i.e., the dcsA product is necessary, but not sufficient for cellulose synthesis); (3) the continued exploration of the developmental context of cellulose synthesis and DcsA; (4) the isolation of a GFP-DcsA-expressing strain (work in progress); and (5) the identification of Dictyostelium homologues for plant genes whose products play roles in cellulose biosynthesis. Although our progress was slow and many of our results negative, we did develop a number of promising avenues of investigation that can serve as the foundation for future projects.

  12. Contractile injection systems of bacteriophages and related systems

    DEFF Research Database (Denmark)

    Taylor, Nicholas M I; van Raaij, Mark J; Leiman, Petr G

    2018-01-01

    Contractile tail bacteriophages, or myobacteriophages, use a sophisticated biomolecular structure to inject their genome into the bacterial host cell. This structure consists of a contractile sheath enveloping a rigid tube that is sharpened by a spike-shaped protein complex at its tip. The spike ...

  13. Cytoplasmic pH and the regulation of the dictyostelium cell cycle

    NARCIS (Netherlands)

    Aerts, R.J.; Durston, A.J.; Moolenaar, W.H.

    1985-01-01

    Cytoplasmic pH (pHl) was monitored during the cell cycle of synchronous populations of Dictyostelium discoideum by means of a pH “null point” method. There is a cycle of pHl that closely corresponds to the DNA replication cycle, with a minimum of pH 7.20 in interphase and a peak of pH 7.45 during S

  14. Two distinct sensing pathways allow recognition of Klebsiella pneumoniae by Dictyostelium amoebae.

    Science.gov (United States)

    Lima, Wanessa C; Balestrino, Damien; Forestier, Christiane; Cosson, Pierre

    2014-03-01

    Recognition of bacteria by metazoans is mediated by receptors that recognize different types of microorganisms and elicit specific cellular responses. The soil amoebae Dictyostelium discoideum feeds upon a variable mixture of environmental bacteria, and it is expected to recognize and adapt to various food sources. To date, however, no bacteria-sensing mechanisms have been described. In this study, we isolated a Dictyostelium mutant (fspA KO) unable to grow in the presence of non-capsulated Klebsiella pneumoniae bacteria, but growing as efficiently as wild-type cells in the presence of other bacteria, such as Bacillus subtilis. fspA KO cells were also unable to respond to K. pneumoniae and more specifically to bacterially secreted folate in a chemokinetic assay, while they responded readily to B. subtilis. Remarkably, both WT and fspA KO cells were able to grow in the presence of capsulated LM21 K. pneumoniae, and responded to purified capsule, indicating that capsule recognition may represent an alternative, FspA-independent mechanism for K. pneumoniae sensing. When LM21 capsule synthesis genes were deleted, growth and chemokinetic response were lost for fspA KO cells, but not for WT cells. Altogether, these results indicate that Dictyostelium amoebae use specific recognition mechanisms to respond to different K. pneumoniae elements. © 2013 John Wiley & Sons Ltd.

  15. Genome sequence of Haloplasma contractile, an unusual contractile bacterium from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre; Alam, Intikhab; El Dorry, Hamza; Siam, Rania; Robertson, Anthony J.; Bajic, Vladimir B.; Stingl, Ulrich

    2011-01-01

    We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutes and Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

  16. Genome sequence of Haloplasma contractile, an unusual contractile bacterium from a deep-sea anoxic brine lake.

    KAUST Repository

    Antunes, Andre

    2011-09-01

    We present the draft genome of Haloplasma contractile, isolated from a deep-sea brine and representing a new order between Firmicutes and Mollicutes. Its complex morphology with contractile protrusions might be strongly influenced by the presence of seven MreB/Mbl homologs, which appears to be the highest copy number ever reported.

  17. Considerations for Contractile Electroactive Materials and Actuators

    Energy Technology Data Exchange (ETDEWEB)

    Lenore Rasmussen, David Schramm, Paul Rasmussen, Kevin Mullaly, Ras Labs, LLC, Intelligent Materials for Prosthetics & Automation, Lewis D. Meixler, Daniel Pearlman and Alice Kirk

    2011-05-23

    Ras Labs produces contractile electroactive polymer (EAP) based materials and actuators that bend, swell, ripple, and contract (new development) with low electric input. In addition, Ras Labs produces EAP materials that quickly contract and expand, repeatedly, by reversing the polarity of the electric input, which can be cycled. This phenomenon was explored using molecular modeling, followed by experimentation. Applied voltage step functions were also investigated. High voltage steps followed by low voltage steps produced a larger contraction followed by a smaller contraction. Actuator control by simply adjusting the electric input is extremely useful for biomimetic applications. Muscles are able to partially contract. If muscles could only completely contract, nobody could hold an egg, for example, without breaking it. A combination of high and low voltage step functions could produce gross motor function and fine manipulation within the same actuator unit. Plasma treated electrodes with various geometries were investigated as a means of providing for more durable actuation.

  18. The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

    Science.gov (United States)

    Alvarez, Diego E; Agaisse, Hervé

    2016-06-01

    Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  19. Role of phospholipase C in Dictyostelium : Formation of inositol 1,4,5-trisphosphate and normal development in cells lacking phospholipase C activity

    NARCIS (Netherlands)

    Drayer, A. Lyndsay; Kaay, Jeroen van der; Mayr, Georg W.; Haastert, Peter J.M. van

    1994-01-01

    The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphabidylinositol-specific phospholipase C

  20. Xpf and not the Fanconi anaemia proteins or Rev3 accounts for the extreme resistance to cisplatin in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Xiao-Yin Zhang

    2009-09-01

    Full Text Available Organisms like Dictyostelium discoideum, often referred to as DNA damage "extremophiles", can survive exposure to extremely high doses of radiation and DNA crosslinking agents. These agents form highly toxic DNA crosslinks that cause extensive DNA damage. However, little is known about how Dictyostelium and the other "extremophiles" can tolerate and repair such large numbers of DNA crosslinks. Here we describe a comprehensive genetic analysis of crosslink repair in Dictyostelium discoideum. We analyse three gene groups that are crucial for a replication-coupled repair process that removes DNA crosslinks in higher eukarya: The Fanconi anaemia pathway (FA, translesion synthesis (TLS, and nucleotide excision repair. Gene disruption studies unexpectedly reveal that the FA genes and the TLS enzyme Rev3 play minor roles in tolerance to crosslinks in Dictyostelium. However, disruption of the Xpf nuclease subcomponent results in striking hypersensitivity to crosslinks. Genetic interaction studies reveal that although Xpf functions with FA and TLS gene products, most Xpf mediated repair is independent of these two gene groups. These results suggest that Dictyostelium utilises a distinct Xpf nuclease-mediated repair process to remove crosslinked DNA. Other DNA damage-resistant organisms and chemoresistant cancer cells might adopt a similar strategy to develop resistance to DNA crosslinking agents.

  1. Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles

    Science.gov (United States)

    Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E.

    2015-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol. PMID:25822753

  2. Genetic fuzzy system predicting contractile reactivity patterns of small arteries

    DEFF Research Database (Denmark)

    Tang, J; Sheykhzade, Majid; Clausen, B F

    2014-01-01

    strategies. Results show that optimized fuzzy systems (OFSs) predict contractile reactivity of arteries accurately. In addition, OFSs identified significant differences that were undetectable using conventional analysis in the responses of arteries between groups. We concluded that OFSs may be used...

  3. Regional gastrointestinal contractility parameters using the wireless motility capsule

    DEFF Research Database (Denmark)

    Farmer, A D; Wegeberg, A-M L; Brock, B

    2018-01-01

    BACKGROUND: The wireless motility capsule concurrently measures temperature, pH and pressure as it traverses the gastrointestinal tract. AIMS: To describe normative values for motility/contractility parameters across age, gender and testing centres. METHODS: Healthy participants underwent...

  4. Considerations for Contractile Electroactive Materials and Actuators

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Lenore; Erickson, Carl J.; Meixler, Lewis D.; Ascione, George; Gentile, Charles A.; Tilson, Carl; Bernasek, Stephen L.; Abelev, Esta

    2010-02-19

    Ras Labs produces electroactive polymer (EAP) based materials and actuators that bend, swell, ripple and now contract (new development) with low electric input. This is an important attribute because of the ability of contraction to produce life-like motion. The mechanism of contraction is not well understood. Radionuclide-labeled experiments were conducted to follow the movement of electrolytes and water in these EAPs when activated. Extreme temperature experiments were performed on the contractile EAPs with very favorable results. One of the biggest challenges in developing these actuators, however, is the electrode-EAP interface because of the pronounced movement of the EAP. Plasma treatments of metallic electrodes were investigated in order to improve the attachment of the embedded electrodes to the EAP material. Surface analysis, adhesive testing, and mechanical testing were conducted to test metal surfaces and metal-polymer interfaces. The nitrogen plasma treatment of titanium produced a strong metal-polymer interface; however, oxygen plasma treatment of both stainless steel and titanium produced even stronger metal-polymer interfaces. Plasma treatment of the electrodes allows for the embedded electrodes and the EAP material of the actuator to work and move as a unit, with no detachment, by significantly improving the metal-polymer interface.

  5. Operative contractility: a functional concept of the inotropic state.

    Science.gov (United States)

    Curiel, Roberto; Perez-Gonzalez, Juan; Torres, Edwar; Landaeta, Ruben; Cerrolaza, Miguel

    2005-10-01

    1. Initial unsuccessful attempts to evaluate ventricular function in terms of the 'heart as a pump' led to focusing on the 'heart as a muscle' and to the concept of myocardial contractility. However, no clinically ideal index exists to assess the contractile state. The aim of the present study was to develop a mathematical model to assess cardiac contractility. 2. A tri-axial system was conceived for preload (PL), afterload (AL) and contractility, where stroke volume (SV) was represented as the volume of the tetrahedron. Based on this model, 'operative' contractility ('OperCon') was calculated from the readily measured values of PL, AL and SV. The model was tested retrospectively under a variety of different experimental and clinical conditions, in 71 studies in humans and 29 studies in dogs. A prospective echocardiographic study was performed in 143 consecutive subjects to evaluate the ability of the model to assess contractility when SV and PL were measured volumetrically (mL) or dimensionally (cm). 3. With inotropic interventions, OperCon changes were comparable to those of ejection fraction (EF), velocity of shortening (Vcf) and dP/dt-max. Only with positive inotropic interventions did elastance (Ees) show significantly larger changes. With load manipulations, OperCon showed significantly smaller changes than EF and Ees and comparable changes to Vcf and dP/dt-max. Values of OperCon were similar when AL was represented by systolic blood pressure or wall stress and when volumetric or dimensional values were used. 4. Operative contractility is a reliable, simple and versatile method to assess cardiac contractility.

  6. eIF2α Kinases Control Chalone Production in Dictyostelium discoideum ▿

    Science.gov (United States)

    Bowman, Robert L.; Xiong, Yanhua; Kirsten, Janet H.; Singleton, Charles K.

    2011-01-01

    Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α. PMID:21278229

  7. A retinoblastoma orthologue is required for the sensing of a chalone in Dictyostelium discoideum.

    Science.gov (United States)

    Bakthavatsalam, Deenadayalan; White, Michael J V; Herlihy, Sarah E; Phillips, Jonathan E; Gomer, Richard H

    2014-03-01

    Retinoblastoma-like proteins regulate cell differentiation and inhibit cell proliferation. The Dictyostelium discoideum retinoblastoma orthologue RblA affects the differentiation of cells during multicellular development, but it is unclear whether RblA has a significant effect on Dictyostelium cell proliferation, which is inhibited by the secreted proteins AprA and CfaD. We found that rblA⁻ cells in shaking culture proliferate to a higher density, die faster after reaching stationary density, and, after starvation, have a lower spore viability than wild-type cells, possibly because in shaking culture, rblA⁻ cells have both increased cytokinesis and lower extracellular accumulation of CfaD. However, rblA⁻ cells have abnormally slow proliferation on bacterial lawns. Recombinant AprA inhibits the proliferation of wild-type cells but not that of rblA⁻ cells, whereas CfaD inhibits the proliferation of both wild-type cells and rblA⁻ cells. Similar to aprA⁻ cells, rblA⁻ cells have a normal mass and protein accumulation rate on a per-nucleus basis, indicating that RblA affects cell proliferation but not cell growth. AprA also functions as a chemorepellent, and RblA is required for proper AprA chemorepellent activity despite the fact that RblA does not affect cell speed. Together, our data indicate that an autocrine proliferation-inhibiting factor acts through RblA to regulate cell density in Dictyostelium, suggesting that such factors may signal through retinoblastoma-like proteins to control the sizes of structures such as developing organs or tumors.

  8. Extracellular calmodulin regulates growth and cAMP-mediated chemotaxis in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    O’Day, Danton H.; Huber, Robert J.; Suarez, Andres

    2012-01-01

    Highlights: ► Extracellular calmodulin is present throughout growth and development in Dictyostelium. ► Extracellular calmodulin localizes within the ECM during development. ► Extracellular calmodulin inhibits cell proliferation and increases chemotaxis. ► Extracellular calmodulin exists in eukaryotic microbes. ► Extracellular calmodulin may be functionally as important as intracellular calmodulin. -- Abstract: The existence of extracellular calmodulin (CaM) has had a long and controversial history. CaM is a ubiquitous calcium-binding protein that has been found in every eukaryotic cell system. Calcium-free apo-CaM and Ca 2+ /CaM exert their effects by binding to and regulating the activity of CaM-binding proteins (CaMBPs). Most of the research done to date on CaM and its CaMBPs has focused on their intracellular functions. The presence of extracellular CaM is well established in a number of plants where it functions in proliferation, cell wall regeneration, gene regulation and germination. While CaM has been detected extracellularly in several animal species, including frog, rat, rabbit and human, its extracellular localization and functions are less well established. In contrast the study of extracellular CaM in eukaryotic microbes remains to be done. Here we show that CaM is constitutively expressed and secreted throughout asexual development in Dictyostelium where the presence of extracellular CaM dose-dependently inhibits cell proliferation but increases cAMP mediated chemotaxis. During development, extracellular CaM localizes within the slime sheath where it coexists with at least one CaMBP, the matricellular CaM-binding protein CyrA. Coupled with previous research, this work provides direct evidence for the existence of extracellular CaM in the Dictyostelium and provides insight into its functions in this model amoebozoan.

  9. Differentiation-inducing factor-1 and -2 function also as modulators for Dictyostelium chemotaxis.

    Directory of Open Access Journals (Sweden)

    Hidekazu Kuwayama

    Full Text Available BACKGROUND: In the early stages of development of the cellular slime mold Dictyostelium discoideum, chemotaxis toward cAMP plays a pivotal role in organizing discrete cells into a multicellular structure. In this process, a series of signaling molecules, such as G-protein-coupled cell surface receptors for cAMP, phosphatidylinositol metabolites, and cyclic nucleotides, function as the signal transducers for controlling dynamics of cytoskeleton. Differentiation-inducing factor-1 and -2 (DIF-1 and DIF-2 were originally identified as the factors (chlorinated alkylphenones that induce Dictyostelium stalk cell differentiation, but it remained unknown whether the DIFs had any other physiologic functions. METHODOLOGY/PRINCIPAL FINDINGS: To further elucidate the functions of DIFs, in the present study we investigated their effects on chemotaxis under various conditions. Quite interestingly, in shallow cAMP gradients, DIF-1 suppressed chemotaxis whereas DIF-2 promoted it greatly. Analyses with various mutants revealed that DIF-1 may inhibit chemotaxis, at least in part, via GbpB (a phosphodiesterase and a decrease in the intracellular cGMP concentration ([cGMP](i. DIF-2, by contrast, may enhance chemotaxis, at least in part, via RegA (another phosphodiesterase and an increase in [cGMP](i. Using null mutants for DimA and DimB, the transcription factors that are required for DIF-dependent prestalk differentiation, we also showed that the mechanisms for the modulation of chemotaxis by DIFs differ from those for the induction of cell differentiation by DIFs, at least in part. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that DIF-1 and DIF-2 function as negative and positive modulators for Dictyostelium chemotaxis, respectively. To our knowledge, this is the first report in any organism of physiologic modulators (small molecules for chemotaxis having differentiation-inducing activity.

  10. Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

    OpenAIRE

    Choe, Jonathan M; Bakthavatsalam, Deenadayalan; Phillips, Jonathan E; Gomer, Richard H

    2009-01-01

    Abstract Background Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the...

  11. Characterization of a 1,4-. beta. -D-glucan synthase from Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Blanton, R.L.

    1992-01-15

    Various aspects of research concerning Dictyostelium discoideum are presented. The initial focus of this project was upon: the characterization of potential probes for the cellulose synthase (antibody and nucleic acid), the determination of the cultural induction conditions of cellulose synthesis, the solubilization of the enzyme activity, the development of a non-inhibitory disruption buffer, the generation and isolation of mutant strains deficient in cellulose synthesis, and the development of the capability to determine the degree of polymerization of the in vitro product. I have briefly summarized our most significant findings with only selected data sets being shown in this report in the interest of brevity.

  12. The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways

    OpenAIRE

    Knetsch, Menno L.W.; Schäfers, Nicole; Horstmann, Heinz; Manstein, Dietmar J.

    2001-01-01

    Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show t...

  13. Identification of Pentatricopeptide Repeat Proteins in the Model Organism Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Sam Manna

    2013-01-01

    Full Text Available Pentatricopeptide repeat (PPR proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protist Dictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localizes to the mitochondria, and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction.

  14. Integrin Subunit CD18 Is the T-Lymphocyte Receptor for the Helicobacter pylori Vacuolating Cytotoxin

    Czech Academy of Sciences Publication Activity Database

    Sewald, X.; Gebert-Vogl, B.; Prassl, S.; Barwig, I.; Weiss, E.; Fabbri, M.; Osička, Radim; Schiemann, M.; Busch, D. H.; Semmrich, M.; Holzmann, B.; Šebo, Peter; Haas, R.

    2008-01-01

    Roč. 3, č. 1 (2008), s. 20-29 ISSN 1931-3128 R&D Projects: GA ČR GP204/07/P105 Institutional research plan: CEZ:AV0Z50200510 Keywords : helicobacter pylori * vacuolating cytotoxin * adenocarcinoma Subject RIV: EE - Microbiology, Virology Impact factor: 7.436, year: 2008

  15. Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

    Science.gov (United States)

    Puchkov, Evgeny O

    2010-06-01

    In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

  16. Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation

    Czech Academy of Sciences Publication Activity Database

    Liu, Q.; Vain, T.; Viotti, C.; Doyle, S. M.; Tarkowská, Danuše; Novák, Ondřej; Zipfel, C.; Sitbon, F.; Robert, S.; Hofius, D.

    2018-01-01

    Roč. 11, č. 4 (2018), s. 553-567 ISSN 1674-2052 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis * brassinosteroid signaling * DUF300 proteins * tonoplast * vacuole integrity Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 8.827, year: 2016

  17. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  18. Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles

    Science.gov (United States)

    Cna'ani, Alon; Shavit, Reut; Ravid, Jasmin; Aravena-Calvo, Javiera; Skaliter, Oded; Masci, Tania; Vainstein, Alexander

    2017-01-01

    Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography–mass spectrometry (GC–MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger β-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC–MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites. PMID:29163617

  19. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

    Science.gov (United States)

    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

  20. Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago

    NARCIS (Netherlands)

    Gavrin, A.Y.; Kaiser, B.N.; Geiger, D.; Tyerman, S.D.; Wen, Z.; Bisseling, T.; Fedorova, E.E.

    2014-01-01

    In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected

  1. Effect of drugs on lipid methylation, receptor-adenylate cyclase coupling and cyclic AMP secretion in Dictyostelium discoideum

    NARCIS (Netherlands)

    Van Waarde, Aren; Van Haastert, P.J.M.

    1986-01-01

    Intercellular communication in Dictyostelium discoldeum takes place by means of cyclic AMP-induced cyclic AMP-synthesis and secretion. Since phospholipid methylation has been suggested to play a role in receptor-adenylate cyclase coupling, we examined the effects of transmethylation inhibitors on

  2. Cyclic AMP signalling in Dictyostelium : G-proteins activate separate Ras pathways using specific RasGEFs

    NARCIS (Netherlands)

    Kae, Helmut; Kortholt, Arjan; Rehmann, Holger; Insall, RobertH.; Van Haastert, Peter J. M.; Spiegelman, George B.; Weeks, Gerald

    In general, mammalian Ras guanine nucleotide exchange factors (RasGEFs) show little substrate specificity, although they are often thought to regulate specific pathways. Here, we provide in vitro and in vivo evidence that two RasGEFs can each act on specific Ras proteins. During Dictyostelium

  3. Nucleus-associated phosphorylation of Ins(1,4,5)P3 to InsP6 in Dictyostelium

    NARCIS (Netherlands)

    Kaay, Jeroen van der; Wesseling, Jelle; Haastert, Peter J.M. van

    1995-01-01

    Although many cells contain large amounts of InsP(6), its metabolism and function is still largely unknown. In Dictyostelium lysates, the formation of InsP(6) by sequential phosphorylation of inositol via Ins(3,4,6)P-3 has been described [Stevens and Irvine (1990) Nature (London) 346, 580-583]; the

  4. The role of cGMP and the rear of the cell in Dictyostelium chemotaxis and cell streaming

    NARCIS (Netherlands)

    Veltman, Douwe M.; van Haastert, Peter J. M.

    2008-01-01

    During chemotaxis, pseudopod extensions lead the cell towards the source of attractant. The role of actin-filled pseudopodia at the front of the cell is well recognized, whereas the function of the rear of the cell in chemotaxis and cell-cell interactions is less well known. Dictyostelium cell

  5. A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

    Directory of Open Access Journals (Sweden)

    Sarah E Herlihy

    Full Text Available In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

  6. A Dictyostelium secreted factor requires a PTEN-like phosphatase to slow proliferation and induce chemorepulsion.

    Science.gov (United States)

    Herlihy, Sarah E; Tang, Yitai; Gomer, Richard H

    2013-01-01

    In Dictyostelium discoideum, AprA and CfaD are secreted proteins that inhibit cell proliferation. We found that the proliferation of cells lacking CnrN, a phosphatase and tensin homolog (PTEN)-like phosphatase, is not inhibited by exogenous AprA and is increased by exogenous CfaD. The expression of CnrN in cnrN cells partially rescues these altered sensitivities, suggesting that CnrN is necessary for the ability of AprA and CfaD to inhibit proliferation. Cells lacking CnrN accumulate normal levels of AprA and CfaD. Like cells lacking AprA and CfaD, cnrN cells proliferate faster and reach a higher maximum cell density than wild type cells, tend to be multinucleate, accumulate normal levels of mass and protein per nucleus, and form less viable spores. When cnrN cells expressing myc-tagged CnrN are stimulated with a mixture of rAprA and rCfaD, levels of membrane-associated myc-CnrN increase. AprA also causes chemorepulsion of Dictyostelium cells, and CnrN is required for this process. Combined, these results suggest that CnrN functions in a signal transduction pathway downstream of AprA and CfaD mediating some, but not all, of the effects of AprA and CfaD.

  7. Caffeine sensitive repair and mutation induction in UV- or γ-ray-irradiated Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Kanishi, Nobuji; Kinjo, Yasuhito; Watanabe, Makoto.

    1990-01-01

    It seems that certain kinds of chemical substances increase the distortion in molecules, change the high order microstructures of nuclei and chromosomes, and exert large variation to the function of repairing the damage of genes due to radiation and others, by coupling with DNA, protein or enzyme system. It has been well known that caffeine is one of such compounds, and by coupling with DNA, it increases the damage due to ultraviolet ray and gives the action of obstructing repair in addition to the action of inducing the abnormality of chromosomes and mutation. Dictyostelium discoideum has the simplest nuclear structure, and shows extremely high resistance to radiation by its high restoration ability. The authors have advanced the research by paying attention to its characteristics, and comparing the Dictyostelium discoideum as one model system with the lymphocyte system of higher animals. This time, the authors analyzed the characteristics of two kinds of sensitivity repair process of caffeine, and investigated into their relation with the occurrence of mutation. The experimental method and the results are reported. (K.I.)

  8. Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium.

    Science.gov (United States)

    Li, Cheng-Lin Frank; Santhanam, Balaji; Webb, Amanda Nicole; Zupan, Blaž; Shaulsky, Gad

    2016-09-01

    Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. © 2016 Li et al.; Published by Cold Spring Harbor Laboratory Press.

  9. Dictyostelium discoideum as a novel host system to study the interaction between phagocytes and yeasts

    Directory of Open Access Journals (Sweden)

    Barbara Koller

    2016-10-01

    Full Text Available The social amoeba Dictyostelium discoideum is a well-established model organism to study the interaction between bacteria and phagocytes. In contrast, research using D. discoideum as a host model for fungi is rare. We describe a comprehensive study, which uses D. discoideum as a host model system to investigate the interaction with apathogenic (Saccharomyces cerevisiae and pathogenic (Candida sp. yeast. We show that Dictyostelium can be co-cultivated with yeasts on solid media, offering a convenient test to study the interaction between fungi and phagocytes. We demonstrate that a number of D. discoideum mutants increase (atg1-, kil1-, kil2- or decrease (atg6- the ability of the amoebae to predate yeast cells. On the yeast side, growth characteristics, reduced phagocytosis rate, as well as known virulence factors of C. albicans (EFG1, CPH1, HGC1, ICL1 contribute to the resistance of yeast cells against predation by the amoebae. Investigating haploid C. albicans strains, we suggest using the amoebae plate test for screening purposes after random mutagenesis. Finally, we discuss the potential of our adapted amoebae plate test to use D. discoideum for risk assessment of yeast strains.

  10. Nanovesicles released by Dictyostelium cells: a potential carrier for drug delivery.

    Science.gov (United States)

    Lavialle, Françoise; Deshayes, Sophie; Gonnet, Florence; Larquet, Eric; Kruglik, Sergei G; Boisset, Nicolas; Daniel, Régis; Alfsen, Annette; Tatischeff, Irène

    2009-10-01

    Nanovesicles released by Dictyostelium discoideum cells grown in the presence of the DNA-specific dye Hoechst 33342 have been previously shown to mediate the transfer of the dye into the nuclei of Hoechst-resistant cells. The present investigation extends this work by conducting experiments in the presence of hypericin, a fluorescent therapeutic photosensitizer assayed for antitumoral photodynamic therapy. Nanovesicles released by Dictyostelium cells exhibit an averaged diameter between 50 and 150 nm, as measured by transmission cryoelectron microscopy. A proteomic analysis reveals a predominance of actin and actin-related proteins. The detection of a lysosomal membrane protein (LIMP II) indicates that these vesicles are likely generated in the late endosomal compartment. The use of the hypericin-containing nanovesicles as nanodevices for in vitro drug delivery was investigated by fluorescence microscopy. The observed signal was almost exclusively located in the perinuclear area of two human cell lines, skin fibroblasts (HS68) and cervix carcinoma (HeLa) cells. Studies by confocal microscopy with specific markers of cell organelles, provided evidence that hypericin was accumulated in the Golgi apparatus. All these data shed a new light on in vitro drug delivery by using cell-released vesicles as carriers.

  11. Caffeine sensitive repair and mutation induction in UV- or. gamma. -ray-irradiated Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Kanishi, Nobuji (Tokyo Metropolitan Research Lab. of Public Health (Japan)); Kinjo, Yasuhito; Watanabe, Makoto

    1990-01-01

    It seems that certain kinds of chemical substances increase the distortion in molecules, change the high order microstructures of nuclei and chromosomes, and exert large variation to the function of repairing the damage of genes due to radiation and others, by coupling with DNA, protein or enzyme system. It has been well known that caffeine is one of such compounds, and by coupling with DNA, it increases the damage due to ultraviolet ray and gives the action of obstructing repair in addition to the action of inducing the abnormality of chromosomes and mutation. Dictyostelium discoideum has the simplest nuclear structure, and shows extremely high resistance to radiation by its high restoration ability. The authors have advanced the research by paying attention to its characteristics, and comparing the Dictyostelium discoideum as one model system with the lymphocyte system of higher animals. This time, the authors analyzed the characteristics of two kinds of sensitivity repair process of caffeine, and investigated into their relation with the occurrence of mutation. The experimental method and the results are reported. (K.I.).

  12. Determination of inositol 1,4,5-trisphosphate levels in Dictyostelium by isotope dilution assay

    International Nuclear Information System (INIS)

    Van Haastert, P.J.

    1989-01-01

    A commercial isotope dilution assay was used for the determination of Ins(1,4,5)P3 levels in the microorganism Dictyostelium discoideum. Cross-reactivity in the assay was detected with extracts from cells and the medium. The compound which induced this cross-reactivity was tentatively identified as Ins(1,4,5)P3 by (i) codegradation with authentic [ 32 P]Ins(1,4,5)P3 by three specific Ins(1,4,5)P3 phosphatases, and (ii) co-chromatography with authentic [ 32 P]Ins(1,4,5)P3 on HPLC columns. The cellular concentration was estimated as 165 +/- 42 pmol/10(8) cells, yielding a mean intracellular Ins(1,4,5)P3 concentration of 3.3 microM. Dictyostelium cells secrete large amounts of Ins(1,4,5)P3 at a rate of about 10% of the cellular content per minute, yielding about 0.13 microM extracellular Ins(1,4,5)P3 after 15 min in a suspension of 10(8) cells/ml. The chemoattractant cAMP induced a transient increase of the Ins(1,4,5)P3 concentration; the data suggest an intracacellular rise from 3.3 to 5.5 microM with a maximum at 6 s after stimulation

  13. Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

    LENUS (Irish Health Repository)

    Vlahou, Georgia

    2009-07-14

    Abstract Background All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops). Results The Yersinia pseudotuberculosis virulence factors YopE, YopH, YopM and YopJ were expressed de novo within Dictyostelium and their effects on growth in axenic medium and on bacterial lawns were analyzed. No severe effect was observed for YopH, YopJ and YopM, but expression of YopE, which is a GTPase activating protein for Rho GTPases, was found to be highly detrimental. GFP-tagged YopE expressing cells had less conspicuous cortical actin accumulation and decreased amounts of F-actin. The actin polymerization response upon cAMP stimulation was impaired, although chemotaxis was unaffected. YopE also caused reduced uptake of yeast particles. These alterations are probably due to impaired Rac1 activation. We also found that YopE predominantly associates with intracellular membranes including the Golgi apparatus and inhibits the function of moderately overexpressed RacH. Conclusion The phenotype elicited by YopE in Dictyostelium can be explained, at least in part, by inactivation of one or more Rho family GTPases. It further demonstrates that the social amoeba Dictyostelium discoideum can be used as an efficient and easy-to-handle model organism in order to analyze the function of a translocated GAP protein of a human pathogen.

  14. dictyExpress: a web-based platform for sequence data management and analytics in Dictyostelium and beyond.

    Science.gov (United States)

    Stajdohar, Miha; Rosengarten, Rafael D; Kokosar, Janez; Jeran, Luka; Blenkus, Domen; Shaulsky, Gad; Zupan, Blaz

    2017-06-02

    Dictyostelium discoideum, a soil-dwelling social amoeba, is a model for the study of numerous biological processes. Research in the field has benefited mightily from the adoption of next-generation sequencing for genomics and transcriptomics. Dictyostelium biologists now face the widespread challenges of analyzing and exploring high dimensional data sets to generate hypotheses and discovering novel insights. We present dictyExpress (2.0), a web application designed for exploratory analysis of gene expression data, as well as data from related experiments such as Chromatin Immunoprecipitation sequencing (ChIP-Seq). The application features visualization modules that include time course expression profiles, clustering, gene ontology enrichment analysis, differential expression analysis and comparison of experiments. All visualizations are interactive and interconnected, such that the selection of genes in one module propagates instantly to visualizations in other modules. dictyExpress currently stores the data from over 800 Dictyostelium experiments and is embedded within a general-purpose software framework for management of next-generation sequencing data. dictyExpress allows users to explore their data in a broader context by reciprocal linking with dictyBase-a repository of Dictyostelium genomic data. In addition, we introduce a companion application called GenBoard, an intuitive graphic user interface for data management and bioinformatics analysis. dictyExpress and GenBoard enable broad adoption of next generation sequencing based inquiries by the Dictyostelium research community. Labs without the means to undertake deep sequencing projects can mine the data available to the public. The entire information flow, from raw sequence data to hypothesis testing, can be accomplished in an efficient workspace. The software framework is generalizable and represents a useful approach for any research community. To encourage more wide usage, the backend is open

  15. Calcium-responsive contractility during fertilization in sea urchin eggs.

    Science.gov (United States)

    Stack, Christianna; Lucero, Amy J; Shuster, Charles B

    2006-04-01

    Fertilization triggers a reorganization of oocyte cytoskeleton, and in sea urchins, there is a dramatic increase in cortical F-actin. However, the role that myosin II plays during fertilization remains largely unexplored. Myosin II is localized to the cortical cytoskeleton both before and after fertilization and to examine myosin II contractility in living cells, Lytechinus pictus eggs were observed by time-lapse microscopy. Upon sperm binding, a cell surface deflection traversed the egg that was followed by and dependent on the calcium wave. The calcium-dependence of surface contractility could be reproduced in unfertilized eggs, where mobilization of intracellular calcium in unfertilized eggs under compression resulted in a marked contractile response. Lastly, inhibition of myosin II delayed absorption of the fertilization cone, suggesting that myosin II not only responds to the same signals that activate eggs but also participates in the remodeling of the cortical actomyosin cytoskeleton during the first zygotic cell cycle. (c) 2006 Wiley-Liss, Inc.

  16. Blood pressure and the contractility of a human leg muscle.

    Science.gov (United States)

    Luu, Billy L; Fitzpatrick, Richard C

    2013-11-01

    These studies investigate the relationships between perfusion pressure, force output and pressor responses for the contracting human tibialis anterior muscle. Eight healthy adults were studied. Changing the height of tibialis anterior relative to the heart was used to control local perfusion pressure. Electrically stimulated tetanic force output was highly sensitive to physiological variations in perfusion pressure showing a proportionate change in force output of 6.5% per 10 mmHg. This perfusion-dependent change in contractility begins within seconds and is reversible with a 53 s time constant, demonstrating a steady-state equilibrium between contractility and perfusion pressure. These stimulated contractions did not produce significant cardiovascular responses, indicating that the muscle pressor response does not play a major role in cardiovascular regulation at these workloads. Voluntary contractions at forces that would require constant motor drive if perfusion pressure had remained constant generated a central pressor response when perfusion pressure was lowered. This is consistent with a larger cortical drive being required to compensate for the lost contractility with lower perfusion pressure. The relationship between contractility and perfusion for this large postural muscle was not different from that of a small hand muscle (adductor pollicis) and it responded similarly to passive peripheral and active central changes in arterial pressure, but extended over a wider operating range of pressures. If we consider that, in a goal-oriented motor task, muscle contractility determines central motor output and the central pressor response, these results indicate that muscle would fatigue twice as fast without a pressor response. From its extent, timing and reversibility we propose a testable hypothesis that this change in contractility arises through contraction- and perfusion-dependent changes in interstitial K(+) concentration.

  17. Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

    Directory of Open Access Journals (Sweden)

    Tomasz J. Wodzicki

    2015-01-01

    Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

  18. Comparison of contractile and extensile pneumatic artificial muscles

    Science.gov (United States)

    Pillsbury, Thomas E.; Wereley, Norman M.; Guan, Qinghua

    2017-09-01

    Pneumatic artificial muscles (PAMs) are used in robotic and prosthetic applications due to their high power to weight ratio, controllable compliance, and simple design. Contractile PAMs are typically used in traditional hard robotics in place of heavy electric motors. As the field of soft robotics grows, extensile PAMs are beginning to have increased usage. This work experimentally tests, models, and compares contractile and extensile PAMs to demonstrate the advantages and disadvantages of each type of PAM and applications for which they are best suited.

  19. Effect of cadmium on myocardial contractility and calcium fluxes

    International Nuclear Information System (INIS)

    Pilati, C.F.

    1979-01-01

    The effect of cadmium on myocardial mechanical performance and calcium fluxes was studied in kitten isometric papillary muscles and in isovolumic Langendorff-perfused rabbit hearts. Therefore, it is concluded that cadmium-induced decreases in contractility are not primarily the result of cadmium interference with ATP metabolic processes. Furthermore, these results imply that cadmium causes no structural alterations of the contractile proteins. These data suggest that cadmium may be competing with the calcium needed for excitation-contraction coupling. During experiments using radioisotopic calcium, a statistically significant cellular influx of calcium was observed following the onset of 100 μM Cd ++ perfusion of isolated, Langendorff-prepared rabbit hearts

  20. Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2016-02-01

    Full Text Available The glutathione of the red beetroot vacuoles (Beta vulgaris L. was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT; the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB; the high-performance liquid chromatography (HPLC. The content of reduced (GSH and oxidized glutathione (GSSG differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione.

  1. Synergy of cell-cell repulsion and vacuolation in a computational model of lumen formation.

    Science.gov (United States)

    Boas, Sonja E M; Merks, Roeland M H

    2014-03-06

    A key step in blood vessel development (angiogenesis) is lumen formation: the hollowing of vessels for blood perfusion. Two alternative lumen formation mechanisms are suggested to function in different types of blood vessels. The vacuolation mechanism is suggested for lumen formation in small vessels by coalescence of intracellular vacuoles, a view that was extended to extracellular lumen formation by exocytosis of vacuoles. The cell-cell repulsion mechanism is suggested to initiate extracellular lumen formation in large vessels by active repulsion of adjacent cells, and active cell shape changes extend the lumen. We used an agent-based computer model, based on the cellular Potts model, to compare and study both mechanisms separately and combined. An extensive sensitivity analysis shows that each of the mechanisms on its own can produce lumens in a narrow region of parameter space. However, combining both mechanisms makes lumen formation much more robust to the values of the parameters, suggesting that the mechanisms may work synergistically and operate in parallel, rather than in different vessel types.

  2. Synergy of cell–cell repulsion and vacuolation in a computational model of lumen formation

    Science.gov (United States)

    Boas, Sonja E. M.; Merks, Roeland M. H.

    2014-01-01

    A key step in blood vessel development (angiogenesis) is lumen formation: the hollowing of vessels for blood perfusion. Two alternative lumen formation mechanisms are suggested to function in different types of blood vessels. The vacuolation mechanism is suggested for lumen formation in small vessels by coalescence of intracellular vacuoles, a view that was extended to extracellular lumen formation by exocytosis of vacuoles. The cell–cell repulsion mechanism is suggested to initiate extracellular lumen formation in large vessels by active repulsion of adjacent cells, and active cell shape changes extend the lumen. We used an agent-based computer model, based on the cellular Potts model, to compare and study both mechanisms separately and combined. An extensive sensitivity analysis shows that each of the mechanisms on its own can produce lumens in a narrow region of parameter space. However, combining both mechanisms makes lumen formation much more robust to the values of the parameters, suggesting that the mechanisms may work synergistically and operate in parallel, rather than in different vessel types. PMID:24430123

  3. Differential Induction of Cytoplasmic Vacuolization and Methuosis by Novel 2-Indolyl-Substituted Pyridinylpropenones.

    Science.gov (United States)

    Trabbic, Christopher J; Dietsch, Heather M; Alexander, Evan M; Nagy, Peter I; Robinson, Michael W; Overmeyer, Jean H; Maltese, William A; Erhardt, Paul W

    2014-01-09

    Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents that can trigger alternative forms of cell death. Methuosis is a novel form of non-apoptotic cell death characterized by accumulation of vacuoles derived from macropinosomes and endosomes. Previous studies identified an indole-based chalcone, 3-(5-methoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), that induces methuosis in human cancer cells. Herein, we describe the synthesis of related 2-indolyl substituted pyridinylpropenones and their effects on U251 glioblastoma cells. Increasing the size of the 2-indolyl substituent substantially reduces growth inhibitory activity and cytotoxicity, but does not prevent cell vacuolization. Computational models suggest that the results are not due to steric-driven conformational effects. The unexpected uncoupling of vacuolization and cell death implies that the relationship between endosomal perturbations and methuotic cell death is more complex than previously realized. The new series of compounds will be useful in further defining the molecular and cellular mechanisms underlying methuosis.

  4. Mitochondrial Stress Tests Using Seahorse Respirometry on Intact Dictyostelium discoideum Cells.

    Science.gov (United States)

    Lay, Sui; Sanislav, Oana; Annesley, Sarah J; Fisher, Paul R

    2016-01-01

    Mitochondria not only play a critical and central role in providing metabolic energy to the cell but are also integral to the other cellular processes such as modulation of various signaling pathways. These pathways affect many aspects of cell physiology, including cell movement, growth, division, differentiation, and death. Mitochondrial dysfunction which affects mitochondrial bioenergetics and causes oxidative phosphorylation defects can thus lead to altered cellular physiology and manifest in disease. The assessment of the mitochondrial bioenergetics can thus provide valuable insights into the physiological state, and the alterations to the state of the cells. Here, we describe a method to successfully use the Seahorse XF(e)24 Extracellular Flux Analyzer to assess the mitochondrial respirometry of the cellular slime mold Dictyostelium discoideum.

  5. An autoregulatory circuit for long-range self-organization in Dictyostelium cell populations.

    Science.gov (United States)

    Sawai, Satoshi; Thomason, Peter A; Cox, Edward C

    2005-01-20

    Nutrient-deprived Dictyostelium amoebae aggregate to form a multicellular structure by chemotaxis, moving towards propagating waves of cyclic AMP that are relayed from cell to cell. Organizing centres are not formed by founder cells, but are dynamic entities consisting of cores of outwardly rotating spiral waves that self-organize in a homogeneous cell population. Spiral waves are ubiquitously observed in chemical reactions as well as in biological systems. Although feedback control of spiral waves in spatially extended chemical reactions has been demonstrated in recent years, the mechanism by which control is achieved in living systems is unknown. Here we show that mutants of the cyclic AMP/protein kinase A pathway show periodic signalling, but fail to organize coherent long-range wave territories, owing to the appearance of numerous spiral cores. A theoretical model suggests that autoregulation of cell excitability mediated by protein kinase A acts to optimize the number of signalling centres.

  6. Inhalation of Budesonide/Formoterol Increases Diaphragm Muscle Contractility

    Directory of Open Access Journals (Sweden)

    Chiyohiko Shindoh

    2012-01-01

    Conclusions: BUD/FORM inhalation has an inotropic effect on diaphragm muscle, protects diaphragm muscle deterioration after endotoxin injection, and inhibits NO production. Increments in muscle contractility with BUD/FORM inhalation are induced through a synergistic effect of an anti-inflammatory agent and 02-agonist.

  7. Clinical Relationship between Steatocholecystitis and Gallbladder Contractility Measured by Cholescintigraphy

    Directory of Open Access Journals (Sweden)

    Chang Seok Bang

    2015-01-01

    Full Text Available Objective. Contractility of gallbladder is known to be decreased in fatty gallbladder diseases. However, clinical estimation data about this relationship is still lacking. The aim of this study was to investigate the association between steatocholecystitis and contractility of gallbladder. Methods. Patients with cholecystitis (steatocholecystitis versus nonsteatocholecystitis who underwent cholescintigraphy before cholecystectomy were retrospectively evaluated in a single teaching hospital of Korea. The association of steatocholecystitis with contractility of gallbladder, measured by preoperative cholescintigraphy, was assessed by univariable and multivariable analysis. Results. A total of 432 patients were finally enrolled (steatocholecystitis versus nonsteatocholecystitis; 75 versus 357, calculous versus acalculous cholecystitis; 316 versus 116. In the multivariable analysis, age (OR: 0.94, 95% CI: 0.90–0.99, P=0.01 and total serum cholesterol (OR: 1.02, 95% CI: 1.01–1.04, P=0.04 were related to steatocholecystitis in patients with acalculous cholecystitis. Only age (OR: 0.97, 95% CI: 0.94–0.99, P=0.004 was significantly related to steatocholecystitis in patients with calculous cholecystitis. However, ejection fraction of gallbladder reflecting contractility measured by cholescintigraphy was not related to steatocholecystitis irrespective of presence of gallbladder stone in patients with cholecystitis. Conclusion. Ejection fraction of gallbladder measured by cholescintigraphy cannot be used for the detection or confirmation of steatocholecystitis.

  8. Myocardial contractile function in survived neonatal piglets after cardiopulmonary bypass

    Directory of Open Access Journals (Sweden)

    Popov Aron-Frederik

    2010-11-01

    Full Text Available Abstract Background Hemodynamic function may be depressed in the early postoperative stages after cardiac surgery. The aim of this study was the analysis of the myocardial contractility in neonates after cardiopulmonary bypass (CPB and mild hypothermia. Methods Three indices of left ventricular myocardial contractile function (dP/dt, (dP/dt/P, and wall thickening were studied up to 6 hours after CPB in neonatal piglets (CPB group; n = 4. The contractility data were analysed and then compared to the data of newborn piglets who also underwent median thoracotomy and instrumentation for the same time intervals but without CPB (non-CPB group; n = 3. Results Left ventricular dP/dtmax and (dP/dtmax/P remained stable in CPB group, while dP/dtmax decreased in non-CPB group 5 hours postoperatively (1761 ± 205 mmHg/s at baseline vs. 1170 ± 205 mmHg/s after 5 h; p max and (dP/dtmax/P there were no statistically significant differences between the two groups. Comparably, although myocardial thickening decreased in the non-CPB group the differences between the two groups were not statistically significant. Conclusions The myocardial contractile function in survived neonatal piglets remained stable 6 hours after cardiopulmonary bypass and mild hypothermia probably due to regional hypercontractility.

  9. Stalk-length-dependence of the contractility of Vorticella convallaria

    Science.gov (United States)

    Gul Chung, Eun; Ryu, Sangjin

    2017-12-01

    Vorticella convallaria is a sessile protozoan of which the spasmoneme contracts on a millisecond timescale. Because this contraction is induced and powered by the binding of calcium ions (Ca2+), the spasmoneme showcases Ca2+-powered cellular motility. Because the isometric tension of V. convallaria increases linearly with its stalk length, it is hypothesized that the contractility of V. convallaria during unhindered contraction depends on the stalk length. In this study, the contractile force and energetics of V. convallaria cells of different stalk lengths were evaluated using a fluid dynamic drag model which accounts for the unsteadiness and finite Reynolds number of the water flow caused by contracting V. convallaria and the wall effect of the no-slip substrate. It was found that the contraction displacement, peak contraction speed, peak contractile force, total mechanical work, and peak power depended on the stalk length. The observed stalk-length-dependencies were simulated using a damped spring model, and the model estimated that the average spring constant of the contracting stalk was 1.34 nN µm-1. These observed length-dependencies of Vorticella’s key contractility parameters reflect the biophysical mechanism of the spasmonemal contraction, and thus they should be considered in developing a theoretical model of the Vorticella spasmoneme.

  10. RNAi silenced Dd-grp94 (Dictyostelium discoideum glucose-regulated protein 94 kDa) cell lines in Dictyostelium exhibit marked reduction in growth rate and delay in development.

    Science.gov (United States)

    Baviskar, Sandhya N; Shields, Malcolm S

    2010-01-01

    Glucose-regulated 94 kDa protein (Grp94) is a resident of the endoplasmic reticulum (ER) of multicellular eukaryotes. It is a constitutively expressed protein that is overexpressed in certain abnormal conditions of the cell such as depletion of glucose and calcium, and low oxygen and pH. The protein is also implicated in diseased conditions like cancer and Alzheimer's disease. In this study, the consequences of downregulation of Grp94 were investigated at both unicellular and multicellular stages of Dictyostelium discoideum. Previous studies have shown the expression of Dd-Grp94 (Dictyostelium discoideum glucose-regulated 94 kDa protein) in wild-type cells varies during development, and overexpression of Dd-Grp94 leads to abnormal cell shape and inhibition of development (i.e., formation of fruiting bodies). Grp94 is a known calcium binding protein and an efficient calcium buffer. Therefore, in the present study we hypothesized that downregulation of Dd-Grp94 protein would affect Dictyostelium cell structure, growth, and development. We found that Dd-grp94 RNAi recombinants exhibited reduced growth rate, cell size, and a subtle change in cell motility compared to the parental cells. The recombinants also exhibited a delay in development and small fruiting bodies. These results establish that Dd-grp94 plays a crucial role in determining normal cell structure, growth and differentiation.

  11. Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

    International Nuclear Information System (INIS)

    Isobe, Ichiro; Maeno, Yoshitaka; Nagao, Masataka; Iwasa, Mineo; Koyama, Hiroyoshi; Seko-Nakamura, Yoshimi; Monma-Ohtaki, Jun

    2003-01-01

    There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

  12. Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

    Science.gov (United States)

    Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

    2009-01-01

    To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

  13. Curcumin inhibits development and cell adhesion in Dictyostelium discoideum: Implications for YakA signaling and GST enzyme function

    Energy Technology Data Exchange (ETDEWEB)

    Garige, Mamatha; Walters, Eric, E-mail: ewalters@howard.edu

    2015-11-13

    The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.

  14. Curcumin inhibits development and cell adhesion in Dictyostelium discoideum: Implications for YakA signaling and GST enzyme function

    International Nuclear Information System (INIS)

    Garige, Mamatha; Walters, Eric

    2015-01-01

    The molecular basis for nutraceutical properties of the polyphenol curcumin (Curcuma longa, Turmeric) is complex, affecting multiple factors that regulate cell signaling and homeostasis. Here, we report the effect of curcumin on cellular and developmental mechanisms in the eukaryotic model, Dictyostelium discoideum. Dictyostelium proliferation was inhibited in the presence of curcumin, which also suppressed the prestarvation marker, discoidin I, members of the yakA-mediated developmental signaling pathway, and expression of the extracellular matrix/cell adhesion proteins (DdCAD and csA). This resulted in delayed chemotaxis, adhesion, and development of the organism. In contrast to the inhibitory effects on developmental genes, curcumin induced gstA gene expression, overall GST activity, and generated production of reactive oxygen species. These studies expand our knowledge of developmental and biochemical signaling influenced by curcumin, and lends greater consideration of GST enzyme function in eukaryotic cell signaling, development, and differentiation.

  15. Scanning the available Dictyostelium discoideum proteome for O-linked GlcNAc glycosylation sitesusing neural networks

    DEFF Research Database (Denmark)

    Gupta, Ramneek; Jung, Eva; Gooley, Andrew A

    1999-01-01

    Dictyostelium discoideum has been suggested as a eukaryotic model organism for glycobiology studies. Presently, the characteristics of acceptor sites for the N-acetylglucosaminyl-transferases in Dictyostelium discoideum, which link GlcNAc in an alpha linkage to hydroxyl residues, are largely...... unknown. This motivates the development of a species specific method for prediction of O-linked GlcNAc glycosylation sites in secreted and membrane proteins of D. discoideum. The method presented here employs a jury of artificial neural networks. These networks were trained to recognize the sequence...... context and protein surface accessibility in 39 experimentally determined O-alpha-GlcNAc sites found in D. discoideum glycoproteins expressed in vivo. Cross-validation of the data revealed a correlation in which 97% of the glycosylated and nonglycosylated sites were correctly identified. Based...

  16. The Parkinson's disease-associated protein DJ-1 plays a positive nonmitochondrial role in endocytosis in Dictyostelium cells

    Directory of Open Access Journals (Sweden)

    Suwei Chen

    2017-10-01

    Full Text Available The loss of function of DJ-1 caused by mutations in DJ1 causes a form of familial Parkinson's disease (PD. However, the role of DJ-1 in healthy and in PD cells is poorly understood. Even its subcellular localization in mammalian cells is uncertain, with both cytosolic and mitochondrial locations having been reported. We show here that DJ-1 is normally located in the cytoplasm in healthy Dictyostelium discoideum cells. With its unique life cycle, straightforward genotype-phenotype relationships, experimental accessibility and genetic tractability, D. discoideum offers an attractive model to investigate the roles of PD-associated genes. Furthermore, the study of mitochondrial biology, mitochondrial genome transcription and AMP-activated protein kinase-mediated cytopathologies in mitochondrial dysfunction have been well developed in this organism. Unlike mammalian systems, Dictyostelium mitochondrial dysfunction causes a reproducible and readily assayed array of aberrant phenotypes: defective phototaxis, impaired growth, normal rates of endocytosis and characteristic defects in multicellular morphogenesis. This makes it possible to study whether the underlying cytopathological mechanisms of familial PD involve mitochondrial dysfunction. DJ-1 has a single homologue in the Dictyostelium genome. By regulating the expression level of DJ-1 in D. discoideum, we show here that in unstressed cells, DJ-1 is required for normal rates of endocytic nutrient uptake (phagocytosis and, to a lesser extent, pinocytosis and thus growth. Reduced expression of DJ-1 had no effect on phototaxis in the multicellular migratory ‘slug’ stage of the life cycle, but resulted in thickened stalks in the final fruiting bodies. This pattern of phenotypes is distinct from that observed in Dictyostelium to result from mitochondrial dyfunction. Direct measurement of mitochondrial respiratory function in intact cells revealed that DJ-1 knockdown stimulates whereas DJ-1

  17. A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1

    OpenAIRE

    Neumann, Christopher S.; Walsh, Christopher T.; Kay, Robert R.

    2010-01-01

    Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate ...

  18. Increasing cyclic electron flow is related to Na+ sequestration into vacuoles for salt tolerance in soybean.

    Science.gov (United States)

    He, Yi; Fu, Junliang; Yu, Chenliang; Wang, Xiaoman; Jiang, Qinsu; Hong, Jian; Lu, Kaixing; Xue, Gangping; Yan, Chengqi; James, Andrew; Xu, Ligen; Chen, Jianping; Jiang, Dean

    2015-11-01

    In land plants, the NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and drives cyclic electron flow (CEF) around PSI. It also produces extra ATP for photosynthesis and improves plant fitness under conditions of abiotic environmental stress. To elucidate the role of CEF in salt tolerance of the photosynthetic apparatus, Na(+) concentration, chlorophyll fluorescence, and expression of NDH B and H subunits, as well as of genes related to cellular and vacuolar Na(+) transport, were monitored. The salt-tolerant Glycine max (soybean) variety S111-9 exhibited much higher CEF activity and ATP accumulation in light than did the salt-sensitive variety Melrose, but similar leaf Na(+) concentrations under salt stress. In S111-9 plants, ndhB and ndhH were highly up-regulated under salt stress and their corresponding proteins were maintained at high levels or increased significantly. Under salt stress, S111-9 plants accumulated Na(+) in the vacuole, but Melrose plants accumulated Na(+) in the chloroplast. Compared with Melrose, S111-9 plants also showed higher expression of some genes associated with Na(+) transport into the vacuole and/or cell, such as genes encoding components of the CBL10 (calcineurin B-like protein 10)-CIPK24 (CBL-interacting protein kinase 24)-NHX (Na(+)/H(+) antiporter) and CBL4 (calcineurin B-like protein 4)-CIPK24-SOS1 (salt overly sensitive 1) complexes. Based on the findings, it is proposed that enhanced NDH-dependent CEF supplies extra ATP used to sequester Na(+) in the vacuole. This reveals an important mechanism for salt tolerance in soybean and provides new insights into plant resistance to salt stress. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  19. Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: Procainamide-induced autophagic cell vacuolization

    International Nuclear Information System (INIS)

    Morissette, Guillaume; Lodge, Robert; Marceau, Francois

    2008-01-01

    Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent K M 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (≥ 24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking

  20. Clues to γ-secretase, huntingtin and Hirano body normal function using the model organism Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Myre Michael A

    2012-04-01

    Full Text Available Abstract Many neurodegenerative disorders, although related by their destruction of brain function, display remarkable cellular and/or regional pathogenic specificity likely due to a deregulated functionality of the mutant protein. However, neurodegenerative disease genes, for example huntingtin (HTT, the ataxins, the presenilins (PSEN1/PSEN2 are not simply localized to neurons but are ubiquitously expressed throughout peripheral tissues; it is therefore paramount to properly understand the earliest precipitating events leading to neuronal pathogenesis to develop effective long-term therapies. This means, in no unequivocal terms, it is crucial to understand the gene's normal function. Unfortunately, many genes are often essential for embryogenesis which precludes their study in whole organisms. This is true for HTT, the β-amyloid precursor protein (APP and presenilins, responsible for early onset Alzheimer's disease (AD. To better understand neurological disease in humans, many lower and higher eukaryotic models have been established. So the question arises: how reasonable is the use of organisms to study neurological disorders when the model of choice does not contain neurons? Here we will review the surprising, and novel emerging use of the model organism Dictyostelium discoideum, a species of soil-living amoeba, as a valuable biomedical tool to study the normal function of neurodegenerative genes. Historically, the evidence on the usefulness of simple organisms to understand the etiology of cellular pathology cannot be denied. But using an organism without a central nervous system to understand diseases of the brain? We will first introduce the life cycle of Dictyostelium, the presence of many disease genes in the genome and how it has provided unique opportunities to identify mechanisms of disease involving actin pathologies, mitochondrial disease, human lysosomal and trafficking disorders and host-pathogen interactions. Secondly, I will

  1. Protein kinase Cα deletion causes hypotension and decreased vascular contractility.

    Science.gov (United States)

    Wynne, Brandi M; McCarthy, Cameron G; Szasz, Theodora; Molina, Patrick A; Chapman, Arlene B; Webb, R Clinton; Klein, Janet D; Hoover, Robert S

    2018-03-01

    Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.

  2. Methuosis: nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments.

    Science.gov (United States)

    Maltese, William A; Overmeyer, Jean H

    2014-06-01

    Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  3. Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria.

    Science.gov (United States)

    Kodama, Yuuki; Fujishima, Masahiro

    2012-07-01

    Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding. Copyright © 2011 Elsevier GmbH. All rights reserved.

  4. Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

    International Nuclear Information System (INIS)

    Johnson-Lyles, Denise N.; Peifley, Kimberly; Lockett, Stephen; Neun, Barry W.; Hansen, Matthew; Clogston, Jeffrey; Stern, Stephan T.; McNeil, Scott E.

    2010-01-01

    Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C 60 OH x ), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

  5. Vacuolization of mucolipidosis type II mouse exocrine gland cells represents accumulation of autolysosomes.

    Science.gov (United States)

    Boonen, Marielle; van Meel, Eline; Oorschot, Viola; Klumperman, Judith; Kornfeld, Stuart

    2011-04-15

    We previously reported that mice deficient in UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (mucolipidosis type II or Gnptab -/- mice), the enzyme that initiates the addition of the mannose 6-phosphate lysosomal sorting signal on acid hydrolases, exhibited extensive vacuolization of their exocrine gland cells, while the liver, brain, and muscle appeared grossly unaffected. Similar pathological findings were observed in several exocrine glands of patients with mucolipidosis II. To understand the basis for this cell type-specific abnormality, we analyzed these tissues in Gnptab -/- mice using a combined immunoelectron microscopy and biochemical approach. We demonstrate that the vacuoles in the exocrine glands are enlarged autolysosomes containing undigested cytoplasmic material that accumulate secondary to deficient lysosomal function. Surprisingly, the acid hydrolase levels in these tissues ranged from normal to modestly decreased, in contrast to skin fibroblasts, which accumulate enlarged lysosomes and/or autolysosomes also but exhibit very low levels of acid hydrolases. We propose that the lysosomal defect in the exocrine cells is caused by the combination of increased secretion of the acid hydrolases via the constitutive pathway along with their entrapment in secretory granules. Taken together, our results provide new insights into the mechanisms of the tissue-specific abnormalities seen in mucolipidosis type II.

  6. New views of the Toxoplasma gondii parasitophorous vacuole as revealed by Helium Ion Microscopy (HIM).

    Science.gov (United States)

    de Souza, Wanderley; Attias, Marcia

    2015-07-01

    The Helium Ion Microscope (HIM) is a new technology that uses a highly focused helium ion beam to scan and interact with the sample, which is not coated. The images have resolution and depth of field superior to field emission scanning electron microscopes. In this paper, we used HIM to study LLC-MK2 cells infected with Toxoplasma gondii. These samples were chemically fixed and, after critical point drying, were scraped with adhesive tape to expose the inner structure of the cell and parasitophorous vacuoles. We confirmed some of the previous findings made by field emission-scanning electron microscopy and showed that the surface of the parasite is rich in structures suggestive of secretion, that the nanotubules of the intravacuolar network (IVN) are not always straight, and that bifurcations are less frequent than previously thought. Fusion of the tubules with the parasite membrane or the parasitophorous vacuole membrane (PVM) was also infrequent. Tiny adhesive links were observed for the first time connecting the IVN tubules. The PVM showed openings of various sizes that even allowed the observation of endoplasmic reticulum membranes in the cytoplasm of the host cell. These findings are discussed in relation to current knowledge on the cell biology of T. gondii. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Recently active contractile deformation in the forearc of southern Peru

    Science.gov (United States)

    Hall, S. R.; Farber, D.; Audin, L.; Finkel, R. C.

    2010-12-01

    In the Precordillera and Western Cordillera of southern Peru (14°-18°S), vast pediment surfaces have been abandoned through drainage diversion and river incision, with the major drainages carving deep canyons. Within this region, we have identified range-sub-parallel contractile structures that accommodate significant distributed crustal deformation. Young geomorphic features document both the presence and youthfulness of these contractile structures. Here, we determine exposure ages on geomorphic features such as pediment surfaces and fluvial terraces using in situ produced cosmogenic radionuclides, in conjunction with field and remote mapping. This chronologic data reveals that ancient surfaces have been preserved as a result of very low erosion rates. We measure this rate to be chronology and geomorphic mapping, we calculate a Pleistocene river incision rate of ~0.3mm/yr determined from data collected along exoreic rivers. This rate is consistent with longer-term incision rates measured in other localities along this margin. We suggest that, in this region of southern Peru, the steep western wedge of the Andean margin supports the high topography of the Altiplano through a combination of uplift along steeply dipping contractile west-vergent structures and isostatic responses to the focused removal of large amounts of crustal material through canyon incision. Further, that these range sub-parallel structures are related at depth to a thrust system that plays a role in not only the maintenance of the Andean margin, but potentially in its formation as well.

  8. Effects of hypoxia and hypercapnia on geniohyoid contractility and endurance.

    Science.gov (United States)

    Salmone, R J; Van Lunteren, E

    1991-08-01

    Sleep apnea and other respiratory diseases produce hypoxemia and hypercapnia, factors that adversely affect skeletal muscle performance. To examine the effects of these chemical alterations on force production by an upper airway dilator muscle, the contractile and endurance characteristics of the geniohyoid muscle were examined in situ during severe hypoxia (arterial PO2 less than 40 Torr), mild hypoxia (PO2 45-65 Torr), and hypercapnia (PCO2 55-80 Torr) and compared with hyperoxic-normocapnic conditions in anesthetized cats. Muscles were studied at optimal length, and contractile force was assessed in response to supramaximal electrical stimulation of the hypoglossal nerve (n = 7 cats) or geniohyoid muscle (n = 2 cats). There were no significant changes in the twitch kinetics or force-frequency curve of the geniohyoid muscle during hypoxia or hypercapnia. However, the endurance of the geniohyoid, as reflected in the fatigue index (ratio of force at 2 min to initial force in response to 40-Hz stimulation at a duty cycle 0.33), was significantly reduced by severe hypoxia but not by hypercapnia or mild hypoxia. In addition, the downward shift in the force-frequency curve after the repetitive stimulation protocol was greater during hypoxia than hyperoxia, especially at higher frequencies. In conclusion, the ability of the geniohyoid muscle to maintain force output during high levels of activation is adversely affected by severe hypoxia but not mild hypoxia or hypercapnia. However, none of these chemical perturbations affected muscle contractility acutely.

  9. Acidification of the parasitophorous vacuole containing Toxoplasma gondii in the presence of hydroxyurea

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    Cristiane S. Carvalho

    2006-09-01

    Full Text Available Toxoplasma gondii multiplies within parasitophorous vacuole that is not recognized by the primary no oxidative defense of host cells, mainly represented by the fusion with acidic organelles. Recent studies have already shown that hydroxyurea arrested the intracellular parasites leading to its destruction. In the present work we investigated the cellular mechanism involved in the destruction of intracellular Toxoplasma gondii. Fluorescent vital stains were used in order to observe possible acidification of parasitophorous vacuole-containing Toxoplasma gondii in presence of hydroxyurea. Vero cells infected with tachyzoites were treated with hydroxyurea for 12, 24 or 48 hours. Fluorescence, indicative of acidification, was observed in the parasitophorous vacuole when the cultures were incubated in presence of acridine orange. LysoTracker red was used in order to determine whether lysosomes were involved in the acidification process. An intense fluorescence was observed after 12 and 24 hours of incubation with hydroxyurea, achieving it is highly intensity after 48 hours of treatment. Ultrastructural cytochemistry for localization of the acid phosphatase lysosomal enzyme was realized. Treated infected cultures showed reaction product in vesicles fusing with vacuole or associated with intravacuolar parasites. These results suggest that fusion with lysosomes and acidification of parasitophorous vacuole leads to parasites destruction in the presence pf hydroxyurea.Toxoplasma gondii se multiplica dentro do vacúolo parasitóforo que não é reconhecido pela defesa primária não oxidativa de células hospedeiras: a fusão com organelas ácidas. Estudos anteriores mostraram que hidroxiuréia interrompeu a multiplicação dos parasitos intracelulares causando sua eliminação. No presente trabalho nós investigamos o mecanismo celular envolvido na destruição do Toxoplasma gondii intracelular. Marcadores vitais fluorescentes foram usados para observar a

  10. Inorganic Polyphosphate Is Essential for Salmonella Typhimurium Virulence and Survival in Dictyostelium discoideum

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    Macarena A. Varas

    2018-01-01

    Full Text Available Inorganic polyphosphate (polyP deficiency in enteric bacterial pathogens reduces their ability to invade and establish systemic infections in different hosts. For instance, inactivation of the polyP kinase gene (ppk encoding the enzyme responsible for polyP biosynthesis reduces invasiveness and intracellular survival of Salmonella enterica serovar Typhimurium (S. Typhimurium in epithelial cells and macrophages in vitro. In addition, the virulence in vivo of a S. Typhimurium Δppk mutant is significantly reduced in a murine infection model. In spite of these observations, the role played by polyP during the Salmonella-host interaction is not well understood. The social amoeba Dictyostelium discoideum has proven to be a useful model for studying relevant aspects of the host-pathogen interaction. In fact, many intracellular pathogens can survive within D. discoideum cells using molecular mechanisms also required to survive within macrophages. Recently, we established that S. Typhimurium is able to survive intracellularly in D. discoideum and identified relevant genes linked to virulence that are crucial for this process. The aim of this study was to determine the effect of a polyP deficiency in S. Typhimurium during its interaction with D. discoideum. To do this, we evaluated the intracellular survival of wild-type and Δppk strains of S. Typhimurium in D. discoideum and the ability of these strains to delay the social development of the amoeba. In contrast to the wild-type strain, the Δppk mutant was unable to survive intracellularly in D. discoideum and enabled the social development of the amoeba. Both phenotypes were complemented using a plasmid carrying a copy of the ppk gene. Next, we simultaneously evaluated the proteomic response of both S. Typhimurium and D. discoideum during host-pathogen interaction via global proteomic profiling. The analysis of our results allowed the identification of novel molecular signatures that give insight into

  11. A Tonoplast P3B-ATPase Mediates Fusion of Two Types of Vacuoles in Petal Cells

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    Marianna Faraco

    2017-06-01

    Full Text Available It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

  12. Sperm with large nuclear vacuoles and semen quality in the evaluation of male infertility.

    Science.gov (United States)

    Komiya, Akira; Watanabe, Akihiko; Kawauchi, Yoko; Fuse, Hideki

    2013-02-01

    This study compared the sperm nuclear vacuoles and semen quality in the evaluation of male infertility. One hundred and forty-two semen samples were obtained from patients who visited the Male Infertility Clinic at Toyama University Hospital. Semen samples were evaluated by conventional semen analyses and the Sperm Motility Analysis System (SMAS). In addition, spermatozoa were analyzed at 3,700-6,150x magnification on an inverted microscope equipped with DIC/Nomarski differential interference contrast optics. A large nuclear vacuole (LNV) was defined as one or more vacuoles with the maximum diameter showing > 50% width of the sperm head. The percentage of spermatozoa with LNV (% LNV) was calculated for each sample. Correlations between the % LNV and parameters in SMAS and conventional semen analyses were analyzed. Processed motile spermatozoa from each sample were evaluated. The mean age of patients was 35 years old. Semen volume was 2.9 ± 1.6mL (0.1-11.0; mean ± standard deviation, minimum-maximum), sperm count was 39.3 ± 54.9 (x10(6)/mL, 0.01-262.0), sperm motility was 25.1 ± 17.8% (0-76.0), and normal sperm morphology was 10.3 ± 10.1% (0-49.0). After motile spermatozoa selection, we could evaluate % LNV in 125 ejaculates (88.0%) and at least one spermatozoon with LNV was observed in 118 ejaculates (94.4%). The percentage of spermatozoa with LNV was 28.0 ± 22.4% (0-100) and % LNV increased significantly when semen quality decreased. The correlation between the % LNV and the semen parameters was weak to moderate; correlation coefficients were -0.3577 in sperm count (p sperm motility (p = 0.0084), -0.2769 in motile sperm count (p = 0.019), -0.2419 in total motile sperm count (p = 0.0070), and -0.1676 in normal sperm morphology (p = 0.0639). The % LNV did not show a significant correlation with the SMAS parameters except for weak correlation to beat/cross frequency (r = -0.2414, p = 0.0071). The percentage of

  13. Comparing the Dictyostelium and Entamoeba genomes reveals an ancient split in the Conosa lineage.

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    Jie Song

    2005-12-01

    Full Text Available The Amoebozoa are a sister clade to the fungi and the animals, but are poorly sampled for completely sequenced genomes. The social amoeba Dictyostelium discoideum and amitochondriate pathogen Entamoeba histolytica are the first Amoebozoa with genomes completely sequenced. Both organisms are classified under the Conosa subphylum. To identify Amoebozoa-specific genomic elements, we compared these two genomes to each other and to other eukaryotic genomes. An expanded phylogenetic tree built from the complete predicted proteomes of 23 eukaryotes places the two amoebae in the same lineage, although the divergence is estimated to be greater than that between animals and fungi, and probably happened shortly after the Amoebozoa split from the opisthokont lineage. Most of the 1,500 orthologous gene families shared between the two amoebae are also shared with plant, animal, and fungal genomes. We found that only 42 gene families are distinct to the amoeba lineage; among these are a large number of proteins that contain repeats of the FNIP domain, and a putative transcription factor essential for proper cell type differentiation in D. discoideum. These Amoebozoa-specific genes may be useful in the design of novel diagnostics and therapies for amoebal pathologies.

  14. PKC-Mediated ZYG1 Phosphorylation Induces Fusion of Myoblasts as well as of Dictyostelium Cells

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    Aiko Amagai

    2012-01-01

    Full Text Available We have previously demonstrated that a novel protein ZYG1 induces sexual cell fusion (zygote formation of Dictyostelium cells. In the process of cell fusion, involvements of signal transduction pathways via Ca2+ and PKC (protein kinase C have been suggested because zygote formation is greatly enhanced by PKC activators. In fact, there are several deduced sites phosphorylated by PKC in ZYG1 protein. Thereupon, we designed the present work to examine whether or not ZYG1 is actually phosphorylated by PKC and localized at the regions of cell-cell contacts where cell fusion occurs. These were ascertained, suggesting that ZYG1 might be the target protein for PKC. A humanized version of zyg1 cDNA (mzyg1 was introduced into myoblasts to know if ZYG1 is also effective in cell fusion of myoblasts. Quite interestingly, enforced expression of ZYG1 in myoblasts was found to induce markedly their cell fusion, thus strongly suggesting the existence of a common signaling pathway for cell fusion beyond the difference of species.

  15. Effects of Nickel, Chlorpyrifos and Their Mixture on the Dictyostelium discoideum Proteome

    Science.gov (United States)

    Boatti, Lara; Robotti, Elisa; Marengo, Emilio; Viarengo, Aldo; Marsano, Francesco

    2012-01-01

    Mixtures of chemicals can have additive, synergistic or antagonistic interactions. We investigated the effects of the exposure to nickel, the organophosphate insecticide chlorpyrifos at effect concentrations (EC) of 25% and 50% and their binary mixture (Ec25 + EC25) on Dictyostelium discoideum amoebae based on lysosomal membrane stability (LMS). We treated D. discoideum with these compounds under controlled laboratory conditions and evaluated the changes in protein levels using a two-dimensional gel electrophoresis (2DE) proteomic approach. Nickel treatment at EC25 induced changes in 14 protein spots, 12 of which were down-regulated. Treatment with nickel at EC50 resulted in changes in 15 spots, 10 of which were down-regulated. Treatment with chlorpyrifos at EC25 induced changes in six spots, all of which were down-regulated; treatment with chlorpyrifos at EC50 induced changes in 13 spots, five of which were down-regulated. The mixture corresponding to EC25 of each compound induced changes in 19 spots, 13 of which were down-regulated. The data together reveal that a different protein expression signature exists for each treatment, and that only a few proteins are modulated in multiple different treatments. For a simple binary mixture, the proteomic response does not allow for the identification of each toxicant. The protein spots that showed significant differences were identified by mass spectrometry, which revealed modulations of proteins involved in metal detoxification, stress adaptation, the oxidative stress response and other cellular processes. PMID:23443088

  16. Cytochemical study of the nucleolus of the slime mold Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Benichou, J.C.; Quiviger, B.; Ryter, A.

    1983-01-01

    The nucleus of the slime mold Dictyostelium discoideum is characterized by the presence of several large dense masses which are all in tight contact with the nuclear membrane. These dense masses, considered as nucleoli, present a rather homogeneous texture, in which dense chromatin, fibrillar, and granular material are not easily detected. The autoradiographic study of [ 3 H]uridine pulse-labeled cells showed that the majority of the silver grains were located inside these masses. The use of EDTA regressive-staining, acetylation and enzymatic digestion indicated that they are mostly composed of RNP and are totally devoid of dense chromatin as the rest of the nucleus is. After treatment with actinomycin D, fibrillar and granular material segregated but no chromatin could be found. All these observations confirmed that the dense masses correspond to nucleoli despite their peculiar ultrastructure. It can also be concluded that this type of nucleoli cannot be considered as a taxonomic character of the slime molds because it does not exist in all slime molds and was observed in some dinoflagellates, and ascomycetes

  17. The human homologue of Dictyostelium discoideum phg1A is expressed by human metastatic melanoma cells.

    Science.gov (United States)

    Lozupone, Francesco; Perdicchio, Maurizio; Brambilla, Daria; Borghi, Martina; Meschini, Stefania; Barca, Stefano; Marino, Maria Lucia; Logozzi, Mariantonia; Federici, Cristina; Iessi, Elisabetta; de Milito, Angelo; Fais, Stefano

    2009-12-01

    Tumour cannibalism is a characteristic of malignancy and metastatic behaviour. This atypical phagocytic activity is a crucial survival option for tumours in conditions of low nutrient supply, and has some similarities to the phagocytic activity of unicellular microorganisms. In fact, Dictyostelium discoideum has been used widely as a model to study phagocytosis. Recently, phg1A has been described as a protein that is primarily involved in the phagocytic process of this microorganism. The closest human homologue to phg1A is transmembrane 9 superfamily protein member 4 (TM9SF4). Here, we report that TM9SF4 is highly expressed in human malignant melanoma cells deriving from metastatic lesions, whereas it is undetectable in healthy human tissues and cells. TM9SF4 is predominantly expressed in acidic vesicles of melanoma cells, in which it co-localizes with the early endosome antigens Rab5 and early endosome antigen 1. TM9SF4 silencing induced marked inhibition of cannibal activity, which is consistent with a derangement of intracellular pH gradients, with alkalinization of acidic vesicles and acidification of the cell cytosol. We propose TM9SF4 as a new marker of malignancy, representing a potential new target for anti-tumour strategies with a specific role in tumour cannibalism and in the establishment of a metastatic phenotype.

  18. Nucleocytoplasmic protein translocation during mitosis in the social amoebozoan Dictyostelium discoideum.

    Science.gov (United States)

    O'Day, Danton H; Budniak, Aldona

    2015-02-01

    Mitosis is a fundamental and essential life process. It underlies the duplication and survival of all cells and, as a result, all eukaryotic organisms. Since uncontrolled mitosis is a dreaded component of many cancers, a full understanding of the process is critical. Evolution has led to the existence of three types of mitosis: closed, open, and semi-open. The significance of these different mitotic species, how they can lead to a full understanding of the critical events that underlie the asexual duplication of all cells, and how they may generate new insights into controlling unregulated cell division remains to be determined. The eukaryotic microbe Dictyostelium discoideum has proved to be a valuable biomedical model organism. While it appears to utilize closed mitosis, a review of the literature suggests that it possesses a form of mitosis that lies in the middle between truly open and fully closed mitosis-it utilizes a form of semi-open mitosis. Here, the nucleocytoplasmic translocation patterns of the proteins that have been studied during mitosis in the social amoebozoan D. discoideum are detailed followed by a discussion of how some of them provide support for the hypothesis of semi-open mitosis. © 2014 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical Society.

  19. Inhibitors of Mycobacterium marinum virulence identified in a Dictyostelium discoideum host model.

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    Hajer Ouertatani-Sakouhi

    Full Text Available Tuberculosis remains one of the major threats to public health worldwide. Given the prevalence of multi drug resistance (MDR in Mycobacterium tuberculosis strains, there is a strong need to develop new anti-mycobacterial drugs with modes of action distinct from classical antibiotics. Inhibitors of mycobacterial virulence might target new molecular processes and may represent a potential new therapeutic alternative. In this study, we used a Dictyostelium discoideum host model to assess virulence of Mycobacterium marinum and to identify compounds inhibiting mycobacterial virulence. Among 9995 chemical compounds, we selected 12 inhibitors of mycobacterial virulence that do not inhibit mycobacterial growth in synthetic medium. Further analyses revealed that 8 of them perturbed functions requiring an intact mycobacterial cell wall such as sliding motility, bacterial aggregation or cell wall permeability. Chemical analogs of two compounds were analyzed. Chemical modifications altered concomitantly their effect on sliding motility and on mycobacterial virulence, suggesting that the alteration of the mycobacterial cell wall caused the loss of virulence. We characterized further one of the selected compounds and found that it inhibited the ability of mycobacteria to replicate in infected cells. Together these results identify new antimycobacterial compounds that represent new tools to unravel the molecular mechanisms controlling mycobacterial pathogenicity. The isolation of compounds with anti-virulence activity is the first step towards developing new antibacterial treatments.

  20. High fidelity information processing in folic acid chemotaxis of Dictyostelium amoebae.

    Science.gov (United States)

    Segota, Igor; Mong, Surin; Neidich, Eitan; Rachakonda, Archana; Lussenhop, Catherine J; Franck, Carl

    2013-11-06

    Living cells depend upon the detection of chemical signals for their existence. Eukaryotic cells can sense a concentration difference as low as a few per cent across their bodies. This process was previously suggested to be limited by the receptor-ligand binding fluctuations. Here, we first determine the chemotaxis response of Dictyostelium cells to static folic acid gradients and show that they can significantly exceed this sensitivity, responding to gradients as shallow as 0.2% across the cell body. Second, using a previously developed information theory framework, we compare the total information gained about the gradient (based on the cell response) to its upper limit: the information gained at the receptor-ligand binding step. We find that the model originally applied to cAMP sensing fails as demonstrated by the violation of the data processing inequality, i.e. the total information exceeds the information at the receptor-ligand binding step. We propose an extended model with multiple known receptor types and with cells allowed to perform several independent measurements of receptor occupancy. This does not violate the data processing inequality and implies the receptor-ligand binding noise dominates both for low- and high-chemoattractant concentrations. We also speculate that the interplay between exploration and exploitation is used as a strategy for accurate sensing of otherwise unmeasurable levels of a chemoattractant.

  1. Cooperation induces other cooperation: Fruiting bodies promote the evolution of macrocysts in Dictyostelium discoideum.

    Science.gov (United States)

    Shibasaki, Shota; Shirokawa, Yuka; Shimada, Masakazu

    2017-05-21

    Biological studies of the evolution of cooperation are challenging because this process is vulnerable to cheating. Many mechanisms, including kin discrimination, spatial structure, or by-products of self-interested behaviors, can explain this evolution. Here we propose that the evolution of cooperation can be induced by other cooperation. To test this idea, we used a model organism Dictyostelium discoideum because it exhibits two cooperative dormant phases, the fruiting body and the macrocyst. In both phases, the same chemoattractant, cyclic AMP (cAMP), is used to collect cells. This common feature led us to hypothesize that the evolution of macrocyst formation would be induced by coexistence with fruiting bodies. Before forming a mathematical model, we confirmed that macrocysts coexisted with fruiting bodies, at least under laboratory conditions. Next, we analyzed our evolutionary game theory-based model to investigate whether coexistence with fruiting bodies would stabilize macrocyst formation. The model suggests that macrocyst formation represents an evolutionarily stable strategy and a global invader strategy under this coexistence, but is unstable if the model ignores the fruiting body formation. This result indicates that the evolution of macrocyst formation and maintenance is attributable to coexistence with fruiting bodies. Therefore, macrocyst evolution can be considered as an example of evolution of cooperation induced by other cooperation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. RNA synthesis during germination of UV-irradiated Dictyostelium discoideum spores

    International Nuclear Information System (INIS)

    Okaichi, Kumio

    1987-01-01

    UV irradiation to the spores of Dictyostelium discoideum NC4 resulted in a more prolonged delay of amoeba-emergence from swollen spores with increasing UV fluence. During the germination, an inhibition of total RNA synthesis and a shift of stage of maximum RNA synthesis to the later period were observed. The maximum poly(A) + RNA synthetic activity was found on an early stage of amoeba-emergence prior about 1 h to the beginning of rRNA synthesis in unirradiated spore germination; but, in UV-irradiated spore germination, the stage of maximum poly(A) + RNA synthesis shifted to the later stage of germination with increasing UV fluence. A decreased synthesis of poly(A) + RNA and a severe inhibition of rRNA synthesis were observed on UV-irradiated and germinated spores, but no significant inhibition of 4 - 5 S RNA synthesis was detected. Actinomycin D suppressed almost completely the rRNA synthesis of emerged amoebae but the drug apparently did not affect the emergence of amoebae at any stage of germination. It was postulated that the delay of amoeba-emergence in UV-irradiated spore must be mainly due to the shift of the stage of maximum synthesis of poly(A) + RNA to the later stage of germination. (author)

  3. The rate and effects of spontaneous mutation on fitness traits in the social amoeba, Dictyostelium discoideum.

    Science.gov (United States)

    Hall, David W; Fox, Sara; Kuzdzal-Fick, Jennie J; Strassmann, Joan E; Queller, David C

    2013-07-08

    We performed a mutation accumulation (MA) experiment in the social amoeba Dictyostelium discoideum to estimate the rate and distribution of effects of spontaneous mutations affecting eight putative fitness traits. We found that the per-generation mutation rate for most fitness components is 0.0019 mutations per haploid genome per generation or larger. This rate is an order of magnitude higher than estimates for fitness components in the unicellular eukaryote Saccharomyces cerevisiae, even though the base-pair substitution rate is two orders of magnitude lower. The high rate of fitness-altering mutations observed in this species may be partially explained by a large mutational target relative to S. cerevisiae. Fitness-altering mutations also may occur primarily at simple sequence repeats, which are common throughout the genome, including in coding regions, and may represent a target that is particularly likely to give fitness effects upon mutation. The majority of mutations had deleterious effects on fitness, but there was evidence for a substantial fraction, up to 40%, being beneficial for some of the putative fitness traits. Competitive ability within the multicellular slug appears to be under weak directional selection, perhaps reflecting the fact that slugs are sometimes, but not often, comprised of multiple clones in nature. Evidence for pleiotropy among fitness components across MA lines was absent, suggesting that mutations tend to act on single fitness components.

  4. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Science.gov (United States)

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  5. Influence of fast advective flows on pattern formation of Dictyostelium discoideum

    Science.gov (United States)

    Bae, Albert; Zykov, Vladimir; Bodenschatz, Eberhard

    2018-01-01

    We report experimental and numerical results on pattern formation of self-organizing Dictyostelium discoideum cells in a microfluidic setup under a constant buffer flow. The external flow advects the signaling molecule cyclic adenosine monophosphate (cAMP) downstream, while the chemotactic cells attached to the solid substrate are not transported with the flow. At high flow velocities, elongated cAMP waves are formed that cover the whole length of the channel and propagate both parallel and perpendicular to the flow direction. While the wave period and transverse propagation velocity are constant, parallel wave velocity and the wave width increase linearly with the imposed flow. We also observe that the acquired wave shape is highly dependent on the wave generation site and the strength of the imposed flow. We compared the wave shape and velocity with numerical simulations performed using a reaction-diffusion model and found excellent agreement. These results are expected to play an important role in understanding the process of pattern formation and aggregation of D. discoideum that may experience fluid flows in its natural habitat. PMID:29590179

  6. Overexpression of TOR (target of rapamycin) inhibits cell proliferation in Dictyostelium discoideum.

    Science.gov (United States)

    Swer, Pynskhem Bok; Mishra, Himanshu; Lohia, Rakhee; Saran, Shweta

    2016-05-01

    TOR (target of rapamycin) protein kinase acts as a central controller of cell growth and development of an organism. Present study was undertaken to find the expression pattern and role of TOR during growth and development of Dictyostelium discoideum. Failures to generate either knockout and/or knockdown mutants indicate that interference with its levels led to cellular defects. Thus, the effects of TOR (DDB_G0281569) overexpression specifically, cells expressing Dd(Δ211-TOR)-Eyfp mutant was analyzed. Elevated expression of (Δ211-TOR)-Eyfp reduced both cell size and cell proliferation. DdTOR was found to be closer to fungus. mRNA level of TOR was found maximally in the freshly starved/aggregate cells that gradually declined. This was also strengthened by the expression patterns observed by in situ and the analysis of β-galactosidase reporter driven by the putative TOR promoter. The TOR protein was found to be highest at the aggregate stage. The fusion protein, (Δ211-TOR)-Eyfp was localized to the cell membrane, cytosol, and the nucleus. We suggest, DdTOR to be an essential protein and high TOR expression inhibits cell proliferation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

    International Nuclear Information System (INIS)

    Schwartz, M.A.; Luna, E.J.

    1986-01-01

    The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

  8. Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium amoebae and food bacteria.

    Science.gov (United States)

    DiSalvo, Susanne; Haselkorn, Tamara S; Bashir, Usman; Jimenez, Daniela; Brock, Debra A; Queller, David C; Strassmann, Joan E

    2015-09-08

    Symbiotic associations can allow an organism to acquire novel traits by accessing the genetic repertoire of its partner. In the Dictyostelium discoideum farming symbiosis, certain amoebas (termed "farmers") stably associate with bacterial partners. Farmers can suffer a reproductive cost but also gain beneficial capabilities, such as carriage of bacterial food (proto-farming) and defense against competitors. Farming status previously has been attributed to amoeba genotype, but the role of bacterial partners in its induction has not been examined. Here, we explore the role of bacterial associates in the initiation, maintenance, and phenotypic effects of the farming symbiosis. We demonstrate that two clades of farmer-associated Burkholderia isolates colonize D. discoideum nonfarmers and infectiously endow them with farmer-like characteristics, indicating that Burkholderia symbionts are a major driver of the farming phenomenon. Under food-rich conditions, Burkholderia-colonized amoebas produce fewer spores than uncolonized counterparts, with the severity of this reduction being dependent on the Burkholderia colonizer. However, the induction of food carriage by Burkholderia colonization may be considered a conditionally adaptive trait because it can confer an advantage to the amoeba host when grown in food-limiting conditions. We observed Burkholderia inside and outside colonized D. discoideum spores after fruiting body formation; this observation, together with the ability of Burkholderia to colonize new amoebas, suggests a mixed mode of symbiont transmission. These results change our understanding of the D. discoideum farming symbiosis by establishing that the bacterial partner, Burkholderia, is an important causative agent of the farming phenomenon.

  9. Differential compartmentation of sucrose and gentianose in the cytosol and vacuoles of storage root protoplasts from Gentiana Lutea L.

    Science.gov (United States)

    Keller, F; Wiemken, A

    1982-12-01

    The storage roots of perennial Gentiana lutea L.plants contain several sugars. The predominant carbohydrate reserve is gentianose (β-D-glucopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1 ↔ 2)-β-D-fructofuranoside). Vacuoles were isolated from root protoplasts and purified through a betaine density gradient. The yield was about 75%. Gentianose and gentiobiose were localized to 100% in the vacuoles, fructose and glucose to about 80%, and sucrose to only about 50%. Taking the volumes of the vacuolar and extravacuolar (cytosolic) compartments into account it is inferred that gentianose is located exclusively in the vacuoles, whilst sucrose is much more concentrated in the cytosol where it may play a role as a cryoprotectant. The concentration of fructose and glucose appeared to be similar on both sides of the tonoplast.

  10. Mucolipin co-deficiency causes accelerated endolysosomal vacuolation of enterocytes and failure-to-thrive from birth to weaning.

    Directory of Open Access Journals (Sweden)

    Natalie N Remis

    2014-12-01

    Full Text Available During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3-/-;Trpml1-/- vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3-/-;Trpml1-/- mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns

  11. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

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    Florante Ricarte

    Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

  12. Examining the impact of grazing on iron remineralization: effect of prey type on digestive vacuole pH

    Science.gov (United States)

    Pritchard, K. R.; Nuester, J.; Twining, B.

    2012-12-01

    Most of the iron available to phytoplankton in high-nutrient, low-chlorophyll areas is regenerated by zooplankton grazers. The extent to which the bioavailability of this regenerated iron is a function of prey-type and the chemical conditions within digestive systems of zooplankton is unknown. The chemical composition of the prey, including silica frustules of diatoms and calcium carbonate coccoliths of cocolithophores, might buffer the acidity within a digestive vacuole and thereby influencing the resulting speciation and bioavailability of regenerated iron. In order to test the effect of prey-type on the chemical condition in the digestive vacuole of the heterotrophic dinoflagellate Oxyrrhis marina, we used the ratiometric fluorescent dye Lysosensor Yellow/Blue DND-160 in conjunction with confocal microscopy to measure and compare digestive vacuole acidity after feeding O. marina with either the diatom Thalassiosira pseudonana, the coccolithophore Emiliana huxleyi, or the chlorophyte Dunaliella tertiolecta. After feeding and loading O. marina with the Lysosensor dye, we recorded the total fluorescence (f) of the wavelength regions λ1=500-555 nm and λ2=410-490 nm using an excitation wavelength of 405 nm, and calculated the Lysosensor fluorescence ratio r=f(λ1)/f(λ2). External calibration curves show that this ratio (r) is inversely related to pH. In addition, we also measured the emission of chlorophyll fluorescence above 640 nm in order to identify prey within the grazers and study the timing chlorophyll degradation in conjunction with vacuole pH. After the initial addition of either prey, O. marina consumed 10 times and 2 times more D. tertiolecta cells than E. huxleyi and T. pseudonana cells, respectively. The clearance of the digestive vacuole measured as the disappearance of chlorophyll fluorescence is ca. twice as long for O. marina feeding on D. tertiolecta than on E. huxleyi or T. pseudonana. Initial r was inversely proportional to prey preference

  13. Chlamydia trachomatis Is Responsible for Lipid Vacuolation in the Amniotic Epithelium of Fetal Gastroschisis.

    Science.gov (United States)

    Feldkamp, Marcia L; Ward, Diane M; Pysher, Theodore J; Chambers, Christina T

    2017-07-17

    Vacuolated amniotic epithelium with lipid droplets in gastroschisis placentas is an unusual finding. Mass spectrometry of lipid droplets identified triglycerides, ester-linked to an unusual pattern of fatty acids. We hypothesize that these findings result from a Chlamydia trachomatis infection during the periconceptional period. The rising incidence of chlamydia infections has paralleled the increasing prevalence of gastroschisis among women less than 25 years of age. Histologically, young women are at greatest risk for a chlamydia infection due to their immature columnar epithelium, the preferential site for attachment of Chlamydia trachomatis infectious particle (elementary body). Chlamydia trachomatis survive in an inclusion, relying on its host to acquire essential nutrients, amino acids, and nucleotides for survival and replication. If essential nutrients are not available, the bacteria cannot replicate and may be trafficked to the lysosome for degradation or remain quiescent, within the inclusion, subverting innate immunologic clearance. Chlamydiae synthesize several lipids (phosphatidylethanolamine, phosphatidylserine, and phosphoatidylglycerol); however, their lipid content reveal eukaryotic lipids (sphingomyelin, cholesterol, phosphatidylcholine, and phosphatidylinositol), evidence that chlamydiae "hijack" host lipids for expansion and replication. The abnormal amniotic epithelial findings are supported by experimental evidence of the trafficking of host lipids into the chlamydiae inclusion. If not lethal, what harm will elementary bodies inflict to the developing embryo? Do these women have a greater pro-inflammatory response to an environmental exposure, whether cigarette smoking, change in partner, or a pathogen? Testing the hypothesis that Chlamydia trachomatis is responsible for amniotic epithelium vacuoles will be a critical first step. Birth Defects Research 109:1003-1010, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. A RabGAP regulates life-cycle duration via trimeric G-protein cascades in Dictyostelium discoideum.

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    Hidekazu Kuwayama

    Full Text Available BACKGROUND: The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3-deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.

  15. The ROCO Kinase QkgA Is Necessary for Proliferation Inhibition by Autocrine Signals in Dictyostelium discoideum▿

    OpenAIRE

    Phillips, Jonathan E.; Gomer, Richard H.

    2010-01-01

    AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA− cells accumulate normal levels of ex...

  16. An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH

    OpenAIRE

    Yu Tang; Yuantai Wu; Sarah E. Herlihy; Francisco J. Brito-Aleman; Jose H. Ting; Chris Janetopoulos; Richard H. Gomer; Scott D. Emr

    2018-01-01

    In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins...

  17. Characterization of a 1,4-{beta}-D-glucan synthase from Dictyostelium discoideum. Progress report, May 1990--January 1992

    Energy Technology Data Exchange (ETDEWEB)

    Blanton, R.L.

    1992-01-15

    Various aspects of research concerning Dictyostelium discoideum are presented. The initial focus of this project was upon: the characterization of potential probes for the cellulose synthase (antibody and nucleic acid), the determination of the cultural induction conditions of cellulose synthesis, the solubilization of the enzyme activity, the development of a non-inhibitory disruption buffer, the generation and isolation of mutant strains deficient in cellulose synthesis, and the development of the capability to determine the degree of polymerization of the in vitro product. I have briefly summarized our most significant findings with only selected data sets being shown in this report in the interest of brevity.

  18. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects

    NARCIS (Netherlands)

    Korstanje, Ron; Desai, Jigar; Lazar, Gloria; King, Benjamin; Rollins, Jarod; Spurr, Melissa; Joseph, Jamie; Kadambi, Sindhuja; Li, Yang; Cherry, Allison; Matteson, Paul G.; Paigen, Beverly; Millonig, James H.

    Korstanje R, Desai J, Lazar G, King B, Rollins J, Spurr M, Joseph J, Kadambi S, Li Y, Cherry A, Matteson PG, Paigen B, Millonig JH. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects. Physiol Genomics 35:

  19. Resolving the role of actoymyosin contractility in cell microrheology.

    Directory of Open Access Journals (Sweden)

    Christopher M Hale

    2009-09-01

    Full Text Available Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells-stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM and particle-tracking microrheology measurements of living cells.

  20. Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa.

    Science.gov (United States)

    Salih, Osman; He, Shaoda; Planamente, Sara; Stach, Lasse; MacDonald, James T; Manoli, Eleni; Scheres, Sjors H W; Filloux, Alain; Freemont, Paul S

    2018-02-06

    Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. Effect of serotonin on small intestinal contractility in healthy volunteers

    DEFF Research Database (Denmark)

    Hansen, M.B.; Arif, F.; Gregersen, H.

    2008-01-01

    The physiological significance of serotonin released into the intestinal lumen for the regulation of motility is unknown in humans. The aim of this study was to evaluate the effect of serotonin infused into the lumen of the gastric antrum, duodenum or the jejunum, on antro-duodeno-jejunal contrac......The physiological significance of serotonin released into the intestinal lumen for the regulation of motility is unknown in humans. The aim of this study was to evaluate the effect of serotonin infused into the lumen of the gastric antrum, duodenum or the jejunum, on antro......-duodeno-jejunal contractility in healthy human volunteers. Manometric recordings were obtained and the effects of either a standard meal, continuous intravenous infusion of serotonin (20 nmol/kg/min) or intraluminal bolus infusions of graded doses of serotonin (2.5, 25 or 250 nmol) were compared. In addition, platelet......-depleted plasma levels of serotonin, blood pressure, heart rate and electrocardiogram were evaluated. All subjects showed similar results. Intravenous serotonin increased migrating motor complex phase In frequency 3-fold and migrating velocity 2-fold. Intraluminal infusion of serotonin did not change contractile...

  2. Variation, sex, and social cooperation: molecular population genetics of the social amoeba Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Jonathan M Flowers

    2010-07-01

    Full Text Available Dictyostelium discoideum is a eukaryotic microbial model system for multicellular development, cell-cell signaling, and social behavior. Key models of social evolution require an understanding of genetic relationships between individuals across the genome or possibly at specific genes, but the nature of variation within D. discoideum is largely unknown. We re-sequenced 137 gene fragments in wild North American strains of D. discoideum and examined the levels and patterns of nucleotide variation in this social microbial species. We observe surprisingly low levels of nucleotide variation in D. discoideum across these strains, with a mean nucleotide diversity (pi of 0.08%, and no strong population stratification among North American strains. We also do not find any clear relationship between nucleotide divergence between strains and levels of social dominance and kin discrimination. Kin discrimination experiments, however, show that strains collected from the same location show greater ability to distinguish self from non-self than do strains from different geographic areas. This suggests that a greater ability to recognize self versus non-self may arise among strains that are more likely to encounter each other in nature, which would lead to preferential formation of fruiting bodies with clonemates and may prevent the evolution of cheating behaviors within D. discoideum populations. Finally, despite the fact that sex has rarely been observed in this species, we document a rapid decay of linkage disequilibrium between SNPs, the presence of recombinant genotypes among natural strains, and high estimates of the population recombination parameter rho. The SNP data indicate that recombination is widespread within D. discoideum and that sex as a form of social interaction is likely to be an important aspect of the life cycle.

  3. Colchicine affects cell motility, pattern formation and stalk cell differentiation in Dictyostelium by altering calcium signaling.

    Science.gov (United States)

    Poloz, Yekaterina; O'Day, Danton H

    2012-04-01

    Previous work, verified here, showed that colchicine affects Dictyostelium pattern formation, disrupts morphogenesis, inhibits spore differentiation and induces terminal stalk cell differentiation. Here we show that colchicine specifically induces ecmB expression and enhances accumulation of ecmB-expressing cells at the posterior end of multicellular structures. Colchicine did not induce a nuclear translocation of DimB, a DIF-1 responsive transcription factor in vitro. It also induced terminal stalk cell differentiation in a mutant strain that does not produce DIF-1 (dmtA-) and after the treatment of cells with DIF-1 synthesis inhibitor cerulenin (100 μM). This suggests that colchicine induces the differentiation of ecmB-expressing cells independent of DIF-1 production and likely through a signaling pathway that is distinct from the one that is utilized by DIF-1. Depending on concentration, colchicine enhanced random cell motility, but not chemotaxis, by 3-5 fold (10-50 mM colchicine, respectively) through a Ca(2+)-mediated signaling pathway involving phospholipase C, calmodulin and heterotrimeric G proteins. Colchicine's effects were not due to microtubule depolymerization as other microtubule-depolymerizing agents did not have these effects. Finally normal morphogenesis and stalk and spore cell differentiation of cells treated with 10 mM colchicine were rescued through chelation of Ca2+ by BAPTA-AM and EDTA and calmodulin antagonism by W-7 but not PLC inhibition by U-73122. Morphogenesis or spore cell differentiation of cells treated with 50 mM colchicine could not be rescued by the above treatments but terminal stalk cell differentiation was inhibited by BAPTA-AM, EDTA and W-7, but not U-73122. Thus colchicine disrupts morphogenesis and induces stalk cell differentiation through a Ca(2+)-mediated signaling pathway involving specific changes in gene expression and cell motility. Copyright © 2011 International Society of Differentiation. Published by Elsevier B

  4. Evaluating Different Virulence Traits of Klebsiella pneumoniae Using Dictyostelium discoideum and Zebrafish Larvae as Host Models

    Directory of Open Access Journals (Sweden)

    Andrés E. Marcoleta

    2018-02-01

    Full Text Available Multiresistant and invasive hypervirulent Klebsiella pneumoniae strains have become one of the most urgent bacterial pathogen threats. Recent analyses revealed a high genomic plasticity of this species, harboring a variety of mobile genetic elements associated with virulent strains, encoding proteins of unknown function whose possible role in pathogenesis have not been addressed. K. pneumoniae virulence has been studied mainly in animal models such as mice and pigs, however, practical, financial, ethical and methodological issues limit the use of mammal hosts. Consequently, the development of simple and cost-effective experimental approaches with alternative host models is needed. In this work we described the use of both, the social amoeba and professional phagocyte Dictyostelium discoideum and the fish Danio rerio (zebrafish as surrogate host models to study K. pneumoniae virulence. We compared three K. pneumoniae clinical isolates evaluating their resistance to phagocytosis, intracellular survival, lethality, intestinal colonization, and innate immune cells recruitment. Optical transparency of both host models permitted studying the infective process in vivo, following the Klebsiella-host interactions through live-cell imaging. We demonstrated that K. pneumoniae RYC492, but not the multiresistant strains 700603 and BAA-1705, is virulent to both host models and elicits a strong immune response. Moreover, this strain showed a high resistance to phagocytosis by D. discoideum, an increased ability to form biofilms and a more prominent and irregular capsule. Besides, the strain 700603 showed the unique ability to replicate inside amoeba cells. Genomic comparison of the K. pneumoniae strains showed that the RYC492 strain has a higher overall content of virulence factors although no specific genes could be linked to its phagocytosis resistance, nor to the intracellular survival observed for the 700603 strain. Our results indicate that both zebrafish

  5. Sociogenomics of self vs. non-self cooperation during development of Dictyostelium discoideum.

    Science.gov (United States)

    Li, Si I; Buttery, Neil J; Thompson, Christopher R L; Purugganan, Michael D

    2014-07-21

    Dictyostelium discoideum, a microbial model for social evolution, is known to distinguish self from non-self and show genotype-dependent behavior during chimeric development. Aside from a small number of cell-cell recognition genes, however, little is known about the genetic basis of self/non-self recognition in this species. Based on the key hypothesis that there should be differential expression of genes if D. discoideum cells were interacting with non-clone mates, we performed transcriptomic profiling study in this species during clonal vs. chimeric development. The transcriptomic profiles of D. discoideum cells in clones vs. different chimeras were compared at five different developmental stages using a customized microarray. Effects of chimerism on global transcriptional patterns associated with social interactions were observed. We find 1,759 genes significantly different between chimera and clone, 1,144 genes associated significant strain differences, and 6,586 genes developmentally regulated over time. Principal component analysis showed a small amount of the transcriptional variance to chimerism-related factors (Chimerism: 0.18%, Chimerism × Timepoint: 0.03%). There are 162 genes specifically regulated under chimeric development, with continuous small differences between chimera vs. clone over development. Almost 60% of chimera-associated differential genes were differentially expressed at the 4 h aggregate stage, which corresponds to the initial transition of D. discoideum from solitary life to a multicellular phase. A relatively small proportion of over-all variation in gene expression is explained by differences between chimeric and clonal development. The relatively small modifications in gene expression associated with chimerism is compatible with the high level of cooperation observed among different strains of D. discoideum; cells of distinct genetic backgrounds will co-aggregate indiscriminately and co-develop into fruiting bodies. Chimeric

  6. Correlated waves of actin filaments and PIP3 in Dictyostelium cells.

    Science.gov (United States)

    Asano, Yukako; Nagasaki, Akira; Uyeda, Taro Q P

    2008-12-01

    Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct movements: (1) they extend protrusions randomly without net displacements; (2) they migrate persistently and unidirectionally in a keratocyte-like manner. Here, we monitored the intracellular distribution of phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)) to gain insight into roles PIP(3) plays in those spontaneous motilities. In keratocyte-like cells, PIP(3) showed convex distribution over the basal membrane, with no anterior enrichment. In stalled cells, as well as in wild type cells, PIP(3) repeated wave-like changes, including emergence, expansion and disappearance, on the basal membrane. The waves induced lamellipodia when they approached the cell edge, and the advancing speed of the waves was comparable to the migration speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase, abolished PIP(3) waves in stalled cells and stopped keratocyte-like cells. These results together suggested that keratocyte-like cells are "surfing" on the PIP(3) waves by coupling steady lamellipodial protrusions to the PIP(3) waves. Simultaneous live observation of actin filaments and PIP(3) in wild type or stalled amiB(-) cells indicated that the PIP(3) waves were correlated with wave-like distributions of actin filaments. Most notably, PIP(3) waves often followed actin waves, suggesting that PIP(3) induces local depolymerization of actin filaments. Consistent with this idea, cortical accumulation of PIP(3) was often correlated with local retraction of the periphery. We propose that the waves of PIP(3) and actin filaments are loosely coupled with each other and play important roles in generating spontaneous cell polarity. Copyright 2008 Wiley-Liss, Inc.

  7. A Simple Retroelement Based Knock-Down System in Dictyostelium: Further Insights into RNA Interference Mechanisms.

    Science.gov (United States)

    Friedrich, Michael; Meier, Doreen; Schuster, Isabelle; Nellen, Wolfgang

    2015-01-01

    We have previously shown that the most abundant Dictyostelium discoideum retroelement DIRS-1 is suppressed by RNAi mechanisms. Here we provide evidence that both inverted terminal repeats have strong promoter activity and that bidirectional expression apparently generates a substrate for Dicer. A cassette containing the inverted terminal repeats and a fragment of a gene of interest was sufficient to activate the RNAi response, resulting in the generation of ~21 nt siRNAs, a reduction of mRNA and protein expression of the respective endogene. Surprisingly, no transitivity was observed on the endogene. This was in contrast to previous observations, where endogenous siRNAs caused spreading on an artificial transgene. Knock-down was successful on seven target genes that we examined. In three cases a phenotypic analysis proved the efficiency of the approach. One of the target genes was apparently essential because no knock-out could be obtained; the RNAi mediated knock-down, however, resulted in a very slow growing culture indicating a still viable reduction of gene expression. ADVANTAGES OF THE DIRS-1–RNAI SYSTEM: The knock-down system required a short DNA fragment (~400 bp) of the target gene as an initial trigger. Further siRNAs were generated by RdRPs since we have shown some siRNAs with a 5'-triphosphate group. Extrachromosomal vectors facilitate the procedure and allowed for molecular and phenotypic analysis within one week. The system provides an efficient and rapid method to reduce protein levels including those of essential genes.

  8. Regulation of Spatiotemporal Patterns by Biological Variability: General Principles and Applications to Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Miriam Grace

    2015-11-01

    Full Text Available Spatiotemporal patterns often emerge from local interactions in a self-organizing fashion. In biology, the resulting patterns are also subject to the influence of the systematic differences between the system's constituents (biological variability. This regulation of spatiotemporal patterns by biological variability is the topic of our review. We discuss several examples of correlations between cell properties and the self-organized spatiotemporal patterns, together with their relevance for biology. Our guiding, illustrative example will be spiral waves of cAMP in a colony of Dictyostelium discoideum cells. Analogous processes take place in diverse situations (such as cardiac tissue, where spiral waves occur in potentially fatal ventricular fibrillation so a deeper understanding of this additional layer of self-organized pattern formation would be beneficial to a wide range of applications. One of the most striking differences between pattern-forming systems in physics or chemistry and those in biology is the potential importance of variability. In the former, system components are essentially identical with random fluctuations determining the details of the self-organization process and the resulting patterns. In biology, due to variability, the properties of potentially very few cells can have a driving influence on the resulting asymptotic collective state of the colony. Variability is one means of implementing a few-element control on the collective mode. Regulatory architectures, parameters of signaling cascades, and properties of structure formation processes can be "reverse-engineered" from observed spatiotemporal patterns, as different types of regulation and forms of interactions between the constituents can lead to markedly different correlations. The power of this biology-inspired view of pattern formation lies in building a bridge between two scales: the patterns as a collective state of a very large number of cells on the one hand

  9. Semiautomated Segmentation and Measurement of Cytoplasmic Vacuoles in a Neutrophil With General-Purpose Image Analysis Software.

    Science.gov (United States)

    Mizukami, Maki; Yamada, Misaki; Fukui, Sayaka; Fujimoto, Nao; Yoshida, Shigeru; Kaga, Sanae; Obata, Keiko; Jin, Shigeki; Miwa, Keiko; Masauzi, Nobuo

    2016-11-01

    Morphological observation of blood or marrow film is still described nonquantitatively. We developed a semiautomatic method for segmenting vacuoles from the cytoplasm using Photoshop (PS) and Image-J (IJ), called PS-IJ, and measured the relative entire cell area (rECA) and relative areas of vacuoles (rAV) in the cytoplasm of neutrophil with PS-IJ. Whole-blood samples were stored at 4°C with ethylenediaminetetraacetate and in two different preserving manners (P1 and P2). Color-tone intensity levels of neutrophil images were semiautomatically compensated using PS, and then vacuole portions were automatically segmented by IJ. The rAV and rECA were measured by counting pixels by IJ. For evaluating the accuracy in segmentations of vacuoles with PS-IJ, the rAV/rECA ratios calculated with results from PS-IJ were compared with those calculated with human eye and IJ (HE-IJ). The rECA and rAV/ in P1 significantly (P < 0.05, P < 0.05) were enlarged and increased, but did not significantly (P = 0.46, P = 0.21) change in P2. The rAV/rECA ratios by PS-IJ were significantly correlated (r = 0.90, P < 0.01) with those by HE-IJ. PS-IJ method can successfully segment vacuoles and measure the rAV and rECA, becoming a useful tool for quantitative description of morphological observation of blood and marrow film. © 2016 Wiley Periodicals, Inc.

  10. Using a two-layered sphere model to investigate the impact of gas vacuoles on the inherent optical properties of Microcystis aeruginosa

    CSIR Research Space (South Africa)

    Matthews, MW

    2013-01-01

    Full Text Available A two-layered sphere model is used to investigate the impact of gas vacuoles on the inherent optical properties (IOPs) of the cyanophyte Microcystis aeruginosa. Enclosing a vacuole-like particle within a chromatoplasm shell layer significantly...

  11. Down-regulation of Cell Surface Cyclic AMP Receptors and Desensitization of Cyclic AMP-stimulated Adenylate Cyclase by Cyclic AMP in Dictyostelium discoideum. Kinetics and Concentration Dependence

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylate cyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of

  12. Effect of sodium fluoride on the amount of polyribosomes, single ribosomes and ribosomal subunits in a cellular slime mold, Dictyostelium discoideum

    Energy Technology Data Exchange (ETDEWEB)

    Sameshima, M; Ito, K; Iwabuchi, M

    1972-01-01

    In the slime mold, Dictyostelium discoideum, when the rate of protein synthesis was decreased by NaF, free 80-S ribosomes accumulated at the expense of polyribosomes, while 60-S and 40-S ribosomal subunits remained almost constant. The same level of ribosomal subunits was also maintained in cells after incubation with cycloheximide or at the stationary phase of growth.

  13. Specificity of the Cyclic GMP-Binding Activity and of a Cyclic GMP-Dependent Cyclic GMP Phosphodiesterase in Dictyostelium discoideum

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Walsum, Hans van; Meer, Rob C. van der; Bulgakov, Roman; Konijn, Theo M.

    1982-01-01

    The nucleotide specificity of the cyclic GMP-binding activity in a homogenate of Dictyostelium discoideum was determined by competition of cyclic GMP derivatives with [8-3H] cyclic GMP for the binding sites. The results indicate that cyclic GMP is bound to the binding proteins by hydrogen bonds at

  14. Functional similarities between the dictyostelium protein AprA and the human protein dipeptidyl-peptidase IV.

    Science.gov (United States)

    Herlihy, Sarah E; Tang, Yu; Phillips, Jonathan E; Gomer, Richard H

    2017-03-01

    Autocrine proliferation repressor protein A (AprA) is a protein secreted by Dictyostelium discoideum cells. Although there is very little sequence similarity between AprA and any human protein, AprA has a predicted structural similarity to the human protein dipeptidyl peptidase IV (DPPIV). AprA is a chemorepellent for Dictyostelium cells, and DPPIV is a chemorepellent for neutrophils. This led us to investigate if AprA and DPPIV have additional functional similarities. We find that like AprA, DPPIV is a chemorepellent for, and inhibits the proliferation of, D. discoideum cells, and that AprA binds some DPPIV binding partners such as fibronectin. Conversely, rAprA has DPPIV-like protease activity. These results indicate a functional similarity between two eukaryotic chemorepellent proteins with very little sequence similarity, and emphasize the usefulness of using a predicted protein structure to search a protein structure database, in addition to searching for proteins with similar sequences. © 2016 The Protein Society.

  15. Functional similarities between the dictyostelium protein AprA and the human protein dipeptidyl‐peptidase IV

    Science.gov (United States)

    Herlihy, Sarah E.; Tang, Yu; Phillips, Jonathan E.

    2017-01-01

    Abstract Autocrine proliferation repressor protein A (AprA) is a protein secreted by Dictyostelium discoideum cells. Although there is very little sequence similarity between AprA and any human protein, AprA has a predicted structural similarity to the human protein dipeptidyl peptidase IV (DPPIV). AprA is a chemorepellent for Dictyostelium cells, and DPPIV is a chemorepellent for neutrophils. This led us to investigate if AprA and DPPIV have additional functional similarities. We find that like AprA, DPPIV is a chemorepellent for, and inhibits the proliferation of, D. discoideum cells, and that AprA binds some DPPIV binding partners such as fibronectin. Conversely, rAprA has DPPIV‐like protease activity. These results indicate a functional similarity between two eukaryotic chemorepellent proteins with very little sequence similarity, and emphasize the usefulness of using a predicted protein structure to search a protein structure database, in addition to searching for proteins with similar sequences. PMID:28028841

  16. A retinoblastoma orthologue is a major regulator of S-phase, mitotic, and developmental gene expression in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Kimchi Strasser

    Full Text Available The retinoblastoma tumour suppressor, Rb, has two major functions. First, it represses genes whose products are required for S-phase entry and progression thus stabilizing cells in G1. Second, Rb interacts with factors that induce cell-cycle exit and terminal differentiation. Dictyostelium lacks a G1 phase in its cell cycle but it has a retinoblastoma orthologue, rblA.Using microarray analysis and mRNA-Seq transcriptional profiling, we show that RblA strongly represses genes whose products are involved in S phase and mitosis. Both S-phase and mitotic genes are upregulated at a single point in late G2 and again in mid-development, near the time when cell cycling is reactivated. RblA also activates a set of genes unique to slime moulds that function in terminal differentiation.Like its mammalian counterpart Dictyostelium, RblA plays a dual role, regulating cell-cycle progression and transcriptional events leading to terminal differentiation. In the absence of a G1 phase, however, RblA functions in late G2 controlling the expression of both S-phase and mitotic genes.

  17. dictyExpress: a Dictyostelium discoideum gene expression database with an explorative data analysis web-based interface

    Science.gov (United States)

    Rot, Gregor; Parikh, Anup; Curk, Tomaz; Kuspa, Adam; Shaulsky, Gad; Zupan, Blaz

    2009-01-01

    Background Bioinformatics often leverages on recent advancements in computer science to support biologists in their scientific discovery process. Such efforts include the development of easy-to-use web interfaces to biomedical databases. Recent advancements in interactive web technologies require us to rethink the standard submit-and-wait paradigm, and craft bioinformatics web applications that share analytical and interactive power with their desktop relatives, while retaining simplicity and availability. Results We have developed dictyExpress, a web application that features a graphical, highly interactive explorative interface to our database that consists of more than 1000 Dictyostelium discoideum gene expression experiments. In dictyExpress, the user can select experiments and genes, perform gene clustering, view gene expression profiles across time, view gene co-expression networks, perform analyses of Gene Ontology term enrichment, and simultaneously display expression profiles for a selected gene in various experiments. Most importantly, these tasks are achieved through web applications whose components are seamlessly interlinked and immediately respond to events triggered by the user, thus providing a powerful explorative data analysis environment. Conclusion dictyExpress is a precursor for a new generation of web-based bioinformatics applications with simple but powerful interactive interfaces that resemble that of the modern desktop. While dictyExpress serves mainly the Dictyostelium research community, it is relatively easy to adapt it to other datasets. We propose that the design ideas behind dictyExpress will influence the development of similar applications for other model organisms. PMID:19706156

  18. Contractility of the guinea pig bladder measured in situ and in vitro

    NARCIS (Netherlands)

    J.M. Groen (Jan); R. van Mastrigt (Ron); J.L.H.R. Bosch (Ruud)

    1994-01-01

    textabstractTo study the relative importance of neurogenic factors in detrusor contractility and to relate a total bladder in vitro contractility model to a previously described bladder wall strip model, active intravesical pressure values were compared in situ and in vitro in eight male guinea

  19. Free radicals in hypoxic rat diaphragm contractility: no role for xanthine oxidase.

    NARCIS (Netherlands)

    Heunks, L.M.A.; Machiels, H.A.; Abreu, R.A. de; Zhu, X.; Heijden, E. van der; Dekhuijzen, P.N.R.

    2001-01-01

    Recent evidence indicates that hypoxia enhances the generation of oxidants. Little is known about the role of free radicals in contractility of the rat diaphragm during hypoxia. We hypothesized that antioxidants improve contractility of the hypoxic rat diaphragm and that xanthine oxidase (XO) is an

  20. Effects of ageing on single muscle fibre contractile function following short-term immobilisation

    DEFF Research Database (Denmark)

    Hvid, Lars G; Ørtenblad, Niels; Aagaard, Per

    2011-01-01

    Very little attention has been given to the combined effect of healthy ageing and short-term disuse on the contractile function of human single muscle fibres. Therefore, the present study investigated the effects of 2 weeks of lower limb cast immobilisation (i.e. disuse) on selected contractile...

  1. Transient impairments in single muscle fibre contractile function after prolonged cycling in elite endurance athletes

    DEFF Research Database (Denmark)

    Hvid, L G; Gejl, Kasper Degn; Bech, R D

    2013-01-01

    Prolonged muscle activity impairs whole-muscle performance and function. However, little is known about the effects of prolonged muscle activity on the contractile function of human single muscle fibres. The purpose of this study was to investigate the effects of prolonged exercise and subsequent...... recovery on the contractile function of single muscle fibres obtained from elite athletes....

  2. Detecting cardiac contractile activity in the early mouse embryo using multiple modalities

    Directory of Open Access Journals (Sweden)

    Chiann-mun eChen

    2015-01-01

    Full Text Available The heart is one of the first organs to develop during mammalian embryogenesis. In the mouse, it starts to form shortly after gastrulation, and is derived primarily from embryonic mesoderm. The embryonic heart is unique in having to perform a mechanical contractile function while undergoing complex morphogenetic remodelling. Approaches to imaging the morphogenesis and contractile activity of the developing heart are important in understanding not only how this remodelling is controlled but also the origin of congenital heart defects. Here, we describe approaches for visualising contractile activity in the developing mouse embryo, using brightfield time lapse microscopy and confocal microscopy of calcium transients. We describe an algorithm for enhancing this image data and quantifying contractile activity from it. Finally we describe how atomic force microscopy can be used to record contractile activity prior to it being microscopically visible.

  3. The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging.

    Directory of Open Access Journals (Sweden)

    Fernando Real

    Full Text Available An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i hosting amastigotes of either L. major or L. amazonensis and ii loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

  4. New challenges for the design of high value plant products: stabilization of anthocyanins in plant vacuoles

    Directory of Open Access Journals (Sweden)

    Valentina ePasseri

    2016-02-01

    Full Text Available In the last decade plant biotechnologists and breeders have made several attempt to improve the antioxidant content of plant-derived food. Most efforts concentrated on increasing the synthesis of antioxidants, in particular anthocyanins, by inducing the transcription of genes encoding the synthesizing enzymes. We present here an overview of economically interesting plant species, both food crops and ornamentals, in which anthocyanin content was improved by traditional breeding or transgenesis. Old genetic studies in petunia and more recent biochemical work in brunfelsia, have shown that after synthesis and compartmentalization in the vacuole, anthocyanins need to be stabilized to preserve the color of the plant tissue over time. The final yield of antioxidant molecules is the result of the balance between synthesis and degradation. Therefore the understanding of the mechanism that determine molecule stabilization in the vacuolar lumen is the next step that needs to be taken to further improve the anthocyanin content in food.In several species a phenomenon known as fading is responsible for the disappearance of pigmentation which in some case can be nearly complete. We discuss the present knowledge about the genetic and biochemical factors involved in pigment preservation/destabilization in plant cells.The improvement of our understanding of the fading process will supply new tools for both biotechnological approaches and marker-assisted breeding.

  5. 'Candidatus Liberibacter asiaticus' Accumulates inside Endoplasmic Reticulum Associated Vacuoles in the Gut Cells of Diaphorina citri.

    Science.gov (United States)

    Ghanim, Murad; Achor, Diann; Ghosh, Saptarshi; Kontsedalov, Svetlana; Lebedev, Galina; Levy, Amit

    2017-12-05

    Citrus greening disease known also as Huanglongbing (HLB) caused by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus' (CLas) has resulted in tremendous losses and the death of millions of trees worldwide. CLas is transmitted by the Asian citrus psyllid Diaphorina citri. The closely-related bacteria 'Candidatus Liberibacter solanacearum' (CLso), associated with vegetative disorders in carrots, is transmitted by the carrot psyllid Bactericera trigonica. A promising approach to prevent the transmission of these pathogens is to interfere with the vector-pathogen interactions, but our understanding of these processes is limited. It was recently reported that CLas induced changes in the nuclear architecture, and activated programmed cell death, in D. citri midgut cells. Here, we used electron and fluorescent microscopy and show that CLas induces the formation of endoplasmic reticulum (ER)-associated bodies. The bacterium recruits those ER structures into Liberibacter containing vacuoles (LCVs), in which bacterial cells seem to propagate. ER- associated LCV formation was unique to CLas, as we could not detect these bodies in B. trigonica infected with CLso. ER recruitment is hypothesized to generate a safe replicative body to escape cellular immune responses in the insect gut. Understanding the molecular interactions that undelay these responses will open new opportunities for controlling CLas.

  6. Cardiac contractility, central haemodynamics and blood pressure regulation during semistarvation

    DEFF Research Database (Denmark)

    Stokholm, K H; Breum, L; Astrup, A

    1991-01-01

    pressure (BP) declined. The fall in BP was caused by the reduction in cardiac output as the total peripheral resistance was unchanged. Finally, the decline in total blood volume was not significant. These findings together with a reduction in heart rate indicated that a reduced sympathetic tone via......Eight obese patients were studied before and after 2 weeks of treatment by a very-low-calorie diet (VLCD). Cardiac output and central blood volume (pulmonary blood volume and left atrial volume) were determined by indicator dilution (125I-albumin) and radionuclide angiocardiography (first pass...... and equilibrium technique by [99Tcm]red blood cells). Cardiac output decreased concomitantly with the reduction in oxygen uptake as the calculated systemic arteriovenous difference of oxygen was unaltered. There were no significant decreases in left ventricular contractility indices, i.e. the ejection fraction...

  7. Mucolipin Co-deficiency Causes Accelerated Endolysosomal Vacuolation of Enterocytes and Failure-to-Thrive from Birth to Weaning

    Science.gov (United States)

    Castiglioni, Andrew J.; Flores, Emma N.; Cantú, Jorge A.; García-Añoveros, Jaime

    2014-01-01

    During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3−/−;Trpml1−/−) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3−/−;Trpml1−/− mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns

  8. Matrix elasticity regulates the optimal cardiac myocyte shape for contractility

    Science.gov (United States)

    McCain, Megan L.; Yuan, Hongyan; Pasqualini, Francesco S.; Campbell, Patrick H.

    2014-01-01

    Concentric hypertrophy is characterized by ventricular wall thickening, fibrosis, and decreased myocyte length-to-width aspect ratio. Ventricular thickening is considered compensatory because it reduces wall stress, but the functional consequences of cell shape remodeling in this pathological setting are unknown. We hypothesized that decreases in myocyte aspect ratio allow myocytes to maximize contractility when the extracellular matrix becomes stiffer due to conditions such as fibrosis. To test this, we engineered neonatal rat ventricular myocytes into rectangles mimicking the 2-D profiles of healthy and hypertrophied myocytes on hydrogels with moderate (13 kPa) and high (90 kPa) elastic moduli. Actin alignment was unaffected by matrix elasticity, but sarcomere content was typically higher on stiff gels. Microtubule polymerization was higher on stiff gels, implying increased intracellular elastic modulus. On moderate gels, myocytes with moderate aspect ratios (∼7:1) generated the most peak systolic work compared with other cell shapes. However, on stiffer gels, low aspect ratios (∼2:1) generated the most peak systolic work. To compare the relative contributions of intracellular vs. extracellular elasticity to contractility, we developed an analytical model and used our experimental data to fit unknown parameters. Our model predicted that matrix elasticity dominates over intracellular elasticity, suggesting that the extracellular matrix may potentially be a more effective therapeutic target than microtubules. Our data and model suggest that myocytes with lower aspect ratios have a functional advantage when the elasticity of the extracellular matrix decreases due to conditions such as fibrosis, highlighting the role of the extracellular matrix in cardiac disease. PMID:24682394

  9. Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

    Directory of Open Access Journals (Sweden)

    Ivan Kulich

    2014-04-01

    Full Text Available Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER to vacuole (and also, apoplast transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast, exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA and salicylic acid (SA degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

  10. Multinodular and Vacuolating Neuronal Tumor of the Cerebrum: A New "Leave Me Alone" Lesion with a Characteristic Imaging Pattern.

    Science.gov (United States)

    Nunes, R H; Hsu, C C; da Rocha, A J; do Amaral, L L F; Godoy, L F S; Watkins, T W; Marussi, V H; Warmuth-Metz, M; Alves, H C; Goncalves, F G; Kleinschmidt-DeMasters, B K; Osborn, A G

    2017-10-01

    Multinodular and vacuolating neuronal tumor of the cerebrum is a recently reported benign, mixed glial neuronal lesion that is included in the 2016 updated World Health Organization classification of brain neoplasms as a unique cytoarchitectural pattern of gangliocytoma. We report 33 cases of presumed multinodular and vacuolating neuronal tumor of the cerebrum that exhibit a remarkably similar pattern of imaging findings consisting of a subcortical cluster of nodular lesions located on the inner surface of an otherwise normal-appearing cortex, principally within the deep cortical ribbon and superficial subcortical white matter, which is hyperintense on FLAIR. Only 4 of our cases are biopsy-proven because most were asymptomatic and incidentally discovered. The remaining were followed for a minimum of 24 months (mean, 3 years) without interval change. We demonstrate that these are benign, nonaggressive lesions that do not require biopsy in asymptomatic patients and behave more like a malformative process than a true neoplasm. © 2017 by American Journal of Neuroradiology.

  11. Progesterone Metabolites Produced by Cytochrome P450 3A Modulate Uterine Contractility in a Murine Model

    Science.gov (United States)

    Patil, Avinash S.; Swamy, Geeta K.; Murtha, Amy P.; Heine, R. Phillips; Zheng, Xiaomei; Grotegut, Chad A.

    2015-01-01

    Objective: We seek to characterize the effect of progesterone metabolites on spontaneous and oxytocin-induced uterine contractility. Study Design: Spontaneous contractility was studied in mouse uterine horns after treatment with progesterone, 2α-hydroxyprogesterone, 6β-hydroxyprogesterone (6β-OHP), 16α-hydroxyprogesterone (16α-OHP), or 17-hydroxyprogesterone caproate (17-OHPC) at 10−9 to 10−6 mol/L. Uterine horns were exposed to progestins (10−6 mol/L), followed by increasing concentrations of oxytocin (1-100 nmol/L) to study oxytocin-induced contractility. Contraction parameters were compared for each progestin and matched vehicle control using repeated measures 2-way analysis of variance. In vitro metabolism of progesterone by recombinant cytochrome P450 3A (CYP3A) microsomes (3A5, 3A5, and 3A7) identified major metabolites. Results: Oxytocin-induced contractile frequency was decreased by 16α-OHP (P = .03) and increased by 6β-OHP (P = .05). Progesterone and 17-OHPC decreased oxytocin-induced contractile force (P = .02 and P = .04, respectively) and frequency (P = .02 and P = .03, respectively). Only progesterone decreased spontaneous contractile force (P = .02). Production of 16α-OHP and 6β-OHP metabolites were confirmed in all CYP3A isoforms tested. Conclusion: Progesterone metabolites produced by maternal or fetal CYP3A enzymes influence uterine contractility. PMID:26037300

  12. Chronic ingestion of 2-deoxy-D-glucose induces cardiac vacuolization and increases mortality in rats

    International Nuclear Information System (INIS)

    Minor, Robin K.; Smith, Daniel L.; Sossong, Alex M.; Kaushik, Susmita; Poosala, Suresh; Spangler, Edward L.; Roth, George S.; Lane, Mark; Allison, David B.; Cabo, Rafael de; Ingram, Donald K.; Mattison, Julie A.

    2010-01-01

    Calorie restriction (CR), the purposeful reduction of energy intake with maintenance of adequate micronutrient intake, is well known to extend the lifespan of laboratory animals. Compounds like 2-deoxy-D-glucose (2DG) that can recapitulate the metabolic effects of CR are of great interest for their potential to extend lifespan. 2DG treatment has been shown to have potential therapeutic benefits for treating cancer and seizures. 2DG has also recapitulated some hallmarks of the CR phenotype including reduced body temperature and circulating insulin in short-term rodent trials, but one chronic feeding study in rats found toxic effects. The present studies were performed to further explore the long-term effects of 2DG in vivo. First we demonstrate that 2DG increases mortality of male Fischer-344 rats. Increased incidence of pheochromocytoma in the adrenal medulla was also noted in the 2DG treated rats. We reconfirm the cardiotoxicity of 2DG in a 6-week follow-up study evaluating male Brown Norway rats and a natural form of 2DG in addition to again examining effects in Fischer-344 rats and the original synthetic 2DG. High levels of both 2DG sources reduced weight gain secondary to reduced food intake in both strains. Histopathological analysis of the hearts revealed increasing vacuolarization of cardiac myocytes with dose, and tissue staining revealed the vacuoles were free of both glycogen and lipid. We did, however, observe higher expression of both cathepsin D and LC3 in the hearts of 2DG-treated rats which indicates an increase in autophagic flux. Although a remarkable CR-like phenotype can be reproduced with 2DG treatment, the ultimate toxicity of 2DG seriously challenges 2DG as a potential CR mimetic in mammals and also raises concerns about other therapeutic applications of the compound.

  13. Identification of vacuoles containing extraintestinal differentiated forms of Legionella pneumophila in colonized Caenorhabditis elegans soil nematodes.

    Science.gov (United States)

    Hellinga, Jacqueline R; Garduño, Rafael A; Kormish, Jay D; Tanner, Jennifer R; Khan, Deirdre; Buchko, Kristyn; Jimenez, Celine; Pinette, Mathieu M; Brassinga, Ann Karen C

    2015-08-01

    Legionella pneumophila, a causative agent of Legionnaires' disease, is a facultative intracellular parasite of freshwater protozoa. Legionella pneumophila features a unique developmental network that involves several developmental forms including the infectious cyst forms. Reservoirs of L. pneumophila include natural and man-made freshwater systems; however, recent studies have shown that isolates of L. pneumophila can also be obtained directly from garden potting soil suggesting the presence of an additional reservoir. A previous study employing the metazoan Caenorhabditis elegans, a member of the Rhabditidae family of free-living soil nematodes, demonstrated that the intestinal lumen can be colonized with L. pneumophila. While both replicative forms and differentiated forms were observed in C. elegans, these morphologically distinct forms were initially observed to be restricted to the intestinal lumen. Using live DIC imaging coupled with focused transmission electron microscopy analyses, we report here that L. pneumophila is able to invade and establish Legionella-containing vacuoles (LCVs) in the intestinal cells. In addition, LCVs containing replicative and differentiated cyst forms were observed in the pseudocoelomic cavity and gonadal tissue of nematodes colonized with L. pneumophila. Furthermore, establishment of LCVs in the gonadal tissue was Dot/Icm dependent and required the presence of the endocytic factor RME-1 to gain access to maturing oocytes. Our findings are novel as this is the first report, to our knowledge, of extraintestinal LCVs containing L. pneumophila cyst forms in C. elegans tissues, highlighting the potential of soil-dwelling nematodes as an alternate environmental reservoir for L. pneumophila. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  14. Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection.

    Directory of Open Access Journals (Sweden)

    Eva Rothmeier

    2013-09-01

    Full Text Available The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS to form in phagocytes a distinct "Legionella-containing vacuole" (LCV, which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.

  15. Natural history of lesions with the MR imaging appearance of multinodular and vacuolating neuronal tumor

    Energy Technology Data Exchange (ETDEWEB)

    Alsufayan, Reema [University of Toronto, Toronto (Canada); Alcaide-Leon, Paula; De Tilly, Lyne Noel [University of Toronto, St. Michael' s Hospital, Toronto (Canada); Mandell, Daniel M.; Krings, Timo [University of Toronto, Toronto Western Hospital, UHN Division of Neuroradiology, Toronto, ON (Canada)

    2017-09-15

    Multinodular and vacuolating neuronal tumor (MVNT) have been recently added to the WHO classification of CNS tumors and has not been extensively reported upon in the radiological literature. We report the first radiological and the largest series of cases, aiming to highlight the natural history of lesions with the imaging appearance of MVNT with long follow-up time. In this retrospective study, we collected cases with the imaging appearance of MVNT. All lesions were evaluated by using routine MR imaging, with follow-up of up to 93 months. Patient demographics, clinical course, and MRI features of the lesions were recorded. Twenty-four subjects were enrolled, f/m = 16:8, age range 24-59 years, with a median age of 45 years. The patients' symptoms were often episodic and most frequently due to headaches in 12 (50%), visual symptoms in 6 (25%), seizures in 5 ± 1 (20-25%), paresthesia in 4 (∝17%), cognitive difficulties in 4 (∝17%), in addition to other variable neurological symptoms, or incidental. A total of 30 lesions identified, 77% of the lesions had gadolinium-enhanced MRI and only 13% showed enhancement. A 6.7% of the lesions that had MRI followed up showed progression, while the rest remained stable up to 93 months interval. All patients had intact neurological examinations (except one case that was diagnosed with optic neuritis), were managed conservatively, and did well. The natural history of lesions with imaging features of MVNT is overall stable from a clinical and imaging appearance over time. (orig.)

  16. Natural history of lesions with the MR imaging appearance of multinodular and vacuolating neuronal tumor

    International Nuclear Information System (INIS)

    Alsufayan, Reema; Alcaide-Leon, Paula; De Tilly, Lyne Noel; Mandell, Daniel M.; Krings, Timo

    2017-01-01

    Multinodular and vacuolating neuronal tumor (MVNT) have been recently added to the WHO classification of CNS tumors and has not been extensively reported upon in the radiological literature. We report the first radiological and the largest series of cases, aiming to highlight the natural history of lesions with the imaging appearance of MVNT with long follow-up time. In this retrospective study, we collected cases with the imaging appearance of MVNT. All lesions were evaluated by using routine MR imaging, with follow-up of up to 93 months. Patient demographics, clinical course, and MRI features of the lesions were recorded. Twenty-four subjects were enrolled, f/m = 16:8, age range 24-59 years, with a median age of 45 years. The patients' symptoms were often episodic and most frequently due to headaches in 12 (50%), visual symptoms in 6 (25%), seizures in 5 ± 1 (20-25%), paresthesia in 4 (∝17%), cognitive difficulties in 4 (∝17%), in addition to other variable neurological symptoms, or incidental. A total of 30 lesions identified, 77% of the lesions had gadolinium-enhanced MRI and only 13% showed enhancement. A 6.7% of the lesions that had MRI followed up showed progression, while the rest remained stable up to 93 months interval. All patients had intact neurological examinations (except one case that was diagnosed with optic neuritis), were managed conservatively, and did well. The natural history of lesions with imaging features of MVNT is overall stable from a clinical and imaging appearance over time. (orig.)

  17. A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles.

    Directory of Open Access Journals (Sweden)

    Shira Ninio

    2009-01-01

    Full Text Available Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.

  18. A flavin-dependent halogenase catalyzes the chlorination step in the biosynthesis of Dictyostelium differentiation-inducing factor 1.

    Science.gov (United States)

    Neumann, Christopher S; Walsh, Christopher T; Kay, Robert R

    2010-03-30

    Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate gene for this transformation. In this work, we present in vivo and in vitro evidence that chlA from Dd encodes a flavin-dependent halogenase capable of catalyzing both chlorinations in the biosynthesis of DIF-1. The results provide in vitro characterization of a eukaryotic oxygen-dependent halogenase and demonstrate a broad reach in biology for this molecular tailoring strategy, notably its involvement in the differentiation program of a social amoeba.

  19. Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

    International Nuclear Information System (INIS)

    Prem Das, O.; Henderson, E.J.

    1986-01-01

    Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum

  20. Predicting the distribution of spiral waves from cell properties in a developmental-path model of Dictyostelium pattern formation.

    Directory of Open Access Journals (Sweden)

    Daniel Geberth

    2009-07-01

    Full Text Available The slime mold Dictyostelium discoideum is one of the model systems of biological pattern formation. One of the most successful answers to the challenge of establishing a spiral wave pattern in a colony of homogeneously distributed D. discoideum cells has been the suggestion of a developmental path the cells follow (Lauzeral and coworkers. This is a well-defined change in properties each cell undergoes on a longer time scale than the typical dynamics of the cell. Here we show that this concept leads to an inhomogeneous and systematic spatial distribution of spiral waves, which can be predicted from the distribution of cells on the developmental path. We propose specific experiments for checking whether such systematics are also found in data and thus, indirectly, provide evidence of a developmental path.

  1. Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins

    Directory of Open Access Journals (Sweden)

    Escalante Ricardo

    2008-01-01

    Full Text Available Abstract Background The social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation. Results Subtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N, that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87–89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development. Conclusion A large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.

  2. Quantifying esophagogastric junction contractility with a novel HRM topographic metric, the EGJ-Contractile Integral: normative values and preliminary evaluation in PPI non-responders.

    Science.gov (United States)

    Nicodème, F; Pipa-Muniz, M; Khanna, K; Kahrilas, P J; Pandolfino, J E

    2014-03-01

    Despite its obvious pathophysiological relevance, the clinical utility of measures of esophagogastric junction (EGJ) contractility is unsubstantiated. High-resolution manometry (HRM) may improve upon this with its inherent ability to integrate the magnitude of contractility over time and length of the EGJ. This study aimed to develop a novel HRM metric summarizing EGJ contractility and test its ability distinguish among subgroups of proton pump inhibitor non-responders (PPI-NRs). 75 normal controls and 88 PPI-NRs were studied. All underwent HRM. PPI-NRs underwent pH-impedance monitoring on PPI therapy scored in terms of acid exposure, number of reflux events, and reflux-symptom correlation and grouped as meeting all criteria, some criteria, or no criteria of abnormality. Control HRM studies were used to establish normal values for candidate EGJ contractility metrics, which were then compared in their ability to differentiate among PPI-NR subgroups. The EGJ contractile integral (EGJ-CI), a metric integrating contractility across the EGJ for three respiratory cycles, best distinguished the All Criteria PPI-NR subgroup from controls and other PPI-NR subgroups. Normal values (median, [IQR]) for this measure were 39 mmHg-cm [25-55 mmHg-cm]. The correlation between the EGJ-CI and a previously proposed metric, the lower esophageal sphincter-pressure integral, that used a fixed 10 s time frame and an atmospheric as opposed to gastric pressure reference was weak. Among HRM metrics tested, the EGJ-CI was best in distinguishing PPI-NRs meeting all criteria of abnormality on pH-impedance testing. Future prospective studies are required to explore its utility in management of broader groups of gastroesophageal reflux disease patients. © 2013 John Wiley & Sons Ltd.

  3. Computed tomographical findings of skeletal muscles in rimmed vacuole type distal myopathy

    International Nuclear Information System (INIS)

    Kunimoto, Masanari; Kawai, Mitsuru; Goto, Jun; Nakano, Imaharu

    1987-01-01

    Skeletal muscle CT scans of three patients with biopsy-proven rimmed vacuole type distal myopathy(RVDM) from two unrelated families showed unique involvement pattern of the lower extremities. Parents of the patients in both families are first cousins. T.Y. is a 36-year-old woman who noticed mild difficulty in walking at 34 years of age. Now she shows waddling but still indpendent gait. Manual muscle test (MMT) revealed the following results: fair (3+/5) for hip flexion, 3 for hip extension, 3 for knee flexion, normal (5/5) for knee extension, poor (2/5) for ankle dorsi-flexion, good (4/5) for ankle plantar flexion. T.M., a 34-year-old younger sister of T.Y., started dragging her feet on gait at age 27. She could walk neither on toes nor heels. At present, she can walk only with support. MMT showed the following: 3- and trace (1/5) for hip flexion and extension, 3- and 4 for knee flexion and extension, 1 for ankle plantar- and dorsiflexion. K.W., a 33-year-old woman, began to drag her foot tips in walk and became unable to walk on toes at age 21. The leg weakness progressed into the wheelchair-ridden state at age 30. MMT gave the following results: 1 for hip flexion and extension, 2 and 4 for knee flexion and extension, zero for plantar- and dorsi-flexion. The most impressive CT findings common to these three patients are prominent contrast between the quadriceps muscles and the adductor- and hamstring-group: the former is markedly well preserved even in the most advanced patient (K.W.) while the latter are diffusely and severely affected even in the least affected patient (T.Y.). This finding well coincides with the results of MMT: the knee extensor (quadriceps muscles) fairly well keeps its strength even in the most advanced patient but the knee flexors (adductors and hamstrings) are definitely affected in the early stage of the condition. (J.P.N.)

  4. Computed tomographical findings of skeletal muscles in rimmed vacuole type distal myopathy

    Energy Technology Data Exchange (ETDEWEB)

    Kunimoto, Masanari; Kawai, Mitsuru; Goto, Jun; Nakano, Imaharu

    1987-03-01

    Skeletal muscle CT scans of three patients with biopsy-proven rimmed vacuole type distal myopathy(RVDM) from two unrelated families showed unique involvement pattern of the lower extremities. Parents of the patients in both families are first cousins. T.Y. is a 36-year-old woman who noticed mild difficulty in walking at 34 years of age. Now she shows waddling but still indpendent gait. Manual muscle test (MMT) revealed the following results: fair (3+/5) for hip flexion, 3 for hip extension, 3 for knee flexion, normal (5/5) for knee extension, poor (2/5) for ankle dorsi-flexion, good (4/5) for ankle plantar flexion. T.M., a 34-year-old younger sister of T.Y., started dragging her feet on gait at age 27. She could walk neither on toes nor heels. At present, she can walk only with support. MMT showed the following: 3- and trace (1/5) for hip flexion and extension, 3- and 4 for knee flexion and extension, 1 for ankle plantar- and dorsiflexion. K.W., a 33-year-old woman, began to drag her foot tips in walk and became unable to walk on toes at age 21. The leg weakness progressed into the wheelchair-ridden state at age 30. MMT gave the following results: 1 for hip flexion and extension, 2 and 4 for knee flexion and extension, zero for plantar- and dorsi-flexion. The most impressive CT findings common to these three patients are prominent contrast between the quadriceps muscles and the adductor- and hamstring-group: the former is markedly well preserved even in the most advanced patient (K.W.) while the latter are diffusely and severely affected even in the least affected patient (T.Y.). This finding well coincides with the results of MMT: the knee extensor (quadriceps muscles) fairly well keeps its strength even in the most advanced patient but the knee flexors (adductors and hamstrings) are definitely affected in the early stage of the condition. (J.P.N.).

  5. The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

    Science.gov (United States)

    Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

    2015-01-01

    Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. PMID:25713145

  6. Adipose-derived stem cells inhibit the contractile myofibroblast in Dupuytren's disease.

    NARCIS (Netherlands)

    Verhoekx, J.S.; Mudera, V.; Walbeehm, E.T.; Hovius, S.E.

    2013-01-01

    BACKGROUND: In an attempt to provide minimally invasive treatment for Dupuytren's disease, percutaneous disruption of the affected tissue followed by lipografting is being tested. Contractile myofibroblasts drive this fibroproliferative disorder, whereas stem cells have recently been implicated in

  7. Considerations for Contractile Electroactive Polymeric Materials and Actuators

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Lenore; Erickson, Carl J.; Meixler, Lewis D.; Ascione, George; Gentile, Charles A.; Tilson, Charles; Bernasek, Stephen L.; Abelev, Esta

    2009-06-16

    Ras Labs produces electroactive polymer (EAP) based materials and actuators that bend, swell, ripple and now contract (new development) with low electric input. This is an important attribute because of the ability of contraction to produce life-like motion. The mechanism of contraction is not well understood. Radionuclide-labeled experiments were conducted to follow the movement of electrolytes and water in these EAPs when activated. Extreme temperature experiments were performed on the contractile EAPs with very favorable results. One of the biggest challenges in developing these actuators, however, is the electrode-EAP interface because of the pronounced movement of the EAP. Plasma treatments of metallic electrodes were investigated in order to improve the attachment of the embedded electrodes to the EAP material. Surface analysis, adhesive testing, and mechanical testing were conducted to test metal surfaces and metal-polymer interfaces. The nitrogen plasma treatment of titanium produced a strong metal-polymer interface; however, oxygen plasma treatment of both stainless steel and titanium produced even stronger metal-polymer interfaces. Plasma treatment of the electrodes allows for the embedded electrodes and the EAP material of the actuator to work and move as a unit, with no detachment, by significantly improving the metal-polymer interface.

  8. Mechanism of action of ethanol on heart contractility

    International Nuclear Information System (INIS)

    Oquendo-Muriente, I.; De Mello, W.C.

    1986-01-01

    Ethanol depresses heart contractility. To investigate the mechanism of the negative inotropic action of ethanol, rat ventricular strips were dissected and mounted vertically in a transparent chamber. The preparation was superfused initially with normal oxygenated Tyrode solution (32.5 0 C) and electrically stimulated (1 Hz). After 1 hour of equilibration, contractures were elicited by exposing the muscle strips to high K + (100 mM) solution. Studies on the influence of (Ca 2+ ) 0 on K + contractures showed that the first rapid component of the contracture (58 mg/sec - S.E. +/- 8; n = 8) was greatly dependent upon (Ca 2+ ) 0 while the second slow component (20 mg/sec - S.E. +/- 5; n = 8) was slightly altered. The addition of ethanol (400 mg/100 ml) to high K solution abolished the fast component and reduced the amplitude of the second phase of K contractures. Similar results were obtained with verapamil (10 -5 M). These results, as well as studies on the effect of the drug on 45 Ca fluxes support the view that ethanol decreases the permeability of the heart cell membrane to Ca

  9. Mechanism of action of ethanol on heart contractility

    Energy Technology Data Exchange (ETDEWEB)

    Oquendo-Muriente, I.; De Mello, W.C.

    1986-03-05

    Ethanol depresses heart contractility. To investigate the mechanism of the negative inotropic action of ethanol, rat ventricular strips were dissected and mounted vertically in a transparent chamber. The preparation was superfused initially with normal oxygenated Tyrode solution (32.5/sup 0/C) and electrically stimulated (1 Hz). After 1 hour of equilibration, contractures were elicited by exposing the muscle strips to high K/sup +/ (100 mM) solution. Studies on the influence of (Ca/sup 2 +/)/sub 0/ on K/sup +/ contractures showed that the first rapid component of the contracture (58 mg/sec - S.E. +/- 8; n = 8) was greatly dependent upon (Ca/sup 2 +/)/sub 0/ while the second slow component (20 mg/sec - S.E. +/- 5; n = 8) was slightly altered. The addition of ethanol (400 mg/100 ml) to high K solution abolished the fast component and reduced the amplitude of the second phase of K contractures. Similar results were obtained with verapamil (10/sup -5/ M). These results, as well as studies on the effect of the drug on /sup 45/Ca fluxes support the view that ethanol decreases the permeability of the heart cell membrane to Ca.

  10. Contractile Force of Human Extraocular Muscle: A Theoretical Analysis

    Directory of Open Access Journals (Sweden)

    Hongmei Guo

    2016-01-01

    Full Text Available Aim. The length-contractile force relationships of six human extraocular muscles (EOMs in primary innervations should be determined during eye movement modeling and surgery of clinical EOMs. This study aims to investigate these relationships. Method. The proposal is based on the assumption that six EOMs have similar constitutive relationships, with the eye suspended in the primary position. The constitutive relationships of EOMs are obtained by optimizing from previous experimental data and the theory of mechanical equilibrium using traditional model. Further, simulate the existing experiment of resistance force, and then compare the simulated results with the existing experimental results. Finally, the mechanical constitutive relationships of EOMs are obtained. Results. The results show that the simulated resistance forces from the other four EOMs except for the horizontal recti well agree with previous experimental results. Conclusion. The mechanical constitutive relationships of six EOMs in primary innervations are obtained, and the rationality of the constitutive relationships is verified. Whereafter, the active stress-strain relationships of the six EOMs in the primary innervations are obtained. The research results can improve the eye movement model to predict the surgical amounts of EOMs before EOM surgery more precisely.

  11. Postextrasystolic potentiation and contractile reserve: requirements and restrictions.

    Science.gov (United States)

    Lust, R M; Lutherer, L O; Gardner, M E; Cooper, M W

    1982-12-01

    These studies were conducted to examine the basic characteristics of postextrasystolic potentiation (PESP) and the relationship of loading effects to PESP. Measurements of left ventricular (LV) and aortic pressures, the rate of pressure rise, and echocardiographically determined LV dimensions were made in anesthetized open-chest dogs. The hearts were paced, and timed extrasystoles were introduced that were followed by postextrasystoles (PES). PES's were elicited after an interval equal to either a full compensatory pause or a time when the diastolic properties of the LV could not be distinguished from control (isolength). Potentiation of contraction for the PES's introduced after an isolength pause was dependent on both the heart rate and the extrasystolic interval, whereas the PES's that occurred after a full pause showed no dependence on either of these intervals. PESP elicited during the isolength period was not dependent on either preload and afterload. It is concluded that PESP depends on the combination of heart rate and extrasystolic and postextrasystolic intervals. Further, PESP may be inaccurate in assessing contractile reserve unless the heart rate and extrasystolic interval are known and the PES is introduced after an isolength pause.

  12. Considerations for Contractile Electroactive Polymeric Materials and Actuators

    International Nuclear Information System (INIS)

    Rasmussen, L.; Erickson, Carl J.; Meixler, Lewis D.; Ascione, G.; Gentile, Charles A.; Tilson, C.; Bernasek, Stephen L.; Abelev, E.

    2009-01-01

    Ras Labs produces electroactive polymer (EAP) based materials and actuators that bend, swell, ripple and now contract (new development) with low electric input. This is an important attribute because of the ability of contraction to produce life-like motion. The mechanism of contraction is not well understood. Radionuclide-labeled experiments were conducted to follow the movement of electrolytes and water in these EAPs when activated. Extreme temperature experiments were performed on the contractile EAPs with very favorable results. One of the biggest challenges in developing these actuators, however, is the electrode-EAP interface because of the pronounced movement of the EAP. Plasma treatments of metallic electrodes were investigated in order to improve the attachment of the embedded electrodes to the EAP material. Surface analysis, adhesive testing, and mechanical testing were conducted to test metal surfaces and metal-polymer interfaces. The nitrogen plasma treatment of titanium produced a strong metal-polymer interface; however, oxygen plasma treatment of both stainless steel and titanium produced even stronger metal-polymer interfaces. Plasma treatment of the electrodes allows for the embedded electrodes and the EAP material of the actuator to work and move as a unit, with no detachment, by significantly improving the metal-polymer interface

  13. Cellular Architecture Regulates Collective Calcium Signaling and Cell Contractility.

    Directory of Open Access Journals (Sweden)

    Jian Sun

    2016-05-01

    Full Text Available A key feature of multicellular systems is the ability of cells to function collectively in response to external stimuli. However, the mechanisms of intercellular cell signaling and their functional implications in diverse vascular structures are poorly understood. Using a combination of computational modeling and plasma lithography micropatterning, we investigate the roles of structural arrangement of endothelial cells in collective calcium signaling and cell contractility. Under histamine stimulation, endothelial cells in self-assembled and microengineered networks, but not individual cells and monolayers, exhibit calcium oscillations. Micropatterning, pharmacological inhibition, and computational modeling reveal that the calcium oscillation depends on the number of neighboring cells coupled via gap junctional intercellular communication, providing a mechanistic basis of the architecture-dependent calcium signaling. Furthermore, the calcium oscillation attenuates the histamine-induced cytoskeletal reorganization and cell contraction, resulting in differential cell responses in an architecture-dependent manner. Taken together, our results suggest that endothelial cells can sense and respond to chemical stimuli according to the vascular architecture via collective calcium signaling.

  14. Modeling the dispersion effects of contractile fibers in smooth muscles

    Science.gov (United States)

    Murtada, Sae-Il; Kroon, Martin; Holzapfel, Gerhard A.

    2010-12-01

    Micro-structurally based models for smooth muscle contraction are crucial for a better understanding of pathological conditions such as atherosclerosis, incontinence and asthma. It is meaningful that models consider the underlying mechanical structure and the biochemical activation. Hence, a simple mechanochemical model is proposed that includes the dispersion of the orientation of smooth muscle myofilaments and that is capable to capture available experimental data on smooth muscle contraction. This allows a refined study of the effects of myofilament dispersion on the smooth muscle contraction. A classical biochemical model is used to describe the cross-bridge interactions with the thin filament in smooth muscles in which calcium-dependent myosin phosphorylation is the only regulatory mechanism. A novel mechanical model considers the dispersion of the contractile fiber orientations in smooth muscle cells by means of a strain-energy function in terms of one dispersion parameter. All model parameters have a biophysical meaning and may be estimated through comparisons with experimental data. The contraction of the middle layer of a carotid artery is studied numerically. Using a tube the relationships between the internal pressure and the stretches are investigated as functions of the dispersion parameter, which implies a strong influence of the orientation of smooth muscle myofilaments on the contraction response. It is straightforward to implement this model in a finite element code to better analyze more complex boundary-value problems.

  15. Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation

    Directory of Open Access Journals (Sweden)

    Phillips Jonathan E

    2009-02-01

    Full Text Available Abstract Background Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA. Results We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 μg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA significantly slows proliferation at 0.1 μg/ml and higher concentrations. From 4 to 64 μg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 ± 11,000 cell-surface receptors with a KD of 0.03 ± 0.02 μg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Ki of 0.03 μg/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA. Conclusion Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a Cfa

  16. Dictyostelium cells bind a secreted autocrine factor that represses cell proliferation.

    Science.gov (United States)

    Choe, Jonathan M; Bakthavatsalam, Deenadayalan; Phillips, Jonathan E; Gomer, Richard H

    2009-02-02

    Dictyostelium cells secrete the proteins AprA and CfaD. Cells lacking either AprA or CfaD proliferate faster than wild type, while AprA or CfaD overexpressor cells proliferate slowly, indicating that AprA and CfaD are autocrine factors that repress proliferation. CfaD interacts with AprA and requires the presence of AprA to slow proliferation. To determine if CfaD is necessary for the ability of AprA to slow proliferation, whether AprA binds to cells, and if so whether the binding requires the presence of CfaD, we examined the binding and effect on proliferation of recombinant AprA. We find that the extracellular accumulation of AprA increases with cell density and reaches a concentration of 0.3 microg/ml near a stationary cell density. When added to wild-type or aprA- cells, recombinant AprA (rAprA) significantly slows proliferation at 0.1 microg/ml and higher concentrations. From 4 to 64 microg/ml, the effect of rAprA is at a plateau, slowing but not stopping proliferation. The proliferation-inhibiting activity of rAprA is roughly the same as that of native AprA in conditioned growth medium. Proliferating aprA- cells show saturable binding of rAprA to 92,000 +/- 11,000 cell-surface receptors with a KD of 0.03 +/- 0.02 microg/ml. There appears to be one class of binding site, and no apparent cooperativity. Native AprA inhibits the binding of rAprA to aprA- cells with a Ki of 0.03 mug/ml, suggesting that the binding kinetics of rAprA are similar to those of native AprA. The proliferation of cells lacking CrlA, a cAMP receptor-like protein, or cells lacking CfaD are not affected by rAprA. Surprisingly, both cell types still bind rAprA. Together, the data suggest that AprA functions as an autocrine proliferation-inhibiting factor by binding to cell surface receptors. Although AprA requires CfaD for activity, it does not require CfaD to bind to cells, suggesting the possibility that cells have an AprA receptor and a CfaD receptor, and activation of both receptors is

  17. A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

    OpenAIRE

    Lee, Yan Quan; Goh, Amanda S. P.; Ch'ng, Jun Hong; Nosten, François H.; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar; Tan, Kevin S. W.

    2014-01-01

    Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of in...

  18. Modifying influence of incorporated 137Cs upon the mechanisms of adrenergic control over contractile myucard function

    International Nuclear Information System (INIS)

    Lobanok, L.M.; Bulanova, K.Ya.; Gerasimovich, N.V.; Sineleva, M.V.; Milyutin, A.A.

    1994-01-01

    Incorporated 137 Cs (absorbed dose of 0.26 Gy) causes decrease of myocard's contractile function and intropic response to β-adrenagonists effect, isoproterenol-stimulated adenylate cyclase activity and β-adrenoreceptors affinity. Adrenergic effects, mediated by α-adrenergic structures on heart contractile function, on the contrary, become stronger, that is due to the increase of the receptors' dencity on sarcolemma surface

  19. Reliability of contractile properties of the knee extensor muscles in individuals with post-polio syndrome.

    Directory of Open Access Journals (Sweden)

    Eric L Voorn

    Full Text Available To assess the reliability of contractile properties of the knee extensor muscles in 23 individuals with post-polio syndrome (PPS and 18 age-matched healthy individuals.Contractile properties of the knee extensors were assessed from repeated electrically evoked contractions on 2 separate days, with the use of a fixed dynamometer. Reliability was determined for fatigue resistance, rate of torque development (MRTD, and early and late relaxation time (RT50 and RT25, using the intraclass correlation coefficient (ICC and standard error of measurement (SEM, expressed as % of the mean.In both groups, reliability for fatigue resistance was good, with high ICCs (>0.90 and small SEM values (PPS: 7.1%, healthy individuals: 7.0%. Reliability for contractile speed indices varied, with the best values found for RT50 (ICCs>0.82, SEM values <2.8%. We found no systematic differences between test and retest occasions, except for RT50 in healthy subjects (p = 0.016.In PPS and healthy individuals, the reliability of fatigue resistance, as obtained from electrically evoked contractions is high. The reliability of contractile speed is only moderate, except for RT50 in PPS, demonstrating high reliability.This was the first study to examine the reliability of electrically evoked contractile properties in individuals with PPS. Our results demonstrate its potential to study mechanisms underlying muscle fatigue in PPS and to evaluate changes in contractile properties over time in response to interventions or from natural course.

  20. Thick filament length and isoform composition determine self-organized contractile units in actomyosin bundles.

    Science.gov (United States)

    Thoresen, Todd; Lenz, Martin; Gardel, Margaret L

    2013-02-05

    Diverse myosin II isoforms regulate contractility of actomyosin bundles in disparate physiological processes by variations in both motor mechanochemistry and the extent to which motors are clustered into thick filaments. Although the role of mechanochemistry is well appreciated, the extent to which thick filament length regulates actomyosin contractility is unknown. Here, we study the contractility of minimal actomyosin bundles formed in vitro by mixtures of F-actin and thick filaments of nonmuscle, smooth, and skeletal muscle myosin isoforms with varied length. Diverse myosin II isoforms guide the self-organization of distinct contractile units within in vitro bundles with shortening rates similar to those of in vivo myofibrils and stress fibers. The tendency to form contractile units increases with the thick filament length, resulting in a bundle shortening rate proportional to the length of constituent myosin thick filament. We develop a model that describes our data, providing a framework in which to understand how diverse myosin II isoforms regulate the contractile behaviors of disordered actomyosin bundles found in muscle and nonmuscle cells. These experiments provide insight into physiological processes that use dynamic regulation of thick filament length, such as smooth muscle contraction. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. A comparative study of contractility of the heart ventricle in some ectothermic vertebrates

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    Sergey Kharin

    2009-07-01

    Full Text Available The purpose of this study was to analyze contractility of the heart ventricle in selected reptilian and amphibian species having the same ventricular excitation pattern. Systolic time intervals and indices of contractility of the heart ventricle were measured in anaesthetized frogs, snakes, and tortoises by use of polycardiography. The electromechanical delay was significantly shorter in tortoises compared with the other two species. The isovolumetric contraction time in frogs was approximately twofold longer than in reptiles. The pre-ejection period was the longest in frogs and the shortest in tortoises, whereas snakes were intermediate. The ejection time was slightly longer in tortoises compared with the other two species. The greatest isovolumetric contraction index and the smallest myocardial tension index corresponded to the frog and tortoise heart ventricle, respectively. The intrasystolic index in tortoises was significantly greater than in frogs, whereas quite similar to that in snakes. The frog ventricle had lower contractility compared with the reptilian one. Although ventricular contractility tended to be lower in snakes compared with tortoises, this difference was not statistically significant. Possible causes for these differences are discussed. We suppose a large variety in ventricular contractility among amphibian and reptilian species having the same ventricular activation pattern. This variety may be conditioned by heart anatomy, intracardiac shunting, lifestyles, and habitats. It can only be hypothesized that on the average, ventricular contractility is higher in reptiles compared with amphibians and in chelonians compared with snakes.

  2. Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19.

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    Anton R Dluzewski

    2008-08-01

    Full Text Available Plasmodium falciparum Merozoite Surface Protein 1 (MSP1 is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19, which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19 and the chloroquine resistance transporter (CRT as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

  3. A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium, fungi and metazoa

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    Uyeda Taro QP

    2005-03-01

    Full Text Available Abstract Background Formins are multidomain proteins defined by a conserved FH2 (formin homology 2 domain with actin nucleation activity preceded by a proline-rich FH1 (formin homology 1 domain. Formins act as profilin-modulated processive actin nucleators conserved throughout a wide range of eukaryotes. Results We present a detailed sequence analysis of the 10 formins (ForA to J identified in the genome of the social amoeba Dictyostelium discoideum. With the exception of ForI and ForC all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD, where DAD is the Diaphanous autoinhibition domain and GBD/FH3 is the Rho GTPase-binding domain/formin homology 3 domain that we propose to represent a single domain. ForC lacks a FH1 domain, ForI lacks recognizable GBD/FH3 and DAD domains and ForA, E and J have additional unique domains. To establish the relationship between formins of Dictyostelium and other organisms we constructed a phylogenetic tree based on the alignment of FH2 domains. Real-time PCR was used to study the expression pattern of formin genes. Expression of forC, D, I and J increased during transition to multi-cellular stages, while the rest of genes displayed less marked developmental variations. During sexual development, expression of forH and forI displayed a significant increase in fusion competent cells. Conclusion Our analysis allows some preliminary insight into the functionality of Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and to regulation by Rho GTPases. The architecture GBD/FH3-FH1-FH2-DAD appears common to almost all Dictyostelium, fungal and metazoan formins, for which we propose the denomination of conventional formins, and implies a common regulatory mechanism.

  4. A comparative sequence analysis reveals a common GBD/FH3-FH1-FH2-DAD architecture in formins from Dictyostelium, fungi and metazoa.

    Science.gov (United States)

    Rivero, Francisco; Muramoto, Tetsuya; Meyer, Ann-Kathrin; Urushihara, Hideko; Uyeda, Taro Q P; Kitayama, Chikako

    2005-03-01

    Formins are multidomain proteins defined by a conserved FH2 (formin homology 2) domain with actin nucleation activity preceded by a proline-rich FH1 (formin homology 1) domain. Formins act as profilin-modulated processive actin nucleators conserved throughout a wide range of eukaryotes. We present a detailed sequence analysis of the 10 formins (ForA to J) identified in the genome of the social amoeba Dictyostelium discoideum. With the exception of ForI and ForC all other formins conform to the domain structure GBD/FH3-FH1-FH2-DAD, where DAD is the Diaphanous autoinhibition domain and GBD/FH3 is the Rho GTPase-binding domain/formin homology 3 domain that we propose to represent a single domain. ForC lacks a FH1 domain, ForI lacks recognizable GBD/FH3 and DAD domains and ForA, E and J have additional unique domains. To establish the relationship between formins of Dictyostelium and other organisms we constructed a phylogenetic tree based on the alignment of FH2 domains. Real-time PCR was used to study the expression pattern of formin genes. Expression of forC, D, I and J increased during transition to multi-cellular stages, while the rest of genes displayed less marked developmental variations. During sexual development, expression of forH and forI displayed a significant increase in fusion competent cells. Our analysis allows some preliminary insight into the functionality of Dictyostelium formins: all isoforms might display actin nucleation activity and, with the exception of ForI, might also be susceptible to autoinhibition and to regulation by Rho GTPases. The architecture GBD/FH3-FH1-FH2-DAD appears common to almost all Dictyostelium, fungal and metazoan formins, for which we propose the denomination of conventional formins, and implies a common regulatory mechanism.

  5. 1H, 15N and 13C assignments of domain 5 of Dictyostelium discoideum gelation factor (ABP-120) in its native and 8M urea-denatured states.

    Science.gov (United States)

    Hsu, Shang-Te Danny; Cabrita, Lisa D; Christodoulou, John; Dobson, Christopher M

    2009-06-01

    The gelation factor from Dictyostelium discoideum (ABP-120) is an actin binding protein consisting of six immunoglobulin (Ig) domains in the C-terminal rod domain. We have recently used the pair of domains 5 and 6 of ABP-120 as a model system for studying multi-domain nascent chain folding on the ribosome. Here we present the NMR assignments of domain 5 in its native and 8M urea-denatured states.

  6. The Functional Lumen Imaging Probe Detects Esophageal Contractility not Observed with Manometry in Patients with Achalasia

    Science.gov (United States)

    Carlson, Dustin A.; Lin, Zhiyue; Kahrilas, Peter J.; Sternbach, Joel; Donnan, Erica N.; Friesen, Laurel; Listernick, Zoe; Mogni, Benjamin; Pandolfino, John E.

    2015-01-01

    Background & Aims The functional lumen imaging probe (FLIP) could improve characterization of achalasia subtypes by detecting non-occlusive esophageal contractions not observed with standard manometry. We aimed to evaluate for esophageal contractions during volumetric distention in patients with achalasia using FLIP topography. Methods Fifty one treatment-naïve patients with achalasia, defined and sub-classified by high-resolution esophageal pressure topography, and 10 asymptomatic individuals (controls) were evaluated with the FLIP during endoscopy. During stepwise distension, simultaneous intra-bag pressures and 16 channels of cross-sectional areas were measured; data were exported to software that generated FLIP topography plots. Esophageal contractility was identified by noting periods of reduced luminal diameter. Esophageal contractions were further characterized by propagation direction, repetitiveness, and based on whether they were occluding or non-occluding. Results Esophageal contractility was detected in all 10 controls: 8/10 had repetitive, antegrade, contractions and 9/10 had occluding contractions. Contractility was detected in 27% (4/15) of patients with type I achalasia and 65% (18/26, including 9 with occluding contractions) of patients with type II achalasia. Contractility was detected in all 10 patients with type III achalasia; 8 of these patients had a pattern of contractility not observed in controls (repetitive, retrograde contractions). Conclusions Esophageal contractility not observed with manometry can be detected in patients with achalasia using FLIP topography. The presence and patterns of contractility detected with FLIP topography may represent variations in pathophysiology, such as mechanisms of pan-esophageal pressurization in patients with type II achalasia. These findings could have implications for additional sub-classification to supplement prediction of the achalasia disease course. PMID:26278501

  7. Effects of endothelin, calcium channel blockade and EDRF inhibition on the contractility of human uteroplacental arteries.

    Science.gov (United States)

    Fried, G; Liu, Y A

    1994-08-01

    In order to examine the possibility that endothelin might be important in the regulation of placental blood flow, human uteroplacental vessels were superfused in vitro to study the contractile effect of endothelin as compared with a known strong contractor of placental blood vessels, serotonin (5-HT). The contractile responses were compared in the presence and absence of calcium channel blocking agents, as well as in the presence of L-NMA, an inhibitor of EDRF/nitric oxide. Endothelin (ET, 10(-10)-10(-6) M) and 5-HT (10(-8)-10(-4) M) induced contractions in the vessels. Maximal contractions in the presence of endothelin were elicited at 10(-7) M, whereas 5-HT elicited maximal contractions at 10(-5) M. At 10(-7) M, ET was more potent than 5-HT. The calcium-channel blocking agents nifedipine, diltiazem and NiCl2 relaxed the vessels by 5-15% from baseline. The contractile response to ET in the presence of nifedipine or diltiazem was reduced by 55 and 67%, respectively. The response of 5-HT in the presence of nifedipine was reduced by 58%. The contractile response to 5-HT as well as ET in the presence of both nifedipine and NiCl2 was not significantly lower than in the presence of nifedipine only. The EDRF-inhibiting agent L-NMA caused a small contractile response at concentrations of 10(-6)-10(-5) M. ET as well as 5-HT added after pretreatment with L-NMA produced a larger contractile response than ET or 5-HT alone. The results show that ET has a strong contractile effect on placental blood vessels at concentrations likely to occur during labor and delivery. The mechanism whereby ET as well as 5-HT contracts placental vessel smooth muscle appears to partly involve nifedipine- and diltiazem-sensitive calcium channels, but almost half of the response depends on mobilization of calcium through other means.

  8. Amino acid transport across the tonoplast of vacuoles isolated from barley mesophyll protoplasts: Uptake of alanine, leucine, and glutamine

    International Nuclear Information System (INIS)

    Dietz, K.J.; Jaeger, R.; Kaiser, G.; Martinoia, E.

    1990-01-01

    Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using 14 C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors

  9. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

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    María Milagros López de Armentia

    2016-03-01

    Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

  10. A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium.

    Science.gov (United States)

    Lee, S; Parent, C A; Insall, R; Firtel, R A

    1999-09-01

    We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras

  11. Derivatives of Dictyostelium differentiation-inducing factors inhibit lysophosphatidic acid–stimulated migration of murine osteosarcoma LM8 cells

    Energy Technology Data Exchange (ETDEWEB)

    Kubohara, Yuzuru, E-mail: ykuboha@juntendo.ac.jp [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Health Science, Juntendo University Graduate School of Health and Sports Science, Inzai 270-1695 (Japan); Komachi, Mayumi [Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation (IMCR), Gunma University, Maebashi 371-8512 (Japan); Department of Radiation Oncology, Gunma University Graduate School of Medicine, Maebashi 371-8511 (Japan); Homma, Yoshimi [Department of Biomolecular Science, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295 (Japan); Kikuchi, Haruhisa; Oshima, Yoshiteru [Laboratory of Natural Product Chemistry, Tohoku University Graduate School of Pharmaceutical Sciences, Aoba-yama, Aoba-ku, Sendai 980-8578 (Japan)

    2015-08-07

    Osteosarcoma is a common metastatic bone cancer that predominantly develops in children and adolescents. Metastatic osteosarcoma remains associated with a poor prognosis; therefore, more effective anti-metastatic drugs are needed. Differentiation-inducing factor-1 (DIF-1), −2, and −3 are novel lead anti-tumor agents that were originally isolated from the cellular slime mold Dictyostelium discoideum. Here we investigated the effects of a panel of DIF derivatives on lysophosphatidic acid (LPA)-induced migration of mouse osteosarcoma LM8 cells by using a Boyden chamber assay. Some DIF derivatives such as Br-DIF-1, DIF-3(+2), and Bu-DIF-3 (5–20 μM) dose-dependently suppressed LPA-induced cell migration with associated IC{sub 50} values of 5.5, 4.6, and 4.2 μM, respectively. On the other hand, the IC{sub 50} values of Br-DIF-1, DIF-3(+2), and Bu-DIF-3 versus cell proliferation were 18.5, 7.2, and 2.0 μM, respectively, in LM8 cells, and >20, 14.8, and 4.3 μM, respectively, in mouse 3T3-L1 fibroblasts (non-transformed). Together, our results demonstrate that Br-DIF-1 in particular may be a valuable tool for the analysis of cancer cell migration, and that DIF derivatives such as DIF-3(+2) and Bu-DIF-3 are promising lead anti-tumor agents for the development of therapies that suppress osteosarcoma cell proliferation, migration, and metastasis. - Highlights: • LPA induces cell migration (invasion) in murine osteosarcoma LM8 cells. • DIFs are novel lead anti-tumor agents found in Dictyostelium discoideum. • We examined the effects of DIF derivatives on LPA-induced LM8 cell migration in vitro. • Some of the DIF derivatives inhibited LPA-induced LM8 cell migration.

  12. 17 alpha-hydroxyprogesterone caproate vehicle, castor oil, enhances the contractile effect of oxytocin in human myometrium in pregnancy.

    Science.gov (United States)

    O'Sullivan, Michael D; Hehir, Mark P; O'Brien, Yvonne M; Morrison, John J

    2010-05-01

    The possibility exists that the vehicle for 17-alpha-hydroxyprogesterone caproate, castor oil, exerts an effect on human uterine contractility. The aim of this study was to evaluate its effects on contractility of myometrial preparations that were obtained during pregnancy. Myometrial strips were suspended under isometric conditions. Contractility was induced with oxytocin. Strips were incubated in castor oil or physiologic salt solution and suspended for a further oxytocin challenge. Contractile integrals were compared between both groups. Strips that were exposed to castor oil demonstrated increased contractile activity that was elicited by oxytocin (mean contractility value, 165.53%+/-17.03%; n=8; P=.004), compared with control strips (mean contractility value, 72.57%+/-7.48%; n=8; P=.003). There was a significant increase in contractile activity of the castor oil-exposed strips, compared with those that were exposed to physiologic salt solution (n=8; Pcastor oil results in enhanced oxytocin-induced contractility. Copyright (c) 2010 Mosby, Inc. All rights reserved.

  13. The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

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    Abel Peter W

    2007-11-01

    Full Text Available Abstract Background Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv channels and large-conductance, calcium-activated potassium (BK channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium. Methods Functional assays which evaluated the effects of channel blockers and various contractile agonists were accomplished by quantifying contractility of isolated uterine smooth muscle obtained from nonpregnant mice as well as mice at various stages of pregnancy. Expression of Kv channel proteins in isolated uterine smooth muscle was evaluated by Western blots. Results The Kv channel blocker 4-aminopyridine (4-AP caused contractions in nonpregnant mouse myometrium (EC50 = 54 micromolar, maximal effect at 300 micromolar but this effect disappeared in pregnant mice; similarly, the Kv4.2/Kv4.3 blocker phrixotoxin-2 caused contractions in nonpregnant, but not pregnant, myometrium. Contractile responses to 4-AP were not dependent upon nerves, as neither tetrodotoxin nor storage of tissues at room temperature significantly altered these responses, nor were responses dependent upon the presence of the endometrium. Spontaneous contractions and contractions in response to 4-AP did not appear to be mediated by BK, as the BK channel-selective blockers iberiotoxin, verruculogen, or tetraethylammonium failed to affect either spontaneous contractions or 4-AP-elicited responses. A number of different Kv channel alpha subunit proteins were

  14. Impact of Cardiac Contractility during Cerebral Blood Flow in Ischemia

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    Silver, Brian

    2011-05-01

    Full Text Available Objective: In cerebral regions affected by ischemia, intrinsic vascular autoregulation is often lost. Blood flow delivery depends upon cardiac function and may be influenced by neuro-endocrine mediated myocardial suppression. Our objective is to evaluate the relation between ejection fraction (EF and transcranial doppler (TCD peak systolic velocities (PSV in patients with cerebral ischemic events.Methods: We conducted a retrospective cohort study from an existing TCD registry. We evaluated patients admitted within 24 hours of onset of a focal neurological deficit who had an echocardiogram and TCD performed within 72 hours of admission.Results: We identified 58 patients from March to October 2003. Eighty-one percent (n=47 had a hospital discharge diagnosis of ischemic stroke and 18.9% (n=11 had a diagnosis of transient ischemic attack. Fourteen patients had systolic dysfunction (EF50% compared to those with systolic dysfunction (EF<50% was as follows: middle cerebral artery 62.0 + 28.6 cm/s vs. 51.0 + 23.3 cm/s, p=0.11; anterior cerebral artery 52.1 + 21.6 cm/s vs. 45.9 + 22.7 cm/s, p=0.28; internal carotid artery 56.5 + 20.1 cm/s vs. 46.4 + 18.4 cm/s, p=0.04; ophthalmic artery 18.6 + 7.2 cm/s vs. 15.3 + 5.2 cm/s, p=0.11; vertebral artery 34.0 + 13.9 cm/s vs. 31.6 + 15.0 cm/s, p=0.44.Conclusion: Cerebral blood flow in the internal carotid artery territory appears to be higher in cerebral ischemia patients with preserved left ventricular contractility. Our study was unable to differentiate pre-existing cardiac dysfunction from neuro-endocrine mediated myocardial stunning. Future research is necessary to better understand heart-brain interactions in this setting and to further explore the underlying mechanisms and consequences of neuro-endocrine mediated cardiac dysfunction. [West J Emerg Med. 2011;12(2:227-232.

  15. Effect of heat stress on contractility of tissue-engineered artificial skeletal muscle.

    Science.gov (United States)

    Takagi, Shunya; Nakamura, Tomohiro; Fujisato, Toshia

    2018-01-23

    The effects of heat stress on tissue like skeletal muscle have been widely studied. However, the mechanism responsible for the effect of heat stress is still unclear. A useful experimental tissue model is necessary because muscle function in cell culture may differ from native muscle and measuring its contractility is difficult. We previously reported three-dimensional tissue-engineered artificial skeletal muscle (TEM) that can be easily set in a measurement apparatus for quantitative evaluation of contractility. We have now applied TEM to the investigation of heat stress. We analyzed contractility immediately after thermal exposure at 39 °C for 24 or 48 h to evaluate the acute effects and after thermal exposure followed by normal culture to evaluate the aftereffects. Peak twitch contractile force and time-to-peak twitch were used as contractile parameters. Heat stress increased the TCF in the early stage (1 week) after normal culture; the TCF decreased temporarily in the middle to late stages (2-3 weeks). These results suggest that heat stress may affect both myoblast fusion and myotube differentiation in the early stage of TEM culture, but not myotube maturation in the late stage. The TCF increase rate with thermal exposure was significantly higher than that without thermal exposure. Although detailed analysis at the molecular level is necessary for further investigation, our artificial skeletal muscle may be a promising tool for heat stress investigation.

  16. Inhibition of isolated human myometrium contractility by minoxidil and reversal by glibenclamide.

    Science.gov (United States)

    Prabhakaran, S S; Dhanasekar, K R; Thomas, E; Jose, R; Peedicayil, J; Samuel, P

    2010-03-01

    This study investigated the ability of the antihypertensive drug minoxidil to inhibit potassium chloride (KCl)-induced contractility of the isolated human myometrium. Twelve strips of myometrium obtained from 12 patients who underwent hysterectomy were triggered to contract with 55 mM KCl before and after incubation with 3 concentrations (1, 3 and 10 microM) of minoxidil. The percent inhibition by minoxidil on the extent of contraction, and the area under the contractile curve of KCl-induced contraction of the myometrial strips was determined. Furthermore, the effect of 10 microM glibenclamide on the inhibition generated by 3 microM minoxidil on KCl-induced contractility was studied. It was found that minoxidil produced a concentration-dependent inhibition of KCl-induced contractility of the myometrium and that glibenclamide reversed this inhibitory effect. These results suggest that the inhibitory effect of minoxidil on isolated human myometrium contractility may prove useful in clinical conditions requiring relaxation of the myometrium. 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

  17. Slack length reduces the contractile phenotype of the Swine carotid artery.

    Science.gov (United States)

    Rembold, Christopher M; Garvey, Sean M; Tejani, Ankit D

    2013-01-01

    Contraction is the primary function of adult arterial smooth muscle. However, in response to vessel injury or inflammation, arterial smooth muscle is able to phenotypically modulate from the contractile state to several 'synthetic' states characterized by proliferation, migration and/or increased cytokine secretion. We examined the effect of tissue length (L) on the phenotype of intact, isometrically held, initially contractile swine carotid artery tissues. Tissues were studied (1) without prolonged incubation at the optimal length for force generation (1.0 Lo, control), (2) with prolonged incubation for 17 h at 1.0 Lo, or (3) with prolonged incubation at slack length (0.6 Lo) for 16 h and then restoration to 1.0 Lo for 1 h. Prolonged incubation at 1.0 Lo minimally reduced the contractile force without substantially altering the mediators of contraction (crossbridge phosphorylation, shortening velocity or stimulated actin polymerization). Prolonged incubation of tissues at slack length (0.6 Lo), despite return of length to 1.0 Lo, substantially reduced contractile force, reduced crossbridge phosphorylation, nearly abolished crossbridge cycling (shortening velocity) and abolished stimulated actin polymerization. These data suggest that (1) slack length treatment significantly alters the contractile phenotype of arterial tissue, and (2) slack length treatment is a model to study acute phenotypic modulation of intact arterial smooth muscle. Copyright © 2013 S. Karger AG, Basel.

  18. Functional characterisation of parvulin-type peptidyl prolyl cis-trans isomerase, PinA in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Haokip, Nemneineng; Naorem, Aruna

    2017-01-01

    Pin1-type parvulins are unique among PPIases that can catalyse an otherwise slow cis-trans isomerisation of phosphorylated peptide bond preceding proline in target proteins. This prolyl isomerisation process can regulate activity, stability and localisation of target proteins and thus control cellular processes like eukaryotic cell proliferation, cell cycle progression and gene regulation. Towards understanding the function of Pin1-type prolyl isomerisation in Dictyostelium discoideum, a slime mould with distinct growth and developmental phases, we identified PinA as a novel Pin1-type parvulin by its ability to complement the temperature sensitivity phenotype associated with a mutation in ESS1 in S. cerevisiae. In D. discoideum, pinA is temporally and spatially regulated during growth and development. PinA is both nuclear as well as cytoplasmic in the growing cells. We further show that loss of pinA (pinA − ) leads to decreased growth rate, reduced spore formation and abnormal prespore-prestalk patterning. We conclude that PinA is required for normal growth as well as development in D. discoideum. - Highlights: • PinA is a bona fide homologue of S. cerevisiae Ess1. • PinA is required for normal cell proliferation of D. discoideum. • PinA is spatially localised in developmental structures. • PinA is important for cell differentiation and patterning.

  19. Characterization of PEBBLEs as a Tool for Real-Time Measurement of Dictyostelium discoideum Endosomal pH

    Directory of Open Access Journals (Sweden)

    Everett Moding

    2009-01-01

    Full Text Available The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum. Oregon Green 514-CdSe Quantum Dot PEBBLEs were used to measure real-time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

  20. An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH

    Directory of Open Access Journals (Sweden)

    Yu Tang

    2018-02-01

    Full Text Available In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH{macron} cells show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH{macron} cells (grlH{macron}/grlHOE rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum.

  1. Dictyostelium discoideum: mutants in the biosynthesis of the lipid-linked precursor of N-linked oligosaccharides

    International Nuclear Information System (INIS)

    Freeze, H.; Willies, L.; Hamilton, S.

    1986-01-01

    The lysosomal enzymes of Dictyostelium discoideum share highly immunogenic oligosaccharides which contain multiple Man-6-SO 4 residues. Two mutant strains which lack the shared antigenic determinant were analyzed in an attempt to identify the primary defect in each. [ 3 H]Man labelled N-linked oligosaccharides of secreted glycoproteins were released by Endo/PNGaseF digestion and analyzed. Both of the mutant strains produced smaller, less sulfated oligosaccharides compared to the wild-type, yet both still contained considerable amounts of Man-6-SO 4 . The size of the precursor lipid-linked oligosaccharide from the wild-type is consistent with a Glc 3 Man 9 GlcNAc 2 structure, while those from both of the mutants have an oligosaccharide the size of Man 5 GlcNAc 2 . The authors conclude that both of the mutants are defective in the biosynthesis of the precursor oligosaccharide. Both oligosaccharides from the mutants contain a tri-mannosyl core and are not glucosylated. Two of the five Man residues are released by a 1,2 specific α mannosidase. Based on the size and mannosidase digestions the authors conclude that 4/5 of the Man residues on the α1,6 branch of the β-linked Man residues are missing. Thus, these residues must be required to define the shared antigenic determinant

  2. The TOM Complex of Amoebozoans: the Cases of the Amoeba Acanthamoeba castellanii and the Slime Mold Dictyostelium discoideum.

    Science.gov (United States)

    Wojtkowska, Małgorzata; Buczek, Dorota; Stobienia, Olgierd; Karachitos, Andonis; Antoniewicz, Monika; Slocinska, Małgorzata; Makałowski, Wojciech; Kmita, Hanna

    2015-07-01

    Protein import into mitochondria requires a wide variety of proteins, forming complexes in both mitochondrial membranes. The TOM complex (translocase of the outer membrane) is responsible for decoding of targeting signals, translocation of imported proteins across or into the outer membrane, and their subsequent sorting. Thus the TOM complex is regarded as the main gate into mitochondria for imported proteins. Available data indicate that mitochondria of representative organisms from across the major phylogenetic lineages of eukaryotes differ in subunit organization of the TOM complex. The subunit organization of the TOM complex in the Amoebozoa is still elusive, so we decided to investigate its organization in the soil amoeba Acanthamoeba castellanii and the slime mold Dictyostelium discoideum. They represent two major subclades of the Amoebozoa: the Lobosa and Conosa, respectively. Our results confirm the presence of Tom70, Tom40 and Tom7 in the A. castellanii and D. discoideum TOM complex, while the presence of Tom22 and Tom20 is less supported. Interestingly, the Tom proteins display the highest similarity to Opisthokonta cognate proteins, with the exception of Tom40. Thus representatives of two major subclades of the Amoebozoa appear to be similar in organization of the TOM complex, despite differences in their lifestyle. Copyright © 2015 The Authors. Published by Elsevier GmbH.. All rights reserved.

  3. Systematic Analysis of γ-Aminobutyric Acid (GABA) Metabolism and Function in the Social Amoeba Dictyostelium discoideum*

    Science.gov (United States)

    Wu, Yuantai; Janetopoulos, Chris

    2013-01-01

    While GABA has been suggested to regulate spore encapsulation in the social amoeba Dictyostelium discoideum, the metabolic profile and other potential functions of GABA during development remain unclear. In this study, we investigated the homeostasis of GABA metabolism by disrupting genes related to GABA metabolism and signaling. Extracellular levels of GABA are tightly regulated during early development, and GABA is generated by the glutamate decarboxylase, GadB, during growth and in early development. However, overexpression of the prespore-specific homologue, GadA, in the presence of GadB reduces production of extracellular GABA. Perturbation of extracellular GABA levels delays the process of aggregation. Cytosolic GABA is degraded by the GABA transaminase, GabT, in the mitochondria. Disruption of a putative vesicular GABA transporter (vGAT) homologue DdvGAT reduces secreted GABA. We identified the GABAB receptor-like family member GrlB as the major GABA receptor during early development, and either disruption or overexpression of GrlB delays aggregation. This delay is likely the result of an abolished pre-starvation response and late expression of several “early” developmental genes. Distinct genes are employed for GABA generation during sporulation. During sporulation, GadA alone is required for generating GABA and DdvGAT is likely responsible for GABA secretion. GrlE but not GrlB is the GABA receptor during late development. PMID:23548898

  4. Uncovering a role for the tail of the Dictyostelium discoideum SadA protein in cell-substrate adhesion.

    Science.gov (United States)

    Kowal, Anthony S; Chisholm, Rex L

    2011-05-01

    Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesion deficiency, and overexpression of the SadA tail domain reduced adhesion in wild-type cells. We also show that SadA is closely associated with the actin cytoskeleton. Mutagenesis studies suggested that four serine residues in the tail, S924/S925 and S940/S941, may regulate association of SadA with the actin cytoskeleton. Glutathione S-transferase pull-down assays identified at least one likely interaction partner of the SadA tail, cortexillin I, a known actin bundling protein. Thus, our data demonstrate an important role for the carboxy-terminal cytoplasmic tail in SadA function and strongly suggest that a phosphorylation event in this tail regulates an interaction with cortexillin I. Based on our data, we propose a model for the function of SadA.

  5. Evidence for a functional link between Dd-STATa and Dd-PIAS, a Dictyostelium PIAS homologue.

    Science.gov (United States)

    Kawata, Takefumi; Hirano, Tatsunori; Ogasawara, Shun; Aoshima, Ryota; Yachi, Ayako

    2011-09-01

    Several mammalian protein families inhibit the activity of signal transducer and activator of transcription (STAT) proteins. The protein inhibitor of activated STAT (PIAS) was initially identified through its ability to interact with human STAT proteins. We isolated a gene (pisA) encoding a Dictyostelium orthologue of PIAS, Dd-PIAS, which possesses almost all the representative motifs and domains of mammalian PIAS proteins. A Dd-PIAS null mutant strain displays a normal terminal morphology but with accelerated development once cells are aggregated. In contrast, Dd-PIAS overexpressor strains demonstrate delayed aggregation, almost no slug phototaxis, impaired slug motility, and a prolonged slug migration period. This strain is a near phenocopy of the Dd-STATa null mutant, although it eventually forms a fruiting body, albeit inefficiently. The expression of several Dd-STATa-activated genes is upregulated in the Dd-PIAS null mutant and there is ectopic expression of pstAB makers. The concentration of a PIAS-green fluorescent protein (GFP) fusion protein, expressed under the PIAS promoter, is greatest in the pstO cells and gradually decreases with proximity to the tip of the slug and culminant: a pattern diametrically opposite to that of Dd-STATa. Our results suggest a functional interrelationship between Dd-PIAS and Dd-STATa that influences gene expression and development. © 2011 The Authors. Development, Growth & Differentiation © 2011 Japanese Society of Developmental Biologists.

  6. PsB multiprotein complex of Dictyostelium discoideum. Demonstration of cellulose binding activity and order of protein subunit assembly.

    Science.gov (United States)

    McGuire, V; Alexander, S

    1996-06-14

    The differentiated spores of Dictyostelium are surrounded by an extracellular matrix, the spore coat, which protects them from environmental factors allowing them to remain viable for extended periods of time. This presumably is a major evolutionary advantage. This unique extracellular matrix is composed of cellulose and glycoproteins. Previous work has shown that some of these spore coat glycoproteins exist as a preassembled multiprotein complex (the PsB multiprotein complex) which is stored in the prespore vesicles (Watson, N., McGuire, V., and Alexander, S (1994) J. Cell Sci. 107, 2567-2579). Later in development, the complex is synchronously secreted from the prespore vesicles and incorporated into the spore coat. We now have shown that the PsB complex has a specific in vitro cellulose binding activity. The analysis of mutants lacking individual subunits of the PsB complex revealed the relative order of assembly of the subunit proteins and demonstrated that the protein subunits must be assembled for cellulose binding activity. These results provide a biochemical explanation for the localization of this multiprotein complex in the spore coat.

  7. Desynchronization of cells on the developmental path triggers the formation of spiral waves of cAMP during Dictyostelium aggregation.

    Science.gov (United States)

    Lauzeral, J; Halloy, J; Goldbeter, A

    1997-08-19

    Whereas it is relatively easy to account for the formation of concentric (target) waves of cAMP in the course of Dictyostelium discoideum aggregation after starvation, the origin of spiral waves remains obscure. We investigate a physiologically plausible mechanism for the spontaneous formation of spiral waves of cAMP in D. discoideum. The scenario relies on the developmental path associated with the continuous changes in the activity of enzymes such as adenylate cyclase and phosphodiesterase observed during the hours that follow starvation. These changes bring the cells successively from a nonexcitable state to an excitable state in which they relay suprathreshold cAMP pulses, and then to autonomous oscillations of cAMP, before the system returns to an excitable state. By analyzing a model for cAMP signaling based on receptor desensitization, we show that the desynchronization of cells on this developmental path triggers the formation of fully developed spirals of cAMP. Developmental paths that do not correspond to the sequence of dynamic transitions no relay-relay-oscillations-relay are less able or fail to give rise to the formation of spirals.

  8. Contractile properties are disrupted in Becker muscular dystrophy, but not in limb girdle type 2I.

    Science.gov (United States)

    Løkken, Nicoline; Hedermann, Gitte; Thomsen, Carsten; Vissing, John

    2016-09-01

    We investigated whether a linear relationship between muscle strength and cross-sectional area (CSA) is preserved in calf muscles of patients with Becker muscular dystrophy (BMD, n = 14) and limb-girdle type 2I muscular dystrophy (LGMD2I, n = 11), before and after correcting for muscle fat infiltration. The Dixon magnetic resonance imaging technique was used to quantify fat and calculate a fat-free contractile CSA. Strength was assessed by dynamometry. Muscle strength/CSA relationships were significantly lower in patients versus controls. The strength/contractile-CSA relationship was still severely lowered in BMD, but was almost normalized in LGMD2I. Our findings suggest close to intact contractile properties in LGMD2I, which are severely disrupted in BMD. Ann Neurol 2016;80:466-471. © 2016 American Neurological Association.

  9. Suppression of guinea pig ileum induced contractility by plasma albumin of hibernators

    Science.gov (United States)

    Bruce, David S.; Ambler, Douglas L.; Henschel, Timothy M.; Oeltgen, Peter R.; Nilekani, Sita P.; Amstrup, Steven C.

    1992-01-01

    Previous studies suggest that hibernation may be regulated by internal opioids and that the putative “hibernation induction trigger” (HIT) may itself be an opioid. This study examined the effect of plasma albumin (known to bind HIT) on induced contractility of the guinea pig ileum muscle strip. Morphine (400 nM) depressed contractility and 100 nM naloxone restored it. Ten milligrams of lyophilized plasma albumin fractions from hibernating ground squirrels, woodchucks, black bears, and polar bears produced similar inhibition, with partial reversal by naloxone. Five hundredths mg of d-Ala2-d-Leu5-enkephalin (DADLE) also inhibited contractility and naloxone reversed it. Conclusions are that hibernating individuals of these species contain an HIT substance that is opioid in nature and summer animals do not; an endogenous opioid similar to leu-enkephalin may be the HIT compound or give rise to it.

  10. Mechanobiological induction of long-range contractility by diffusing biomolecules and size scaling in cell assemblies

    Science.gov (United States)

    Dasbiswas, K.; Alster, E.; Safran, S. A.

    2016-06-01

    Mechanobiological studies of cell assemblies have generally focused on cells that are, in principle, identical. Here we predict theoretically the effect on cells in culture of locally introduced biochemical signals that diffuse and locally induce cytoskeletal contractility which is initially small. In steady-state, both the concentration profile of the signaling molecule as well as the contractility profile of the cell assembly are inhomogeneous, with a characteristic length that can be of the order of the system size. The long-range nature of this state originates in the elastic interactions of contractile cells (similar to long-range “macroscopic modes” in non-living elastic inclusions) and the non-linear diffusion of the signaling molecules, here termed mechanogens. We suggest model experiments on cell assemblies on substrates that can test the theory as a prelude to its applicability in embryo development where spatial gradients of morphogens initiate cellular development.

  11. Plasticity of TOM complex assembly in skeletal muscle mitochondria in response to chronic contractile activity.

    Science.gov (United States)

    Joseph, Anna-Maria; Hood, David A

    2012-03-01

    We investigated the assembly of the TOM complex within skeletal muscle under conditions of chronic contractile activity-induced mitochondrial biogenesis. Tom40 import into mitochondria was increased by chronic contractile activity, as was its time-dependent assembly into the TOM complex. These changes coincided with contractile activity-induced augmentations in the expression of key protein import machinery components Tim17, Tim23, and Tom22, as well as the cytosolic chaperone Hsp90. These data indicate the adaptability of the TOM protein import complex and suggest a regulatory role for the assembly of this complex in exercise-induced mitochondrial biogenesis. Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved. All rights reserved.

  12. Effects of minoxidil and nitroprusside on reflex increases in myocardial contractility.

    Science.gov (United States)

    Robie, N W

    1978-01-01

    1 The effects of nitroprusside and minoxidil on increases in myocardial contractility resulting from carotid artery occlusion were investigated in anaesthetized dogs. The results were compared with those produced by intravenous influsion of noradrenaline. 2 Nitroprusside and minoxidil attenuated the pressor responses produced by carotid artery occlusion. 3 Nitroprusside, but not minoxidil, attenuated the maximal myocardial contractility resulting from carotid occlusion. 4 The pressor and contractility responses to noradrenaline infusion were unaffected by either agent. 5 Nitroprusside failed to alter the myocardial responses produced by dimethylphenylpiperazinium. 6 These results, in conjunction with those of other investigators who have demonstrated that nitroprusside does not affect the release of noradrenaline from adrenergic neurons, suggest that nitroprusside may inhibit sympathetic nervous system reflex activity via an afferent and/or central component. PMID:620094

  13. Sex differences and the effects of ovariectomy on the β-adrenergic contractile response

    Science.gov (United States)

    McIntosh, Victoria J.; Chandrasekera, P. Charukeshi

    2011-01-01

    The presence of sex differences in myocardial β-adrenergic responsiveness is controversial, and limited studies have addressed the mechanism underlying these differences. Studies were performed using isolated perfused hearts from male, intact female and ovariectomized female mice to investigate sex differences and the effects of ovarian hormone withdrawal on β-adrenergic receptor function. Female hearts exhibited blunted contractile responses to the β-adrenergic receptor agonist isoproterenol (ISO) compared with males but not ovariectomized females. There were no sex differences in β1-adrenergic receptor gene or protein expression. To investigate the role of adenylyl cyclase, phosphodiesterase, and the cAMP-signaling cascade in generating sex differences in the β-adrenergic contractile response, dose-response studies were performed in isolated perfused male and female hearts using forskolin, 3-isobutyl-1-methylxanthine (IBMX), and 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate (CPT-cAMP). Males showed a modestly enhanced contractile response to forskolin at 300 nM and 5 μM compared with females, but there were no sex differences in the response to IBMX or CPT-cAMP. The role of the A1 adenosine receptor (A1AR) in antagonizing the β-adrenergic contractile response was investigated using both the A1AR agonist 2-chloro-N6-cyclopentyl-adenosine and A1AR knockout (KO) mice. Intact females showed an enhanced A1AR anti-adrenergic effect compared with males and ovariectomized females. The β-adrenergic contractile response was potentiated in both male and female A1ARKO hearts, with sex differences no longer present above 1 nM ISO. The β-adrenergic contractile response is greater in male hearts than females, and minor differences in the action of adenylyl cyclase or the A1AR may contribute to these sex differences. PMID:21685268

  14. Clinical Characteristics and Associated Systemic Diseases in Patients With Esophageal "Absent Contractility"-A Clinical Algorithm.

    Science.gov (United States)

    Laique, Sobia; Singh, Tavankit; Dornblaser, David; Gadre, Abhishek; Rangan, Vikram; Fass, Ronnie; Kirby, Donald; Chatterjee, Soumya; Gabbard, Scott

    2018-01-19

    This study was carried out to assess the clinical characteristics and associated systemic diseases seen in patients diagnosed with absent contractility as per the Chicago Classification version 3.0, allowing us to propose a diagnostic algorithm for their etiologic testing. The Chicago Classification version 3.0 has redefined major and minor esophageal motility disorders using high-resolution esophageal manometry. There is a dearth of publications based on research on absent contractility, which historically has been associated with myopathic processes such as systemic sclerosis (SSc). We conducted a retrospective, multicenter study. Data of patients diagnosed with absent contractility were pooled from Cleveland Clinic, Cleveland, OH (January 2006 to July 2016) and Metrohealth Medical Center, Cleveland, OH (July 2014 to July 2016) and included: age, gender, associated medical conditions, surgical history, medications, and specific antibody testing. A total of 207 patients, including 57 male individuals and 150 female individuals, with mean age of 56.1 and 60.0 years, respectively, were included. Disease distribution was as follows: SSc (diffuse or limited cutaneous) 132, overlap syndromes 7, systemic lupus erythematosus17, Sjögren syndrome 4, polymyositis 3, and dermatomyositis 3. Various other etiologies including gastroesophageal reflux disease, postradiation esophagitis, neuromuscular disorders, and surgical complications were seen in the remaining cohort. Most practitioners use the term "absent contractility" interchangeably with "scleroderma esophagus"; however, only 63% of patients with absent contractility had SSc. Overall, 20% had another systemic autoimmune rheumatologic disease and 16% had a nonrheumatologic etiology for absent contractility. Therefore, alternate diagnosis must be sought in these patients. We propose an algorithm for their etiologic evaluation.

  15. Accessory left atrial diverticulae: contractile properties depicted with 64-slice cine-cardiac CT.

    LENUS (Irish Health Repository)

    Killeen, Ronan P

    2012-02-01

    To assess the contractility of accessory left atrial appendages (LAAs) using multiphasic cardiac CT. We retrospectively analyzed the presence, location, size and contractile properties of accessory LAAs using multiphasic cardiac 64-slice CT in 102 consecutive patients (63 males, 39 females, mean age 57). Multiplanar reformats were used to create image planes in axial oblique, sagittal oblique and coronal oblique planes. For all appendages with an orifice diameter >or= 10 mm, axial and sagittal diameters and appendage volumes were recorded in atrial diastole and systole. Regression analysis was performed to assess which imaging appearances best predicted accessory appendage contractility. Twenty-three (23%) patients demonstrated an accessory LAA, all identified along the anterior LA wall. Dimensions for axial oblique (AOD) and sagittal oblique (SOD) diameters and sagittal oblique length (SOL) were 6.3-19, 3.4-20 and 5-21 mm, respectively. All appendages (>or=10 mm) demonstrated significant contraction during atrial systole (greatest diameter reduction was AOD [3.8 mm, 27%]). Significant correlations were noted between AOD-contraction and AOD (R = 0.57, P < 0.05) and SOD-contraction and AOD, SOD and SOL (R = 0.6, P < 0.05). Mean diverticulum volume in atrial diastole was 468.4 +\\/- 493 mm(3) and in systole was 171.2 +\\/- 122 mm(3), indicating a mean change in volume of 297.2 +\\/- 390 mm(3), P < 0.0001. Stepwise multiple regression analysis revealed SOL to be the strongest independent predictor of appendage contractility (R(2) = 0.86, P < 0.0001) followed by SOD (R(2) = 0.91, P < 0.0001). Accessory LAAs show significant contractile properties on cardiac CT. Those accessory LAAs with a large sagittal height or depth should be evaluated for contractile properties, and if present should be examined for ectopic activity during electrophysiological studies.

  16. Validation of an in vitro contractility assay using canine ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Harmer, A.R., E-mail: alex.harmer@astrazeneca.com; Abi-Gerges, N.; Morton, M.J.; Pullen, G.F.; Valentin, J.P.; Pollard, C.E.

    2012-04-15

    Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ∼ 36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration–effect curves were constructed for each compound in 4–30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6–8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds. -- Highlights: ► Cardiac contractility is an important physiological function of the heart. ► Assessment of contractility is a logical part of pre-clinical drug safety testing. ► There are limited validated assays that predict effects of compounds on contractility. ► Using dog myocytes, we have developed an in vitro cardiac contractility assay. ► The assay predicted the in vivo contractility with a good level of accuracy.

  17. Spontaneous and α-adrenoceptor-induced contractility in human collecting lymphatic vessels require chloride

    DEFF Research Database (Denmark)

    Mohanakumar, Sheyanth; Majgaard, Jens; Telinius, Niklas

    2018-01-01

    - with the impermeant anion aspartate and inhibition of Cl- transport and channels with the clinical diuretics furosemide and bendroflumethiazide, as well as DIDS and NPPB. The molecular expression of calcium-activated chloride channels was investigated by RT-PCR and proteins localized using immunoreactivity....... Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl- substitution with aspartate. 100‒300µM DIDS or NPPB inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200µM DIDS. Furosemide lowered...

  18. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  19. Specific binding of Ulex europaeus agglutinin I lectin to sarcolemma of distal myopathy with rimmed vacuole formation.

    Science.gov (United States)

    Yatabe, K; Kawai, M

    1997-08-01

    Ulex europaeus agglutinin I (UEA I) binding was studied in 83 patients with various neuromuscular disorders. UEA I labelled endomysial capillaries and endothelial cells of perimysial blood vessels in all the examined muscles. There was no UEA I binding to muscle fibres except for all (9) cases of distal myopathy with rimmed vacuole formation (DMRV), 1 of 5 cases of inclusion body myositis and 1 of 36 cases of inflammatory myopathies. The UEA I binding was completely eliminated by preincubation of UEA I solution with L-fucose. Using electron microscopy, the UEA I binding was localized to sarcolemma and intrasarco-plasmic membranous organelles other than mitochondria. Myosatellite cells were not labelled. These findings revealed the existence of fucosylated proteins or lipids in a subset of skeletal muscles suffering from DMRV. Biochemical identification of the fucosylated substance and further detailed study on subcellular localization of UEA I binding may yield important clues to the unknown pathogenesis of DMRV.

  20. Neuronal vacuolation of the trigeminal nuclei in goats caused by ingestion of Prosopis juliflora pods (mesquite beans).

    Science.gov (United States)

    Tabosa, I M; Souza, J C; Graça, D L; Barbosa-Filho, J M; Almeida, R N; Riet-Correa, F

    2000-06-01

    Three groups of 6 goats each were fed a ration containing 30, 60, or 90%, on a dry matter base, of Prosopis juliflora pods. A control group of 4 goats ingested only the basic ration. Two hundred and ten days after the start of the experiment 3 goats that ingested 60% pods in and 4 that ingested 90% had mandibular tremors, mainly during chewing. All animals were killed after 270 d of ingestion. No gross lesions were observed. Histologic lesions were characterized by fine vacuolation of the pericaryon of neurons from the trigeminal nuclei. Occasionally neurons of the oculomotor nuclei were also affected. Wallerian degeneration was occasionally observed in the mandibular and trigeminal nerves. Denervation atrophy of the masseter, temporal, hypoglossus, genioglossus, styloglossus, medial pterygoid and lateral pterygoid muscles was seen. The clinical signs from feeding the P juliflora pods were caused by a selective toxicity to neurons of some cranial nerve nuclei.

  1. Vacúolos de gás e flutuação em Difflugia mitriformis Wallich (Protista, Rhizopoda, Testaceolobosea Gas vacuoles and flotation in Diffugia mitriformis Wallich (Protista, Rhizopoda, Testaceolobosea

    Directory of Open Access Journals (Sweden)

    Vladimir Stolzenberg Torres

    1996-01-01

    Full Text Available The natural formation of gas vacuoles as a method of locomotion is described for Difflugia mitriformis Wallich, 1984. These vacuoles may contain different compositions of gases, basicly carbodioxyde or oxigen, with a membranous limitation similar or identical to other types of vacuoles. Those vacuoles are utilised by the organism as a mode of dislocation frorn the bottom to the water surface by flotation permiting better conditions for the survival of the individual, with the consequence of the perpetuance of the taxon.

  2. The use of CaCl2 and other salts to improve surface finish and eliminate vacuoles in ICF microencapsulated shells

    International Nuclear Information System (INIS)

    McQuillan, B.W.; Elsner, F.H.; Stephens, R.B.; Brown, L.C.

    1999-01-01

    Polystyrene and poly(α-methylstyrene) (PAMS) shells made by microencapsulation are prone to having vacuoles in the walls and a concomitant surface roughness. These defects can be detrimental to the implosion required for ICF shots. The authors have found that adding sufficient salt (typically CaCl 2 or NH 4 Cl) to the exterior polyvinylalcohol (PVA) solution during the drying phase inhibits the formation of vacuoles and decreases the surface roughness of the shells. The use of such salts does affect other shell specifications, for which other process variables must be adjusted

  3. 2-Deoxyadenosine triphosphate restores the contractile function of cardiac myofibril from adult dogs with naturally occurring dilated cardiomyopathy

    OpenAIRE

    Cheng, Yuanhua; Hogarth, Kaley A.; O'Sullivan, M. Lynne; Regnier, Michael; Pyle, W. Glen

    2015-01-01

    1) First report to characterize and define the contractile kinetics and defects associated with naturally occurring dilated cardiomyopathy (DCM) in dogs. 2) Novel findings that dATP is able to reverse the contractile defects associated with naturally occurring canine DCM.

  4. Psychromonas boydii sp. nov., a gas-vacuolate, psychrophilic bacterium isolated from an Arctic sea-ice core.

    Science.gov (United States)

    Auman, Ann J; Breezee, Jennifer L; Gosink, John J; Schumann, Peter; Barnes, Carmen R; Kämpfer, Peter; Staley, James T

    2010-01-01

    A gas-vacuolate bacterium, strain 174(T), was isolated from a sea-ice core collected from Point Barrow, Alaska, USA. Comparative analysis of 16S rRNA gene sequences showed that this bacterium was most closely related to Psychromonas ingrahamii 37(T), with a similarity of >99 %. However, strain 174(T) could be clearly distinguished from closely related species by DNA-DNA hybridization; relatedness values determined by two different methods between strain 174(T) and P. ingrahamii 37(T) were 58.4 and 55.7 % and those between strain 174(T) and Psychromonas antarctica DSM 10704(T) were 46.1 and 33.1 %, which are well below the 70 % level used to define a distinct species. Phenotypic analysis, including cell size (strain 174(T) is the largest member of the genus Psychromonas, with rod-shaped cells, 8-18 microm long), further differentiated strain 174(T) from other members of the genus Psychromonas. Strain 174(T) could be distinguished from its closest relative, P. ingrahamii, by its utilization of D-mannose and D-xylose as sole carbon sources, its ability to ferment myo-inositol and its inability to use fumarate and glycerol as sole carbon sources. In addition, strain 174(T) contained gas vacuoles of two distinct morphologies and grew at temperatures ranging from below 0 to 10 degrees C and its optimal NaCl concentration for growth was 3.5 %. The DNA G+C content was 40 mol%. Whole-cell fatty acid analysis showed that 16 : 1omega7c and 16 : 0 comprised 44.9 and 26.4 % of the total fatty acid content, respectively. The name Psychromonas boydii sp. nov. is proposed for this novel species, with strain 174(T) (=DSM 17665(T) =CCM 7498(T)) as the type strain.

  5. Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

    Science.gov (United States)

    Silva, Victor Diogenes A; Pitanga, Bruno P S; Nascimento, Ravena P; Souza, Cleide S; Coelho, Paulo Lucas C; Menezes-Filho, Noélio; Silva, André Mário M; Costa, Maria de Fátima D; El-Bachá, Ramon S; Velozo, Eudes S; Costa, Silvia L

    2013-12-16

    Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 μg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 μg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 μg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in β-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

  6. Partial genetic suppression of a loss-of-function mutant of the neuronal ceroid lipofuscinosis-associated protease TPP1 in Dictyostelium discoideum

    Directory of Open Access Journals (Sweden)

    Jonathan E. Phillips

    2015-02-01

    Full Text Available Neuronal ceroid lipofuscinosis (NCL is the most common childhood-onset neurodegenerative disease. NCL is inevitably fatal, and there is currently no treatment available. Children with NCL show a progressive decline in movement, vision and mental abilities, and an accumulation of autofluorescent deposits in neurons and other cell types. Late-infantile NCL is caused by mutations in the lysosomal protease tripeptidyl peptidase 1 (TPP1. TPP1 cleaves tripeptides from the N-terminus of proteins in vitro, but little is known about the physiological function of TPP1. TPP1 shows wide conservation in vertebrates but it is not found in Drosophila, Caenorhabditis elegans or Saccharomyces cerevisiae. Here, we characterize ddTpp1, a TPP1 ortholog present in the social amoeba Dictyostelium discoideum. Lysates from cells lacking ddTpp1 show a reduced but not abolished ability to cleave a TPP1 substrate, suggesting that other Dictyostelium enzymes can perform this cleavage. ddTpp1 and human TPP1 localize to the lysosome in Dictyostelium, indicating conserved function and trafficking. Cells that lack ddTpp1 show precocious multicellular development and a reduced ability to form spores during development. When cultured in autophagy-stimulating conditions, cells lacking ddTpp1 rapidly decrease in size and are less viable than wild-type cells, suggesting that one function of ddTpp1 could be to limit autophagy. Cells that lack ddTpp1 exhibit strongly impaired development in the presence of the lysosome-perturbing drug chloroquine, and this phenotype can be suppressed through a secondary mutation in the gene that we name suppressor of tpp1− A (stpA, which encodes a protein with some similarity to mammalian oxysterol-binding proteins (OSBPs. Taken together, these results suggest that targeting specific proteins could be a viable way to suppress the effects of loss of TPP1 function.

  7. Uncovering a Role for the Tail of the Dictyostelium discoideum SadA Protein in Cell-Substrate Adhesion ▿ †

    OpenAIRE

    Kowal, Anthony S.; Chisholm, Rex L.

    2011-01-01

    Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, demonstrating an important role for the C-terminal cytoplasmic tail in SadA function. We found that a SadA tailless mutant was unable to rescue the sadA adhesi...

  8. In vitro contractile effects of agents used in the clinical management of postpartum haemorrhage.

    Science.gov (United States)

    Morrison, John J; Crosby, David A; Crankshaw, Denis J

    2016-10-15

    Uterine atony is a major cause of postpartum haemorrhage and maternal mortality. However, the comparative pharmacology of agents used to treat this condition is poorly understood. This study evaluates, using human pregnant myometrium in vitro, a range of contractile parameters for agents used in the clinical treatment of atonic postpartum haemorrhage. The effects of oxytocin, carbetocin, ergometrine, carboprost, syntometrine and misoprostol were investigated in 146 myometrial strips from 19 donors. The potency and maximal response values were obtained, and compared, using both maximal amplitude and mean contractile force as indices of contraction. Single, EC50 concentrations of the agents were administered and both force and contraction peak parameters were compared during a 15-min exposure. Differences were considered significant when Poxytocin and carbetocin being the most potent. The most important difference between the agents was in their ability to increase the mean contractile force, with oxytocin superior to all agents except syntometrine. In single dose experiments, mean contractile force was the parameter that separated the agents. In this respect, oxytocin was not statistically different from carboprost or syntometrine, but was superior to all other agents. These findings support a clear role for oxytocin as the first line agent for treatment of postpartum haemorrhage and raise doubts about the potential clinical usefulness of misoprostol. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Smoothelin-B deficiency results in reduced arterial contractility, hypertension, and cardiac hypertrophy in mice

    NARCIS (Netherlands)

    Rensen, Sander S.; Niessen, Petra M.; van Deursen, Jan M.; Janssen, Ben J.; Heijman, Edwin; Hermeling, Evelien; Meens, Merlijn; Lie, Natascha; Gijbels, Marion J.; Strijkers, Gustav J.; Doevendans, Pieter A.; Hofker, Marten H.; de Mey, Jo G. R.; van Eys, Guillaume J.

    2008-01-01

    Smoothelins are actin-binding proteins that are abundantly expressed in healthy visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. Their expression is strongly associated with the contractile phenotype of smooth muscle cells. Analysis of mice lacking both smoothelins (Smtn-A/B(-/-)

  10. Acute hypoxia limits endurance but does not affect muscle contractile properties.

    NARCIS (Netherlands)

    Degens, H.; Sanchez Horneros, J.M.; Hopman, M.T.E.

    2006-01-01

    Acute hypoxia causes skeletal muscle dysfunction in vitro, but little is known about its effect on muscle function in vivo. In 10 healthy male subjects, isometric contractile properties and fatigue resistance of the quadriceps muscle were determined during normoxia and hypoxia using electrically

  11. Effect of a Carbohydrate-Rich Diet on Rat Detrusor Smooth Muscle Contractility: An Experimental Study

    Directory of Open Access Journals (Sweden)

    Mustafa Suat Bolat

    2017-01-01

    Full Text Available Objectives. We aimed to investigate the effect of a carbohydrate-rich diet on detrusor contractility in rats. Materials and Methods. Sprague-Dawley rats were randomized into two groups. The control group received regular food and water. The study group received carbohydrate-rich diet for six weeks. The rats’ detrusor muscle was isolated for pharmacological and histopathological examinations. Results. In the control and study groups, mean body weights were 431.5 ± 27.6 g and 528.0 ± 36.2 g, respectively (p < 0.001. Electrical stimulation of the detrusor strips of the control group resulted in gradual contraction. A decreased contractile response was shown in the study group. Acetylcholine in 10-7-10-3 molar concentration produced a decreased contractile response in the study group, compared to the control group (p < 0.01. The study group showed marked subepithelial and intermuscular fibrosis in the bladder. Conclusion. Carbohydrate-rich diet causes marked subepithelial and extracellular fibrosis and changes in contractility in the detrusor within a six-week period. Changes have higher costs in therapeutic choices and correction of these changes remains difficult. Putting an end to carbohydrate-rich diet would seem to be more cost-effective than dealing with the effects of consuming it in high proportions which should be the national policy worldwide.

  12. Prolonged ischemic heart disease and coronary artery bypass - relation to contractile reserve

    DEFF Research Database (Denmark)

    Kofoed, Klaus F; Bangsgaard, Regitze; Carstensen, Steen

    2002-01-01

    OBJECTIVE: A major effect of coronary artery bypass grafting (CABG) in patients with ischemic heart disease and impaired left ventricular (LV) contractile function is believed to be an improvement in LV function due to recovery of dysfunctional, but viable myocardium. However, recent studies have...

  13. Effect of short-term outlet obstruction on rat bladder nerve density and contractility

    NARCIS (Netherlands)

    Barendrecht, M. M.; Chichester, P.; Michel, M. C.; Levin, R. M.

    2007-01-01

    1 The present study was designed to investigate the relationship between innervation density and contractile responses to field stimulation and exogenous agonists at early time points after induction of bladder outlet obstruction (BOO) in rats. 2 When compared with sham-operated animals, 1, 3 and 7

  14. Contractile responses to ergotamine and dihydroergotamine in the perfused middle cerebral artery of rat

    DEFF Research Database (Denmark)

    Tfelt-Hansen, Peer; Nilsson, Elisabeth; Edvinsson, Lars

    2007-01-01

    mmHg and luminally perfused. All vessels used attained spontaneous contractile tone (34.9+/-1.8% of resting tone) and responded to luminal adenosine triphosphate (ATP) with dilatation (24.1+/-4.0%), which showed functioning endothelium. Luminally added ergotamine or DHE induced maximal contractions...

  15. Gallbladder contractility and mucus secretion after cholesterol feeding in the prairie dog

    NARCIS (Netherlands)

    Li, Y. F.; Moody, F. G.; Weisbrodt, N. W.; Zalewsky, C. A.; Coelho, J. C.; Senninger, N.; Gouma, D.

    1986-01-01

    The purpose of our study was to evaluate changes in gallbladder contractility and mucus secretion in vitro during the early stages of gallstone formation in prairie dogs. Thirty-two animals were divided into five groups. Control animals were fed a trace cholesterol diet. Experimental animals were

  16. Hydrogen ion changes and contractile behavior in the perfused rat heart

    NARCIS (Netherlands)

    Cingolani, H.E.; Maas, A.H.J.; Zimmerman, A.N.E.; Meijler, F.L.

    1975-01-01

    The effect of acid-base alterations was analyzed using isolated rat hearts perfused at constant coronary perfusion pressure, and stimulated to contract at constant rate. The amount of shortening in the major axis and its derivative were measured to assess myocardial contractility. Both the

  17. The importance of myocardial contractile reserve in predicting response to cardiac resynchronization therapy

    NARCIS (Netherlands)

    Kloosterman, Mariëlle; Damman, Kevin; Van Veldhuisen, Dirk J; Rienstra, Michiel; Maass, Alexander H

    AimTo perform a meta-analysis and systematic review of published data to assess the relationship between contractile reserve and response to cardiac resynchronization therapy (CRT) in patients with heart failure. Methods and resultsWe searched MEDLINE/PubMed and Cochrane for all papers published up

  18. ATP-induced changes in rat skeletal muscle contractility.

    Science.gov (United States)

    Gabdrakhmanov, A I; Khayrullin, A E; Grishin, C H; Ziganshin, A U

    2015-01-01

    considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated.It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown.It is known that the body's adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress.We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred. To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes. Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under

  19. Gene transfer of heterologous G protein-coupled receptors to cardiomyocytes: differential effects on contractility.

    Science.gov (United States)

    Laugwitz, K L; Weig, H J; Moretti, A; Hoffmann, E; Ueblacker, P; Pragst, I; Rosport, K; Schömig, A; Ungerer, M

    2001-04-13

    In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to beta-adrenergic stimulation. Parathyroid hormone (PTH)-related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective beta-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V(2) vasopressin receptors (rV(2)-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant beta(2)-adrenergic receptors (rbeta(2)-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V(2)-Rs are uniquely coupled to Gs, PTH1-Rs and beta(2)-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or rbeta(2)-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV(2)-R-expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of beta(2)-ARS: In the absence of receptor agonists, rPTH1-Rs and rbeta(2)-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV(2)-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V(2)-Rs might be more suited to test the effects of c

  20. An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH.

    Science.gov (United States)

    Tang, Yu; Wu, Yuantai; Herlihy, Sarah E; Brito-Aleman, Francisco J; Ting, Jose H; Janetopoulos, Chris; Gomer, Richard H

    2018-02-13

    In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum AprA is a chemorepellent for and inhibits the proliferation of D. discoideum We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH ( grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells ( grlH¯/grlH OE ) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum IMPORTANCE Little is known about how eukaryotic cells can count themselves and thus regulate the size of a tissue or density of cells. In addition, little is known about how eukaryotic cells can sense a repellant signal and move away from the source of the repellant, for instance, to organize the movement of cells in a developing embryo or to move immune cells out of a tissue. In this study, we found that a eukaryotic microbe uses G protein-coupled receptors to mediate both cell density sensing and chemorepulsion. Copyright © 2018 Tang et al.

  1. The ROCO kinase QkgA is necessary for proliferation inhibition by autocrine signals in Dictyostelium discoideum.

    Science.gov (United States)

    Phillips, Jonathan E; Gomer, Richard H

    2010-10-01

    AprA and CfaD are secreted proteins that function as autocrine signals to inhibit cell proliferation in Dictyostelium discoideum. Cells lacking AprA or CfaD proliferate rapidly, and adding AprA or CfaD to cells slows proliferation. Cells lacking the ROCO kinase QkgA proliferate rapidly, with a doubling time 83% of that of the wild type, and overexpression of a QkgA-green fluorescent protein (GFP) fusion protein slows cell proliferation. We found that qkgA(-) cells accumulate normal levels of extracellular AprA and CfaD. Exogenous AprA or CfaD does not slow the proliferation of cells lacking qkgA, and expression of QkgA-GFP in qkgA(-) cells rescues this insensitivity. Like cells lacking AprA or CfaD, cells lacking QkgA tend to be multinucleate, accumulate nuclei rapidly, and show a mass and protein accumulation per nucleus like those of the wild type, suggesting that QkgA negatively regulates proliferation but not growth. Despite their rapid proliferation, cells lacking AprA, CfaD, or QkgA expand as a colony on bacteria less rapidly than the wild type. Unlike AprA and CfaD, QkgA does not affect spore viability following multicellular development. Together, these results indicate that QkgA is necessary for proliferation inhibition by AprA and CfaD, that QkgA mediates some but not all of the effects of AprA and CfaD, and that QkgA may function downstream of these proteins in a signal transduction pathway regulating proliferation.

  2. Whole genome sequencing of mutation accumulation lines reveals a low mutation rate in the social amoeba Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Gerda Saxer

    Full Text Available Spontaneous mutations play a central role in evolution. Despite their importance, mutation rates are some of the most elusive parameters to measure in evolutionary biology. The combination of mutation accumulation (MA experiments and whole-genome sequencing now makes it possible to estimate mutation rates by directly observing new mutations at the molecular level across the whole genome. We performed an MA experiment with the social amoeba Dictyostelium discoideum and sequenced the genomes of three randomly chosen lines using high-throughput sequencing to estimate the spontaneous mutation rate in this model organism. The mitochondrial mutation rate of 6.76×10(-9, with a Poisson confidence interval of 4.1×10(-9 - 9.5×10(-9, per nucleotide per generation is slightly lower than estimates for other taxa. The mutation rate estimate for the nuclear DNA of 2.9×10(-11, with a Poisson confidence interval ranging from 7.4×10(-13 to 1.6×10(-10, is the lowest reported for any eukaryote. These results are consistent with low microsatellite mutation rates previously observed in D. discoideum and low levels of genetic variation observed in wild D. discoideum populations. In addition, D. discoideum has been shown to be quite resistant to DNA damage, which suggests an efficient DNA-repair mechanism that could be an adaptation to life in soil and frequent exposure to intracellular and extracellular mutagenic compounds. The social aspect of the life cycle of D. discoideum and a large portion of the genome under relaxed selection during vegetative growth could also select for a low mutation rate. This hypothesis is supported by a significantly lower mutation rate per cell division in multicellular eukaryotes compared with unicellular eukaryotes.

  3. The carboxy-terminal domain of Dictyostelium C-module-binding factor is an independent gene regulatory entity.

    Directory of Open Access Journals (Sweden)

    Jörg Lucas

    Full Text Available The C-module-binding factor (CbfA is a multidomain protein that belongs to the family of jumonji-type (JmjC transcription regulators. In the social amoeba Dictyostelium discoideum, CbfA regulates gene expression during the unicellular growth phase and multicellular development. CbfA and a related D. discoideum CbfA-like protein, CbfB, share a paralogous domain arrangement that includes the JmjC domain, presumably a chromatin-remodeling activity, and two zinc finger-like (ZF motifs. On the other hand, the CbfA and CbfB proteins have completely different carboxy-terminal domains, suggesting that the plasticity of such domains may have contributed to the adaptation of the CbfA-like transcription factors to the rapid genome evolution in the dictyostelid clade. To support this hypothesis we performed DNA microarray and real-time RT-PCR measurements and found that CbfA regulates at least 160 genes during the vegetative growth of D. discoideum cells. Functional annotation of these genes revealed that CbfA predominantly controls the expression of gene products involved in housekeeping functions, such as carbohydrate, purine nucleoside/nucleotide, and amino acid metabolism. The CbfA protein displays two different mechanisms of gene regulation. The expression of one set of CbfA-dependent genes requires at least the JmjC/ZF domain of the CbfA protein and thus may depend on chromatin modulation. Regulation of the larger group of genes, however, does not depend on the entire CbfA protein and requires only the carboxy-terminal domain of CbfA (CbfA-CTD. An AT-hook motif located in CbfA-CTD, which is known to mediate DNA binding to A+T-rich sequences in vitro, contributed to CbfA-CTD-dependent gene regulatory functions in vivo.

  4. Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

    Directory of Open Access Journals (Sweden)

    Chenyu Zhang

    2009-05-01

    Full Text Available Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial

  5. Lack of Ecological and Life History Context Can Create the Illusion of Social Interactions in Dictyostelium discoideum.

    Science.gov (United States)

    Martínez-García, Ricardo; Tarnita, Corina E

    2016-12-01

    Studies of social microbes often focus on one fitness component (reproductive success within the social complex), with little information about or attention to other stages of the life cycle or the ecological context. This can lead to paradoxical results. The life cycle of the social amoeba Dictyostelium discoideum includes a multicellular stage in which not necessarily clonal amoebae aggregate upon starvation to form a possibly chimeric (genetically heterogeneous) fruiting body made of dead stalk cells and spores. The lab-measured reproductive skew in the spores of chimeras indicates strong social antagonism that should result in low genotypic diversity, which is inconsistent with observations from nature. Two studies have suggested that this inconsistency stems from the one-dimensional assessment of fitness (spore production) and that the solution lies in tradeoffs between multiple life-history traits, e.g.: spore size versus viability; and spore-formation (via aggregation) versus staying vegetative (as non-aggregated cells). We develop an ecologically-grounded, socially-neutral model (i.e. no social interactions between genotypes) for the life cycle of social amoebae in which we theoretically explore multiple non-social life-history traits, tradeoffs and tradeoff-implementing mechanisms. We find that spore production comes at the expense of time to complete aggregation, and, depending on the experimental setup, spore size and viability. Furthermore, experimental results regarding apparent social interactions within chimeric mixes can be qualitatively recapitulated under this neutral hypothesis, without needing to invoke social interactions. This allows for simple potential resolutions to the previously paradoxical results. We conclude that the complexities of life histories, including social behavior and multicellularity, can only be understood in the appropriate multidimensional ecological context, when considering all stages of the life cycle.

  6. Structure, dynamics and folding of an immunoglobulin domain of the gelation factor (ABP-120) from Dictyostelium discoideum.

    Science.gov (United States)

    Hsu, Shang-Te Danny; Cabrita, Lisa D; Fucini, Paola; Dobson, Christopher M; Christodoulou, John

    2009-05-15

    We have carried out a detailed structural and dynamical characterisation of the isolated fifth repeat of the gelation factor (ABP-120) from Dictyostelium discoideum (ddFLN5) by NMR spectroscopy to provide a basis for studies of co-translational folding on the ribosome of this immunoglobulin-like domain. The isolated ddFLN5 can fold autonomously in solution into a structure that resembles very closely the crystal structure of the domain in a construct in which the adjacent sixth repeat (ddFLN6) is covalently linked to its C-terminus in tandem but deviates locally from a second crystal structure in which ddFLN5 is flanked by ddFLN4 and ddFLN6 at both N- and C-termini. Conformational fluctuations were observed via (15)N relaxation methods and are primarily localised in the interstrand loops that encompass the C-terminal hemisphere. These fluctuations are distinct in location from the region where line broadening is observed in ddFLN5 when attached to the ribosome as part of a nascent chain. This observation supports the conclusion that the broadening is associated with interactions with the ribosome surface [Hsu, S. T. D., Fucini, P., Cabrita, L. D., Launay, H., Dobson, C. M. & Christodoulou, J. (2007). Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy. Proc. Natl. Acad. Sci. USA, 104, 16516-16521]. The unfolding of ddFLN5 induced by high concentrations of urea shows a low population of a folding intermediate, as inferred from an intensity-based analysis, a finding that differs from that of ddFLN5 as a ribosome-bound nascent chain. These results suggest that interesting differences in detail may exist between the structure of the domain in isolation and when linked to the ribosome and between protein folding in vitro and the folding of a nascent chain as it emerges from the ribosome.

  7. The actinome of Dictyostelium discoideum in comparison to actins and actin-related proteins from other organisms.

    Directory of Open Access Journals (Sweden)

    Jayabalan M Joseph

    Full Text Available Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps. To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases developmentally regulated and coding for identical proteins (Act8-group. According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8 as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes.

  8. 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

  9. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    Energy Technology Data Exchange (ETDEWEB)

    Morton, M.J., E-mail: michael.morton@astrazeneca.com [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Armstrong, D.; Abi Gerges, N. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Bridgland-Taylor, M. [Discovery Sciences, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom); Pollard, C.E.; Bowes, J.; Valentin, J.-P. [Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG (United Kingdom)

    2014-09-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

  10. Thrombopoietin modulates cardiac contractility in vitro and contributes to myocardial depressing activity of septic shock serum.

    Science.gov (United States)

    Lupia, Enrico; Spatola, Tiziana; Cuccurullo, Alessandra; Bosco, Ornella; Mariano, Filippo; Pucci, Angela; Ramella, Roberta; Alloatti, Giuseppe; Montrucchio, Giuseppe

    2010-09-01

    Thrombopoietin (TPO) is a humoral growth factor that has been shown to increase platelet activation in response to several agonists. Patients with sepsis have increased circulating TPO levels, which may enhance platelet activation, potentially participating to the pathogenesis of multi-organ failure. Aim of this study was to investigate whether TPO affects myocardial contractility and participates to depress cardiac function during sepsis. We showed the expression of the TPO receptor c-Mpl on myocardial cells and tissue by RT-PCR, immunofluorescence and western blotting. We then evaluated the effect of TPO on the contractile function of rat papillary muscle and isolated heart. TPO did not change myocardial contractility in basal conditions, but, when followed by epinephrine (EPI) stimulation, it blunted the enhancement of contractile force induced by EPI both in papillary muscle and isolated heart. An inhibitor of TPO prevented TPO effect on cardiac inotropy. Treatment of papillary muscle with pharmacological inhibitors of phosphatidylinositol 3-kinase, NO synthase, and guanilyl cyclase abolished TPO effect, indicating NO as the final mediator. We finally studied the role of TPO in the negative inotropic effect exerted by human septic shock (HSS) serum and TPO cooperation with TNF-alpha and IL-1beta. Pre-treatment with the TPO inhibitor prevented the decrease in contractile force induced by HSS serum. Moreover, TPO significantly amplified the negative inotropic effect induced by TNF-alpha and IL-1beta in papillary muscle. In conclusion, TPO negatively modulates cardiac inotropy in vitro and contributes to the myocardial depressing activity of septic shock serum.

  11. Retosiban Prevents Stretch-Induced Human Myometrial Contractility and Delays Labor in Cynomolgus Monkeys.

    Science.gov (United States)

    Aye, Irving L M H; Moraitis, Alexandros A; Stanislaus, Dinesh; Charnock-Jones, D Stephen; Smith, Gordon C S

    2018-03-01

    Stretch of the myometrium promotes its contractility and is believed to contribute to the control of parturition at term and to the increased risk of preterm birth in multiple pregnancies. To determine the effects of the putative oxytocin receptor (OTR) inverse agonist retosiban on (1) the contractility of human myometrial explants and (2) labor in nonhuman primates. Human myometrial biopsies were obtained at planned term cesarean, and explants were exposed to stretch in the presence and absence of a range of drugs, including retosiban. The in vivo effects of retosiban were determined in cynomolgus monkeys. Prolonged mechanical stretch promoted myometrial extracellular signal-regulated kinase (ERK)1/2 phosphorylation. Moreover, stretch-induced stimulation of myometrial contractility was prevented by ERK1/2 inhibitors. Retosiban (10 nM) prevented stretch-induced stimulation of myometrial contractility and phosphorylation of ERK1/2. Moreover, the inhibitory effect of retosiban on stretch-induced ERK1/2 phosphorylation was prevented by coincubation with a 100-fold excess of a peptide OTR antagonist, atosiban. Compared with vehicle-treated cynomolgus monkeys, treatment with oral retosiban (100 to 150 days of gestational age) reduced the risk of spontaneous delivery (hazard ratio = 0.07, 95% confidence interval 0.01 to 0.60, P = 0.015). The OTR acts as a uterine mechanosensor, whereby stretch increases myometrial contractility through agonist-free activation of the OTR. Retosiban prevents this through inverse agonism of the OTR and, in vivo, reduced the likelihood of spontaneous labor in nonhuman primates. We hypothesize that retosiban may be an effective preventative treatment of preterm birth in high-risk multiple pregnancies, an area of unmet clinical need.

  12. Dynamic MR cholangiography after fatty meal loading. Cystic contractility and dynamic evaluation of biliary stasis

    International Nuclear Information System (INIS)

    Omata, Takayuki; Saito, Kazuhiro; Kotake, Fumio; Mizokami, Yuji; Matsuoka, Takeshi; Abe, Kimihiko

    2002-01-01

    Dynamic MR cholangiography was conducted on patients with cholelithiasis or choledocholithiasis who had consumed a fatty test meal (Molyork) and the cystic contractility and dynamics of biliary stasis was evaluated. The subjects were 25 with intracystic cholelithiasis, 10 with choledocholithiasis and 10 normal controls. For an imaging sequence, the rapid acquisition with relaxation enhancement (RARE) method was employed and imaging was conducted for 40 min (every 30 s following Molyork administration) without breath-holding. The gallbladder contraction ratio was computed and the contractile ratio for the common bile duct was calculated. To determine the bile flow to the duodenum, the high-intensity signal, indicating the flow from the lower common bile duct, and perfusion of the duodenum were observed in dynamic mode on the monitor with the naked eye and interpreted as positive bile flow. The frequency of this flow was visually monitored. The gallbladder contractile ratio was significantly reduced in patients with cholelithiasis or choledocholithiasis compared with the controls. In a comparison with the normal controls, no sequential changes were noted in the mean contractile ratio of the common bile duct of the patients with cholelithiasis or choledocholithiasis. The mean frequency of bile flow observed for each 40 min period was 13±2.4, 6±2.2, and 4±1.3 times for the controls, those with intracystic cholelithiasis, and those with choledocholithiasis, respectively. Compared with the controls, the latter two patient groups showed evident reductions in the frequency of bile flow to the duodenum (p<0.001). Dynamic MRC combined with Molyork loading makes it possible to compute cystic contractile ratios and perform a dynamic examination of bile flow under non-invasive, near-physiological conditions. (author)

  13. Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration.

    Science.gov (United States)

    Gregorevic, Paul; Plant, David R; Stupka, Nicole; Lynch, Gordon S

    2004-07-15

    Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca(2+)- and Sr(2+)-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had approximately 10% of the maximal force producing capacity (P(o)) of control (uninjured) fibres, and an altered sensitivity to Ca(2+) and Sr(2+) at 7 days post-injury. Increased force production and a shift in Ca(2+) sensitivity consistent with fibre maturation were observed during regeneration such that P(o) was restored to 36-45% of that in control fibres by 21 days, and sensitivity to Ca(2+) and Sr(2+) was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.

  14. Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

    International Nuclear Information System (INIS)

    Morton, M.J.; Armstrong, D.; Abi Gerges, N.; Bridgland-Taylor, M.; Pollard, C.E.; Bowes, J.; Valentin, J.-P.

    2014-01-01

    Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility

  15. Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole

    Directory of Open Access Journals (Sweden)

    Lara J. Kohler

    2016-07-01

    Full Text Available Coxiella burnetii replicates in an acidified lysosome-derived vacuole. Biogenesis of the Coxiella-containing vacuole (CCV requires bacterial effector proteins delivered into host cells by the Dot/Icm secretion system. Genetic and cell biological analysis revealed that an effector protein called Cig2 promotes constitutive fusion of autophagosomes with the CCV to maintain this compartment in an autolysosomal stage of maturation. This distinguishes the CCV from other pathogen-containing vacuoles that are targeted by the host autophagy pathway, which typically confers host resistance to infection by delivering the pathogen to a toxic lysosomal environment. By maintaining the CCV in an autolysosomal stage of maturation, Cig2 enabled CCV homotypic fusion and enhanced bacterial virulence in the Galleria mellonella (wax moth model of infection by a mechanism that decreases host tolerance. Thus, C. burnetii residence in an autolysosomal organelle alters host tolerance of infection, which indicates that Cig2-dependent manipulation of a lysosome-derived vacuole influences the host response to infection.

  16. The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

    Science.gov (United States)

    Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

    2015-04-10

    Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Phg1/TM9 proteins control intracellular killing of bacteria by determining cellular levels of the Kil1 sulfotransferase in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Marion Le Coadic

    Full Text Available Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing.

  18. Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium.

    Science.gov (United States)

    Ginsburg, G T; Kimmel, A R

    1997-08-15

    Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4- cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by another cAMP receptor, CAR1 and/or CAR3. Additional data indicate that these CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by an ancient mechanism that is shared by the Wnt/Fz circuits for dorsoventral patterning during early Xenopus development and establishing Drosophila segment polarity.

  19. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

    Directory of Open Access Journals (Sweden)

    Katherine Maringer

    2014-08-01

    Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  20. TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium.

    Science.gov (United States)

    Froquet, Romain; le Coadic, Marion; Perrin, Jackie; Cherix, Nathalie; Cornillon, Sophie; Cosson, Pierre

    2012-02-01

    TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. In this study, we found that genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule in Dictyostelium amoebae. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits nine transmembrane domains and is essential for cellular adhesion. A contact site A (csA)-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of the Dictyostelium surface protein csA also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface.

  1. A long-range foresight for the medical application of apoptosis specifically induced by Dd-MRP4, Dictyostelium mitochondrial ribosomal protein S4, to cancer therapy.

    Science.gov (United States)

    Maeda, Yasuo

    2015-02-10

    Apoptosis (programmed cell death) is regarded as ultimate differentiation of the cell. We have recently demonstrated that a targeted delivery of Dd-MRP4 (Dictyostelium mitochondrial ribosomal protein S4) suppresses specifically the proliferation of the human cancer cells, by inducing their apoptotic cell death (Chida et al., 2014, doi:10.1186/1475-2867-14-56). This amazing fact was discovered, simply based on the finding that Dd-MRP4 expression is absolutely required for transition of Dictyostelium cells from growth to differentiation (Chida et al., 2008, doi:10.1186/1471-2156-9-25; Maeda et al., 2013, doi:10.3390/biom3040943). Dd-MRP4 protein has quite unique structural characters, in that it is highly basic (pI: about 11.5) and interestingly has several nuclear-localization signals within the molecule. In this review, we introduce briefly the efficacy of several apoptosis-inducing substances reported thus far for cancer therapy, and speculate the possible mechanisms, by which apoptosis is specifically induced by Dd-MRP4, on the basis of its structural uniqueness. We also discuss several issues to be solved for the medical application of ectopically expressed Dd-MRP4 in human cancer cells.

  2. Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

    Directory of Open Access Journals (Sweden)

    Heather E Miller

    2018-04-01

    Full Text Available Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1 and cationic-independent mannose-6-phosphate receptor (CI-M6PR, respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the

  3. [Gallbladder contractility in children with functional abdominal pain or irritable bowel syndrome].

    Science.gov (United States)

    Iwańczak, Franciszek; Siedlecka-Dawidko, Jolanta; Iwanczak, Barbara

    2013-07-01

    III Rome Criteria of functional gastrointestinal disorders in children, distinguished the disturbances with abdominal pain, to which irritable bowel syndrome, functional abdominal pains, functional dyspepsia and abdominal migraine were included. THE AIM OF THE STUDY was sonographic assessment of the gallbladder and its contractility in functional abdominal pain and irritable bowel syndrome in children. The study comprised 96 children aged 6 to 18 years, 59 girls and 37 boys. Depending on diagnosis, the children were divided into three groups. 38 children with functional abdominal pain constituted the first group, 26 children with irritable bowel syndrome were included to the second group, the third group consisted of 32 healthy children (control group). Diagnosis of functional abdominal pain and irritable bowel syndrome was made based on the III Rome Criteria. In irritable bowel syndrome both forms with diarrhea (13) and with constipation (13) were observed. Anatomy and contractility of the gallbladder were assessed by ultrasound examination. The presence of septum, wall thickness, thick bile, vesicle volume in fasting state and 30th and 60th minute after test meal were taken into consideration. Test meal comprised about 15% of caloric requirement of moderate metabolism. Children with bile stones and organic diseases were excluded from the study. Thickened vesicle wall and thick bile were present more frequently in children with irritable bowel syndrome and functional abdominal pain than in control group (p functional abdominal pain than in irritable bowel syndrome and control group (p = 0.003, p = 0.05). Vesicle contractility after test meal was greatest in children with functional abdominal pain. Evaluation of diminished (smaller than 30%) and enlarged (greater then 80%) gallbladder contractility at 30th and 60th minute after test meal demonstrated disturbances of contractility in children with irritable bowel syndrome and functional abdominal pain. In children

  4. Expression of Hsp27 and Hsp70 and vacuolization in the pituitary glands in cases of fatal hypothermia.

    Science.gov (United States)

    Doberentz, Elke; Markwerth, Philipp; Wagner, Rebecca; Madea, Burkhard

    2017-09-01

    Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.

  5. The Effect of Herbicides on Hydrogen Peroxide Generation in Isolated Vacuoles of Red Beet Root (Beta vulgaris L.

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2015-12-01

    Full Text Available Influence of herbicides on the hydrogen peroxide generation in vacuolar extracts of red beet root (Beta vulgaris L. was investigated. Belonging to different chemical classes of herbicide compounds have been used. Herbicides differ from each other in the mechanism of effects on plants. Clopyralid (aromatic acid herbicide, derivative of picolinic acid and 2.4-D (phenoxyacetic herbicide, characterized by hormone-like effects, contributed to the formation of H2O2 in vacuolar extracts. Fluorodifen (nitrophenyl ether herbicide and diuron (urea herbicide also have increased contents H2O2. These compounds inhibit the electron transport, photosynthesis, and photorespiration in sensitive plants. Herbicidal effect of glyphosate (organophosphorus herbicide is due to the inhibition of amino acid synthesis in plant cells. Glyphosate did not affect the content of H2O2 in vacuolar extracts. Herbicide dependent H2O2-generation did not occur with oxidoreductase inhibitors, potassium cyanide and sodium azide. The results suggest that the formation of ROS in the vacuoles due to activity of oxidoreductases, which could interact with herbicides.

  6. Cell survival under nutrient stress is dependent on metabolic conditions regulated by Akt and not by autophagic vacuoles.

    Science.gov (United States)

    Bruno, P; Calastretti, A; Priulla, M; Asnaghi, L; Scarlatti, F; Nicolin, A; Canti, G

    2007-10-01

    Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

  7. Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

    Science.gov (United States)

    Colombo, María Isabel; García Véscovi, Eleonora

    2011-01-01

    Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell. PMID:21901159

  8. Live Cell Imaging During Germination Reveals Dynamic Tubular Structures Derived from Protein Storage Vacuoles of Barley Aleurone Cells

    Directory of Open Access Journals (Sweden)

    Verena Ibl

    2014-09-01

    Full Text Available The germination of cereal seeds is a rapid developmental process in which the endomembrane system undergoes a series of dynamic morphological changes to mobilize storage compounds. The changing ultrastructure of protein storage vacuoles (PSVs in the cells of the aleurone layer has been investigated in the past, but generally this involved inferences drawn from static pictures representing different developmental stages. We used live cell imaging in transgenic barley plants expressing a TIP3-GFP fusion protein as a fluorescent PSV marker to follow in real time the spatially and temporally regulated remodeling and reshaping of PSVs during germination. During late-stage germination, we observed thin, tubular structures extending from PSVs in an actin-dependent manner. No extensions were detected following the disruption of actin microfilaments, while microtubules did not appear to be involved in the process. The previously-undetected tubular PSV structures were characterized by complex movements, fusion events and a dynamic morphology. Their function during germination remains unknown, but might be related to the transport of solutes and metabolites.

  9. Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole 1

    Science.gov (United States)

    Tucker, Joseph E.; Mauzerall, David; Tucker, Edward B.

    1989-01-01

    The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water. PMID:16666864

  10. Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole.

    Science.gov (United States)

    Tucker, J E; Mauzerall, D; Tucker, E B

    1989-07-01

    The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water.

  11. Effect of changes in contractility on the index of myocardial performance in the dysfunctional left ventricle

    Directory of Open Access Journals (Sweden)

    Lavine Steven J

    2006-11-01

    Full Text Available Abstract Background The index of myocardial performance has prognostic power in patients with cardiomyopathy and following myocardial infarction. As the index of myocardial performance has been shown to be preload and afterload dependent, the effect of altering contractility on IMP and its components with left ventricular dysfunction has been incompletely delineated. Methods Chronic left ventricular dysfunction was induced in 10 canines using coronary microsphere embolization. Each dog was instrumented and imaged with 2D echo and Doppler. At the same atrially paced rate, contractility was increased with a dobutamine infusion and then following 4 weeks of oral digoxin. Results With chronic left ventricular dysfunction, a reduced left ventricular ejection fraction (42 ± 3%, p Conclusion Increased inotropy with digoxin and dobutamine reduced the index of myocardial performance in dogs with left ventricular dysfunction. Shortened isovolumic contraction time, increased diastolic filling period, and reduced left ventricular end diastolic pressure with digoxin may provide insight into its efficacy in heart failure.

  12. An Estimating Method of Contractile State Changes Come From Continuous Isometric Contraction of Skeletal Muscle

    Energy Technology Data Exchange (ETDEWEB)

    Park, H.J.; Lee, S.J. [Wonkwang University, Iksan (Korea)

    2003-01-01

    In this study was proposed that a new estimating method for investigation of contractile state changes which generated from continuous isometric contraction of skeletal muscle. The physiological changes (EMG, ECG) and the psychological changes by CNS(central nervous system) were measured by experiments, while the muscle of subjects contracted continuously with isometric contraction in constant load. The psychological changes were represented as three-step-change named 'fatigue', 'pain' and 'sick(greatly pain)' from oral test, and the method which compared physiological change with psychological change on basis of these three steps was developed. The result of analyzing the physiological signals, EMG and ECG signal changes were observed at the vicinity of judging point in time of psychological changes. Namely, it is supposed that contractile states have three kind of states pattern (stable, fatigue, pain) instead of two states (stable, fatigue). (author). 24 refs., 7 figs.

  13. Measuring the Contractile Response of Isolated Tissue Using an Image Sensor

    Directory of Open Access Journals (Sweden)

    David Díaz-Martín

    2015-04-01

    Full Text Available Isometric or isotonic transducers have traditionally been used to study the contractile/relaxation effects of drugs on isolated tissues. However, these mechanical sensors are expensive and delicate, and they are associated with certain disadvantages when performing experiments in the laboratory. In this paper, a method that uses an image sensor to measure the contractile effect of drugs on blood vessel rings and other luminal organs is presented. The new method is based on an image-processing algorithm, and it provides a fast, easy and non-expensive way to analyze the effects of such drugs. In our tests, we have obtained dose-response curves from rat aorta rings that are equivalent to those achieved with classical mechanic sensors.

  14. Numerical investigation of perforated polymer microcantilever sensor for contractile behavior of cardiomyocytes

    Science.gov (United States)

    Khoa Nguyen, Trieu; Lee, Dong-Weon; Lee, Bong-Kee

    2017-06-01

    In this study, a numerical investigation of microcantilever sensors for detecting the contractile behavior of cardiomyocytes (CMs) was performed. Recently, a novel surface-patterned perforated SU-8 microcantilever sensor has been developed for the preliminary screening of cardiac toxicity. From the contractile motion of the CMs cultured on the microcantilever surface, a macroscopic bending of the microcantilever was obtained, which is considered to reflect a physiological change. As a continuation of the previous research, a novel numerical method based on a surface traction model was proposed and verified to further understand the bending behavior of the microcantilevers. Effects of various factors, including surface traction magnitude, focal area of CMs, and stiffness of microcantilever, on the bending displacement were investigated. From static and transient analyses, the focal area was found to be the most crucial factor. In addition, the current result can provide a design guideline for various micromechanical devices based on the same principle.

  15. Effect of a crude sulfated polysaccharide from Halymenia floresia (Rhodophyta on gastrointestinal smooth muscle contractility

    Directory of Open Access Journals (Sweden)

    José Ronaldo Vasconcelos Graça

    2011-10-01

    Full Text Available The aim of this work was to study the effect of Halymenia floresia (Hf on duodenum contractility, and on experimental protocols of gastric compliance (GC in rats. Fraction Hf2s exhibited a concentration-dependent myocontractile effect (EC50 12.48 µg/ml, and an inhibitory effect after consecutive washing. The contractile response promoted by Hf2s in the duodenum strips was completely inhibited by verapamil, and the effects were prevented in the presence of Ca2+-free medium. The pretreatment with atropine prevented the Hf2s myocontractile effect. Hf2s was also capable to decrease the GC (from 3.8±0.06 to 3.4±0.13 ml, P<0.05, which did not return to basal levels after more 50 min of observation. These results indicated that the algal polysaccharide possessed in vitro and in vivo gastrointestinal effects.

  16. Cell density and actomyosin contractility control the organization of migrating collectives within an epithelium

    Science.gov (United States)

    Loza, Andrew J.; Koride, Sarita; Schimizzi, Gregory V.; Li, Bo; Sun, Sean X.; Longmore, Gregory D.

    2016-01-01

    The mechanisms underlying collective migration are important for understanding development, wound healing, and tumor invasion. Here we focus on cell density to determine its role in collective migration. Our findings show that increasing cell density, as might be seen in cancer, transforms groups from broad collectives to small, narrow streams. Conversely, diminishing cell density, as might occur at a wound front, leads to large, broad collectives with a distinct leader–follower structure. Simulations identify force-sensitive contractility as a mediator of how density affects collectives, and guided by this prediction, we find that the baseline state of contractility can enhance or reduce organization. Finally, we test predictions from these data in an in vivo epithelium by using genetic manipulations to drive collective motion between predicted migratory phases. This work demonstrates how commonly altered cellular properties can prime groups of cells to adopt migration patterns that may be harnessed in health or exploited in disease. PMID:27605707

  17. Effects of ageing on single muscle fibre contractile function following short-term immobilisation

    DEFF Research Database (Denmark)

    Hvid, Lars G; Ortenblad, Niels; Aagaard, Per

    2011-01-01

    Very little attention has been given to the combined effects of healthy ageing and short-term disuse on the contractile function of human single muscle fibres. Therefore, the present study investigated the effects of 2 weeks of lower limb cast immobilisation (i.e. disuse) on selected contractile...... IIa: young 18% and old 25%; P selective decrease in Ca(2+) sensitivity in MHC IIa fibres of young (P ....05), respectively. In conclusion, 2 weeks of lower limb immobilisation caused greater impairments in single muscle fibre force and specific force in MHC IIa than MHC I fibres independently of age. In contrast, immobilisation-induced changes in Ca(2+) sensitivity that were dependent on age and MHC isoform....

  18. The PsB glycoprotein complex is secreted as a preassembled precursor of the spore coat in Dictyostelium discoideum.

    Science.gov (United States)

    Watson, N; McGuire, V; Alexander, S

    1994-09-01

    The PsB glycoprotein in Dictyostelium discoideum is one of a diverse group of developmentally regulated, prespore-cell-specific proteins, that contain a common O-linked oligosaccharide. This post-translational modification is dependent on the wild-type modB allele. The PsB protein exists as part of a multiprotein complex of six different proteins, which have different post-translational modifications and are held together by both covalent and non-covalent interactions (Watson et al. (1993). J. Biol. Chem. 268, 22634-22641). In this study we have used microscopic and biochemical analyses to examine the cellular localization and function of the PsB complex during development. We found that the PsB complex first accumulates in prespore vesicles in slug cells and is secreted later during culmination and becomes localized to both the extracellular matrix of the apical spore mass of mature fruiting bodies and to the inner layer of the spore coat. The PsB associated with the spore coat is covalently bound by disulfide bridges. The PsB protein always exists in a multiprotein complex, but the composition of the PsB complex changes during secretion and spore maturation. Some of the PsB complex proteins have been identified as spore coat proteins. These data demonstrate that some of the proteins that form the spore coat exist as a preassembled precursor complex. The PsB complex is secreted in a developmentally regulated manner during the process of spore differentiation, at which time proteins of the complex, as well as additional spore coat proteins, become covalently associated in at least two forms of extracellular matrix: the interspore matrix and the spore coat. These and other studies show that proteins with modB dependent O-linked oligosaccharides are involved in a wide variety of processes underlying morphogenesis in this organism. These developmental processes are the direct result of cellular mechanisms regulating protein targeting, assembly and secretion, and the

  19. Levothyroxine treatment generates an abnormal uterine contractility patterns in an in vitro animal model.

    Science.gov (United States)

    Corriveau, Stéphanie; Blouin, Simon; Raiche, Évelyne; Nolin, Marc-Antoine; Rousseau, Éric; Pasquier, Jean-Charles

    2015-12-01

    Abnormal uterine contraction patterns were recently demonstrated in uterine strips from pregnant women treated with Levothyroxine (T4). These abnormalities were correlated with an increased risk of C-section delivery and associated surgical complications. To date, no study has investigated whether uterine contractility is modified by hypothyroidism or T4 treatment. Herein, we analyze the physiological role of T4 on uterine contractions. Female non-pregnant Sprague-Dawley rats ( N  = 22) were used and divided into four groups: 1) control, 2) hypothyroidism, 3) hypothyroidism treated with low T4 doses (20 μg/kg/day) and 4) with high T4 doses (100 μg/kg/day). Hypothyroidism was induced by an iodine-deficient diet. Isometric tension measurements were performed in vitro on myometrium tissues in isolated organ baths. Contractile activity parameters were quantified (amplitude, duration, frequency and area under the curve) using pharmacological tools to assess their effect. Screening of thyroid function confirmed a hypothyroid state for all rats under iodine-free diet to which T4 was subsequently administered to counterbalance hypothyroidism. Results demonstrate that hypothyroidism significantly decreased contractile duration (-17%) and increased contractile frequency (+26%), while high doses of T4 increased duration (+200%) and decreased frequency (-51%). These results thus mimic the pattern of abnormal contractions previously observed in uterine tissue from T4-treated hypothyroid pregnant women. Our data suggest that changes in myometrial reactivity are induced by T4 treatment. Thus, in conjunction with our previous observations on human myometrial strips, management of hypothyroidism should be improved to reduce the rate of C-sections in this group of patients.

  20. Functions of nonmuscle myosin II in assembly of the cellular contractile system.

    Directory of Open Access Journals (Sweden)

    Maria Shutova

    Full Text Available The contractile system of nonmuscle cells consists of interconnected actomyosin networks and bundles anchored to focal adhesions. The initiation of the contractile system assembly is poorly understood structurally and mechanistically, whereas system's maturation heavily depends on nonmuscle myosin II (NMII. Using platinum replica electron microscopy in combination with fluorescence microscopy, we characterized the structural mechanisms of the contractile system assembly and roles of NMII at early stages of this process. We show that inhibition of NMII by a specific inhibitor, blebbistatin, in addition to known effects, such as disassembly of stress fibers and mature focal adhesions, also causes transformation of lamellipodia into unattached ruffles, loss of immature focal complexes, loss of cytoskeleton-associated NMII filaments and peripheral accumulation of activated, but unpolymerized NMII. After blebbistatin washout, assembly of the contractile system begins with quick and coordinated recovery of lamellipodia and focal complexes that occurs before reappearance of NMII bipolar filaments. The initial formation of focal complexes and subsequent assembly of NMII filaments preferentially occurred in association with filopodial bundles and concave actin bundles formed by filopodial roots at the lamellipodial base. Over time, accumulating NMII filaments help to transform the precursor structures, focal complexes and associated thin bundles, into stress fibers and mature focal adhesions. However, semi-sarcomeric organization of stress fibers develops at much slower rate. Together, our data suggest that activation of NMII motor activity by light chain phosphorylation occurs at the cell edge and is uncoupled from NMII assembly into bipolar filaments. We propose that activated, but unpolymerized NMII initiates focal complexes, thus providing traction for lamellipodial protrusion. Subsequently, the mechanical resistance of focal complexes activates a

  1. Influence of genotype on contractile protein differentiation in different bovine muscles during foetal life

    OpenAIRE

    Gagnière , Hélène; Ménissier , François; Geay , Yves; Picard , Brigitte

    2000-01-01

    International audience; The purpose of this work was to compare muscle fibre differentiation in two genetic types: "normal charolais" and double-muscled (DM) "INRA 95" cattles displaying muscle hypertrophy. Six muscles with different contractile and metabolic characteristics in adult animal: Masseter, Diaphragma (Di), Biceps femoris (BF), Longissimus thoracis, Semitendinosus and Cutaneus trunci (CT) were excised from 60 to 260-day-old fœtuses of both genotypes. These muscles present different...

  2. Chronic clenbuterol treatment compromises force production without directly altering skeletal muscle contractile machinery

    Science.gov (United States)

    Py, G; Ramonatxo, C; Sirvent, P; Sanchez, A M J; Philippe, A G; Douillard, A; Galbès, O; Lionne, C; Bonnieu, A; Chopard, A; Cazorla, O; Lacampagne, A; Candau, R B

    2015-01-01

    Clenbuterol is a β2-adrenergic receptor agonist known to induce skeletal muscle hypertrophy and a slow-to-fast phenotypic shift. The aim of the present study was to test the effects of chronic clenbuterol treatment on contractile efficiency and explore the underlying mechanisms, i.e. the muscle contractile machinery and calcium-handling ability. Forty-three 6-week-old male Wistar rats were randomly allocated to one of six groups that were treated with either subcutaneous equimolar doses of clenbuterol (4 mg kg−1 day−1) or saline solution for 9, 14 or 21 days. In addition to the muscle hypertrophy, although an 89% increase in absolute maximal tetanic force (Po) was noted, specific maximal tetanic force (sPo) was unchanged or even depressed in the slow twitch muscle of the clenbuterol-treated rats (P muscle contraction and relaxation force kinetics indicated that clenbuterol treatment significantly reduced the rate constant of force development and the slow and fast rate constants of relaxation in extensor digitorum longus muscle (P fast rate constant of relaxation in soleus muscle (P fibres (fast twitch fibres) from clenbuterol-treated animals demonstrated decreased amplitude after 14 days (−19%, P < 0.01) and 21 days (−25%, P < 0.01). In conclusion, we showed that chronic clenbuterol treatment reduces contractile efficiency, with altered contraction and relaxation kinetics, but without directly altering the contractile machinery. Lower Ca2+ release during contraction could partially explain these deleterious effects. PMID:25656230

  3. Matching of sarcoplasmic reticulum and contractile properties in rat fast- and slow-twitch muscle fibres.

    Science.gov (United States)

    Trinh, Huong H; Lamb, Graham D

    2006-07-01

    1. The twitch characteristics (fast-twitch or slow-twitch) of skeletal muscle fibres are determined not only by the contractile apparatus properties of the fibre, but also by the time-course of Ca2+ release and re-uptake by the sarcoplasmic reticulum (SR). The present study examined, in individual fibres from non-transforming muscle of the rat, whether particular SR properties are matched to the contractile apparatus properties of the fibre, in particular in the case of fibres with fast-twitch contractile apparatus located in a slow-twitch muscle, namely the soleus. 2. Force was recorded in single, mechanically skinned fibres from extensor digitorum longus (EDL), gastrocnemius, peroneus longus and soleus muscles. Using repeated cycles in which the SR was emptied of all releasable Ca2+ and then reloaded, it was possible to determine the relative amount of Ca2+ present in the SR endogenously, the maximum SR capacity and the rate of Ca2+ loading. The sensitivity of the contractile apparatus to Ca2+ and Sr2+ was used to classify the fibres as fast-twitch (FT), slow-twitch (ST) or mixed (fibres examined) and thereby identify the likely troponin C and myosin heavy chain types present. 3. There was no significant difference in SR properties between the groups of FT fibres obtained from the four different muscles, including soleus. Despite some overlap in the SR properties of individual fibres between the FT and ST groups, the properties of the FT fibres in all four muscles studied were significantly different from those of the ST and mixed fibres. 4. In general, in FT fibres the SR had a larger capacity and the endogenous Ca2+ content was a relatively lower percentage of maximum compared with ST fibres. Importantly, in terms of their SR properties, FT fibres from soleus muscle more closely resembled FT fibres from other muscles than they did ST fibres from soleus muscle.

  4. Myosin phosphorylation improves contractile economy of mouse fast skeletal muscle during staircase potentiation.

    Science.gov (United States)

    Bunda, Jordan; Gittings, William; Vandenboom, Rene

    2018-01-30

    Phosphorylation of the myosin regulatory light chain (RLC) by skeletal myosin light chain kinase (skMLCK) potentiates rodent fast twitch muscle but is an ATP-requiring process. Our objective was to investigate the effect of skMLCK-catalyzed RLC phosphorylation on the energetic cost of contraction and the contractile economy (ratio of mechanical output to metabolic input) of mouse fast twitch muscle in vitro (25°C). To this end, extensor digitorum longus (EDL) muscles from wild-type (WT) and from skMLCK-devoid (skMLCK -/- ) mice were subjected to repetitive low-frequency stimulation (10 Hz for 15 s) to produce staircase potentiation of isometric twitch force, after which muscles were quick frozen for determination of high-energy phosphate consumption (HEPC). During stimulation, WT muscles displayed significant potentiation of isometric twitch force while skMLCK -/- muscles did not (i.e. 23% versus 5% change, respectively). Consistent with this, RLC phosphorylation was increased ∼3.5-fold from the unstimulated control value in WT but not in skMLCK -/- muscles. Despite these differences, the HEPC of WT muscles was not greater than that of skMLCK -/- muscles. As a result of the increased contractile output relative to HEPC, the calculated contractile economy of WT muscles was greater than that of skMLCK -/- muscles. Thus, our results suggest that skMLCK-catalyzed phosphorylation of the myosin RLC increases the contractile economy of WT mouse EDL muscle compared with skMLCK -/- muscles without RLC phosphorylation. © 2018. Published by The Company of Biologists Ltd.

  5. Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: role of autophagy.

    Science.gov (United States)

    Turdi, Subat; Han, Xuefeng; Huff, Anna F; Roe, Nathan D; Hu, Nan; Gao, Feng; Ren, Jun

    2012-09-15

    Lipopolysaccharide (LPS) from gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complications in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis, and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity, and carbonyl formation. A Kaplan-Meier curve was constructed for survival after LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice after LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O(2)(-), and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury after LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by the antioxidant N-acetylcysteine and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Cardiac-Specific Overexpression of Catalase Attenuates Lipopolysaccharide-Induced Myocardial Contractile Dysfunction: Role of Autophagy

    OpenAIRE

    Turdi, Subat; Han, Xuefeng; Huff, Anna F.; Roe, Nathan D.; Hu, Nan; Gao, Feng; Ren, Jun

    2012-01-01

    Lipopolysaccharide (LPS) from Gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complication in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged ...

  7. A thermodynamical model for stress-fiber organization in contractile cells

    OpenAIRE

    Foucard, Louis; Vernerey, Franck J.

    2012-01-01

    Cell mechanical adaptivity to external stimuli is vital to many of its biological functions. A critical question is therefore to understand the formation and organization of the stress fibers from which emerge the cell’s mechanical properties. By accounting for the mechanical aspects and the viscoelastic behavior of stress fibers, we here propose a thermodynamic model to predict the formation and orientation of stress fibers in contractile cells subjected to constant or cyclic stretch and dif...

  8. Cardiac-Specific Overexpression of Catalase Attenuates Lipopolysaccharide-Induced Myocardial Contractile Dysfunction: Role of Autophagy

    Science.gov (United States)

    Turdi, Subat; Han, Xuefeng; Huff, Anna F.; Roe, Nathan D.; Hu, Nan; Gao, Feng; Ren, Jun

    2012-01-01

    Lipopolysaccharide (LPS) from Gram-negative bacteria is a major initiator of sepsis, leading to cardiovascular collapse. Accumulating evidence has indicated a role of reactive oxygen species (ROS) in cardiovascular complication in sepsis. This study was designed to examine the effect of cardiac-specific overexpression of catalase in LPS-induced cardiac contractile dysfunction and the underlying mechanism(s) with a focus on autophagy. Catalase transgenic and wild-type FVB mice were challenged with LPS (6 mg/kg) and cardiac function was evaluated. Levels of oxidative stress, autophagy, apoptosis and protein damage were examined using fluorescence microscopy, Western blot, TUNEL assay, caspase-3 activity and carbonyl formation. Kaplan-Meier curve was constructed for survival following LPS treatment. Our results revealed a lower mortality in catalase mice compared with FVB mice following LPS challenge. LPS injection led to depressed cardiac contractile capacity as evidenced by echocardiography and cardiomyocyte contractile function, the effect of which was ablated by catalase overexpression. LPS treatment induced elevated TNF-α level, autophagy, apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), production of ROS and O2−, and protein carbonyl formation, the effects of which were significantly attenuated by catalase overexpression. Electron microscopy revealed focal myocardial damage characterized by mitochondrial injury following LPS treatment, which was less severe in catalase mice. Interestingly, LPS-induced cardiomyocyte contractile dysfunction was prevented by antioxidant NAC and the autophagy inhibitor 3-methyladenine. Taken together, our data revealed that catalase protects against LPS-induced cardiac dysfunction and mortality, which may be associated with inhibition of oxidative stress and autophagy. PMID:22902401

  9. Cardiac myofibrillar contractile properties during the progression from hypertension to decompensated heart failure.

    Science.gov (United States)

    Hanft, Laurin M; Emter, Craig A; McDonald, Kerry S

    2017-07-01

    Heart failure arises, in part, from a constellation of changes in cardiac myocytes including remodeling, energetics, Ca 2+ handling, and myofibrillar function. However, little is known about the changes in myofibrillar contractile properties during the progression from hypertension to decompensated heart failure. The aim of the present study was to provide a comprehensive assessment of myofibrillar functional properties from health to heart disease. A rodent model of uncontrolled hypertension was used to test the hypothesis that myocytes in compensated hearts exhibit increased force, higher rates of force development, faster loaded shortening, and greater power output; however, with progression to overt heart failure, we predicted marked depression in these contractile properties. We assessed contractile properties in skinned cardiac myocyte preparations from left ventricles of Wistar-Kyoto control rats and spontaneous hypertensive heart failure (SHHF) rats at ~3, ~12, and >20 mo of age to evaluate the time course of myofilament properties associated with normal aging processes compared with myofilaments from rats with a predisposition to heart failure. In control rats, the myofilament contractile properties were virtually unchanged throughout the aging process. Conversely, in SHHF rats, the rate of force development, loaded shortening velocity, and power all increased at ~12 mo and then significantly fell at the >20-mo time point, which coincided with a decrease in left ventricular fractional shortening. Furthermore, these changes occurred independent of changes in β-myosin heavy chain but were associated with depressed phosphorylation of myofibrillar proteins, and the fall in loaded shortening and peak power output corresponded with the onset of clinical signs of heart failure. NEW & NOTEWORTHY This novel study systematically examined the power-generating capacity of cardiac myofilaments during the progression from hypertension to heart disease. Previously

  10. Robust gap repair in the contractile ring ensures timely completion of cytokinesis.

    OpenAIRE

    Silva, AM; Osório, DS; Pereira, AJ; Maiato, H; Pinto, IM; Rubinstein, B; Gassmann, R; Telley, IA; Carvalho, AX

    2016-01-01

    Cytokinesis in animal cells requires the constriction of an actomyosin contractile ring, whose architecture and mechanism remain poorly understood. We use laser microsurgery to explore the biophysical properties of constricting rings in Caenorhabditis elegans embryos. Laser cutting causes rings to snap open. However, instead of disintegrating, ring topology recovers and constriction proceeds. In response to severing, a finite gap forms and is repaired by recruitment of new material in an acti...

  11. Changes in contractile properties of muscles receiving repeat injections of botulinum toxin (Botox).

    Science.gov (United States)

    Fortuna, Rafael; Vaz, Marco Aurélio; Youssef, Aliaa Rehan; Longino, David; Herzog, Walter

    2011-01-04

    Botulinum toxin type A (BTX-A) is a frequently used therapeutic tool to denervate muscles in the treatment of neuromuscular disorders. Although considered safe by the US Food and Drug Administration, BTX-A can produce adverse effects in target and non-target muscles. With an increased use of BTX-A for neuromuscular disorders, the effects of repeat injections of BTX-A on strength, muscle mass and structure need to be known. Therefore, the purpose of this study was to investigate the changes in strength, muscle mass and contractile material in New Zealand White (NZW) rabbits. Twenty NZW rabbits were divided into 4 groups: control and 1, 3 and 6 months of unilateral, repeat injections of BTX-A into the quadriceps femoris. Outcome measures included knee extensor torque, muscle mass and the percentage of contractile material in the quadriceps muscles of the target and non-injected contralateral hindlimbs. Strength in the injected muscles was reduced by 88%, 89% and 95% in the 1, 3 and 6 months BTX-A injected hindlimbs compared to controls. Muscle mass was reduced by 50%, 42% and 31% for the vastus lateralis (VL), rectus femoris (RF) and vastus medialis (VM), respectively, at 1 month, by 68%, 51% and 50% at 3 months and by 76%, 44% and 13% at 6 months. The percentage of contractile material was reduced for the 3 and 6 months animals to 80-64%, respectively, and was replaced primarily by fat. Similar, but less pronounced results were also observed for the quadriceps muscles of the contralateral hindlimbs, suggesting that repeat BTX-A injections cause muscle atrophy and loss of contractile tissue in target muscles and also in non-target muscles that are far removed from the injection site. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Impact of tamsulosin and nifedipine on contractility of pregnant rat ureters in vitro.

    Science.gov (United States)

    Haddad, Lisette; Corriveau, Stéphanie; Rousseau, Eric; Blouin, Simon; Pasquier, Jean-Charles; Ponsot, Yves; Roy-Lacroix, Marie-Ève

    2018-01-01

    To evaluate the in vitro effect of tamsulosin and nifedipine on the contractility of pregnant rat ureters and to perform quantitative analysis of the pharmacological effects. Medical expulsive therapy (MET) is commonly used to treat urolithiasis. However, this treatment is seldom used in pregnant women since no studies support this practice. This was an in vitro study on animal tissue derived from pregnant Sprague-Dawley rats. A total of 124 ureteral segments were mounted in an organ bath system and contractile response to methacholine (MCh) was assessed. Tamsulosin or nifedipine were added at cumulative concentrations (0.001-1 μM). The area under the curve (AUC) from isometric tension measurements was calculated. The effect of pharmacological agents and the respective controls were assessed by calculating the AUC for each 5-min interval. Statistical analyses were performed using the Mann-Whitney-Wilcoxon nonparametric test. Both drugs displayed statistically significant inhibitory activity at concentrations of 0.1 and 1 μM for tamsulosin and 1 μM for nifedipine when calculated as the AUC as compared to DMSO controls. Tamsulosin and nifedipine directly inhibit MCh-induced contractility of pregnant rat ureters. Further work is needed to determine the clinical efficacy of these medications for MET in pregnancy.

  13. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    Directory of Open Access Journals (Sweden)

    Ana E. González Wusener

    2016-01-01

    Full Text Available Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO cells and PTP1B reconstituted (WT cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration.

  14. PTP1B triggers integrin-mediated repression of myosin activity and modulates cell contractility

    Science.gov (United States)

    González Wusener, Ana E.; González, Ángela; Nakamura, Fumihiko; Arregui, Carlos O.

    2016-01-01

    ABSTRACT Cell contractility and migration by integrins depends on precise regulation of protein tyrosine kinase and Rho-family GTPase activities in specific spatiotemporal patterns. Here we show that protein tyrosine phosphatase PTP1B cooperates with β3 integrin to activate the Src/FAK signalling pathway which represses RhoA-myosin-dependent contractility. Using PTP1B null (KO) cells and PTP1B reconstituted (WT) cells, we determined that some early steps following cell adhesion to fibronectin and vitronectin occurred robustly in WT cells, including aggregation of β3 integrins and adaptor proteins, and activation of Src/FAK-dependent signalling at small puncta in a lamellipodium. However, these events were significantly impaired in KO cells. We established that cytoskeletal strain and cell contractility was highly enhanced at the periphery of KO cells compared to WT cells. Inhibition of the Src/FAK signalling pathway or expression of constitutive active RhoA in WT cells induced a KO cell phenotype. Conversely, expression of constitutive active Src or myosin inhibition in KO cells restored the WT phenotype. We propose that this novel function of PTP1B stimulates permissive conditions for adhesion and lamellipodium assembly at the protruding edge during cell spreading and migration. PMID:26700725

  15. Changes of contractile responses due to simulated weightlessness in rat soleus muscle

    Science.gov (United States)

    Elkhammari, A.; Noireaud, J.; Léoty, C.

    1994-08-01

    Some contractile and electrophysiological properties of muscle fibers isolated from the slow-twitch soleus (SOL) and fast-twitch extensor digitorum longus (EDL) muscles of rats were compared with those measured in SOL muscles from suspended rats. In suspendede SOL (21 days of tail-suspension) membrane potential (Em), intracellular sodium activity (aiNa) and the slope of the relationship between Em and log [K]o were typical of fast-twitch muscles. The relation between the maximal amplitude of K-contractures vs Em was steeper for control SOL than for EDL and suspended SOL muscles. After suspension, in SOL muscles the contractile threshold and the inactivation curves for K-contractures were shifted to more positive Em. Repriming of K-contractures was unaffected by suspencion. The exposure of isolated fibers to perchlorate (ClO4-)-containing (6-40 mM) solutions resulted ina similar concentration-dependent shift to more negative Em of activation curves for EDL and suspended SOL muscles. On exposure to a Na-free TEA solution, SOL from control and suspended rats, in contrast to EDL muscles, generated slow contractile responses. Suspended SOL showed a reduced sensitivity to the contracture-producing effect of caffeine compared to control muscles. These results suggested that the modification observed due to suspension could be encounted by changes in the characteristics of muscle fibers from slow to fast-twitch type.

  16. A human in vitro model of Duchenne muscular dystrophy muscle formation and contractility.

    Science.gov (United States)

    Nesmith, Alexander P; Wagner, Matthew A; Pasqualini, Francesco S; O'Connor, Blakely B; Pincus, Mark J; August, Paul R; Parker, Kevin Kit

    2016-10-10

    Tongue weakness, like all weakness in Duchenne muscular dystrophy (DMD), occurs as a result of contraction-induced muscle damage and deficient muscular repair. Although membrane fragility is known to potentiate injury in DMD, whether muscle stem cells are implicated in deficient muscular repair remains unclear. We hypothesized that DMD myoblasts are less sensitive to cues in the extracellular matrix designed to potentiate structure-function relationships of healthy muscle. To test this hypothesis, we drew inspiration from the tongue and engineered contractile human muscle tissues on thin films. On this platform, DMD myoblasts formed fewer and smaller myotubes and exhibited impaired polarization of the cell nucleus and contractile cytoskeleton when compared with healthy cells. These structural aberrations were reflected in their functional behavior, as engineered tongues from DMD myoblasts failed to achieve the same contractile strength as healthy tongue structures. These data suggest that dystrophic muscle may fail to organize with respect to extracellular cues necessary to potentiate adaptive growth and remodeling. © 2016 Nesmith et al.

  17. Carboxyl-terminal-dependent recruitment of nonmuscle myosin II to megakaryocyte contractile ring during polyploidization.

    Science.gov (United States)

    Badirou, Idinath; Pan, Jiajia; Legrand, Céline; Wang, Aibing; Lordier, Larissa; Boukour, Siham; Roy, Anita; Vainchenker, William; Chang, Yunhua

    2014-10-16

    Endomitosis is a unique megakaryocyte (MK) differentiation process that is the consequence of a late cytokinesis failure associated with a contractile ring defect. Evidence from in vitro studies has revealed the distinct roles of 2 nonmuscle myosin IIs (NMIIs) on MK endomitosis: only NMII-B (MYH10), but not NMII-A (MYH9), is localized in the MK contractile ring and implicated in mitosis/endomitosis transition. Here, we studied 2 transgenic mouse models in which nonmuscle myosin heavy chain (NMHC) II-A was genetically replaced either by II-B or by a chimeric NMHCII that combined the head domain of II-A with the rod and tail domains of II-B. This study provides in vivo evidence on the specific role of NMII-B on MK polyploidization. It demonstrates that the carboxyl-terminal domain of the heavy chains determines myosin II localization to the MK contractile ring and is responsible for the specific role of NMII-B in MK polyploidization.

  18. A contractile and counterbalancing adhesion system controls the 3D shape of crawling cells.

    Science.gov (United States)

    Burnette, Dylan T; Shao, Lin; Ott, Carolyn; Pasapera, Ana M; Fischer, Robert S; Baird, Michelle A; Der Loughian, Christelle; Delanoe-Ayari, Helene; Paszek, Matthew J; Davidson, Michael W; Betzig, Eric; Lippincott-Schwartz, Jennifer

    2014-04-14

    How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.

  19. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

    Science.gov (United States)

    Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

    2015-11-01

    The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

  20. Cellular contractility and substrate elasticity: a numerical investigation of the actin cytoskeleton and cell adhesion.

    Science.gov (United States)

    Ronan, William; Deshpande, Vikram S; McMeeking, Robert M; McGarry, J Patrick

    2014-04-01

    Numerous experimental studies have established that cells can sense the stiffness of underlying substrates and have quantified the effect of substrate stiffness on stress fibre formation, focal adhesion area, cell traction, and cell shape. In order to capture such behaviour, the current study couples a mixed mode thermodynamic and mechanical framework that predicts focal adhesion formation and growth with a material model that predicts stress fibre formation, contractility, and dissociation in a fully 3D implementation. Simulations reveal that SF contractility plays a critical role in the substrate-dependent response of cells. Compliant substrates do not provide sufficient tension for stress fibre persistence, causing dissociation of stress fibres and lower focal adhesion formation. In contrast, cells on stiffer substrates are predicted to contain large amounts of dominant stress fibres. Different levels of cellular contractility representative of different cell phenotypes are found to alter the range of substrate stiffness that cause the most significant changes in stress fibre and focal adhesion formation. Furthermore, stress fibre and focal adhesion formation evolve as a cell spreads on a substrate and leading to the formation of bands of fibres leading from the cell periphery over the nucleus. Inhibiting the formation of FAs during cell spreading is found to limit stress fibre formation. The predictions of this mutually dependent material-interface framework are strongly supported by experimental observations of cells adhered to elastic substrates and offer insight into the inter-dependent biomechanical processes regulating stress fibre and focal adhesion formation.

  1. EXERCISE PERFORMANCE AND MUSCLE CONTRACTILE PROPERTIES AFTER CREATINE MONOHYDRATE SUPPLEMENTATION IN AEROBIC-ANAEROBIC TRAINING RATS

    Directory of Open Access Journals (Sweden)

    Nickolay Boyadjiev

    2007-12-01

    Full Text Available The purpose of this study was to investigate the effects of creatine monohydrate supplementation on exercise performance and contractile variables in aerobic-anaerobic training rats. Twenty 90-day-old male Sprague Dawley rats were divided into two groups - creatine (Cr and controls (K. The creatine group received creatine monohydrate as a nutritional supplement, whereas the control group was given placebo. Both groups were trained 5 days a week on a treadmill for 20 days in a mixed (aerobic-anaerobic metabolic working regimen (27 m·min-1, 15% elevation for 40 min. The exercise performance (sprint-test, contractile properties (m. tibialis anterior, oxidative enzyme activity (SDH, LDH, NADH2 in m. soleus and blood hematological and chemical variables were assessed in the groups at the end of the experiment. It was found out that creatine supplementation improved the exercise performance after 20 days of administration in a dose of 60 mg per day on the background of a mixed (aerobic-anaerobic exercise training. At the end of the trial the Cr-group demonstrated better values for the variables which characterize the contractile properties of m. tibialis anterior containing predominantly types IIA and IIB muscle fibers. On the other hand, a higher oxidative capacity was found out in m. soleus (type I muscle fibers as a result of 20-day creatine supplementation. No side effects of creatine monohydrate supplementation were assessed by the hematological and blood biochemical indices measured in this study

  2. Robust gap repair in the contractile ring ensures timely completion of cytokinesis.

    Science.gov (United States)

    Silva, Ana M; Osório, Daniel S; Pereira, Antonio J; Maiato, Helder; Pinto, Inês Mendes; Rubinstein, Boris; Gassmann, Reto; Telley, Ivo Andreas; Carvalho, Ana Xavier

    2016-12-19

    Cytokinesis in animal cells requires the constriction of an actomyosin contractile ring, whose architecture and mechanism remain poorly understood. We use laser microsurgery to explore the biophysical properties of constricting rings in Caenorhabditis elegans embryos. Laser cutting causes rings to snap open. However, instead of disintegrating, ring topology recovers and constriction proceeds. In response to severing, a finite gap forms and is repaired by recruitment of new material in an actin polymerization-dependent manner. An open ring is able to constrict, and rings repair from successive cuts. After gap repair, an increase in constriction velocity allows cytokinesis to complete at the same time as controls. Our analysis demonstrates that tension in the ring increases while net cortical tension at the site of ingression decreases throughout constriction and suggests that cytokinesis is accomplished by contractile modules that assemble and contract autonomously, enabling local repair of the actomyosin network. Consequently, cytokinesis is a highly robust process impervious to discontinuities in contractile ring structure. © 2016 Silva et al.

  3. Adaptive responses of mouse skeletal muscle to contractile activity: The effect of age.

    Science.gov (United States)

    Vasilaki, A; McArdle, F; Iwanejko, L M; McArdle, A

    2006-11-01

    This study has characterised the time course of two major transcriptional adaptive responses to exercise (changes in antioxidant defence enzyme activity and heat shock protein (HSP) content) in muscles of adult and old male mice following isometric contractions and has examined the mechanisms involved in the age-related reduction in transcription factor activation. Muscles of B6XSJL mice were subjected to isometric contractions and analysed for antioxidant defence enzyme activities, heat shock protein content and transcription factor DNA binding activity. Data demonstrated a significant increase in superoxide dismutase (SOD) and catalase activity and HSP content of muscles of adult mice following contractile activity which was associated with increased activation of the transcription factors, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and heat shock factor (HSF) following contractions. Significant increases in SOD and catalase activity and heat shock cognate (HSC70) content were seen in quiescent muscles of old mice. The increase in antioxidant defence enzyme activity following contractile activity seen in muscles of adult mice was not seen in muscles of old mice and this was associated with a failure to fully activate NF-kappaB and AP-1 following contractions. In contrast, although the production of HSPs was also reduced in muscles of old mice following contractile activity compared with muscles of adult mice following contractions, this was not due to a gross reduction in the DNA binding activity of HSF.

  4. Galectin-3 and fibulin-1 in systolic heart failure - relation to glucose metabolism and left ventricular contractile reserve

    OpenAIRE

    Holmager, Pernille; Egstrup, Michael; Gustafsson, Ida; Schou, Morten; Dahl, Jordi S.; Rasmussen, Lars Melholt; M?ller, Jacob E.; Tuxen, Christian; Faber, Jens; Kistorp, Caroline

    2017-01-01

    Background: Heart failure (HF) patients with diabetes (DM) have an adverse prognosis and reduced functional capacity, which could be associated with cardiac fibrosis, increased chamber stiffness and reduced left ventricular (LV) contractile reserve. Galectin-3 (Gal-3) and fibulin-1 are circulating biomarkers potentially reflecting cardiac fibrosis. We hypothesize that plasma levels of Gal-3 and fibulin-1 are elevated in HF patients with DM and are associated with reduced LV contractile reserv...

  5. A novel PNPLA2 mutation causes neutral lipid storage disease with myopathy (NLSDM) presenting muscular dystrophic features with lipid storage and rimmed vacuoles.

    Science.gov (United States)

    Chen, J; Hong, D; Wang, Z; Yuan, Y

    2010-01-01

    Neutral lipid storage disease with myopathy (NLSDM) is a type of lipid storage myopathy arising due to a mutation in the PNPLA2 gene encoding an adipose triglyceride lipase responsible for the degradation of intracellular triglycerides. Herein, we report the cases of two siblings manifesting slowly progressive proximal and distal limb weakness in adulthood. One of the patients had dilated cardiomyopathy, hearing loss and short stature. Muscle specimens of the 2 patients revealed muscular dystrophic features with massive lipid droplets and numerous rimmed vacuoles in the fibers. A novel homozygous mutation IVS2+1G > A in the PNPLA2 gene was identified in the 2 cases, but not in the healthy familial individuals. The presence of massive lipid droplets with muscular dystrophic changes and rimmed vacuoles in muscle fibers might be one of the characteristic pathological changes of NLSDM.

  6. Dietary supplementation of antioxidants improves semen quality of IVF patients in terms of motility, sperm count, and nuclear vacuolization.

    Science.gov (United States)

    Wirleitner, Barbara; Vanderzwalmen, Pierre; Stecher, Astrid; Spitzer, Dietmar; Schuff, Maximilian; Schwerda, Delf; Bach, Magnus; Schechinger, Birgit; Herbert Zech, Nicolas

    2012-12-01

    This study aimed to investigate the influence of an oral antioxidative supplementation on sperm quality of in vitro fertilization (IVF) patients, as analyzed by sperm motility according to the WHO criteria and motile sperm organelle morphology examination (MSOME). Semen samples were collected from 147 patients before undergoing an IVF/intracytoplasmic morphologically-selected sperm injection (IMSI) cycle and 2 - 12 months after an antioxidative supplementation. Semen analysis was evaluated according to WHO and MSOME criteria. Spermatozoa were grouped according to the size of nuclear vacuoles within the sperm's heads. Patients were divided into oligoasthenoteratozoospermic (OAT) and non-OAT men. Between first and second semen analysis, patients were supplemented orally with an antioxidative preparation. After the antioxidative therapy we observed a significant reduction in the percentage of immotile sperm cells in the patients. Additionally, the percentage of class I spermatozoa according to MSOME criteria was significantly higher after antioxidative supplementation. In OAT patients the percentage of class I sperm was found to be increased, although not significantly. However, we observed a drastic improvement in sperm motility as well as in total sperm count in this group. The results demonstrated a considerable improvement in semen quality, notably in OAT patients. Considering the putative relationship between semen quality on the one hand and reactive oxygen species on the other, the observed changes in the sperm parameters indicate that a decline in semen quality, and even subtle morphological changes, might be associated with oxidative stress. Our findings suggest that an antioxidative and micronutrient supplementation has a remarkable benefit for IVF patients having restricted sperm parameters, in particular.

  7. Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease.

    Science.gov (United States)

    Ghiara, P; Marchetti, M; Blaser, M J; Tummuru, M K; Cover, T L; Segal, E D; Tompkins, L S; Rappuoli, R

    1995-10-01

    The pathogenic role of Helicobacter pylori virulence factors has been studied with a mouse model of gastric disease. BALB/c mice were treated orally with different amounts of sonic extracts of cytotoxic H. pylori strains (NCTC 11637, 60190, 84-183, and 87A300 [CagA+/Tox+]). The pathological effects on histological sections of gastric mucosae were assessed and were compared with the effects of treatments with extracts from noncytotoxic strains (G21 and G50 [CagA-/Tox-]) and from strains that express either CagA alone (D931 [CagA+/Tox-]) or the cytotoxin alone (G104 [CagA-/Tox+]). The treatment with extracts from cytotoxic strains induced various epithelial lesions (vacuolation, erosions, and ulcerations), recruitment of inflammatory cells in the lamina propria, and a marked reduction of the mucin layer. Extracts of noncytotoxic strains induced mucin depletion but no other significant pathology. Crude extracts of strain D931, expressing CagA alone, caused only mild infiltration of inflammatory cells, whereas extracts of strain G104, expressing cytotoxin alone, induced extensive epithelial damage but little inflammatory reaction. Loss of the mucin layer was not associated with a cytotoxic phenotype, since this loss was observed in mice treated with crude extracts of all strains. The pathogenic roles of CagA, cytotoxin, and urease were further assessed by using extracts of mutant strains of H. pylori defective in the expression of each of these virulence factors. The results obtained suggest that (i) urease activity does not play a significant role in inducing the observed gastric damage, (ii) cytotoxin has an important role in the induction of gastric epithelial cell lesions but not in eliciting inflammation, and (iii) other components present in strains which carry the cagA gene, but distinct from CagA itself, are involved in eliciting the inflammatory response.

  8. A global, myosin light chain kinase-dependent increase in myosin II contractility accompanies the metaphase-anaphase transition in sea urchin eggs.

    Science.gov (United States)

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R; Shuster, Charles B

    2006-09-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase-anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase-anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus.

  9. A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

    Science.gov (United States)

    Lucero, Amy; Stack, Christianna; Bresnick, Anne R.

    2006-01-01

    Myosin II is the force-generating motor for cytokinesis, and although it is accepted that myosin contractility is greatest at the cell equator, the temporal and spatial cues that direct equatorial contractility are not known. Dividing sea urchin eggs were placed under compression to study myosin II-based contractile dynamics, and cells manipulated in this manner underwent an abrupt, global increase in cortical contractility concomitant with the metaphase–anaphase transition, followed by a brief relaxation and the onset of furrowing. Prefurrow cortical contractility both preceded and was independent of astral microtubule elongation, suggesting that the initial activation of myosin II preceded cleavage plane specification. The initial rise in contractility required myosin light chain kinase but not Rho-kinase, but both signaling pathways were required for successful cytokinesis. Last, mobilization of intracellular calcium during metaphase induced a contractile response, suggesting that calcium transients may be partially responsible for the timing of this initial contractile event. Together, these findings suggest that myosin II-based contractility is initiated at the metaphase–anaphase transition by Ca2+-dependent myosin light chain kinase (MLCK) activity and is maintained through cytokinesis by both MLCK- and Rho-dependent signaling. Moreover, the signals that initiate myosin II contractility respond to specific cell cycle transitions independently of the microtubule-dependent cleavage stimulus. PMID:16837551

  10. Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome.

    Science.gov (United States)

    Collins, H L; Schaible, U E; Ernst, J D; Russell, D G

    1997-01-01

    The eukaryotic intracellular pathogen Leishmania mexicana resides inside macrophages contained within a membrane bound parasitophorous vacuole which, as it matures, acquires the characteristics of a late endosomal compartment. This study reports the selectivity of fusion of this compartment with other particle containing vacuoles. Phagosomes containing zymosan or live Listeria monocytogenes rapidly fused with L. mexicana parasitophorous vacuoles, while those containing latex beads or heat killed L. monocytogenes failed to do so. Fusigenicity of phagosomes was not primarily dependent on the receptor utilized for ingestion, as opsonization with defined ligands could not overcome the exclusion of either latex beads or heat killed organisms. However modulation of intracellular pH by pharmacological agents such as chloroquine and ammonium chloride increased delivery of live Listeria and also induced transfer of previously excluded particles. The absence of fusion correlated with the acquisition of annexin I, a putative lysosomal targeting, molecule, on the phagosome membrane. We propose that the acquisition of cellular membrane constituents such as annexin I during phagosome maturation can ultimately direct the fusion pathway of the vesicles formed and have described a model system to further document changes in vesicle fusigenicity within cells.

  11. A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3',4'-dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

    Science.gov (United States)

    Lee, Yan Quan; Goh, Amanda S P; Ch'ng, Jun Hong; Nosten, François H; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar; Tan, Kevin S W

    2014-01-01

    Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

  12. Myocardial contractility in the echo lab: molecular, cellular and pathophysiological basis

    Directory of Open Access Journals (Sweden)

    Bombardini Tonino

    2005-09-01

    Full Text Available Abstract In the standard accepted concept, contractility is the intrinsic ability of heart muscle to generate force and to shorten, independently of changes in the preload or afterload with fixed heart rates. At molecular level the crux of the contractile process lies in the changing concentrations of Ca2+ ions in the myocardial cytosol. Ca2+ ions enter through the calcium channel that opens in response to the wave of depolarization that travels along the sarcolemma. These Ca2+ ions "trigger" the release of more calcium from the sarcoplasmic reticulum (SR and thereby initiate a contraction-relaxation cycle. In the past, several attempts were made to transfer the pure physiological concept of contractility, expressed in the isolated myocardial fiber by the maximal velocity of contraction of unloaded muscle fiber (Vmax, to the in vivo beating heart. Suga and Sagawa achieved this aim by measuring pressure/volume loops in the intact heart: during a positive inotropic intervention, the pressure volume loop reflects a smaller end-systolic volume and a higher end-systolic pressure, so that the slope of the pressure volume relationship moves upward and to the left. The pressure volume relationship is the most reliable index for assessing myocardial contractility in the intact circulation and is almost insensitive to changes in preload and after load. This is widely used in animal studies and occasionally clinically. The limit of the pressure volume relationship is that it fails to take into account the frequency-dependent regulation of contractility: the frequency-dependent control of transmembrane Ca2+ entry via voltage-gated Ca2+ channels provides cardiac cells with a highly sophisticated short-term system for the regulation of intracellular Ca2+ homeostasis. An increased stimulation rate increases the force of contraction: the explanation is repetitive Ca2+ entry with each depolarization and, hence, an accumulation of cytosolic calcium. As the heart

  13. Immobilization of Deoxyadenosine Kinase from Dictyostelium discoideum (DddAK) and Its Application in the 5’-Phosphorylation of Arabinosyladenine and Arabinosyl-2-fluoroadenine

    DEFF Research Database (Denmark)

    Serra, Immacolata; Ubiali, Daniela; Piskur, Jure

    2017-01-01

    Deoxyadenosine kinase from Dictyostelium discoideum (DddAK) phosphorylates its natural substrate (2’‐deoxyadenosine, dAdo) as well as the arabinosyladenine analogues vidarabine (araA) and fludarabine (F‐araA) to their corresponding 5’‐monophosphates. DddAK has been here immobilized by ionic...... interaction on an aminated epoxy‐functionalized support (SepabeadsTM EC‐EP), and cross‐linked with oxidized dextran. The final activity recovery was 33–42 %, depending on the protein loading. Immobilization enhanced the stability of DddAK at pH 10 and, to a lesser extent, at 45 °C. Phosphorylation of d...

  14. Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells of the hippocampal neurogenesis in rat offspring via dysfunction of cholinergic inputs by myelin vacuolation

    International Nuclear Information System (INIS)

    Itahashi, Megu; Abe, Hajime; Tanaka, Takeshi; Mizukami, Sayaka; Kimura, Masayuki; Yoshida, Toshinori; Shibutani, Makoto

    2015-01-01

    Highlights: • The effect of maternal exposure to HCP on rat hippocampal neurogenesis was examined. • HCP induces myelin vacuolation of nerve tracts in the septal–hippocampal pathway. • Myelin changes suppress Chrnb2-mediated cholinergic inputs to the dentate gyrus. • SGZ apoptosis occurs via the mitochondrial pathway and targets type-2b cells. • Dysfunction of cholinergic inputs is related to type-2b SGZ cell apoptosis. - Abstract: Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2 + progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling + apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction

  15. The Functional Lumen Imaging Probe Detects Esophageal Contractility Not Observed With Manometry in Patients With Achalasia.

    Science.gov (United States)

    Carlson, Dustin A; Lin, Zhiyue; Kahrilas, Peter J; Sternbach, Joel; Donnan, Erica N; Friesen, Laurel; Listernick, Zoe; Mogni, Benjamin; Pandolfino, John E

    2015-12-01

    The functional lumen imaging probe (FLIP) could improve the characterization of achalasia subtypes by detecting nonocclusive esophageal contractions not observed with standard manometry. We aimed to evaluate esophageal contractions during volumetric distention in patients with achalasia using FLIP topography. Fifty-one treatment-naive patients with achalasia, defined and subclassified by high-resolution esophageal pressure topography, and 10 asymptomatic individuals (controls) were evaluated with the FLIP during endoscopy. During stepwise distension, simultaneous intrabag pressures and 16 channels of cross-sectional areas were measured; data were exported to software that generated FLIP topography plots. Esophageal contractility was identified by noting periods of reduced luminal diameter. Esophageal contractions were characterized further by propagation direction, repetitiveness, and based on whether they were occluding or nonoccluding. Esophageal contractility was detected in all 10 controls: 8 of 10 had repetitive antegrade contractions and 9 of 10 had occluding contractions. Contractility was detected in 27% (4 of 15) of patients with type I achalasia and in 65% (18 of 26, including 9 with occluding contractions) of patients with type II achalasia. Contractility was detected in all 10 patients with type III achalasia; 8 of these patients had a pattern of contractility that was not observed in controls (repetitive retrograde contractions). Esophageal contractility not observed with manometry can be detected in patients with achalasia using FLIP topography. The presence and patterns of contractility detected with FLIP topography may represent variations in pathophysiology, such as mechanisms of panesophageal pressurization in patients with type II achalasia. These findings could have implications for additional subclassification to supplement prediction of the achalasia disease course. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights

  16. Isolation and maintenance-free culture of contractile myotubes from Manduca sexta embryos.

    Directory of Open Access Journals (Sweden)

    Amanda L Baryshyan

    Full Text Available Skeletal muscle tissue engineering has the potential to treat tissue loss and degenerative diseases. However, these systems are also applicable for a variety of devices where actuation is needed, such as microelectromechanical systems (MEMS and robotics. Most current efforts to generate muscle bioactuators are focused on using mammalian cells, which require exacting conditions for survival and function. In contrast, invertebrate cells are more environmentally robust, metabolically adaptable and relatively autonomous. Our hypothesis is that the use of invertebrate muscle cells will obviate many of the limitations encountered when mammalian cells are used for bioactuation. We focus on the tobacco hornworm, Manduca sexta, due to its easy availability, large size and well-characterized muscle contractile properties. Using isolated embryonic cells, we have developed culture conditions to grow and characterize contractile M. sexta muscles. The insect hormone 20-hydroxyecdysone was used to induce differentiation in the system, resulting in cells that stained positive for myosin, contract spontaneously for the duration of the culture, and do not require media changes over periods of more than a month. These cells proliferate under normal conditions, but the application of juvenile hormone induced further proliferation and inhibited differentiation. Cellular metabolism under normal and low glucose conditions was compared for C2C12 mouse and M. sexta myoblast cells. While differentiated C2C12 cells consumed glucose and produced lactate over one week as expected, M. sexta muscle did not consume significant glucose, and lactate production exceeded mammalian muscle production on a per cell basis. Contractile properties were evaluated using index of movement analysis, which demonstrated the potential of these cells to perform mechanical work. The ability of cultured M. sexta muscle to continuously function at ambient conditions without medium replenishment

  17. Changes in muscle fiber contractility and extracellular matrix production during skeletal muscle hypertrophy.

    Science.gov (United States)

    Mendias, Christopher L; Schwartz, Andrew J; Grekin, Jeremy A; Gumucio, Jonathan P; Sugg, Kristoffer B

    2017-03-01

    Skeletal muscle can adapt to increased mechanical loads by undergoing hypertrophy. Transient reductions in whole muscle force production have been reported during the onset of hypertrophy, but contractile changes in individual muscle fibers have not been previously studied. Additionally, the extracellular matrix (ECM) stores and transmits forces from muscle fibers to tendons and bones, and determining how the ECM changes during hypertrophy is important in understanding the adaptation of muscle tissue to mechanical loading. Using the synergist ablation model, we sought to measure changes in muscle fiber contractility, collagen content, and cross-linking, and in the expression of several genes and activation of signaling proteins that regulate critical components of myogenesis and ECM synthesis and remodeling during muscle hypertrophy. Tissues were harvested 3, 7, and 28 days after induction of hypertrophy, and nonoverloaded rats served as controls. Muscle fiber specific force (sF o ), which is the maximum isometric force normalized to cross-sectional area, was reduced 3 and 7 days after the onset of mechanical overload, but returned to control levels by 28 days. Collagen abundance displayed a similar pattern of change. Nearly a quarter of the transcriptome changed over the course of overload, as well as the activation of signaling pathways related to hypertrophy and atrophy. Overall, this study provides insight into fundamental mechanisms of muscle and ECM growth, and indicates that although muscle fibers appear to have completed remodeling and regeneration 1 mo after synergist ablation, the ECM continues to be actively remodeling at this time point. NEW & NOTEWORTHY This study utilized a rat synergist ablation model to integrate changes in single muscle fiber contractility, extracellular matrix composition, activation of important signaling pathways in muscle adaption, and corresponding changes in the muscle transcriptome to provide novel insight into the basic

  18. The effect of exercise hypertrophy and disuse atrophy on muscle contractile properties: a mechanomyographic analysis.

    Science.gov (United States)

    Than, Christian; Tosovic, Danijel; Seidl, Laura; Mark Brown, J

    2016-12-01

    To determine whether mechanomyographic (MMG) determined contractile properties of the biceps brachii change during exercise-induced hypertrophy and subsequent disuse atrophy. Healthy subjects (mean ± SD, 23.7 ± 2.6 years, BMI 21.8 ± 2.4, n = 19) performed unilateral biceps curls (9 sets × 12 repetitions, 5 sessions per week) for 8 weeks (hypertrophic phase) before ceasing exercise (atrophic phase) for the following 8 weeks (non-dominant limb; treatment, dominant limb; control). MMG measures of muscle contractile properties (contraction time; T c , maximum displacement; D max , contraction velocity; V c ), electromyographic (EMG) measures of muscle fatigue (median power frequency; MPF), strength measures (maximum voluntary contraction; MVC) and measures of muscle thickness (ultrasound) were obtained. Two-way repeated measures ANOVA showed significant differences (P muscle thickness was greater than control, reflecting gross hypertrophy. MMG variables Dmax (weeks 2, 7) and Vc (weeks 7, 8) declined. During the atrophic phase, MVC (weeks 9-12) and muscle thickness (weeks 9, 10) initially remained high before declining to control levels, reflecting gross atrophy. MMG variables D max (weeks 9, 14) and V c (weeks 9, 14, 15) also declined during the atrophic phase. No change in T c was found throughout the hypertrophic or atrophic phases. MMG detects changes in contractile properties during stages of exercise-induced hypertrophy and disuse atrophy suggesting its applicability as a clinical tool in musculoskeletal rehabilitation.

  19. Myocardial mitochondrial and contractile function are preserved in mice lacking adiponectin.

    Directory of Open Access Journals (Sweden)

    Martin Braun

    Full Text Available Adiponectin deficiency leads to increased myocardial infarct size following ischemia reperfusion and to exaggerated cardiac hypertrophy following pressure overload, entities that are causally linked to mitochondrial dysfunction. In skeletal muscle, lack of adiponectin results in impaired mitochondrial function. Thus, it was our objective to investigate whether adiponectin deficiency impairs mitochondrial energetics in the heart. At 8 weeks of age, heart weight-to-body weight ratios were not different between adiponectin knockout (ADQ-/- mice and wildtypes (WT. In isolated working hearts, cardiac output, aortic developed pressure and cardiac power were preserved in ADQ-/- mice. Rates of fatty acid oxidation, glucose oxidation and glycolysis were unchanged between groups. While myocardial oxygen consumption was slightly reduced (-24% in ADQ-/- mice in isolated working hearts, rates of maximal ADP-stimulated mitochondrial oxygen consumption and ATP synthesis in saponin-permeabilized cardiac fibers were preserved in ADQ-/- mice with glutamate, pyruvate or palmitoyl-carnitine as a substrate. In addition, enzymatic activity of respiratory complexes I and II was unchanged between groups. Phosphorylation of AMP-activated protein kinase and SIRT1 activity were not decreased, expression and acetylation of PGC-1α were unchanged, and mitochondrial content of OXPHOS subunits was not decreased in ADQ-/- mice. Finally, increasing energy demands due to prolonged subcutaneous infusion of isoproterenol did not differentially affect cardiac contractility or mitochondrial function in ADQ-/- mice compared to WT. Thus, mitochondrial and contractile function are preserved in hearts of mice lacking adiponectin, suggesting that adiponectin may be expendable in the regulation of mitochondrial energetics and contractile function in the heart under non-pathological conditions.

  20. Separate effects of ischemia, hypoxia, and contractility on thallium-201 kinetics in rabbit myocardium

    International Nuclear Information System (INIS)

    Leppo, J.A.; Macneil, P.B.; Moring, A.F.; Apstein, C.S.

    1986-01-01

    The effects of hypoxia and ischemia, as well as altered contractility, on thallium-201 ( 201 TI) kinetics were evaluated in 42 isolated isovolumetrically contracting rabbit hearts. In Group A, three subgroups (n = 7 each) were studied that had either normal flow and oxygenation, hypoxia and normal flow, or ischemic flow and normal perfusate oxygen content. In Group B, three subgroups (n = 7 each) were studied and all hearts had normal flow but the contractile state was either enhanced with isoproterenol or impaired by hypocalcemia. A hemoglobin-free buffer perfusate was used in all experiments and multiple timed collections of arterial and coronary sinus effluent were used to model myocardial isotope activity during 30 min of constant uptake followed by 30 min of tracer clearance. During ischemia, hypoxia and hypocalcemia peak developed pressure and peak positive and negative dP/dt were all significantly reduced when compared to normal hemodynamic parameters (p less than 0.01). As expected, isoproterenol significantly elevated these parameters (p less than 0.04). Myocardial 201 TI kinetics were adequately described utilizing a bi-exponential model having a fast and slow component. Only ischemic hearts had significantly lower rate constants for 201 TI uptake and clearance than normal hearts (p less than 0.001). The mean (+/- s.d.) myocardial uptake and clearance rates for 201 TI (%/min) varied between 4.86 +/- 0.87 and 7.18 +/- 1.45 for the remaining groups of hearts. Therefore, myocardial 201 TI kinetics appear to be dominated by coronary flow and may not reflect marked alterations in the metabolic and contractile state. These data suggest that normal 201 TI uptake in impaired or hypercontractile cells, receiving normal flow, may not represent normal cellular function

  1. Opportunities to Target Specific Contractile Abnormalities with Smooth Muscle Protein Kinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Annegret Ulke-Lemée

    2010-05-01

    Full Text Available Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine; therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK and integrin-linked kinase (ILK are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.

  2. The role of apical contractility in determining cell morphology in multilayered epithelial sheets and tubes

    Science.gov (United States)

    Zhen Tan, Rui; Lai, Tanny; Chiam, K.-H.

    2017-08-01

    A multilayered epithelium is made up of individual cells that are stratified in an orderly fashion, layer by layer. In such tissues, individual cells can adopt a wide range of shapes ranging from columnar to squamous. From histological images, we observe that, in flat epithelia such as the skin, the cells in the top layer are squamous while those in the middle and bottom layers are columnar, whereas in tubular epithelia, the cells in all layers are columnar. We develop a computational model to understand how individual cell shape is governed by the mechanical forces within multilayered flat and curved epithelia. We derive the energy function for an epithelial sheet of cells considering intercellular adhesive and intracellular contractile forces. We determine computationally the cell morphologies that minimize the energy function for a wide range of cellular parameters. Depending on the dominant adhesive and contractile forces, we find four dominant cell morphologies for the multilayered-layered flat sheet and three dominant cell morphologies for the two-layered curved sheet. We study the transitions between the dominant cell morphologies for the two-layered flat sheet and find both continuous and discontinuous transitions and also the presence of multistable states. Matching our computational results with histological images, we conclude that apical contractile forces from the actomyosin belt in the epithelial cells is the dominant force determining cell shape in multilayered epithelia. Our computational model can guide tissue engineers in designing artificial multilayered epithelia, in terms of figuring out the cellular parameters needed to achieve realistic epithelial morphologies.

  3. Contractile profile of esophageal and gastric fundus strips in experimental doxorubicin-induced esophageal atresia

    Directory of Open Access Journals (Sweden)

    F.A. Capeto

    2015-05-01

    Full Text Available Esophageal atresia (EA is characterized by esophageal and gastric motility changes secondary to developmental and postsurgical damage. This study evaluated the in vitro contractile profile of the distal esophagus and gastric fundus in an experimental model of EA induced by doxorubicin (DOXO. Wistar pregnant rats received DOXO 2.2 mg/kg on the 8th and 9th gestational days. On day 21.5, fetuses were collected, sacrificed, and divided into groups: control, DOXO without EA (DOXO-EA, and DOXO with EA (DOXO+EA. Strips from the distal esophagus and gastric fundus were mounted on a wire myograph and isolated organ-bath system, respectively, and subjected to increasing concentrations of carbamylcholine chloride (carbachol, CCh. The isolated esophagus was also stimulated with increasing concentrations of KCl. In esophagus, the concentration-effect curves were reduced in response to CCh in the DOXO+EA and DOXO-EA groups compared to the control group (P0.05. In response to KCl, the distal esophagus samples in the three groups were not statistically different with regard to Emax or EC50 values (P>0.05. No significant difference was noted for EC50 or Emax values in fundic strips stimulated with CCh (P>0.05. In conclusion, exposure of dams to DOXO during gestation inhibited the contractile behavior of esophageal strips from offspring in response to CCh but not KCl, regardless of EA induction. The gastric fundus of DOXO-exposed offspring did not have altered contractile responsiveness to cholinergic stimulation.

  4. Contractility-afterload mismatch in patients with protein-losing enteropathy after the Fontan operation.

    Science.gov (United States)

    Ozawa, Hideto; Ueno, Takayoshi; Iwai, Shigemitsu; Kawata, Hiroaki; Nishigaki, Kyouichi; Kishimoto, Hidefumi; Sawa, Yoshiki

    2014-10-01

    This study aimed to clarify the relationship between onset of protein-losing enteropathy (PLE) and Fontan circulation, with special reference to the development of contractility-afterload mismatch. The PLE group comprised 9 patients who experienced PLE after undergoing the Fontan operation, and the control group consisted of 32 patients had did not experienced PLE more than 10 years after the Fontan operation. The study compared the pre- and postoperative values of arterial elastance (Ea), end-systolic elastance (Ees), and contractility-afterload mismatch (Ea/Ees). Furthermore, the variations in the values were examined during the preoperative, postoperative, and midterm postoperative periods in seven PLE patients who underwent cardiac catheterization at the onset of PLE and during the pre- and postintervention periods in three PLE patients who underwent surgical intervention to improve the Fontan circulation after the onset of PLE. Comparison of the values obtained before and after Fontan operations showed that the Ea values increased significantly in the PLE group. However, the pre- and postoperative Ees values did not differ in the two groups. During the postoperative period, Ea/Ees increased significantly, and the Ea and Ea/Ees values increased continuously until the onset of PLE in the PLE group. In the patients who underwent surgical intervention to improve the Fontan circulation after the onset of PLE, the Ea/Ees decreased significantly, and the serum albumin levels improved after the intervention. Contractility-afterload mismatch, mainly caused by the increase in the afterload of the systemic ventricle, may have an important role in the development of PLE after the Fontan operation.

  5. Altered Ca fluxes and contractile state during pH changes in cultured heart cells

    International Nuclear Information System (INIS)

    Kim, D.; Smith, T.W.

    1987-01-01

    The authors studied mechanisms underlying changes in myocardial contractile state produced by intracellular (pH/sub i/) or extracellular (pH 0 ) changes in pH using cultured chick embryo ventricular cells. A change in pH 0 of HEPES-buffered medium from 7.4 to 6.0 or to 8.8 changed the amplitude of cell motion by -85 or +60%, and 45 Ca uptake at 10 s by -29 or +22%, respectively. The pH 0 induced change in Ca uptake was not sensitive to nifedipine but was Na gradient dependent. Changes in pH/sub i/ produced by NH 4 Cl or preincubation in media at pH values ranging from 6.0 to 8.8 failed to alter significantly 45 Ca uptake or efflux. However, larger changes in pH/sub i/ were associated with altered Ca uptake. Changes in pH 0 from 7.5 to 6.0 or to 8.8 were associated with initial changes in 45 Ca efflux by +17 or -18%, respectively, and these effects were not Na dependent. Exposure of cells to 20 mM NH 4 Cl produced intracellular alkalinization and a positive inotropic effect, whereas subsequent removal of NH 4 Cl caused intracellular acidification and a negative inotropic effect. There was, however, a lack of close temporal relationships between pH/sub i/ and contractile state. These results indicated that pH 0 -induced changes in contractile state in cultured heart cells are closely correlated with altered transarcolemmal Ca movements and presumably are due to these Ca flux changes

  6. Acute exposure to lead increases myocardial contractility independent of hypertension development

    International Nuclear Information System (INIS)

    Fioresi, M.; Furieri, L.B.; Simões, M.R.; Ribeiro, R.F. Junior; Meira, E.F.; Fernandes, A.A.; Stefanon, I.; Vassallo, D.V.

    2013-01-01

    We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetate iv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na + ,K + -ATPase and myosin Ca 2+ -ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2 vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13 vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na + ,K + -ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension

  7. Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts.

    Science.gov (United States)

    Guo, Rui; Hu, Nan; Kandadi, Machender R; Ren, Jun

    2012-04-01

    Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

  8. Pulmonary artery radiocardiography and rheography in the diagnosis of hemodynamic and contractile function impairments of the right ventricle in patients with obstructive bronchitis

    International Nuclear Information System (INIS)

    Paleev, N.P.; Cherejskaya, N.K.; Tsar'kova, L.N.; Baklykova, S.N.; Novoderezhkina, L.B.; Oblovatskaya, O.G.; Dubinina, E.B.

    1990-01-01

    Radiocardiography and rheography of the pulmonary artery were used to examine impairments in hemodynamics and contractile function of the right ventricle in 40 patients with chronic obstructive bronchitis complicated with persistent hypertension. Right ventricular hemodynamic and contractile impairments were shown to be not equivalent with similar clinical and functional signs of pulmonary hypertension. This fact indicates that the use of special techiques is of practical value in the determination of right ventricular hemodynamics and myocardial contractility in patients with chronic obstructive bronchitis. Radiocardiography and rheography of the pulmonary artery are sufficiently reliable noninvasive techniques for examining the hemodynamics and contractile function of the right ventricular myocardium

  9. Bladder contractility is modulated by Kv7 channels in pig detrusor

    DEFF Research Database (Denmark)

    Svalø, Julie; Bille, Michala; Parameswaran Theepakaran, Neeraja

    2013-01-01

    Kv7 channels are involved in smooth muscle relaxation, and accordingly we believe that they constitute potential targets for the treatment of overactive bladder syndrome. We have therefore used myography to examine the function of Kv7 channels in detrusor, i.e. pig bladder, with a view...... relaxation, suggesting that Kv7.2 and/or Kv7.4 channels constitute the subtypes that are relevant to bladder contractility. The effects of retigabine and ML213 were attenuated by pre-incubation with 10µM XE991 (Kv7.1-7.5 channel blocker) (P...

  10. A thermodynamical model for stress-fiber organization in contractile cells.

    Science.gov (United States)

    Foucard, Louis; Vernerey, Franck J

    2012-01-02

    Cell mechanical adaptivity to external stimuli is vital to many of its biological functions. A critical question is therefore to understand the formation and organization of the stress fibers from which emerge the cell's mechanical properties. By accounting for the mechanical aspects and the viscoelastic behavior of stress fibers, we here propose a thermodynamic model to predict the formation and orientation of stress fibers in contractile cells subjected to constant or cyclic stretch and different substrate stiffness. Our results demonstrate that the stress fibers viscoelastic behavior plays a crucial role in their formation and organization and shows good consistency with various experiments.

  11. Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.

    Directory of Open Access Journals (Sweden)

    Hayley J Newton

    Full Text Available The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into

  12. Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

    Science.gov (United States)

    Connor, Michael G.; Pulsifer, Amanda R.; Ceresa, Brian K.

    2018-01-01

    ABSTRACT Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV. PMID:29463656

  13. Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones

    Science.gov (United States)

    Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

    2015-01-01

    The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production. PMID:26618153

  14. A stepwise procedure to test contractility and susceptibility to injury for the rodent quadriceps muscle

    Directory of Open Access Journals (Sweden)

    Stephen J.P. Pratt

    2014-07-01

    Full Text Available In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies.  Biological markers can also be used in measuring muscle injury, such as increased creatine kinase levels in the blood, but these are not always correlated with loss in muscle function (i.e. loss of force production.  This is even true of histological findings from animals, which provide a “direct measure” of damage, but do not account for loss of function.  The most comprehensive measure of the overall health of the muscle is contractile force.  To date, animal models testing contractile force have been limited to the muscle groups moving the ankle.  Here we describe an in vivo animal model for the quadriceps, with abilities to measure torque, produce a reliable muscle injury, and follow muscle recovery within the same animal over time.  We also describe a second model used for direct measurement of force from an isolated quadriceps muscle in situ. 

  15. Influence of intracellular acidosis on contractile function in the working rat heart

    International Nuclear Information System (INIS)

    Jeffrey, F.M.H.; Malloy, C.R.; Radda, G.K.

    1987-01-01

    The decrease in myocardial contractility during ischemia, hypoxia, and extracellular acidosis has been attributed to intracellular acidosis. Previous studies of the relationship between pH and contractile state have utilized respiratory or metabolic acidosis to alter intracellular pH. The authors developed a model in the working perfused rat heart to study the effects of intracellular acidosis with normal external pH and optimal O 2 delivery. Intracellular pH and high-energy phosphates were monitored by 31 P nuclear magnetic resonance spectroscopy. Hearts were perfused to a steady state with a medium containing 10 mM NH 4 Cl. Acidosis induced a substantial decrease in aortic flow and stroke volume which was associated with little change in peak systolic pressure. It was concluded that (1) for the same intracellular acidosis the influence on tension development was more pronounced with a combined extra- and intracellular acidosis than with an isolated intracellular acidosis, and (2) stroke volume at constant preload was impaired by intracellular acidosis even though changes in developed pressure were minimal. These observations suggest that isolated intracellular acidosis has adverse effects on diastolic compliance and/or relaxation

  16. Oscillatory behaviors and hierarchical assembly of contractile structures in intercalating cells

    International Nuclear Information System (INIS)

    Fernandez-Gonzalez, Rodrigo; Zallen, Jennifer A

    2011-01-01

    Fluctuations in the size of the apical cell surface have been associated with apical constriction and tissue invagination. However, it is currently not known if apical oscillatory behaviors are a unique property of constricting cells or if they constitute a universal feature of the force balance between cells in multicellular tissues. Here, we set out to determine whether oscillatory cell behaviors occur in parallel with cell intercalation during the morphogenetic process of axis elongation in the Drosophila embryo. We applied multi-color, time-lapse imaging of living embryos and SIESTA, an integrated tool for automated and semi-automated cell segmentation, tracking, and analysis of image sequences. Using SIESTA, we identified cycles of contraction and expansion of the apical surface in intercalating cells and characterized them at the molecular, cellular, and tissue scales. We demonstrate that apical oscillations are anisotropic, and this anisotropy depends on the presence of intact cell–cell junctions and spatial cues provided by the anterior–posterior patterning system. Oscillatory cell behaviors during axis elongation are associated with the hierarchical assembly and disassembly of contractile actomyosin structures at the medial cortex of the cell, with actin localization preceding myosin II and with the localization of both proteins preceding changes in cell shape. We discuss models to explain how the architecture of cytoskeletal networks regulates their contractile behavior and the mechanisms that give rise to oscillatory cell behaviors in intercalating cells

  17. Dependence of intramyocardial pressure and coronary flow on ventricular loading and contractility: a model study.

    Science.gov (United States)

    Bovendeerd, Peter H M; Borsje, Petra; Arts, Theo; van De Vosse, Frans N

    2006-12-01

    The phasic coronary arterial inflow during the normal cardiac cycle has been explained with simple (waterfall, intramyocardial pump) models, emphasizing the role of ventricular pressure. To explain changes in isovolumic and low afterload beats, these models were extended with the effect of three-dimensional wall stress, nonlinear characteristics of the coronary bed, and extravascular fluid exchange. With the associated increase in the number of model parameters, a detailed parameter sensitivity analysis has become difficult. Therefore we investigated the primary relations between ventricular pressure and volume, wall stress, intramyocardial pressure and coronary blood flow, with a mathematical model with a limited number of parameters. The model replicates several experimental observations: the phasic character of coronary inflow is virtually independent of maximum ventricular pressure, the amplitude of the coronary flow signal varies about proportionally with cardiac contractility, and intramyocardial pressure in the ventricular wall may exceed ventricular pressure. A parameter sensitivity analysis shows that the normalized amplitude of coronary inflow is mainly determined by contractility, reflected in ventricular pressure and, at low ventricular volumes, radial wall stress. Normalized flow amplitude is less sensitive to myocardial coronary compliance and resistance, and to the relation between active fiber stress, time, and sarcomere shortening velocity.

  18. Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome.

    Science.gov (United States)

    Kranias, Evangelia G; Hajjar, Roger J

    2012-06-08

    Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.

  19. Effects of Silodosin and Tamsulosin on the Seminal Vesicle Contractile Response.

    Science.gov (United States)

    Hayashi, Tokumasa; Takeya, Mitsue; Nakamura, Kei-ichiro; Matsuoka, Kei

    2016-01-01

    To understand the mechanisms underlying ejaculation dysfunction caused by α1A-adrenocetor (AR) antagonists, the effects of α1A-AR antagonists on the contractile responses of the seminal vesicle were investigated. Isolated seminal vesicles from guinea pigs were cannulated and pressurized, and the changes in the intraluminal pressure were recorded. Periodic applications of electrical stimulation (ES) caused biphasic increase in the intraluminal pressure, that is, initial and subsequent contractions. The effects of silodosin and tamsulosin, α1A-AR antagonists, on the contractile responses were examined. The ES-induced biphasic contractions were blocked by tetrodotoxin (TTX). Silodosin and tamsulosin suppressed the initial contractions in a dose-dependent manner, while also exerting various inhibitory effects on the subsequent contractions. Increases in the intraluminal pressure facilitated spontaneous phasic contractions. The spontaneous contractions were not affected by TTX or α1A-AR antagonists, but were abolished by nifedipine. The initial contractions triggered by neuronal excitations were suppressed by silodosin and tamsulosin, suggesting that the ejaculation dysfunction may be attributed to the α1A-AR antagonist-mediated suppression of nerve-evoked contractions in the seminal vesicle. The subsequent contractions may be induced by mechanical stimulation associated with the initial, nerve-evoked contractions. Alternatively, other transmitters may be involved to various degrees in the neuromuscular transmission of the seminal vesicle. © 2014 Wiley Publishing Asia Pty Ltd.

  20. Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

    Science.gov (United States)

    Hernández, Andrés; Schiffer, Tomas A; Ivarsson, Niklas; Cheng, Arthur J; Bruton, Joseph D; Lundberg, Jon O; Weitzberg, Eddie; Westerblad, Håkan

    2012-08-01

    Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

  1. Dynamics of myosin II organization into cortical contractile networks and fibers

    Science.gov (United States)

    Nie, Wei; Wei, Ming-Tzo; Ou-Yang, Daniel; Jedlicka, Sabrina; Vavylonis, Dimitrios

    2014-03-01

    The morphology of adhered cells critically depends on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin which disrupts actomyosin stress fibers. Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared them to studies by other groups. This analysis suggested that the following processes contribute to the assembly of cortical actomyosin into fibers: random myosin mini-filament assembly and disassembly along the cortex; myosin mini-filament aligning and contraction; stabilization of cortical myosin upon increasing contractile tension. We developed simple numerical simulations that include those processes. The results of simulations of cells at steady state and in response to blebbistatin capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness.

  2. Dynamics of myosin II organization into contractile networks and fibers at the medial cell cortex

    Science.gov (United States)

    Nie, Wei

    The cellular morphology of adhered cells depends crucially on the formation of a contractile meshwork of parallel and cross-linked stress fibers along the contacting surface. The motor activity and mini-filament assembly of non-muscle myosin II is an important component of cell-level cytoskeletal remodeling during mechanosensing. To monitor the dynamics of non-muscle myosin II, we used confocal microscopy to image cultured HeLa cells that stably express myosin regulatory light chain tagged with GFP (MRLC-GFP). MRLC-GFP was monitored in time-lapse movies at steady state and during the response of cells to varying concentrations of blebbistatin (which disrupts actomyosin stress fibers). Using image correlation spectroscopy analysis, we quantified the kinetics of disassembly and reassembly of actomyosin networks and compared to studies by other groups. This analysis suggested the following processes: myosin minifilament assembly and disassembly; aligning and contraction; myosin filament stabilization upon increasing contractile tension. Numerical simulations that include those processes capture some of the main features observed in the experiments. This study provides a framework to help interpret how different cortical myosin remodeling kinetics may contribute to different cell shape and rigidity depending on substrate stiffness. We discuss methods to monitor myosin reorganization using non-linear imaging methods.

  3. EFFECT OF HYDROGEN SULFIDE ON ATRIUM CONTRACTILITY IN CONTROL AND DIABETHIC MICE

    Directory of Open Access Journals (Sweden)

    A. S. Lifanova

    2014-01-01

    Full Text Available Hydrogen sulfide (H2S is endogenously synthesized gasotransmitter that has a regulatory effect in cardiovascular system. Diabetes mellitus leads to an increased risk of hypertension and cardiovascular diseases, so the purpose of the study was to analyze the contractility of the atria mice after application of L-cysteine and H2S. Contractile activity of the myocardium was investigated in the experiment on isolated mouse atria. Alloxan was used for modeling diabetes. Intraperitoneal injection of alloxan resulted in a significant increase of glucose concentration in blood, whereas the concentration of glucose didn’t change at the injection of physiological solution. In control, the addition of NaHS resulted in a significant dose-dependent decrease of the amplitude of contraction of the myocardium, whereas the negative inotropic effect of NaHS was significantly lower in terms of modeling diabetes compare to control conditions. In the control, L-cysteine reduced the amplitude contractions significantly, whereas L-cysteine did not lead to significant changes in the amplitude of contractions in terms of modeling diabetes. These data indicate that the sensitivity of mice’s atria reduced for H2S and L-cysteine in diabetes mellitus.

  4. The effectiveness of two novel techniques in establishing the mechanical and contractile responses of biceps femoris

    International Nuclear Information System (INIS)

    Ditroilo, Massimiliano; De Vito, Giuseppe; Hunter, Angus M; Haslam, Samuel

    2011-01-01

    Portable tensiomyography (TMG) and myotonometry (MMT) devices have been developed to measure mechanical and contractile properties of skeletal muscle. The aim of this study was to explore the sensitivity of the aforementioned techniques in detecting a change in passive mechanical properties of the biceps femoris (BF) muscle as a result of change in knee joint angle (i.e. muscle length). BF responses were assessed in 16 young participants (23.4 ± 4.9 years), at three knee joint angles (0°, 45° and 90°), for maximal isometric torque (MIT) along with myo-electrical activity. Contractile and mechanical properties were measured in a relaxed state. Inter-day reliability of the TMG and MMT was also assessed. MIT changed significantly (p < 0.01) across the three angles, so did stiffness and other parameters measured with MMT (p < 0.01). Conversely, TMG could detect changes only at two knee angles (0° and 45°, p < 0.01), when there is enough tension in the muscle. Reliability was overall insufficient for TMG whilst absolute reliability was excellent (coefficient of variation < 5%) for MMT. The ability of MMT more than TMG to detect an inherent change in stiffness can be conceivably exploited in a number of clinical/therapeutic applications that have to do with unnatural changes in passive muscle stiffness

  5. Scintigraphic measurement of the contractile activity of the gastric antrum using factor analysis

    International Nuclear Information System (INIS)

    Bergmann, H.; Hoebart, J.; Kugi, A.; Stacher, G.; Granser, G.V.

    1990-01-01

    The motor activity of the gastric antrum is difficult to record by manometric means and scintigraphic methods have proved unsatisfactory so far as no consistent relationship between antral contractile activity and gastric emptying rate could be detected. We investigated, using data recorded in 16 healthy human subjects after the ingestion of a semisolid standard meal, whether a newly developed method employing factor analysis would yield more meaningful and reproducible results. Factor analysis was applied to sequential scintigraphic images (3-s frame time) of gastric antrum. The computed factor images and the respective factor curves are representative of distinct dynamic structures of the antrum. From the more or less sinusoidal excursions of the factor curves, which exhibited the 3 cycles per minute frequency characteristic for the stomach, amplitude, frequency and propagation velocity of antral contractions can be calculated. The amplitudes of the factor curves were used to calculate a contraction index. This contraction index was found to be correlated significantly negatively with the gastric half-emptying time of the ingested meal. The employed factor analytical approach thus seems a promising tool to further investigate the role of antral contractility in the process of gastric emptying. (Authors)

  6. Izalpinin from fruits of Alpinia oxyphylla with antagonistic activity against the rat bladder contractility.

    Science.gov (United States)

    Yuan, Yuan; Tan, Yin-Feng; Xu, Peng; Li, Hailong; Li, Yong-Hui; Chen, Wen-Ya; Zhang, Jun-Qing; Chen, Feng; Huang, Guo-Jun

    2014-01-01

    Alpinia oxyphylla (Zingiberaceae), an herbaceous perennial plant, its capsular fruit is commonly used in traditional Chinese medicine for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia. These symptoms are similar to the overactive bladder syndrome. In our lab, we found that the 95% ethanol extract of the capsular fruits exhibited significant anti-muscarinic activity. Some constituents in capsular fruits including flavonoids (e.g., izalpinin and tectochrysin), diarylheptanoids (e.g., yakuchinone A and yakuchinone B) and sesquiterpenes (e.g., nootkatone), are regarded as representative chemicals with putative pharmacological activities. This study aimed to evaluate the in vitro antagonistic actions of izalpinin on carbachol-induced contraction of the rat detrusor muscle. In vitro inhibition of rat detrusor contractile response to carbachol was used to study the functional activity of izalpinin. The isolated detrusor strips of rats were mounted in organ baths containing oxygenated Krebs' solution. The cumulative consecutive concentration-response curves to carbachol-evoked contractions in strips of rat bladder were obtained. Carbachol induced concentration-dependent contractions of isolated rat bladder detrusor strips. The vehicle DMSO had no impact on the contraction response. The contraction effects were concentration-dependently antagonized by izalpinin, with a mean EC50 value of 0.35 µM. The corresponding cumulative agonist concentration-response curves shifted right-ward. Izalpinin exhibits inhibitory role of muscarinic receptor-related detrusor contractile activity, and it may be a promising lead compound to treat overactive bladder.

  7. PPARγ Ligands Regulate Noncontractile and Contractile Functions of Airway Smooth Muscle: Implications for Asthma Therapy

    Directory of Open Access Journals (Sweden)

    Chantal Donovan

    2012-01-01

    Full Text Available In asthma, the increase in airway smooth muscle (ASM can contribute to inflammation, airway wall remodeling and airway hyperresponsiveness (AHR. Targetting peroxisome proliferator-activated receptor γ (PPARγ, a receptor upregulated in ASM in asthmatic airways, may provide a novel approach to regulate these contributions. This review summarises experimental evidence that PPARγ ligands, such as rosiglitazone (RGZ and pioglitazone (PGZ, inhibit proliferation and inflammatory cytokine production from ASM in vitro. In addition, inhaled administration of these ligands reduces inflammatory cell infiltration and airway remodelling in mouse models of allergen-induced airways disease. PPARγ ligands can also regulate ASM contractility, with acute treatment eliciting relaxation of mouse trachea in vitro through a PPARγ-independent mechanism. Chronic treatment can protect against the loss of bronchodilator sensitivity to β2-adrenoceptor agonists and inhibit the development of AHR associated with exposure to nicotine in utero or following allergen challenge. Of particular interest, a small clinical trial has shown that oral RGZ treatment improves lung function in smokers with asthma, a group that is generally unresponsive to conventional steroid treatment. These combined findings support further investigation of the potential for PPARγ agonists to target the noncontractile and contractile functions of ASM to improve outcomes for patients with poorly controlled asthma.

  8. Long-term vascular contractility assay using genipin-modified muscular thin films

    International Nuclear Information System (INIS)

    Hald, Eric S; Steucke, Kerianne E; Reeves, Jack A; Win, Zaw; Alford, Patrick W

    2014-01-01

    Vascular disease is a leading cause of death globally and typically manifests chronically due to long-term maladaptive arterial growth and remodeling. To date, there is no in vitro technique for studying vascular function over relevant disease time courses that both mimics in vivo-like tissue structure and provides a simple readout of tissue stress. We aimed to extend tissue viability in our muscular thin film contractility assay by modifying the polydimethylsiloxane (PDMS) substrate with micropatterned genipin, allowing extracellular matrix turnover without cell loss. To achieve this, we developed a microfluidic delivery system to pattern genipin and extracellular matrix proteins on PDMS prior to cell seeding. Tissues constructed using this method showed improved viability and maintenance of in vivo-like lamellar structure. Functional contractility of tissues fabricated on genipin-modified substrates remained consistent throughout two weeks in culture. These results suggest that muscular thin films with genipin-modified PDMS substrates are a viable method for conducting functional studies of arterial growth and remodeling in vascular diseases. (paper)

  9. Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl− cotransporters, participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles

    Czech Academy of Sciences Publication Activity Database

    Petrezsélyová, Silvia; Kinclová-Zimmermannová, Olga; Sychrová, Hana

    2013-01-01

    Roč. 1828, č. 2 (2013), s. 623-631 ISSN 0005-2736 R&D Projects: GA AV ČR(CZ) IAA500110801; GA MŠk(CZ) LC531; GA MŠk(CZ) OC10012 Grant - others:Rada Programu interní podpory projektů mezinárodní spolupráce AV ČR(CZ) M200110901 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : salt tolerance * yeast vacuole * potassium homeostasis * cation- chloride cotransport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.431, year: 2013

  10. Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae.

    OpenAIRE

    Srivastava, A; Woolford, C A; Jones, E W

    2000-01-01

    Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional ...

  11. Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion

    OpenAIRE

    Sundrud, Mark S.; Torres, Victor J.; Unutmaz, Derya; Cover, Timothy L.

    2004-01-01

    Recent evidence indicates that the secreted Helicobacter pylori vacuolating toxin (VacA) inhibits the activation of T cells. VacA blocks IL-2 secretion in transformed T cell lines by suppressing the activation of nuclear factor of activated T cells (NFAT). In this study, we investigated the effects of VacA on primary human CD4+ T cells. VacA inhibited the proliferation of primary human T cells activated through the T cell receptor (TCR) and CD28. VacA-treated Jurkat T cells secreted markedly ...

  12. Endothelin-1 and endothelin-2 initiate and maintain contractile responses by different mechanisms in rat mesenteric and cerebral arteries

    DEFF Research Database (Denmark)

    Compeer, M. G.; Janssen, G. M. J.; De Mey, J. G. R.

    2013-01-01

    , but relaxed ET-1-induced contractions in MRA. A PLC inhibitor prevented contractile responses to ET-1 and ET-2 in MRA and BA, and relaxed ET-1- and ET-2-induced responses in MRA and ET-1 effects in BA. A Rho-kinase inhibitor did not modify sensitivity, maximum and maintenance of responses to both peptides...... in both arteries but relaxed ET-2, but not ET-1, effects in MRA and ET-1 effects in BA. Conclusions and ImplicationsPLC played a key role in arterial contractile responses to ETs, but ET-1 and ET-2 initiated and maintained vasoconstriction through different mechanisms, and these differed between MRA...

  13. The p21-activated kinase (PAK family member PakD is required for chemorepulsion and proliferation inhibition by autocrine signals in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Jonathan E Phillips

    Full Text Available In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.

  14. The p21-activated kinase (PAK) family member PakD is required for chemorepulsion and proliferation inhibition by autocrine signals in Dictyostelium discoideum.

    Science.gov (United States)

    Phillips, Jonathan E; Gomer, Richard H

    2014-01-01

    In Dictyostelium discoideum, the secreted proteins AprA and CfaD function as reporters of cell density and regulate cell number by inhibiting proliferation at high cell densities. AprA also functions to disperse groups of cells at high density by acting as a chemorepellent. However, the signal transduction pathways associated with AprA and CfaD are not clear, and little is known about how AprA affects the cytoskeleton to regulate cell movement. We found that the p21-activated kinase (PAK) family member PakD is required for both the proliferation-inhibiting activity of AprA and CfaD and the chemorepellent activity of AprA. Similar to cells lacking AprA or CfaD, cells lacking PakD proliferate to a higher cell density than wild-type cells. Recombinant AprA and CfaD inhibit the proliferation of wild-type cells but not cells lacking PakD. Like AprA and CfaD, PakD affects proliferation but does not significantly affect growth (the accumulation of mass) on a per-nucleus basis. In contrast to wild-type cells, cells lacking PakD are not repelled from a source of AprA, and colonies of cells lacking PakD expand at a slower rate than wild-type cells, indicating that PakD is required for AprA-mediated chemorepulsion. A PakD-GFP fusion protein localizes to an intracellular punctum that is not the nucleus or centrosome, and PakD-GFP is also occasionally observed at the rear cortex of moving cells. Vegetative cells lacking PakD show excessive actin-based filopodia-like structures, suggesting that PakD affects actin dynamics, consistent with previously characterized roles of PAK proteins in actin regulation. Together, our results implicate PakD in AprA/CfaD signaling and show that a PAK protein is required for proper chemorepulsive cell movement in Dictyostelium.

  15. Fructan active enzymes (FAZY) activities and biosynthesis of fructooligosaccharides in the vacuoles of Agave tequilana Weber Blue variety plants of different age.

    Science.gov (United States)

    Mellado-Mojica, Erika; González de la Vara, Luis E; López, Mercedes G

    2017-02-01

    Biosynthesis of agave fructans occurs in mesontle vacuoles which showed fluctuations in FAZY activities and synthesized a diverse spectrum of fructooligosaccharide isomers. Agave tequilana Weber Blue variety is an important agronomic crop in Mexico. Fructan metabolism in A. tequilana exhibits changes in fructan content, type, degree of polymerization (DP), and molecular structure. Specific activities of vacuolar fructan active enzymes (FAZY) in A. tequilana plants of different age and the biosynthesis of fructooligosaccharides (FOSs) were analyzed in this work. Vacuoles from mesontle (stem) protoplasts were isolated and collected from 2- to 7-year-old plants. For the first time, agave fructans were identified in the vacuolar content by HPAEC-PAD. Several FAZY activities (1-SST, 6-SFT, 6G-FFT, 1-FFT, and FEH) with fluctuations according to the plant age were found in protein vacuolar extracts. Among vacuolar FAZY, 1-SST activities appeared in all plant developmental stages, as well as 1-FFT and FEH activities. The enzymes 6G-FFT and 6-SST showed only minimal activities. Lowest and highest FAZY activities were found in 2- and 6-year-old plants, respectively. Synthesized products (FOS) were analyzed by TLC and HPAEC-PAD. Vacuolar FAZYs yielded large FOS isomers diversity, being 7-year-old plants the ones that synthesized a greater variety of fructans with different DP, linkages, and molecular structures. Based on the above, we are proposing a model for the FAZY activities constituting the FOS biosynthetic pathways in Agave tequilana Weber Blue variety.

  16. Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

    Science.gov (United States)

    Ploemen, Ivo H J; Croes, Huib J; van Gemert, Geert-Jan J; Wijers-Rouw, Mietske; Hermsen, Cornelus C; Sauerwein, Robert W

    2012-01-01

    The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

  17. In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves.

    Science.gov (United States)

    Carrión, Cristian A; Costa, María Lorenza; Martínez, Dana E; Mohr, Christina; Humbeck, Klaus; Guiamet, Juan J

    2013-11-01

    Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

  18. Acute exposure to lead increases myocardial contractility independent of hypertension development

    Energy Technology Data Exchange (ETDEWEB)

    Fioresi, M. [Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Departamento de Enfermagem, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Furieri, L.B.; Simões, M.R.; Ribeiro, R.F. Junior; Meira, E.F.; Fernandes, A.A.; Stefanon, I. [Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Vassallo, D.V. [Programa de Pós-Graduação em Ciências Fisiológicas, Centro de Ciências da Saúde, Universidade Federal do Espírito Santo, Vitória, ES (Brazil); Centro de Ciências da Saúde de Vitória, Escola Superior de Ciências da Santa Casa de Misericórdia de Vitória, Vitória, ES (Brazil)

    2013-02-01

    We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetate iv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na{sup +},K{sup +}-ATPase and myosin Ca{sup 2+}-ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2 vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13 vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na{sup +},K{sup +}-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

  19. Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis.

    Science.gov (United States)

    Clausen, Torben; Nielsen, Ole Bækgaard; Clausen, Johannes D; Pedersen, Thomas Holm; Hayward, Lawrence J

    2011-07-01

    In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K(+) ingestion or rest after exercise. Force can be restored by muscle work or treatment with β(2)-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na(+) channel (Na(v)1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K(+)](o). In resting mutant soleus, tetrodotoxin (TTX)-suppressible (22)Na uptake and [Na(+)](i) were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P Na(+),K(+) pump-mediated (86)Rb uptake was 83% larger than in WT. Salbutamol stimulated (86)Rb uptake and reduced [Na(+)](i) both in mutant and WT soleus. Stimulating Na(+),K(+) pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na(+)](i) with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na(+),K(+) pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na(+)](i) on the synthesis of Na(+),K(+) pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na(+) influx and show that contractility can be restored by acute stimulation of the Na(+),K(+) pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in (86)Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated

  20. Contractile function and motor unit firing rates of the human hamstrings.

    Science.gov (United States)

    Kirk, Eric A; Rice, Charles L

    2017-01-01

    Neuromuscular properties of the lower limb in health, aging, and disease are well described for major lower limb muscles comprising the quadriceps, triceps surae, and dorsiflexors, with the notable exception of the posterior thigh (hamstrings). The purpose of this study was to further characterize major muscles of the lower limb by comprehensively exploring contractile properties in relation to spinal motor neuron output expressed as motor unit firing rates (MUFRs) in the hamstrings of 11 (26.5 ± 3.8) young men. Maximal isometric voluntary contraction (MVC), voluntary activation, stimulated contractile properties including a force-frequency relationship, and MUFRs from submaximal to maximal voluntary contractile intensities were assessed in the hamstrings. Strength and MUFRs were assessed at two presumably different muscle lengths by varying the knee joint angles (90° and 160°). Knee flexion MVCs were 60-70% greater in the extended position (160°). The frequency required to elicit 50% of maximum tetanic torque was 16-17 Hz. Mean MUFRs at 25-50% MVC were 9-31% less in the biceps femoris compared with the semimembranosus-semitendinosus group. Knee joint angle (muscle length) influenced MUFRs such that mean MUFRs were greater in the shortened (90°) position at 50% and 100% MVC. Compared with previous reports, mean maximal MUFRs in the hamstrings are greater than those in the quadriceps and triceps surae and somewhat less than those in the tibialis anterior. Mean maximal MUFRs in the hamstrings are influenced by changes in knee joint angle, with lower firing rates in the biceps femoris compared with the semimembranosus-semitendinosus muscle group. We studied motor unit firing rates (MUFRs) at various voluntary contraction intensities in the hamstrings, one of the only major lower limb muscles to have MUFRs affected by muscle length changes. Within the hamstrings muscle-specific differences have greater impact on MUFRs than length changes, with the biceps femoris

  1. Muscle contractility decrement and correlated morphology during the pathogenesis of streptozotocin-diabetic mice.

    Science.gov (United States)

    Fahim, M A; el-Sabban, F; Davidson, N

    1998-06-01

    Peripheral neuropathy of both motor and sensory nerves has been well documented in diabetes mellitus, but the evidence for physiological and correlated morphological changes during the pathogenesis of myopathy is scarce. In the present report, we have chosen the dorsiflexor muscle of adult male mice as a model for studying in situ muscle contraction and neuromuscular ultrastructure during the pathogenesis of streptozotocin-induced diabetes. Thirty mice (30 g bodyweight) were injected once i.p. with streptozotocin solution (200 mg/Kg) to induce experimental diabetes mellitus. Comparative analyses of in situ muscle isometric contractile characteristics were studied (at 1 Hz, 5 Hz and 30 Hz nerve stimulation) in urethane-anesthetized (2 mg/g, i.p.) control and diabetic mice at three time points, 2 weeks, 4 weeks, and 8 weeks postinjection. Synaptic delay was also recorded in diabetic and age-matched control mice. There was a significant increase in synaptic delay in both 4-week and 8-week diabetic mice compared with control mice (8.9 +/- 1.2 msec and 7.6 +/- 0.6 msec, respectively, compared with 6.1 +/- 0.5 msec). At all three stimulation frequencies, diabetes did not affect muscle contractile speed but significantly reduced the twitch tension after 8 weeks, with no changes at 2 weeks or 4 weeks. The recorded single-twitch tension values were 2.6 +/- 0.3 g, 2.1 +/- 0.6 g, 2.2 +/- 0.7 g, and 1.2 +/- 0.1 g for control, 2 weeks, 4 weeks, and 8 weeks, respectively. At 30 Hz, the recorded tension values were 4.6 +/- 1.6 g, 3.1 +/- 1.2 g, 3.1 +/- 1.1 g, and 2.1 +/- 1.0 g for control, 2 weeks, 4 weeks, and 8 weeks, respectively. Ultrastructural changes in neuromuscular junctions were similar to those that have been described in disuse and aging. These changes were observed after 8 weeks and included serve loss of synaptic vesicles, electron-dense bodies, and myelin-like figures as well as degeneration of mitochondria. The results reveal that streptozotocin-induced diabetes

  2. Beta adrenergic overstimulation impaired vascular contractility via actin-cytoskeleton disorganization in rabbit cerebral artery.

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    Hyoung Kyu Kim

    Full Text Available BACKGROUND AND PURPOSE: Beta adrenergic overstimulation may increase the vascular damage and stroke. However, the underlying mechanisms of beta adrenergic overstimulation in cerebrovascular dysfunctions are not well known. We investigated the possible cerebrovascular dysfunction response to isoproterenol induced beta-adrenergic overstimulation (ISO in rabbit cerebral arteries (CAs. METHODS: ISO was induced in six weeks aged male New Zealand white rabbit (0.8-1.0 kg by 7-days isoproterenol injection (300 μg/kg/day. We investigated the alteration of protein expression in ISO treated CAs using 2DE proteomics and western blot analysis. Systemic properties of 2DE proteomics result were analyzed using bioinformatics software. ROS generation and following DNA damage were assessed to evaluate deteriorative effect of ISO on CAs. Intracellular Ca(2+ level change and vascular contractile response to vasoactive drug, angiotensin II (Ang II, were assessed to evaluate functional alteration of ISO treated CAs. Ang II-induced ROS generation was assessed to evaluated involvement of ROS generation in CA contractility. RESULTS: Proteomic analysis revealed remarkably decreased expression of cytoskeleton organizing proteins (e.g. actin related protein 1A and 2, α-actin, capping protein Z beta, and vimentin and anti-oxidative stress proteins (e.g. heat shock protein 9A and stress-induced-phosphoprotein 1 in ISO-CAs. As a cause of dysregulation of actin-cytoskeleton organization, we found decreased level of RhoA and ROCK1, which are major regulators of actin-cytoskeleton organization. As functional consequences of proteomic alteration, we found the decreased transient Ca(2+ efflux and constriction response to angiotensin II and high K(+ in ISO-CAs. ISO also increased basal ROS generation and induced oxidative damage in CA; however, it decreased the Ang II-induced ROS generation rate. These results indicate that ISO disrupted actin cytoskeleton proteome network

  3. The Effect of Cleft Palate Repair on Contractile Properties of Single Permeabilized Muscle Fibers From Congenitally Cleft Goats Palates

    Science.gov (United States)

    A cleft palate goat model was used to study the contractile properties of the levator veli palatini (LVP) muscle which is responsible for the movement of the soft palate. In 15-25% of patients that undergo palatoplasty, residual velopharyngeal insufficiency (VPI) remains a problem and often require...

  4. Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90

    Directory of Open Access Journals (Sweden)

    Alex Henke

    2016-08-01

    Full Text Available Background: Prostate cancer-associated fibroblasts (CAF can stimulate malignant progression and invasion of prostatic tumour cells via several mechanisms including those active in extracellular matrix; Methods: We isolated CAF from prostate cancer patients of Gleason Score 6–10 and confirmed their cancer-promoting activity using an in vivo tumour reconstitution assay comprised of CAF and BPH1 cells. We tested the effects of heat shock protein 90 (HSP90 inhibitors upon reconstituted tumour growth in vivo. Additionally, CAF contractility was measured in a 3D collagen contraction assay and migration was measured by scratch assay; Results: HSP90 inhibitors dipalmitoyl-radicicol and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG reduced tumour size and proliferation in CAF/BPH1 reconstituted tumours in vivo. We observed that the most contractile CAF were derived from patients with lower Gleason Score and of younger age compared with the least contractile CAF. HSP90 inhibitors radicicol and 17-DMAG inhibited contractility and reduced the migration of CAF in scratch assays. Intracellular levels of HSP70 and HSP90 were upregulated upon treatment with HSP90 inhibitors. Inhibition of HSP90 also led to a specific increase in transforming growth factor beta 2 (TGFβ2 levels in CAF; Conclusions: We suggest that HSP90 inhibitors act not only upon tumour cells, but also on CAF in the tumour microenvironment.

  5. Investigating Cardiac MRI Based Right Ventricular Contractility As A Novel Non-Invasive Metric of Pulmonary Arterial Pressure

    Science.gov (United States)

    Menon, Prahlad G; Adhypak, Srilakshmi M; Williams, Ronald B; Doyle, Mark; Biederman, Robert WW

    2014-01-01

    BACKGROUND We test the hypothesis that cardiac magnetic resonance (CMR) imaging-based indices of four-dimensional (4D) (three dimensions (3D) + time) right ventricle (RV) function have predictive values in ascertaining invasive pulmonary arterial systolic pressure (PASP) measurements from right heart catheterization (RHC) in patients with pulmonary arterial hypertension (PAH). METHODS We studied five patients with idiopathic PAH and two age and sex-matched controls for RV function using a novel contractility index (CI) for amplitude and phase to peak contraction established from analysis of regional shape variation in the RV endocardium over 20 cardiac phases, segmented from CMR images in multiple orientations. RESULTS The amplitude of RV contractility correlated inversely with RV ejection fraction (RVEF; R2 = 0.64, P = 0.03) and PASP (R2 = 0.71, P = 0.02). Phase of peak RV contractility also correlated inversely to RVEF (R2 = 0.499, P = 0.12) and PASP (R2 = 0.66, P = 0.04). CONCLUSIONS RV contractility analyzed from CMR offers promising non-invasive metrics for classification of PAH, which are congruent with invasive pressure measurements. PMID:25624777

  6. 5-HT4 receptors mediating enhancement of contractility in canine stomach; an in vitro and in vivo study

    NARCIS (Netherlands)

    Prins, NH; van der Grijn, A; Lefebvre, RA; Akkermans, LMA; Schuurkes, JAJ

    1 We aimed to study 5-HT4 receptors in canine stomach contractility both in vivo and in vitro. 2 In anaesthetized Beagle dogs, the selective 5-HT4 receptor agonist prucalopride (i.v.) induced dose-dependent tonic stomach contractions under isobaric conditions, an effect that was antagonized by the

  7. Inhibition of MMP-2 Expression with siRNA Increases Baseline Cardiomyocyte Contractility and Protects against Simulated Ischemic Reperfusion Injury

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    Han-Bin Lin

    2014-01-01

    Full Text Available Matrix metalloproteinases (MMPs significantly contribute to ischemia reperfusion (I/R injury, namely, by the degradation of contractile proteins. However, due to the experimental models adopted and lack of isoform specificity of MMP inhibitors, the cellular source and identity of the MMP(s involved in I/R injury remain to be elucidated. Using isolated adult rat cardiomyocytes, subjected to chemically induced I/R-like injury, we show that specific inhibition of MMP-2 expression and activity using MMP-2 siRNA significantly protected cardiomyocyte contractility from I/R-like injury. This was also associated with increased expression of myosin light chains 1 and 2 (MLC1/2 in comparison to scramble siRNA transfection. Moreover, the positive effect of MMP-2 siRNA transfection on cardiomyocyte contractility and MLC1/2 expression levels was also observed under control conditions, suggesting an important additional role for MMP-2 in physiological sarcomeric protein turnover. This study clearly demonstrates that intracellular expression of MMP-2 plays a significant role in sarcomeric protein turnover, such as MLC1 and MLC2, under aerobic (physiological conditions. In addition, this study identifies intracellular/autocrine, cardiomyocyte-produced MMP-2, rather than paracrine/extracellular, as responsible for the degradation of MLC1/2 and consequent contractile dysfunction in cardiomyocytes subjected to I/R injury.

  8. Effect of Propafenone on the Contractile Activity of Latissimus Dorsi Muscle Isolated in an Organ Chamber: Experimental Study in Rats

    Directory of Open Access Journals (Sweden)

    Ricardo Simões

    2002-03-01

    Full Text Available OBJECTIVE: To study the effect of propafenone on the contractile function of latissimus dorsi muscle isolated from rats in an organ chamber. METHODS: We studied 20 latissimus dorsi muscles of Wistar rats and divided them into 2 groups: group I (n=10, or control group - we studied the feasibility of muscle contractility; group II (n=10, in which the contralateral muscles were grouped - we analyzed the effect of propafenone on muscle contractility. After building a muscle ring, 8 periods of sequential 2-minute baths were performed, with intervals of preprogrammed electrical stimulation using a pacemaker of 50 stimuli/min. In group II, propafenone, at the concentration of 9.8 µg/mL, was added to the bath in period 2 and withdrawn in period 4. RESULTS: In group I, no significant depression in muscle contraction occurred up to period 5 (p>0.05. In group II, a significant depression occurred in all periods, except between the last 2 periods (p0.05. CONCLUSION: Propafenone had a depressing effect on the contractile function of latissimus dorsi muscle isolated from rats and studied in an organ chamber.

  9. Effect of acute and chronic simvastatin treatment on post-ischemic contractile dysfunction in isolated rat heart

    Czech Academy of Sciences Publication Activity Database

    Szárszoi, Ondrej; Malý, J.; Ošťádal, P.; Netuka, I.; Bešík, J.; Kolář, František; Ošťádal, Bohuslav

    2008-01-01

    Roč. 57, č. 5 (2008), s. 793-796 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : simvastatin * contractile dysfunction * protection Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.653, year: 2008

  10. [A basis for application of cardiac contractility variability in the Evaluation and assessment of exercise and fitness].

    Science.gov (United States)

    Bu, Bin; Wang, Aihua; Han, Haijun; Xiao, Shouzhong

    2010-06-01

    Cardiac contractility variability (CCV) is a new concept which is introduced in the research field of cardiac contractility in recent years, that is to say, there are some disparities between cardiac contractilities when heart contracts. The changing signals of cardiac contractility contain a plenty of information on the cardiovascular function and disorder. In order to collect and analyze the message, we could quantitatively evaluate the tonicity and equilibrium of cardiac sympathetic nerve and parasympathetic nerve, and the effects of bio-molecular mechanism on the cardiovascular activities. By analyzing CCV, we could further understand the background of human being's heritage characteristics, nerve types, the adjusting mechanism, the molecular biology, and the adjustment of cardiac automatic nerve. With the development of the computing techniques, the digital signal processing method and its application in medical field, this analysis has been progressing greatly. By now, the assessment of CCV, just like the analysis of heart rate variability, is mainly via time domain and frequency domain analysis. CCV is one of the latest research fields in human cardiac signals being scarcely reported in the field of sports medicine; however, its research progresses are of important value for cardiac physiology and pathology in sports medicine and rehabilitation medicine.

  11. Effects of testosterone on contractile properties of sexually dimorphic forelimb muscles in male bullfrogs (Rana catesbeiana, Shaw 1802

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    Aaron R. Kampe

    2013-07-01

    This study examined the effects of testosterone (T on the contractile properties of two sexually dimorphic forelimb muscles and one non-dimorphic muscle in male bullfrogs (Rana catesbeiana, Shaw 1802. The dimorphic muscles in castrated males with testosterone replacement (T+ achieved higher forces and lower fatigability than did castrated males without replaced testosterone (T0 males, but the magnitude of the differences was low and many of the pair-wise comparisons of each muscle property were not statistically significant. However, when taken as a whole, the means of seven contractile properties varied in the directions expected of masculine values in T+ animals in the sexually dimorphic muscles. Moreover, these data, compared with previous data on male and female bullfrogs, show that values for T+ males are similar to normal males and are significantly different from females. The T0 males tended to be intermediate in character between T+ males and females, generally retaining masculine values. This suggests that the exposure of young males to T in their first breeding season produces a masculinizing effect on the sexually dimorphic muscles that is not reversed between breeding seasons when T levels are low. The relatively minor differences in contractile properties between T+ and T0 males may indicate that as circulating T levels rise during breeding season in normal males, contractile properties can be enhanced rapidly to maximal functional levels for breeding success.

  12. Microtubule depolymerization normalizes in vivo myocardial contractile function in dogs with pressure-overload left ventricular hypertrophy

    Science.gov (United States)

    Koide, M.; Hamawaki, M.; Narishige, T.; Sato, H.; Nemoto, S.; DeFreyte, G.; Zile, M. R.; Cooper G, I. V.; Carabello, B. A.

    2000-01-01

    BACKGROUND: Because initially compensatory myocardial hypertrophy in response to pressure overloading may eventually decompensate to myocardial failure, mechanisms responsible for this transition have long been sought. One such mechanism established in vitro is densification of the cellular microtubule network, which imposes a viscous load that inhibits cardiocyte contraction. METHODS AND RESULTS: In the present study, we extended this in vitro finding to the in vivo level and tested the hypothesis that this cytoskeletal abnormality is important in the in vivo contractile dysfunction that occurs in experimental aortic stenosis in the adult dog. In 8 dogs in which gradual stenosis of the ascending aorta had caused severe left ventricular (LV) pressure overloading (gradient, 152+/-16 mm Hg) with contractile dysfunction, LV function was measured at baseline and 1 hour after the intravenous administration of colchicine. Cardiocytes obtained by biopsy before and after in vivo colchicine administration were examined in tandem. Microtubule depolymerization restored LV contractile function both in vivo and in vitro. CONCLUSIONS: These and additional corroborative data show that increased cardiocyte microtubule network density is an important mechanism for the ventricular contractile dysfunction that develops in large mammals with adult-onset pressure-overload-induced cardiac hypertrophy.

  13. Effect of a Periodized Power Training Program on the Functional Performances and Contractile Properties of the Quadriceps in Sprinters

    Science.gov (United States)

    Kamandulis, Sigitas; Skurvydas, Albertas; Brazaitis, Marius; Stanislovaitis, Aleksas; Duchateau, Jacques; Stanislovaitiene, Jurate

    2012-01-01

    Our purpose was to compare the effect of a periodized preparation consisting of power endurance training and high-intensity power training on the contractile properties of the quadriceps muscle and functional performances in well trained male sprinters (n = 7). After 4 weeks of high-intensity power training, 60-m sprint running time improved by an…

  14. Alterations in serotonin receptor-induced contractility of bovine lateral saphenous vein in cattle grazing endophyte-infected tall fescue

    Science.gov (United States)

    As part of a large 2-year study documenting the physiologic impact of grazing endophyte-infected tall fescue on growing cattle, 2 experiments were conducted to characterize and evaluate the effects of grazing 2 levels of toxic endophyte-infected tall fescue pastures on vascular contractility and ser...

  15. Bidirectional Interplay between Vimentin Intermediate Filaments and Contractile Actin Stress Fibers

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    Yaming Jiu

    2015-06-01

    Full Text Available The actin cytoskeleton and cytoplasmic intermediate filaments contribute to cell migration and morphogenesis, but the interplay between these two central cytoskeletal elements has remained elusive. Here, we find that specific actin stress fiber structures, transverse arcs, interact with vimentin intermediate filaments and promote their retrograde flow. Consequently, myosin-II-containing arcs are important for perinuclear localization of the vimentin network in cells. The vimentin network reciprocally restricts retrograde movement of arcs and hence controls the width of flat lamellum at the leading edge of the cell. Depletion of plectin recapitulates the vimentin organization phenotype of arc-deficient cells without affecting the integrity of vimentin filaments or stress fibers, demonstrating that this cytoskeletal cross-linker is required for productive interactions between vimentin and arcs. Collectively, our results reveal that plectin-mediated interplay between contractile actomyosin arcs and vimentin intermediate filaments controls the localization and dynamics of these two cytoskeletal systems and is consequently important for cell morphogenesis.

  16. Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

    Science.gov (United States)

    Lemichez, Emmanuel; Gonzalez-Rodriguez, David; Bassereau, Patricia; Brochard-Wyart, Françoise

    2013-03-01

    Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions. Copyright © 2013 Soçiété Française des Microscopies and Soçiété de Biologie Cellulaire de France.

  17. Nonequilibrium phase transitions, fluctuations and correlations in an active contractile polar fluid.

    Science.gov (United States)

    Gowrishankar, Kripa; Rao, Madan

    2016-02-21

    We study the patterning, fluctuations and correlations of an active polar fluid consisting of contractile polar filaments on a two-dimensional substrate, using a hydrodynamic description. The steady states generically consist of arrays of inward pointing asters and show a continuous transition from a moving lamellar phase, a moving aster street, to a stationary aster lattice with no net polar order. We next study the effect of spatio-temporal athermal noise, parametrized by an active temperature TA, on the stability of the ordered phases. In contrast to its equilibrium counterpart, we find that the active crystal shows true long range order at low TA. On increasing TA, the asters dynamically remodel, concomitantly we find novel phase transitions characterized by bond-orientational and polar order upon "heating".

  18. Image Processing Techniques for Assessing Contractility in Isolated Adult Cardiac Myocytes

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    Carlos Bazan

    2009-01-01

    The physiologic application of the methodology is evaluated by assessing overall contraction in enzymatically dissociated adult rat cardiocytes. Our results demonstrate the effectiveness of the proposed approach in characterizing the true, two-dimensional, “shortening” in the contraction process of adult cardiocytes. We compare the performance of the proposed method to that of a popular edge detection system in the literature. The proposed method not only provides a more comprehensive assessment of the myocyte contraction process but also can potentially eliminate historical concerns and sources of errors caused by myocyte rotation or translation during contraction. Furthermore, the versatility of the image processing techniques makes the method suitable for determining myocyte shortening in cells that usually bend or move during contraction. The proposed method can be utilized to evaluate changes in contractile behavior resulting from drug intervention, disease modeling, transgeneity, or other common applications to mammalian cardiocytes.

  19. A new strain gage method for measuring the contractile strain ratio of Zircaloy tubing

    International Nuclear Information System (INIS)

    Hwang, S.K.; Sabol, G.P.

    1988-01-01

    An improved strain gage method for determining the contractile strain ratio (CSR) of Zircaloy tubing was developed. The new method consists of a number of load-unload cyclings at approximately 0.2% plastic strain interval. With this method the CSR of Zircaloy-4 tubing could be determined accurately because it was possible to separate the plastic strains from the elastic strain involvement. The CSR values determined by use of the new method were in good agreement with those calculated from conventional post-test manual measurements. The CSR of the tubing was found to decrease with the amount of deformation during testing because of uneven plastic flow in the gage section. A new technique of inscribing gage marks by use of a YAG laser is discussed. (orig.)

  20. Leucine elicits myotube hypertrophy and enhances maximal contractile force in tissue engineered skeletal muscle in vitro.

    Science.gov (United States)

    Martin, Neil R W; Turner, Mark C; Farrington, Robert; Player, Darren J; Lewis, Mark P

    2017-10-01

    The amino acid leucine is thought to be important for skeletal muscle growth by virtue of its ability to acutely activate mTORC1 and enhance muscle protein synthesis, yet little data exist regarding its impact on skeletal muscle size and its ability to produce force. We utilized a tissue engineering approach in order to test whether supplementing culture medium with leucine could enhance mTORC1 signaling, myotube growth, and muscle function. Phosphorylation of the mTORC1 target proteins 4EBP-1 and rpS6 and myotube hypertrophy appeared to occur in a dose dependent manner, with 5 and 20 mM of leucine inducing similar effects, which were greater than those seen with 1 mM. Maximal contractile force was also elevated with leucine supplementation; however, although this did not appear to be enhanced with increasing leucine doses, this effect was completely ablated by co-incubation with the mTOR inhibitor rapamycin, showing that the augmented force production in the presence of leucine was mTOR sensitive. Finally, by using electrical stimulation to induce chronic (24 hr) contraction of engineered skeletal muscle constructs, we were able to show that the effects of leucine and muscle contraction are additive, since the two stimuli had cumulative effects on maximal contractile force production. These results extend our current knowledge of the efficacy of leucine as an anabolic nutritional aid showing for the first time that leucine supplementation may augment skeletal muscle functional capacity, and furthermore validates the use of engineered skeletal muscle for highly-controlled investigations into nutritional regulation of muscle physiology. © 2017 The Authors. Journal of Cellular Physiology Published by wiley periodicals, Inc.

  1. Exposure to low mercury concentration in vivo impairs myocardial contractile function

    International Nuclear Information System (INIS)

    Furieri, Lorena Barros; Fioresi, Mirian; Junior, Rogerio Faustino Ribeiro; Bartolome, Maria Visitacion; Fernandes, Aurelia Araujo; Cachofeiro, Victoria; Lahera, Vicente; Salaices, Mercedes; Stefanon, Ivanita; Vassallo, Dalton Valentim

    2011-01-01

    Increased cardiovascular risk after mercury exposure has been described but cardiac effects resulting from controlled chronic treatment are not yet well explored. We analyzed the effects of chronic exposure to low mercury concentrations on hemodynamic and ventricular function of isolated hearts. Wistar rats were treated with HgCl 2 (1st dose 4.6 μg/kg, subsequent dose 0.07 μg/kg/day, im, 30 days) or vehicle. Mercury treatment did not affect blood pressure (BP) nor produced cardiac hypertrophy or changes of myocyte morphometry and collagen content. This treatment: 1) in vivo increased left ventricle end diastolic pressure (LVEDP) without changing left ventricular systolic pressure (LVSP) and heart rate; 2) in isolated hearts reduced LV isovolumic systolic pressure and time derivatives, and β-adrenergic response; 3) increased myosin ATPase activity; 4) reduced Na + -K + ATPase (NKA) activity; 5) reduced protein expression of SERCA and phosphorylated phospholamban on serine 16 while phospholamban expression increased; as a consequence SERCA/phospholamban ratio reduced; 6) reduced sodium/calcium exchanger (NCX) protein expression and α-1 isoform of NKA, whereas α-2 isoform of NKA did not change. Chronic exposure for 30 days to low concentrations of mercury does not change BP, heart rate or LVSP but produces small but significant increase of LVEDP. However, in isolated hearts mercury treatment promoted contractility dysfunction as a result of the decreased NKA activity, reduction of NCX and SERCA and increased PLB protein expression. These findings offer further evidence that mercury chronic exposure, even at small concentrations, is an environmental risk factor affecting heart function. - Highlights: → Unchanges blood pressure, heart rate, systolic pressure. → Increases end diastolic pressure. → Promotes cardiac contractility dysfunction. → Decreases NKA activity, NCX and SERCA, increases PLB protein expression. → Small concentrations constitutes

  2. Tension cost correlates with mechanical and biochemical parameters in different myocardial contractility conditions

    Directory of Open Access Journals (Sweden)

    Cleci M. Moreira

    2012-01-01

    Full Text Available OBJECTIVES: Tension cost, the ratio of myosin ATPase activity to tension, reflects the economy of tension development in the myocardium. To evaluate the mechanical advantage represented by the tension cost, we studied papillary muscle contractility and the activity of myosin ATPase in the left ventricles in normal and pathophysiological conditions. METHODS: Experimental protocols were performed using rat left ventricles from: (1 streptozotocin-induced diabetic and control Wistar rats; (2 N-nitro-L-arginine methyl ester (L-NAME hypertensive and untreated Wistar rats; (3 deoxycorticosterone acetate (DOCA salt-treated, nephrectomized and salt- and DOCA-treated rats; (4 spontaneous hypertensive rats (SHR and Wistar Kyoto (WKY rats; (5 rats with myocardial infarction and shamoperated rats. The isometric force, tetanic tension, and the activity of myosin ATPase were measured. RESULTS: The results obtained from infarcted, diabetic, and deoxycorticosterone acetate-salt-treated rats showed reductions in twitch and tetanic tension compared to the control and sham-operated groups. Twitch and tetanic tension increased in the N-nitro-L-arginine methyl ester-treated rats compared with the Wistar rats. Myosin ATPase activity was depressed in the infarcted, diabetic, and deoxycorticosterone acetate salt-treated rats compared with control and sham-operated rats and was increased in N-nitro-L-arginine methyl ester-treated rats. These parameters did not differ between SHR and WKY rats. In the studied conditions (e.g., post-myocardial infarction, deoxycorticosterone acetate salt-induced hypertension, chronic N-nitro-L-arginine methyl ester treatment, and streptozotocin-induced diabetes, a positive correlation between force or plateau tetanic tension and myosin ATPase activity was observed. CONCLUSION: Our results suggest that the myocardium adapts to force generation by increasing or reducing the tension cost to maintain myocardial contractility with a better

  3. Perfusion-induced changes in cardiac contractility depend on capillary perfusion.

    Science.gov (United States)

    Dijkman, M A; Heslinga, J W; Sipkema, P; Westerhof, N

    1998-02-01

    The perfusion-induced increase in cardiac contractility (Gregg phenomenon) is especially found in heart preparations that lack adequate coronary autoregulation and thus protection of changes in capillary pressure. We determined in the isolated perfused papillary muscle of the rat whether cardiac muscle contractility is related to capillary perfusion. Oxygen availability of this muscle is independent of internal perfusion, and perfusion may be varied or even stopped without loss of function. Muscles contracted isometrically at 27 degrees C (n = 7). During the control state stepwise increases in perfusion pressure resulted in all muscles in a significant increase in active tension. Muscle diameter always increased with increased perfusion pressure, but muscle segment length was unaffected. Capillary perfusion was then obstructed by plastic microspheres (15 microns). Flow, at a perfusion pressure of 66.6 +/- 26.2 cmH2O, reduced from 17.6 +/- 5.4 microliters/min in the control state to 3.2 +/- 1.3 microliters/min after microspheres. Active tension developed by the muscle in the unperfused condition before microspheres and after microspheres did not differ significantly (-12.8 +/- 29.4% change). After microspheres similar perfusion pressure steps as in control never resulted in an increase in active tension. Even at the two highest perfusion pressures (89.1 +/- 28.4 and 106.5 +/- 31.7 cmH2O) that were applied a significant decrease in active tension was found. We conclude that the Gregg phenomenon is related to capillary perfusion.

  4. Troglitazone stimulates β-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1A receptor

    International Nuclear Information System (INIS)

    Tilley, Douglas G.; Nguyen, Anny D.; Rockman, Howard A.

    2010-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPARγ-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPARγ activity, thus we hypothesized that a PPARγ agonist may exert physiologic effects via the angiotensin II type 1 A receptor (AT1 A R). In AT1 A R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPARγ agonist troglitazone (Trog) enhanced AT1 A R internalization and recruitment of endogenous β-arrestin1/2 (βarr1/2) to the AT1 A R. A fluorescence assay to measure diacylglycerol (DAG) accumulation showed that although Ang II induced AT1 A R-G q protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of βarr1/2 was selective to AT1 A R as the response was prevented by an ARB- and Trog-mediated βarr1/2 recruitment to β1-adrenergic receptor (β1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be βarr2-dependent, as cardiomyocytes isolated from βarr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPARγ agonist Trog acts at the AT1 A R to simultaneously block G q protein activation and induce the recruitment of βarr1/2, which leads to an increase in cardiomyocyte contractility.

  5. Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction

    Directory of Open Access Journals (Sweden)

    Mouli Chakraborty

    2015-12-01

    Full Text Available Up to 80% of individuals with myotonic dystrophy type 1 (DM1 will develop cardiac abnormalities at some point during the progression of their disease, the most common of which is heart blockage of varying degrees. Such blockage is characterized by conduction defects and supraventricular and ventricular tachycardia, and carries a high risk of sudden cardiac death. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts, as revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in humans with DM1, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function, reduced heart tube diameters and reduced contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind from the CUG RNA repeats and reduced ribonuclear formation in the Drosophila heart, but also rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats.

  6. Low thermal dependence of the contractile properties of a wing muscle in the bat Carollia perspicillata.

    Science.gov (United States)

    Rummel, Andrea D; Swartz, Sharon M; Marsh, Richard L

    2018-05-29

    Temperature affects contractile rate properties in muscle, which may affect locomotor performance. Endotherms are known to maintain high core body temperatures, but temperatures in the periphery of the body can fluctuate. Such a phenomenon occurs in bats, whose wing musculature is relatively poorly insulated, resulting in substantially depressed temperatures in the distal wing. We examined a wing muscle in the small-bodied tropical bat Carollia perspicillata and a hindlimb muscle in the laboratory mouse at 5°C intervals from 22 to 42°C to determine the thermal dependence of the contractile properties of both muscles. We found that the bat ECRL had low thermal dependence from near body temperature to 10°C lower, with Q 10 values of less than 1.5 for relaxation from contraction and shortening velocities in that interval, and with no significant difference in some rate properties in the interval between 32 and 37°C. In contrast, for all temperature intervals below 37°C, Q 10 values for the mouse EDL were 1.5 or higher, and rate properties differed significantly across successive temperature intervals from 37 to 22°C. An ANCOVA analysis found that the thermal dependencies of all measured isometric and isotonic rate processes were significantly different between the bat and mouse muscles. The relatively low thermal dependence of the bat muscle likely represents a downward shift of its optimal temperature and may be functionally significant in light of the variable operating temperatures of bat wing muscles. © 2018. Published by The Company of Biologists Ltd.

  7. A comparison of the contractile properties of smooth muscle from pig urethra and internal anal sphincter.

    Science.gov (United States)

    Ramalingam, Thanesan; Durlu-Kandilci, N Tugba; Brading, Alison F

    2010-09-01

    Smooth muscles from the urethra and internal anal sphincter (IAS) play an essential role in the maintenance of urinary and fecal continence. Any damage in these muscles may cause serious problems. The aim of this study was to directly compare the contractile properties of pig urethra and IAS taken from the same animal. Smooth muscle strips of urethra and IAS dissected from the same pig were transferred to organ baths superfused with Krebs' solution, loaded with 1 g tension and equilibrated for 1 hr. Carbachol and phenylephrine response curves and EFS responses were elicited in the absence and presence of inhibitors. Both tissues developed tone during the 1 hr equilibration period. Carbachol (3 × 10(-6)-10(-3) M) contracted urethra whilst relaxing IAS. Guanethidine (10(-6) M) inhibited the carbachol responses in both tissues. L-NOARG (10(-4) M) decreased carbachol responses in IAS, but not in urethra. Phenylephrine (3 × 10(-6)-10(-2) M) contracted both tissues. EFS (1-40 Hz) induced a contractile response in urethra which was decreased with guanethidine (10(-6) M) and further blocked by atropine (10(-6) M). In the presence of both, a relaxation response was observed that is sensitive to NOS inhibitors especially at low frequencies. EFS induced a relaxation followed by a contraction in IAS strips. This contraction was blocked by guanethidine but not by atropine, and the remaining relaxation at 20 Hz was decreased with L-NOARG and increased with L-arginine. There are differences between urethra and IAS in terms of muscarinic activation and neural innervation, relevant for pharmacotherapy. © 2010 Wiley-Liss, Inc.

  8. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra.

    Science.gov (United States)

    Cannon, Tracy W; Lee, Ji Youl; Somogyi, George; Pruchnic, Ryan; Smith, Christopher P; Huard, Johnny; Chancellor, Michael B

    2003-11-01

    To study the physiologic outcome of allogenic transplant of muscle-derived progenitor cells (MDPCs) in the denervated female rat urethra. MDPCs were isolated from muscle biopsies of normal 6-week-old Sprague-Dawley rats and purified using the preplate technique. Sciatic nerve-transected rats were used as a model of stress urinary incontinence. The experimental group was divided into three subgroups: control, denervated plus 20 microL saline injection, and denervated plus allogenic MDPCs (1 to 1.5 x 10(6) cells) injection. Two weeks after injection, urethral muscle strips were prepared and underwent electrical field stimulation. The pharmacologic effects of d-tubocurare, phentolamine, and tetrodotoxin on the urethral strips were assessed by contractions induced by electrical field stimulation. The urethral tissues also underwent immunohistochemical staining for fast myosin heavy chain and CD4-activated lymphocytes. Urethral denervation resulted in a significant decrease of the maximal fast-twitch muscle contraction amplitude to only 8.77% of the normal urethra and partial impairment of smooth muscle contractility. Injection of MDPCs into the denervated sphincter significantly improved the fast-twitch muscle contraction amplitude to 87.02% of normal animals. Immunohistochemistry revealed a large amount of new skeletal muscle fiber formation at the injection site of the urethra with minimal inflammation. CD4 staining showed minimal lymphocyte infiltration around the MDPC injection sites. Urethral denervation resulted in near-total abolishment of the skeletal muscle and partial impairment of smooth muscle contractility. Allogenic MDPCs survived 2 weeks in sciatic nerve-transected urethra with minimal inflammation. This is the first report of the restoration of deficient urethral sphincter function through muscle-derived progenitor cell tissue engineering. MDPC-mediated cellular urethral myoplasty warrants additional investigation as a new method to treat stress urinary

  9. The putative bZIP transcription factor BzpN slows proliferation and functions in the regulation of cell density by autocrine signals in Dictyostelium.

    Directory of Open Access Journals (Sweden)

    Jonathan E Phillips

    Full Text Available The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN(- cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation.

  10. The Putative bZIP Transcripton Factor BzpN Slows Proliferation and Functions in the Regulation of Cell Density by Autocrine Signals in Dictyostelium

    Science.gov (United States)

    Phillips, Jonathan E.; Huang, Eryong; Shaulsky, Gad; Gomer, Richard H.

    2011-01-01

    The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN− cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation. PMID:21760904

  11. The putative bZIP transcription factor BzpN slows proliferation and functions in the regulation of cell density by autocrine signals in Dictyostelium.

    Science.gov (United States)

    Phillips, Jonathan E; Huang, Eryong; Shaulsky, Gad; Gomer, Richard H

    2011-01-01

    The secreted proteins AprA and CfaD function as autocrine signals that inhibit cell proliferation in Dictyostelium discoideum, thereby regulating cell numbers by a negative feedback mechanism. We report here that the putative basic leucine zipper transcription factor BzpN plays a role in the inhibition of proliferation by AprA and CfaD. Cells lacking BzpN proliferate more rapidly than wild-type cells but do not reach a higher stationary density. Recombinant AprA inhibits wild-type cell proliferation but does not inhibit the proliferation of cells lacking BzpN. Recombinant CfaD also inhibits wild-type cell proliferation, but promotes the proliferation of cells lacking BzpN. Overexpression of BzpN results in a reduced cell density at stationary phase, and this phenotype requires AprA, CfaD, and the kinase QkgA. Conditioned media from high-density cells stops the proliferation of wild-type but not bzpN(-) cells and induces a nuclear localization of a BzpN-GFP fusion protein, though this localization does not require AprA or CfaD. Together, the data suggest that BzpN is necessary for some but not all of the effects of AprA and CfaD, and that BzpN may function downstream of AprA and CfaD in a signal transduction pathway that inhibits proliferation.

  12. Quadruplexes in 'Dicty': crystal structure of a four-quartet G-quadruplex formed by G-rich motif found in the Dictyostelium discoideum genome.

    Science.gov (United States)

    Guédin, Aurore; Lin, Linda Yingqi; Armane, Samir; Lacroix, Laurent; Mergny, Jean-Louis; Thore, Stéphane; Yatsunyk, Liliya A

    2018-06-01

    Guanine-rich DNA has the potential to fold into non-canonical G-quadruplex (G4) structures. Analysis of the genome of the social amoeba Dictyostelium discoideum indicates a low number of sequences with G4-forming potential (249-1055). Therefore, D. discoideum is a perfect model organism to investigate the relationship between the presence of G4s and their biological functions. As a first step in this investigation, we crystallized the dGGGGGAGGGGTACAGGGGTACAGGGG sequence from the putative promoter region of two divergent genes in D. discoideum. According to the crystal structure, this sequence folds into a four-quartet intramolecular antiparallel G4 with two lateral and one diagonal loops. The G-quadruplex core is further stabilized by a G-C Watson-Crick base pair and a A-T-A triad and displays high thermal stability (Tm > 90°C at 100 mM KCl). Biophysical characterization of the native sequence and loop mutants suggests that the DNA adopts the same structure in solution and in crystalline form, and that loop interactions are important for the G4 stability but not for its folding. Four-tetrad G4 structures are sparse. Thus, our work advances understanding of the structural diversity of G-quadruplexes and yields coordinates for in silico drug screening programs and G4 predictive tools.

  13. Crystallization and preliminary X-ray crystallographic analysis of the NmrA-like DDB-G0286605 protein from the social amoeba Dictyostelium discoideum

    International Nuclear Information System (INIS)

    Kim, Min-Kyu; Yim, Hyung-Soon; Kang, Sa-Ouk

    2010-01-01

    In order to investigate its structure and function, the NmrA-like domain-containing DDB-G0286605 protein from D. discoideum was expressed, purified and crystallized. X-ray diffraction analysis is reported to a resolution of 1.64 Å. The DDB-G0286605 gene product from Dictyostelium discoideum, an NmrA-like protein that belongs to the short-chain dehydrogenase/reductase family, has been crystallized by the hanging-drop vapour-diffusion method at 295 K. A 1.64 Å resolution data set was collected using synchrotron radiation. The DDB-G0286605 protein crystals belonged to space group P2 1 , with unit-cell parameters a = 67.598, b = 54.935, c = 84.219 Å, β = 109.620°. Assuming the presence of two molecules in the asymmetric unit, the solvent content was estimated to be about 43.25% with 99% probability. Molecular-replacement trials were attempted with three NmrA-like proteins, NmrA, HSCARG and QOR2, as search models, but failed. This may be a consequence of the low sequence identity between the DDB-G0286605 protein and the search models (DDB-G0286605 has a primary-sequence identity of 28, 32 and 19% to NmrA, HCARG and QOR2, respectively)

  14. MicroRNAs in Amoebozoa: deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs.

    Science.gov (United States)

    Avesson, Lotta; Reimegård, Johan; Wagner, E Gerhart H; Söderbom, Fredrik

    2012-10-01

    The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.

  15. Uncovering a Role for the Tail of the Dictyostelium discoideum SadA Protein in Cell-Substrate Adhesion ▿ †

    Science.gov (United States)

    Kowal, Anthony S.; Chisholm, Rex L.

    2011-01-01

    Previous work from our laboratory showed that the Dictyostelium discoideum SadA protein plays a central role in cell-substrate adhesion. SadA null cells exhibit a loss of adhesion, a disrupted actin cytoskeleton, and a cytokinesis defect. How SadA mediates these phenotypes is unknown. This work addresses the mechanism of SadA function, dem