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Sample records for cytoplasmic vacuolization responses

  1. Gypenoside L, Isolated from Gynostemma pentaphyllum, Induces Cytoplasmic Vacuolation Death in Hepatocellular Carcinoma Cells through Reactive-Oxygen-Species-Mediated Unfolded Protein Response.

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    Zheng, Kai; Liao, Chenghui; Li, Yan; Fan, Xinmin; Fan, Long; Xu, Hong; Kang, Qiangrong; Zeng, Yong; Wu, Xuli; Wu, Haiqiang; Liu, Lizhong; Xiao, Xiaohua; Zhang, Jian; Wang, Yifei; He, Zhendan

    2016-03-02

    Exploring novel anticancer agents that can trigger non-apoptotic or non-autophagic cell death is urgent for cancer treatment. In this study, we screened and identified an unexplored anticancer activity of gypenoside L (Gyp-L) isolated from Gynostemma pentaphyllum. We showed that treatment with Gyp-L induces non-apoptotic and non-autophagic cytoplasmic vacuolation death in human hepatocellular carcinoma (HCC) cells. Mechanically, Gyp-L initially increased the intracellular reactive oxygen species (ROS) levels, which, in turn, triggered protein ubiquitination and unfolded protein response (UPR), resulting in Ca(2+) release from endoplasm reticulum (ER) inositol trisphosphate receptor (IP3R)-operated stores and finally cytoplasmic vacuolation and cell death. Interruption of the ROS-ER-Ca(2+) signaling pathway by chemical inhibitors significantly prevented Gyp-L-induced vacuole formation and cell death. In addition, Gyp-L-induced ER stress and vacuolation death required new protein synthesis. Overall, our works provide strong evidence for the anti-HCC activity of Gyp-L and suggest a novel therapeutic option by Gyp-L through the induction of a unconventional ROS-ER-Ca(2+)-mediated cytoplasmic vacuolation death in human HCC.

  2. Mitochondrial Extrusion through the cytoplasmic vacuoles during cell death.

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    Nakajima, Akihito; Kurihara, Hidetake; Yagita, Hideo; Okumura, Ko; Nakano, Hiroyasu

    2008-08-29

    Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

  3. The Cytoplasm-to-Vacuole Targeting Pathway: A Historical Perspective

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    Midori Umekawa

    2012-01-01

    Full Text Available From today's perspective, it is obvious that macroautophagy (hereafter autophagy is an important pathway that is connected to a range of developmental and physiological processes. This viewpoint, however, is relatively recent, coinciding with the molecular identification of autophagy-related (Atg components that function as the protein machinery that drives the dynamic membrane events of autophagy. It may be difficult, especially for scientists new to this area of research, to appreciate that the field of autophagy long existed as a “backwater” topic that attracted little interest or attention. Paralleling the development of the autophagy field was the identification and analysis of the cytoplasm-to-vacuole targeting (Cvt pathway, the only characterized biosynthetic route that utilizes the Atg proteins. Here, we relate some of the initial history, including some never-before-revealed facts, of the analysis of the Cvt pathway and the convergence of those studies with autophagy.

  4. Reactive oxygen species (ROS) is not a promotor of taxol-induced cytoplasmic vacuolization

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    Sun, Qingrui; Chen, Tongsheng

    2009-02-01

    we have previously reported that taxol, a potent anticancer agent, induces caspase-independent cell death and cytoplasmic vacuolization in human lung adenocarcinoma (ASTC-a-1) cells. However, the mechanisms of taxol-induced cytoplasmic vacuolization are poorly understood. Reactive oxygen species (ROS) has been reported to be involved in the taxol-induced cell death. Here, we employed confocal fluorescence microscopy imaging to explore the role of ROS in taxol-induced cytoplasmic vacuolization. We found that ROS inhibition by addition of N-acetycysteine (NAC), a total ROS scavenger, did not suppress these vacuolization but instead increased vacuolization. Take together, our results showed that ROS is not a promotor of the taxol-induced cytoplasmic vacuolization.

  5. Early cytoplasmic vacuolization of African green monkey kidney cells by SV40.

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    Miyamura, T; Kitahara, T

    1975-01-01

    As early as 3--4 hours after infection with SV40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10--20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by preincubating the virus with specific antiserum, or by heating the virus with MgCl2. The ECV could be induced by UV-irradiated SV40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV40 virion and the vacuolization is not associated with the replication of SV40.

  6. Early cytoplasmic vacuolization of African green monkey kidney cells by SV40. [uv radiation

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    Miyamura, T.; Kitahara, T.

    1975-01-01

    As early as 3 to 4 hours after infection with SV 40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10 to 20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV 40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by pre-incubating the virus with specific antiserum, or by heating the virus with MgCl/sub 2/. The ECV could be induced by uv-irradiated SV 40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV 40 virion and the vacuolization is not associated with the replication of SV 40.

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

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    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.

  8. Intratumoral injection of taxol in vivo suppresses A549 tumor showing cytoplasmic vacuolization.

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    Wang, Chaoyang; Chen, Tongsheng

    2012-04-01

    Based on our recent in vitro studies, this report was designed to explore the mechanism by which high concentration of taxol (70 µM) induced paraptosis-like cell death in human lung carcinoma (A549) cells, and to evaluate the therapeutic efficacy of taxol using A549 tumor-bearing mice in vivo. Exposure of cells to taxol induced time-dependent cytotoxicity and cytoplasmic vacuolization without the involvement of Bax, Bak, Mcl-1, Bcl-XL, and caspase-3. Although taxol treatment induced activating transcription factor 6 (ATF6) cleavage indicative of endoplasmic reticulum (ER) stress, silencing ATF6 by shATF6 did not prevent taxol-induced both cytotoxcity and cytoplasmic vacuolization, suggesting that taxol-induced cytoplasmic vacuolization and cell death were not due to ER stress. Moreover, taxol-treated cells did not show DNA fragmentation and loss of mitochondrial membrane potential, the typical characteristics of apoptosis. In addition, taxol-induced cytoplasmic vacuolization did not show the cellular lysis, the characteristics of oncosis, and positive of β-galactosidase, the characteristic of senescence, indicating that taxol induced paraptosis-like cell death is neither oncosis nor senescence. Moreover, our in vivo data showed that intratumoral injection of taxol (50 mg/kg) in A549 tumor xenograft mice on day 1 and day 19 potently suppressed tumor growth showing significant ER vacuolization without toxicity. In conclusion, high concentration of taxol exhibits a significant anticancer activity by inducing paraptosis-like cell death in vitro and in vivo, without significant toxicity, suggesting a promising therapeutic strategy for apoptosis-resistance cancer by inducing ER vacuolization.

  9. Mitochondrial Extrusion through the Cytoplasmic Vacuoles during Cell Death*S⃞

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    Nakajima, Akihito; Kurihara, Hidetake; Yagita, Hideo; Okumura, Ko; Nakano, Hiroyasu

    2008-01-01

    Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor α-induced cell death in a caspase-dependent f...

  10. The potential role of kinesin and dynein in Golgi scattering and cytoplasmic vacuole formation during acute experimental pancreatitis

    Institute of Scientific and Technical Information of China (English)

    Ina A Weber; Igor Buchwalow; Daniela Hahn; Wolfram Domschke; Markus M Lerch; Jürgen Schnekenburger

    2010-01-01

    @@ Dear Editor, Acute pancreatitis is initiated in pancreatic acinar cells and characterized by a profound disturbance in intracel-lular vesicle transport. Moreover, secretion of pancreatic zymogens is blocked, paralleled by the formation of cytoplasmic vacuoles with premature protease activation that precedes cellular necrosis and organ damage.

  11. "Autophagy suite": Atg9 cycling in the cytoplasm to vacuole targeting pathway.

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    Munakata, Nobuo; Klionsky, Daniel J

    2010-08-01

    Macroautophagy continues to gather increasing attention because it is connected with a wide range of human pathophysiologies, developmental processes and life span extension. It is also an interesting process from a basic cellular biology standpoint, as it involves dynamic membrane rearrangements and multiple protein-protein interactions. Although macroautophagy can be nonspecific, there are many examples of selective sequestration including pexophagy, mitophagy and the cytoplasm to vacuole targeting (Cvt) pathway. At present, the Cvt pathway is unique in that it is the only example of a biosynthetic use of macroautophagy. Most of the autophagy-related (Atg) proteins are involved in the Cvt pathway, and various types of analyses have placed these proteins at particular stages of the process. For example, Atg9 is the only characterized transmembrane protein that is absolutely required for Cvt vesicle formation, and it is proposed to carry membrane from peripheral donor sites to the phagophore assembly site where the vesicle forms. Additional proteins, including Atg11, Atg23 and Atg27 are involved in this anterograde movement, whereas Atg1-Atg13 and Atg2-Atg18 are required for the retrograde return to the peripheral sites. Even when we illustrate our understanding of these events in a schematic model, however, they are by necessity flat two-dimensional representations, lacking movement and sound. Yet the cell is a living entity that is not well served by this sole method of information display. Accordingly, we decided to present the Cvt pathway as a vibrant, dynamic process by combining science, music and illustration.

  12. Fluorescence imaging analysis of taxol-induced ASTC-a-1 cell death with cell swelling and cytoplasmic vacuolization

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    Chen, Tong-sheng; Sun, Lei; Wang, Longxiang; Wang, Huiying

    2008-02-01

    Taxol (Paclitaxel), an isolated component from the bark of the Pacific yew Taxus brevifolia, exhibits a broad spectrum of clinical activity against human cancers. Taxol can promote microtubule (MT) assembly, inhibit depolymerization, and change MT dynamics, resulting in disruption of the normal reorganization of the microtubule network required for mitosis and cell proliferation. However, the molecular mechanism of taxol-induced cell death is still unclear. In this report, CCK-8 was used to assay the inhibition of taxol on the human lung adenocarcinoma (ASTC-a-1) cells viability, confocal fluorescence microscope was used to monitor the morphology changes of cells with taxol treatment. We for the first time describe the characteristics of taxol-induced cells swelling, cytoplasmic vacuolization and cell death. Taxol induced swelling, cytoplasmatic vacuolization and cell death without cell shrinkage and membrane rupture. These features differ from those of apoptosis and resemble the paraptosis, a novel nonapoptotic PCD.

  13. Ubiquitin, a central component of selective cytoplasmic proteolysis, is linked to proteins residing at the locus of non-selective proteolysis, the vacuole

    NARCIS (Netherlands)

    Simeon, Angela; Klei, Ida J. van der; Veenhuis, Marten; Wolf, Dieter H.

    1992-01-01

    Ubiquitin, an evolutionary highly conserved protein, is known to be involved in selective proteolysis in the cytoplasm. Here we show that ubiquitin-protein conjugates are also found in the yeast vacuole. Mutants defective in the major vacuolar endopeptidases, proteinase yscA and yscB, lead to accumu

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

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    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.

  15. The nucleocytoplasmic microfilament network in protoplasts from cultured soybean cells is a plastic entity that pervades the cytoplasm except the central vacuole.

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    Villanueva, Marco A; Schindler, Melvin; Wang, John L

    2005-11-01

    The microfilament network of cultured Glycine max cells (SB-1 line), and protoplasts was visualized with rhodamine-phalloidin under conditions that lysed the protoplast and changed the cell shape. The whole cell had the typical microfilament distribution of a "cage" around the nucleus, from which the large subcortical cables and transvacuolar strands radiated towards the cortex until it reached the cortical microfilament network. Upon cell wall removal, the network conserved its compartmentalization. Thus, the redistribution of the shape where the vacuole becomes a central entity, made the cytoplasm displace peripherally, but the network distribution was conserved. When protoplasts were lysed in a low osmotic medium, the vacuoles were gradually released intact. Under these conditions, the F-actin staining remained within the ghost of the cell, but none was detected in either emerging or almost completely released vacuoles. Most of the released F-actin was found in debris from the cell lysate in the form of microfilaments. When the ghosts were constrained in a coverslip with an air bubble, the shape of the ghost changed accordingly, but the microfilament network distribution remained constant. These results provide further evidence that the vacuole of plant cells does not have detectable associated F-actin. In addition, we demonstrate that the actin microfilament network is a moldable entity that can change its shape but keeps its distribution under constant conditions, in these cultured cells.

  16. Protein dynamics and proteolysis in plant vacuoles.

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    Müntz, Klaus

    2007-01-01

    Plant cells cannot live without their vacuoles. The tissues and organs of a plant contain a wide variety of differentiated and specialized vacuoles -- even a single plant cell can possess two or more types of vacuoles. Vacuolar proteins are encoded by nuclear genes and synthesized in the cytoplasm. Their transport into the vacuolar compartment is under cytoplasmic control. Transcription seems to be a major control level for differential protein supply to the vacuoles. It is at this level that vacuole differentiation and functions are mainly integrated into cellular processes. Recycling amino acids generated by protein degradation is a major function of the vacuole. This is most evident when storage proteins are mobilized in storage tissues of generative or vegetative organs in order to nourish the embryo of germinating seeds or sprouting buds. When specific proteins are transferred to the vacuole for immediate degradation this compartment contributes to the adaptation of protein complexes in response to changes in developmental or environmental conditions. Vacuolar proteases are involved in protein degradation during reversible senescence and programmed cell death, which is also called irreversible senescence. Vacuoles contribute to defence against pathogens and herbivores by limited and unlimited proteolysis. Our present knowledge on functions and processes of vacuolar protein dynamics in plants is reviewed. Research perspectives are deduced.

  17. Gold nanoparticles administration induces disarray of heart muscle, hemorrhagic, chronic inflammatory cells infiltrated by small lymphocytes, cytoplasmic vacuolization and congested and dilated blood vessels

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    Abdelhalim Mohamed Anwar K

    2011-12-01

    Full Text Available Abstract Background Despite significant research efforts on cancer therapy, diagnostics and imaging, many challenges remain unsolved. There are many unknown details regarding the interaction of nanoparticles (NPs and biological systems. The structure and properties of gold nanoparticles (GNPs make them useful for a wide array of biological applications. However, for the application of GNPs in therapy and drug delivery, knowledge regarding their bioaccumulation and associated local or systemic toxicity is necessary. Information on the biological fate of NPs, including distribution, accumulation, metabolism, and organ specific toxicity is still minimal. Studies specifically dealing with the toxicity of NPs are rare. The aim of the present study was to investigate the effects of intraperitoneal administration of GNPs on histological alterations of the heart tissue of rats in an attempt to identify and understand the toxicity and the potential role of GNPs as a therapeutic and diagnostic tool. Methods A total of 40 healthy male Wistar-Kyoto rats received 50 μl infusions of 10, 20 and 50 nm GNPs for 3 or 7 days. Animals were randomly divided into groups: 6 GNP-treated rats groups and one control group (NG. Groups 1, 2 and 3 received infusions of 50 μl GNPs of size 10 nm (3 or 7 days, 20 nm (3 or 7 days and 50 nm (3 or 7 days, respectively. Results In comparison with the respective control rats, exposure to GNPs doses produced heart muscle disarray with a few scattered chronic inflammatory cells infiltrated by small lymphocytes, foci of hemorrhage with extravasation of red blood cells, some scattered cytoplasmic vacuolization and congested and dilated blood vessels. None of the above alterations were observed in the heart muscle of any member of the control group. Conclusions The alterations induced by intraperitoneal administration of GNPs were size-dependent, with smaller ones inducing greater affects, and were also related to the time exposure to

  18. Coxiella burnetii and Leishmania mexicana residing within similar parasitophorous vacuoles elicit disparate host responses

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    Jess A Millar

    2015-08-01

    Full Text Available Coxiella burnetii is a bacterium that thrives in an acidic parasitophorous vacuole (PV derived from lysosomes. Leishmania mexicana, a eukaryote, has also independently evolved to live in a morphologically similar PV. As Coxiella and Leishmania are highly divergent organisms that cause different diseases, we reasoned that their respective infections would likely elicit distinct host responses despite producing phenotypically similar parasite-containing vacuoles. The objective of this study was to investigate, at the molecular level, the macrophage response to each pathogen. Infection of THP-1 (human monocyte/macrophage cells with Coxiella and Leishmania elicited disparate host responses. At 5 days post-infection, when compared to uninfected cells, 1057 genes were differentially expressed (746 genes up- and 311 genes down-regulated in C. burnetii infected cells, whereas 698 genes (534 genes up- and 164 genes down-regulated were differentially expressed in L. mexicana infected cells. Interestingly, of the 1755 differentially expressed genes identified in this study, only 126 genes (~7% are common to both infections. We also discovered that 1090 genes produced mRNA isoforms at significantly different levels under the two infection conditions, suggesting that alternate proteins encoded by the same gene might have important roles in host response to each infection. Additionally, we detected 257 micro RNAs (miRNAs that were expressed in THP-1 cells and identified miRNAs that were specifically expressed during Coxiella or Leishmania infections. Collectively, this study identified host mRNAs and miRNAs that were influenced by Coxiella and/or Leishmania infections. Intriguingly, our data indicate that although their PVs are morphologically similar, Coxiella and Leishmania have evolved different strategies that perturb distinct host processes to create and thrive within their respective intracellular niches.

  19. Region of Nipah virus C protein responsible for shuttling between the cytoplasm and nucleus.

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    Horie, Ryo; Yoneda, Misako; Uchida, Shotaro; Sato, Hiroki; Kai, Chieko

    2016-10-01

    Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm.

  20. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response

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    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway. PMID:27252702

  1. Accessory Factors of Cytoplasmic Viral RNA Sensors Required for Antiviral Innate Immune Response.

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    Oshiumi, Hiroyuki; Kouwaki, Takahisa; Seya, Tsukasa

    2016-01-01

    Type I interferon (IFN) induces many antiviral factors in host cells. RIG-I-like receptors (RLRs) are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs) and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and, thus, cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  2. Accessory factors of cytoplasmic viral RNA sensors required for antiviral innate immune response

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    Hiroyuki eOshiumi

    2016-05-01

    Full Text Available Type I interferon (IFN induces many antiviral factors in host cells. RIG-I-like receptors (RLRs are cytoplasmic viral RNA sensors that trigger the signal to induce the innate immune response that includes type I IFN production. RIG-I and MDA5 are RLRs that form nucleoprotein filaments along viral double-stranded RNA, resulting in the activation of MAVS adaptor molecule. The MAVS protein forms a prion-like aggregation structure, leading to type I IFN production. RIG-I and MDA5 undergo post-translational modification. TRIM25 and Riplet ubiquitin ligases deliver a K63-linked polyubiquitin moiety to the RIG-I N-terminal caspase activation and recruitment domains (CARDs and C-terminal region; the polyubiquitin chain then stabilizes the two-CARD tetramer structure required for MAVS assembly. MDA5 activation is regulated by phosphorylation. RIOK3 is a protein kinase that phosphorylates the MDA5 protein in a steady state, and PP1α/γ dephosphorylate this protein, resulting in its activation. RIG-I and MDA5 require cytoplasmic RNA helicases for their efficient activation. LGP2, another RLR, is an RNA helicase involved in RLR signaling. This protein does not possess N-terminal CARDs and thus cannot trigger downstream signaling by itself. Recent studies have revealed that this protein modulates MDA5 filament formation, resulting in enhanced type I IFN production. Several other cytoplasmic RNA helicases are involved in RLR signaling. DDX3, DHX29, DHX36, and DDX60 RNA helicases have been reported to be involved in RLR-mediated type I IFN production after viral infection. However, the underlying mechanism is largely unknown. Future studies are required to reveal the role of RNA helicases in the RLR signaling pathway.

  3. Cell vacuolation induced by Haemophilus influenzae supernatants in HEp-2 cells

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    Maria del Rosario Espinoza-Mellado

    2013-12-01

    Full Text Available Haemophilus influenzae belongs to respiratory tract microbiota. We observed vacuoles formation in previous studies with H. influenzae culture supernatants, so in this work we characterised that cytotoxic effect. We observed an abundant production of acidic cytoplasmic vacuoles due to the presence of a “vacuolating factor” in H. influenzae supernatants which was characterised as thermolabile. Greatest vacuolating activity was observed when utilizing the fraction > 50 kDa. The presence of a large number of vacuoles in HEp-2 cells was verified by transmission electron microscopy and some vacuoles were identified with a double membrane and/or being surrounded by ribosomes. These results suggest similar behaviour to that of vacuolating effects described by autotransporter proteins an undescribed cytotoxic effect induced by H. influenzae .

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

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    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.

  5. Balanced nuclear and cytoplasmic activities of EDS1 are required for a complete plant innate immune response.

    Directory of Open Access Journals (Sweden)

    Ana V García

    Full Text Available An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat (NB-LRR receptors recognizing specific microbial effectors. Signaling from activated receptors of the TIR (Toll/Interleukin-1 Receptor-NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility1. In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes. EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus. By enhancing EDS1 export from inside nuclei (through attachment of an additional nuclear export sequence (NES or conditionally releasing EDS1 to the nucleus (by fusion to a glucocorticoid receptor (GR in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming. Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei. Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites. We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack.

  6. Balanced nuclear and cytoplasmic activities of EDS1 are required for a complete plant innate immune response.

    Science.gov (United States)

    García, Ana V; Blanvillain-Baufumé, Servane; Huibers, Robin P; Wiermer, Marcel; Li, Guangyong; Gobbato, Enrico; Rietz, Steffen; Parker, Jane E

    2010-07-01

    An important layer of plant innate immunity to host-adapted pathogens is conferred by intracellular nucleotide-binding/oligomerization domain-leucine rich repeat (NB-LRR) receptors recognizing specific microbial effectors. Signaling from activated receptors of the TIR (Toll/Interleukin-1 Receptor)-NB-LRR class converges on the nucleo-cytoplasmic immune regulator EDS1 (Enhanced Disease Susceptibility1). In this report we show that a receptor-stimulated increase in accumulation of nuclear EDS1 precedes or coincides with the EDS1-dependent induction and repression of defense-related genes. EDS1 is capable of nuclear transport receptor-mediated shuttling between the cytoplasm and nucleus. By enhancing EDS1 export from inside nuclei (through attachment of an additional nuclear export sequence (NES)) or conditionally releasing EDS1 to the nucleus (by fusion to a glucocorticoid receptor (GR)) in transgenic Arabidopsis we establish that the EDS1 nuclear pool is essential for resistance to biotrophic and hemi-biotrophic pathogens and for transcriptional reprogramming. Evidence points to post-transcriptional processes regulating receptor-triggered accumulation of EDS1 in nuclei. Changes in nuclear EDS1 levels become equilibrated with the cytoplasmic EDS1 pool and cytoplasmic EDS1 is needed for complete resistance and restriction of host cell death at infection sites. We propose that coordinated nuclear and cytoplasmic activities of EDS1 enable the plant to mount an appropriately balanced immune response to pathogen attack.

  7. Localization of acid hydrolases in protoplasts. Examination of the proposed lysosomal function of the mature vacuole

    Energy Technology Data Exchange (ETDEWEB)

    Butcher, H.C.; Wagner, G.J.; Siegelman, H.W.

    1977-06-01

    The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, ..beta..-galactosidase, ..cap alpha..-glycosidase and ..beta..-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome.

  8. The peripheral cytoplasm of adrenocortical cells: zone-specific responses to ACTH.

    Science.gov (United States)

    Loesser, K E; Cain, L D; Malamed, S

    1994-05-01

    Differences in the cytoskeletal protein actin in cells from the zona glomerulosa and zona fasciculata would be of considerable interest because there is persuasive evidence that rat corticosteroids are secreted by mechanisms that are somewhat zone-specific. We have previously shown evidence that actin may be involved in steroid secretion, possibly in connection with changes in adrenocortical microvilli. However, the cells upon which the data were based were not separated according to zone of origin. Immunogold electron microscopy and morphometric procedures were used to determine whether ACTH-induced changes in the peripheral cytoplasm of isolated adrenocortical cells occur in both zona fasciculata and zona glomerulosa cells. Actin immunoreactivity was more concentrated in the cytoplasm adjacent to the plasma membrane (including the cytoplasm within the microvilli) than it was in the internal cytoplasm in cells from both zones (4-6 times more concentrated in zona glomerulosa cells and 3-6 times more concentrated in zona fasciculata cells). However, the mean aggregate microvillar surface length (microvillar index) of untreated zona fasciculata cells (previously reported (Loesser and Malamed, 1987)) was 23% greater than that of untreated zona glomerulosa cells. Although ACTH (at a maximal steroidogenic concentration) had no effect on the peripheral cytoplasmic actin concentration of zona glomerulosa cells, there was a 24% increase in the aggregate microvillar length. In contrast, in zona fasciculata cells, ACTH treatment was accompanied by an increase in peripheral cytoplasmic actin concentration of 58-64% and an increase in aggregate microvillar surface length of 40% (previously reported (Loesser and Malamed, 1987)), almost twice that for zona glomerulosa cells. The results suggest that ACTH-induced hormone release from zona fasciculata cells is mediated by increases in peripheral cytoplasmic actin and aggregate microvillar length; in zona glomerulosa cells such

  9. Effects of stereochemistry, saturation, and hydrocarbon chain length on the ability of synthetic constrained azacyclic sphingolipids to trigger nutrient transporter down-regulation, vacuolation, and cell death.

    Science.gov (United States)

    Perryman, Michael S; Tessier, Jérémie; Wiher, Timothy; O'Donoghue, Heather; McCracken, Alison N; Kim, Seong M; Nguyen, Dean G; Simitian, Grigor S; Viana, Matheus; Rafelski, Susanne; Edinger, Aimee L; Hanessian, Stephen

    2016-09-15

    Constrained analogs containing a 2-hydroxymethylpyrrolidine core of the natural sphingolipids sphingosine, sphinganine, N,N-dimethylsphingosine and N-acetyl variants of sphingosine and sphinganine (C2-ceramide and dihydro-C2-ceramide) were synthesized and evaluated for their ability to down-regulate nutrient transporter proteins and trigger cytoplasmic vacuolation in mammalian cells. In cancer cells, the disruptions in intracellular trafficking produced by these sphingolipids lead to cancer cell death by starvation. Structure activity studies were conducted by varying the length of the hydrocarbon chain, the degree of unsaturation and the presence or absence of an aryl moiety on the appended chains, and stereochemistry at two stereogenic centers. In general, cytotoxicity was positively correlated with nutrient transporter down-regulation and vacuolation. This study was intended to identify structural and functional features in lead compounds that best contribute to potency, and to develop chemical biology tools that could be used to isolate the different protein targets responsible for nutrient transporter loss and cytoplasmic vacuolation. A molecule that produces maximal vacuolation and transporter loss is expected to have the maximal anti-cancer activity and would be a lead compound.

  10. Histone H2B-IFI16 Recognition of Nuclear Herpesviral Genome Induces Cytoplasmic Interferon-β Responses

    Science.gov (United States)

    Iqbal, Jawed; Ansari, Mairaj Ahmed; Kumar, Binod; Dutta, Dipanjan; Roy, Arunava; Chikoti, Leela; Pisano, Gina; Dutta, Sujoy; Veettil, Mohanan Valiya; Chandran, Bala

    2016-01-01

    IFI16 (gamma-interferon-inducible protein 16), a predominantly nuclear protein involved in transcriptional regulation, also functions as an innate immune response DNA sensor and induces the IL-1β and antiviral type-1 interferon-β (IFN-β) cytokines. We have shown that IFI16, in association with BRCA1, functions as a sequence independent nuclear sensor of episomal dsDNA genomes of KSHV, EBV and HSV-1. Recognition of these herpesvirus genomes resulted in IFI16 acetylation, BRCA1-IFI16-ASC-procaspase-1 inflammasome formation, cytoplasmic translocation, and IL-1β generation. Acetylated IFI16 also interacted with cytoplasmic STING and induced IFN-β. However, the identity of IFI16 associated nuclear proteins involved in STING activation and the mechanism is not known. Mass spectrometry of proteins precipitated by anti-IFI16 antibodies from uninfected endothelial cell nuclear lysate revealed that histone H2B interacts with IFI16. Single and double proximity ligation microscopy, immunoprecipitation, EdU-genome labeled virus infection, and chromatin immunoprecipitation studies demonstrated that H2B is associated with IFI16 and BRCA1 in the nucleus in physiological conditions. De novo KSHV and HSV-1 infection as well as latent KSHV and EBV infection induces the cytoplasmic distribution of H2B-IFI16, H2B-BRCA1 and IFI16-ASC complexes. Vaccinia virus (dsDNA) cytoplasmic replication didn’t induce the redistribution of nuclear H2B-IFI16 or H2B into the cytoplasm. H2B is critical in KSHV and HSV-1 genome recognition by IFI16 during de novo infection. Viral genome sensing by IFI16-H2B-BRCA1 leads to BRCA1 dependent recruitment of p300, and acetylation of H2B and IFI16. BRCA1 knockdown or inhibition of p300 abrogated the acetylation of H2B-IFI16 or H2B. Ran-GTP protein mediated the translocation of acetylated H2B and IFI16 to the cytoplasm along with BRCA1 that is independent of IFI16-ASC inflammasome. ASC knockdown didn’t affect the acetylation of H2B, its cytoplasmic

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

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    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

  12. Complete mitochondrial genome sequence and identification of a candidate gene responsible for cytoplasmic male sterility in radish (Raphanus sativus L.) containing DCGMS cytoplasm.

    Science.gov (United States)

    Park, Jee Young; Lee, Young-Pyo; Lee, Jonghoon; Choi, Beom-Soon; Kim, Sunggil; Yang, Tae-Jin

    2013-07-01

    A novel cytoplasmic male sterility (CMS) conferred by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its restorer-of-fertility gene (Rfd1) was previously reported in radish (Raphanus sativus L.). Its inheritance of fertility restoration and profiles of mitochondrial DNA (mtDNA)-based molecular markers were reported to be different from those of Ogura CMS, the first reported CMS in radish. The complete mitochondrial genome sequence (239,186 bp; GenBank accession No. KC193578) of DCGMS mitotype is reported in this study. Thirty-four protein-coding genes and three ribosomal RNA genes were identified. Comparative analysis of a mitochondrial genome sequence of DCGMS and previously reported complete sequences of normal and Ogura CMS mitotypes revealed various recombined structures of seventeen syntenic sequence blocks. Short-repeat sequences were identified in almost all junctions between syntenic sequence blocks. Phylogenetic analysis of three radish mitotypes showed that DCGMS was more closely related to the normal mitotype than to the Ogura mitotype. A single 1,551-bp unique region was identified in DCGMS mtDNA sequences and a novel chimeric gene, designated orf463, consisting of 128-bp partial sequences of cox1 gene and 1,261-bp unidentified sequences were found in the unique region. No other genes with a chimeric structure, a major feature of most characterized CMS-associated genes in other plant species, were found in rearranged junctions of syntenic sequence blocks. Like other known CMS-associated mitochondrial genes, the predicted gene product of orf463 contained 12 transmembrane domains. Thus, this gene product might be integrated into the mitochondrial membrane. In total, the results indicate that orf463 is likely to be a casual factor for CMS induction in radish containing the DCGMS cytoplasm.

  13. Vps1 in the late endosome-to-vacuole traffic

    Indian Academy of Sciences (India)

    Jacob Hayden; Michelle Williams; Ann Granich; Hyoeun Ahn; Brandon Tenay; Joshua Lukehart; Chad Highfill; Sarah Dobard; Kyoungtae Kim

    2013-03-01

    Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.

  14. Ultrastructural and autoradiographic studies of the role of nucleolar vacuoles in soybean root meristem.

    Directory of Open Access Journals (Sweden)

    Dariusz Stepiński

    2004-03-01

    Full Text Available Ultrastructural and autoradiographic studies of nucleoli in soybean root meristematic cells in seedlings: (1 grown for 3 days at 25 degrees C (control, (2 grown for three days at 25 degrees C and for 4 days at 10 degrees C, and (3 grown as in (2 and recovered for 1 day at 25 degrees C were carried out. Control nucleoli had dense structure and a few small nucleolar vacuoles. Chilled plant nucleoli had less dense structure and no vacuoles. Nucleoli of plants recovered at 25 degrees C had big nucleolar vacuoles. In autoradiograms of squashed preparations, the labeling of nucleoli and cytoplasm after 20-min incubation in 3H-uridine was 5- and 6-fold stronger, respectively, in control than in chilled roots. Following recovery, the labeling of nucleoli and cytoplasm was much stronger than after chilling or even than in control roots. After 80-min postincubation in non-radioactive medium, average labeling of particular areas of cells was the highest in recovered plants which indicated intensification of rRNA synthesis, maturation and transport into cytoplasm resulting from the resumption of optimal conditions which was correlated with the appearance of big nucleolar vacuoles. In autoradiograms of semi-thin sections from roots of seedlings chilled for 4 days then recovered and incubated for 20 min in 3H-uridine, practically only extravacuolar parts of nucleoli were labeled. After 80-min postincubation, the labeling of nucleolar vacuoles was observed. Thus, during postincubation the labeled molecules were translocated from the nucleolar periphery into nucleolar vacuoles in cells where intensive transport of these molecules to the cytoplasm takes place. On the basis of these results, a hypothesis has been put forward that nucleolar vacuoles may be involved in the intensification of pre-ribosome transport outside nucleolus.

  15. Wolbachia age-sex-specific density in Aedes albopictus: a host evolutionary response to cytoplasmic incompatibility?

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    Pablo Tortosa

    Full Text Available BACKGROUND: Wolbachia bacteria have invaded many arthropod species by inducing Cytoplasmic Incompatibility (CI. These symbionts represent fascinating objects of study for evolutionary biologists, but also powerful potential biocontrol agents. Here, we assess the density dynamics of Wolbachia infections in males and females of the mosquito Aedes albopitcus, an important vector of human pathogens, and interpret the results within an evolutionary framework. METHODOLOGY/PRINCIPAL FINDINGS: Wolbachia densities were measured in natural populations and in age controlled mosquitoes using quantitative PCR. We show that the density dynamics of the wAlbA Wolbachia strain infecting Aedes albopictus drastically differ between males and females, with a very rapid decay of infection in males only. CONCLUSIONS/SIGNIFICANCE: Theory predicts that Wolbachia and its hosts should cooperate to improve the transmission of infection to offspring, because only infected eggs are protected from the effects of CI. However, incompatible matings effectively lower the fertility of infected males, so that selection acting on the host genome should tend to reduce the expression of CI in males, for example, by reducing infection density in males before sexual maturation. The rapid decay of one Wolbachia infection in Aedes albopictus males, but not in females, is consistent with this prediction. We suggest that the commonly observed reduction in CI intensity with male age reflects a similar evolutionary process. Our results also highlight the importance of monitoring infection density dynamics in both males and females to assess the efficiency of Wolbachia-based control strategies.

  16. Cytoplasmic LSM-1 protein regulates stress responses through the insulin/IGF-1 signaling pathway in Caenorhabditis elegans.

    Science.gov (United States)

    Cornes, Eric; Porta-De-La-Riva, Montserrat; Aristizábal-Corrales, David; Brokate-Llanos, Ana María; García-Rodríguez, Francisco Javier; Ertl, Iris; Díaz, Mònica; Fontrodona, Laura; Reis, Kadri; Johnsen, Robert; Baillie, David; Muñoz, Manuel J; Sarov, Mihail; Dupuy, Denis; Cerón, Julián

    2015-09-01

    Genes coding for members of the Sm-like (LSm) protein family are conserved through evolution from prokaryotes to humans. These proteins have been described as forming homo- or heterocomplexes implicated in a broad range of RNA-related functions. To date, the nuclear LSm2-8 and the cytoplasmic LSm1-7 heteroheptamers are the best characterized complexes in eukaryotes. Through a comprehensive functional study of the LSm family members, we found that lsm-1 and lsm-3 are not essential for C. elegans viability, but their perturbation, by RNAi or mutations, produces defects in development, reproduction, and motility. We further investigated the function of lsm-1, which encodes the distinctive protein of the cytoplasmic complex. RNA-seq analysis of lsm-1 mutants suggests that they have impaired Insulin/IGF-1 signaling (IIS), which is conserved in metazoans and involved in the response to various types of stress through the action of the FOXO transcription factor DAF-16. Further analysis using a DAF-16::GFP reporter indicated that heat stress-induced translocation of DAF-16 to the nuclei is dependent on lsm-1. Consistent with this, we observed that lsm-1 mutants display heightened sensitivity to thermal stress and starvation, while overexpression of lsm-1 has the opposite effect. We also observed that under stress, cytoplasmic LSm proteins aggregate into granules in an LSM-1-dependent manner. Moreover, we found that lsm-1 and lsm-3 are required for other processes regulated by the IIS pathway, such as aging and pathogen resistance.

  17. Effective cytoplasmic release of siRNA from liposomal carriers by controlling the electrostatic interaction of siRNA with a charge-invertible peptide, in response to cytoplasmic pH

    Science.gov (United States)

    Itakura, Shoko; Hama, Susumu; Matsui, Ryo; Kogure, Kentaro

    2016-05-01

    Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is effectively released via electrostatic repulsion of siRNA with negatively charged SAPSP at cytoplasmic pH (7.4). The condensed complex of siRNA and positively-charged SAPSP at acidic pH (siRNA/SAPSP) was found to result in almost complete release of siRNA upon charge inversion of SAPSP at pH 7.4, with the resultant negatively-charged SAPSP having no undesirable interactions with endogenous mRNA. Moreover, liposomes encapsulating siRNA/SAPSP demonstrated knockdown efficiencies comparable to those of commercially available siRNA carriers. Taken together, SAPSP may be very useful as a siRNA condenser, as it facilitates effective cytoplasmic release of siRNA, and subsequent induction of specific RNAi effects.Condensing siRNA with cationic polymers is a major strategy used in the development of siRNA carriers that can avoid degradation by nucleases and achieve effective delivery of siRNA into the cytoplasm. However, ineffective release of siRNA from such condensed forms into the cytoplasm is a limiting step for induction of RNAi effects, and can be attributed to tight condensation of siRNA with the cationic polymers, due to potent electrostatic interactions. Here, we report that siRNA condensed with a slightly acidic pH-sensitive peptide (SAPSP), whose total charge is inverted from positive to negative in response to cytoplasmic pH, is

  18. Vacuole-targeting fungicidal activity of amphotericin B

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    Akira eOgita

    2012-03-01

    Full Text Available Invasive fungal infections are recognized as major threats to patients with immune depression as well as those with cancer chemotherapy. Amphotericin B (AmB, a classical antifungal agent with a polyene macrolide structure, is widely used for the control of serious fungal infections. However, the clinical use of this antibiotic is limited by the treatment-associated side effects and the appearance of resistant strains. AmB lethality has been generally elucidated by the alteration of plasma membrane ion permeability due to its specific binding to plasma membrane ergosterol. While, the recent studies with Saccharomyces cerevisiae and Candida albicans reveals the vacuole disruptive action as another cause of AmB lethality on the basis of its marked amplification in combination with allicin, an allyl sulfur compound from garlic. Indeed, AmB causes a serious structural damage to the vacuole membrane at a lethal concentration, and even at a non-lethal concentration in combination with allicin. Such an enhancement effect of allicin is dependent on an inhibition of ergosterol-trafficking from the plasma membrane to the vacuole membrane, which is considered to be a cellular response to protect against the vacuole membrane disintegration. Allicin can also decrease the minimum fungicidal concentration of AmB against the pathogenic fungi C. albicans and Aspergillus fumigatus, as is the case of S. cerevisiae. The synergistic fungicidal activities of AmB and allicin may have significant implications in the development of the vacuole-targeting chemotherapy against fungal infections.

  19. Nucleo-cytoplasmic communication in apoptotic response to genotoxic and inflammatory stress

    Institute of Scientific and Technical Information of China (English)

    Jean Y. J. WANG

    2005-01-01

    Genotoxic agents or inflammatory cytokines activate cellular stress responses and trigger programmed cell death.We have identified a signal transduction module, including three nuclear proteins that participate in the regulation of cell death induced by chemotherapeutic agents and tumor necrosis factor (TNF). In this nuclear signaling module, retinoblastoma protein (Rb) functions as an inhibitor of apoptotic signal transduction. Inactivation of Rb by phosphorylation or caspase-dependent cleavage/degradation is required for cell death to occur. Rb inhibits the Abl tyrosine kinase. Thus,Rb inactivation is a pre-requisite for Abl activation by DNA damage or TNF. Activation of nuclear Abl and its downstream effector p73 induces mitochondriadependent cell death. The involvement of these nuclear signal transducers in TNF induced apoptosis, which does not require new gene expression, indicates that nuclear events other than transcription can contribute to extrinsic apoptotic signal transduction.

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

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    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.

  1. Cytoplasmic Male Sterility in Maize

    Institute of Scientific and Technical Information of China (English)

    RONG Ting-zhao; LI Wan-chen; CAO Mo-ju; HU Chang-yuan

    2002-01-01

    14 isoplasmic and allonuclear cytoplasmic male sterile lines were used as female parents, 8 tester lines as male parents, 101 F1 progenies were obtained. Fertility restoration response of 101 F1 progenies were investigated through field observation and pollen stainability examination under microscope. 14 isoplasmic and allonuclear cytoplasmic male sterile lines were developed by repeated backcross with recurrent male parent lines for more than 8 generations. The result shows: tester line Zifeng1 not only restored the isoplasmic and allonuclear sterile lines of group C backcrossed with Mo17, Yu30 and Heer, but also completely restored the isoplasmic and allonuclear cytoplasm male sterile lines of group T backcrossed with Mo17, HZS , 1792 ,292 and Yu30. Therefore, nuclear background limits the use of Zifeng1 as a tester for identification of cytoplasmic male sterility. Furthermore RFLPs of mitochondrial DNA of 6 isonuclear and alloplasmic cytoplasmic male sterile lines were analyzed with Bam H Ⅰ and Hind Ⅲ restriction endonuclease and mitochondrial DNA probes pBcmH3 and Cox Ⅱ. The same RFLPs were found within sterile cytoplasm of group C, including C,Chuan G, Lei 2 and Lei 3, but a different RFLP pattern was observed among sterile cytoplasm of group S, C,T and the normal cytoplasm. This result suggested that the RFLP markers tightly linked to sterile mitochondrial genes of different groups could be applied in the identifcation of cytoplasmic male sterility.

  2. Calcium Signals from the Vacuole

    Directory of Open Access Journals (Sweden)

    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.

  3. Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation.

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    Krishna Samanta

    Full Text Available Ca2+ flux into mitochondria is an important regulator of cytoplasmic Ca2+ signals, energy production and cell death pathways. Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.

  4. Mitochondrial calcium uniporter MCU supports cytoplasmic Ca2+ oscillations, store-operated Ca2+ entry and Ca2+-dependent gene expression in response to receptor stimulation.

    Science.gov (United States)

    Samanta, Krishna; Douglas, Sophie; Parekh, Anant B

    2014-01-01

    Ca2+ flux into mitochondria is an important regulator of cytoplasmic Ca2+ signals, energy production and cell death pathways. Ca2+ uptake can occur through the recently discovered mitochondrial uniporter channel (MCU) but whether the MCU is involved in shaping Ca2+ signals and downstream responses to physiological levels of receptor stimulation is unknown. Here, we show that modest stimulation of leukotriene receptors with the pro-inflammatory signal LTC4 evokes a series of cytoplasmic Ca2+ oscillations that are rapidly and faithfully propagated into mitochondrial matrix. Knockdown of MCU or mitochondrial depolarisation, to reduce the driving force for Ca2+ entry into the matrix, prevents the mitochondrial Ca2+ rise and accelerates run down of the oscillations. The loss of cytoplasmic Ca2+ oscillations appeared to be a consequence of enhanced Ca2+-dependent inactivation of InsP3 receptors, which arose from the loss of mitochondrial Ca2+ buffering. Ca2+ dependent gene expression in response to leukotriene receptor activation was suppressed following knockdown of the MCU. In addition to buffering Ca2+ release, mitochondria also sequestrated Ca2+ entry through store-operated Ca2+ channels and this too was prevented following loss of MCU. MCU is therefore an important regulator of physiological pulses of cytoplasmic Ca2+.

  5. Vibrio cholerae O1 Strains of Different Ribotypes have Similar hlyA RFLP Patterns but Different Vacuolating Ability

    Directory of Open Access Journals (Sweden)

    Jorge E. Vidal

    2007-01-01

    Full Text Available Extensive cytoplasmic vacuolation on Vero and HeLa cells in vitro by the Vibrio cholerae pore forming toxin HlyA, has been previously reported by our group. Vibrio cholerae O1 and non-O1 pathogenic strains show differences in the potential to induce vacuolation, here we study occurring variations on vacuolating cytotoxic ability, related to changes in the nucleotide sequence of the hlyA-orf. A collection of eight toxigenic strains of V. cholerae O1 El Tor and a non-toxigenic one, all belonging to different ribotypes was tested for their vacuolating ability, and hlyA-orf similarity based on PCR and RFLPs. The strains had extremely different vacuolating capacities, those from the ribotype 2 isolated from the US Gulf Coast, showed the highest vacuolating titer (10240 dil, and the rest of the collection had considerably lower titers ranging among 40 to 360 dilutions. PCR of hlyA-orf, was performed and RFLPs were generated using seven restriction enzymes, this approach later revealed small changes of restriction maps, among the strains. The phenogram constructed from the RFLPs, showed two major branches, one of them included most of the strains, the other separates the only Mexican wild type non-O1 Vibrio cholerae. To test for vacuolating ability out of the Vibrio genetic context, the amplified hlyA-orfs from the collection of strains were cloned in pGEMT- vector system and supernatants from the recombinant E coli DH5-, showed no differences on vacuolating titers, the clones always were low producers. Results from the cloning, together with those from the phenogram indicated that the hlyA gene is mainly conserved and the differences on vacuolating activity are unrelated to minute changes seen in the hlyA-orf. Production of high vacuolating titers on Vibrio strains could be due to transcriptional regulation. Whether the high vacuolating titer would be related to increased virulence, is still to be found.

  6. The role of suppression of p38 MAPK in cellular vacuole formation%阻断p38丝裂原活化蛋白激酶在细胞空泡形成中的作用

    Institute of Scientific and Technical Information of China (English)

    张春燕; 冯春红; 敬健雄; 段春燕; 刘友平; 夏先明; 李洪; 代荣阳; 陈绍坤

    2014-01-01

    目的:探讨p38丝裂原活化蛋白激酶(p38MAPK)通路与细胞空泡形成的关系。方法应用茴香霉素、放线菌酮、p38MAPK抑制剂SB203580、JNK抑制剂SP600125处理HepG2、LM3、QBC939、Hela和A549细胞,光学显微镜和激光共聚焦显微镜观察细胞空泡化情况;Westernblot法检测p38MAPK等通路相关分子的表达水平;内质网红色荧光探针标记内质网,激光共聚焦显微镜观察内质网结构变化;溶酶体红色荧光探针标记溶酶体,激光共聚焦显微镜观察溶酶体荧光染色情况。结果(1)茴香霉素对HepG2细胞空泡有消除作用。(2)茴香霉素通过活化p38MAPK消除细胞空泡。(3)阻断p38MAPK诱导多种肿瘤细胞空泡形成。(4)阻断p38MAPK介导的空泡形成破坏内质网结构的整体性。(5)阻断p38MAPK介导的空泡形成具有可逆性。结论p38MAPK通路在调节细胞空泡形成中发挥了重要作用。%Objective To investigate the role of the p38 MAPK pathway in the formation of cytoplasmic vacuoles .Methods Af-ter treated with Anisomycin ,SB203580 or SP600125 ,images of HepG2 ,LM3 ,QBC939 ,Hela and A549 cells were recorded by light microscopy and taken at a magnification of 400 × .The effects of anisomycin ,SB203580 and SP600125 on the activity of p38 and JNK were measured by Western blot .LM3 and A549 cells were stained with the ER-tracker red and the lyso-tracker red and subjec-ted to confocal microscopy analysis .Results (1)Anisomycin could abolish cytoplasmic vacuolization of HepG2 cells .(2)p38 MAPK activation was responsible for anisomycin-induced cytoplasmic vacuolization abolishment .(3)p38 MAPK blocking initiated cytoplas-mic vacuoles formation in various cancer cell lines .(4)p38 MAPK blocking-induced cytoplasmic vacuoles disrupted the integrity of endoplasmic reticulum .(5)p38 MAPK blocking reversibly induced cytoplasmic vacuoles formation .Conclusion These observations provide direct evidence for a

  7. Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance.

    Science.gov (United States)

    Chan, Yee-Hung M; Reyes, Lorena; Sohail, Saba M; Tran, Nancy K; Marshall, Wallace F

    2016-05-09

    It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model.

  8. Effect of the Vacuolation of Helicobacter Pylori

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cytotoxic test in vitro combined with cytochemical stain, fluorescent stain, transmission electronmicrograph was used to study the vacuolated effect by helicobacter pylori (H.pylori) (Toxin+) and its pathological mechanism. 78.26 % patients with peptic ulcer associated with H.pylori was infected with H.pylori (Toxin+), while 42.86 % patients with gastritis was infected with H.pylori (Toxin+). It was positive in vacuole with acridine orange and acid phosphatase stain. Transmission electronmicrograph of vacuole revealed the presence of abounding membrane. There was a closed relationship between infection with H.pylori (Toxin+) and peptic ulcer disease. The vacuole induced by H.pylori (Toxin+) was autophagosome, which was pathological phenomenon induced by toxin.

  9. The virulence protein SopD2 regulates membrane dynamics of Salmonella-containing vacuoles.

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    Nina Schroeder

    Full Text Available Salmonella enterica serovar Typhimurium is a Gram-negative bacterial pathogen causing gastroenteritis in humans and a systemic typhoid-like illness in mice. The capacity of Salmonella to cause diseases relies on the establishment of its intracellular replication niche, a membrane-bound compartment named the Salmonella-containing vacuole (SCV. This requires the translocation of bacterial effector proteins into the host cell by type three secretion systems. Among these effectors, SifA is required for the SCV stability, the formation of Salmonella-induced filaments (SIFs and plays an important role in the virulence of Salmonella. Here we show that the effector SopD2 is responsible for the SCV instability that triggers the cytoplasmic release of a sifA(- mutant. Deletion of sopD2 also rescued intra-macrophagic replication and increased virulence of sifA(- mutants in mice. Membrane tubular structures that extend from the SCV are the hallmark of Salmonella-infected cells. Until now, these unique structures have not been observed in the absence of SifA. The deletion of sopD2 in a sifA(- mutant strain re-established membrane trafficking from the SCV and led to the formation of new membrane tubular structures, the formation of which is dependent on other Salmonella effector(s. Taken together, our data demonstrate that SopD2 inhibits the vesicular transport and the formation of tubules that extend outward from the SCV and thereby contributes to the sifA(- associated phenotypes. These results also highlight the antagonistic roles played by SopD2 and SifA in the membrane dynamics of the vacuole, and the complex actions of SopD2, SifA, PipB2 and other unidentified effector(s in the biogenesis and maintenance of the Salmonella replicative niche.

  10. 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.

  11. Burkholderia cepacia complex isolates survive intracellularly without replication within acidic vacuoles of Acanthamoeba polyphaga.

    Science.gov (United States)

    Lamothe, Julie; Thyssen, Sandra; Valvano, Miguel A

    2004-12-01

    We have previously demonstrated that isolates of the Burkholderia cepacia complex can survive intracellularly in murine macrophages and in free-living Acanthamoeba. In this work, we show that the clinical isolates B. vietnamiensis strain CEP040 and B. cenocepacia H111 survived but did not replicate within vacuoles of A. polyphaga. B. cepacia-containing vacuoles accumulated the fluid phase marker Lysosensor Blue and displayed strong blue fluorescence, indicating that they had low pH. In contrast, the majority of intracellular bacteria within amoebae treated with the V-ATPse inhibitor bafilomycin A1 localized in vacuoles that did not fluoresce with Lysosensor Blue. Experiments using bacteria fluorescently labelled with chloromethylfluorescein diacetate demonstrated that intracellular bacteria remained viable for at least 24 h. In contrast, Escherichia coli did not survive within amoebae after 2 h post infection. Furthermore, intracellular B. vietnamiensis CEP040 retained green fluorescent protein within the bacterial cytoplasm, while this protein rapidly escaped from the cytosol of phagocytized heat-killed bacteria into the vacuolar lumen. Transmission electron microscopy analysis confirmed that intracellular Burkholderia cells were structurally intact. In addition, both Legionella pneumophila- and B. vietnamiensis-containing vacuoles did not accumulate cationized ferritin, a compound that localizes within the lysosome. Thus, our observations support the notion that B. cepacia complex isolates can use amoebae as a reservoir in the environment by surviving without intracellular replication within an acidic vacuole that is distinct from the lysosomal compartment.

  12. Primary observations of the existence of Fas-like cytoplasmic death factor in plant cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The main activity of Fas is to trigger cytoplasm death program in animal cells. In G2 pea, vacuole plays a pivotal role in inducing cell death in the cytoplasm of longday (LD) grown apical meristem cells. Expression patterns of the Fas in G2 pea cells revealed that the Fas is mainly localized in the vacuole of cells undergoing programmed cell death (PCD). The Fas expression is corresponding to the initiation of menadione-induced PCD in tobacco protoplasts.The results suggest the existence of the Fas-like mediated cytoplasmic death pathway in plant cells.``

  13. Herpesvirus Genome Recognition Induced Acetylation of Nuclear IFI16 Is Essential for Its Cytoplasmic Translocation, Inflammasome and IFN-β Responses.

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    Mairaj Ahmed Ansari

    2015-07-01

    Full Text Available The IL-1β and type I interferon-β (IFN-β molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16 involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1β and IFN-β production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1 episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1β production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-β production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1β production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the

  14. Cytoplasmic continuity revisited: closure of septa of the filamentous fungus Schizophyllum commune in response to environmental conditions.

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    Arend F van Peer

    Full Text Available BACKGROUND: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. CONCLUSIONS/SIGNIFICANCE: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous per se.

  15. Photoreactivation of a Cytoplasmic Virus

    Science.gov (United States)

    Pfefferkorn, E. R.; Boyle, Mary K.

    1972-01-01

    Ultraviolet light-inactivated frog virus 3 is efficiently photoreactivated by chick embryo cells. A cellular enzyme is presumably responsible for this repair of viral deoxyribonucleic acid, for the phenomenon is insensitive to an inhibitor of protein synthesis and is not seen in mammalian cells that are known to lack photoreactivating enzyme. Since frog virus 3 is a cytoplasmic virus, functionally significant amounts of photoreactivating enzyme are probably present in the cytoplasm of chick embryo cells. PMID:5062749

  16. A Glycine soja ABA-responsive receptor-like cytoplasmic kinase, GsRLCK, positively controls plant tolerance to salt and drought stresses.

    Science.gov (United States)

    Sun, XiaoLi; Sun, Mingzhe; Luo, Xiao; Ding, XiaoDong; Ji, Wei; Cai, Hua; Bai, Xi; Liu, XiaoFei; Zhu, YanMing

    2013-06-01

    Receptor such as protein kinases are proposed to work as sensors to initiate signaling cascades in higher plants. However, little is known about the precise functions of receptor such as protein kinases in abiotic stress response in plants, especially in wild soybean. Here, we focused on characterization of the biological functions of a receptor-like cytoplasmic serine/threonine protein kinase gene, GsRLCK, which was previously identified as a putative salt-alkali stress-related gene from the transcriptome profiles of Glycine soja. Bioinformatic analysis showed that GsRLCK protein contained a conserved kinase catalytic domain and two transmembrane domains at the N-terminus, but no typical extracellular domain. Consistently, GsRLCK-eGFP fusion protein was observed on the plasma membrane, but eGFP alone was distributing throughout the cytoplasm in onion epidermal cells. Quantitative real-time PCR analysis revealed the induced expression of GsRLCK by ABA, salt, alkali, and drought stresses. However, the expression levels of GsRLCK seemed to be similar in different tissues, except soybean pod. Phenotypic assays demonstrated that GsRLCK overexpression decreased ABA sensitivity and altered expression levels of ABA-responsive genes. Furthermore, we also found that GsRLCK conferred increased tolerance to salt and drought stresses and increased expression levels of a handful of stress-responsive genes, when overexpressing in Arabidopsis. In a word, we gave exact evidence that GsRLCK was a novel receptor-like cytoplasmic protein kinase and played a crucial role in plant responses to ABA, salt, and drought stresses.

  17. The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles

    Science.gov (United States)

    Vitali, Victoria; Sutka, Moira; Amodeo, Gabriela; Chara, Osvaldo; Ozu, Marcelo

    2016-01-01

    Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf. We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf/Ps), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements

  18. The water to solute permeability ratio governs the osmotic volume dynamics in beetroot vacuoles

    Directory of Open Access Journals (Sweden)

    Victoria Vitali

    2016-09-01

    Full Text Available Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf. We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modelling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf/Ps, which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons or stems that perform extensive rhythmic

  19. Leishmania amazonensis Engages CD36 to Drive Parasitophorous Vacuole Maturation.

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    Kendi Okuda

    2016-06-01

    Full Text Available Leishmania amastigotes manipulate the activity of macrophages to favor their own success. However, very little is known about the role of innate recognition and signaling triggered by amastigotes in this host-parasite interaction. In this work we developed a new infection model in adult Drosophila to take advantage of its superior genetic resources to identify novel host factors limiting Leishmania amazonensis infection. The model is based on the capacity of macrophage-like cells, plasmatocytes, to phagocytose and control the proliferation of parasites injected into adult flies. Using this model, we screened a collection of RNAi-expressing flies for anti-Leishmania defense factors. Notably, we found three CD36-like scavenger receptors that were important for defending against Leishmania infection. Mechanistic studies in mouse macrophages showed that CD36 accumulates specifically at sites where the parasite contacts the parasitophorous vacuole membrane. Furthermore, CD36-deficient macrophages were defective in the formation of the large parasitophorous vacuole typical of L. amazonensis infection, a phenotype caused by inefficient fusion with late endosomes and/or lysosomes. These data identify an unprecedented role for CD36 in the biogenesis of the parasitophorous vacuole and further highlight the utility of Drosophila as a model system for dissecting innate immune responses to infection.

  20. ER and vacuoles: never been closer

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    Corrado eViotti

    2014-02-01

    Full Text Available The endoplasmic reticulum (ER represents the gateway for intracellular trafficking of membrane proteins, soluble cargoes and lipids. In all eukaryotes, the best described mechanism of exiting the ER is via COPII-coated vesicles, which transport both membrane proteins and soluble cargo to the cis-Golgi. The vacuole, together with the plasma membrane, is the most distal point of the secretory pathway, and many vacuolar proteins are transported from the ER through intermediate compartments. However, past results and recent findings demonstrate the presence of alternative transport routes from the ER towards the tonoplast, which are independent of Golgi- and post-Golgi trafficking. Moreover, the transport mechanism of the vacuolar proton pumps VHA-a3 and AVP1 challenges the current model of vacuole biogenesis, pointing to the endoplasmic reticulum for being the main membrane source for the biogenesis of the plant lytic compartment. This review gives an overview of the current knowledge on the transport routes towards the vacuole and discusses the possible mechanism of vacuole biogenesis in plants.

  1. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases.

    Science.gov (United States)

    Meunier, Etienne; Dick, Mathias S; Dreier, Roland F; Schürmann, Nura; Kenzelmann Broz, Daniela; Warming, Søren; Roose-Girma, Merone; Bumann, Dirk; Kayagaki, Nobuhiko; Takeda, Kiyoshi; Yamamoto, Masahiro; Broz, Petr

    2014-05-15

    Lipopolysaccharide from Gram-negative bacteria is sensed in the host cell cytoplasm by a non-canonical inflammasome pathway that ultimately results in caspase-11 activation and cell death. In mouse macrophages, activation of this pathway requires the production of type-I interferons, indicating that interferon-induced genes have a critical role in initiating this pathway. Here we report that a cluster of small interferon-inducible GTPases, the so-called guanylate-binding proteins, is required for the full activity of the non-canonical caspase-11 inflammasome during infections with vacuolar Gram-negative bacteria. We show that guanylate-binding proteins are recruited to intracellular bacterial pathogens and are necessary to induce the lysis of the pathogen-containing vacuole. Lysis of the vacuole releases bacteria into the cytosol, thus allowing the detection of their lipopolysaccharide by a yet unknown lipopolysaccharide sensor. Moreover, recognition of the lysed vacuole by the danger sensor galectin-8 initiates the uptake of bacteria into autophagosomes, which results in a reduction of caspase-11 activation. These results indicate that host-mediated lysis of pathogen-containing vacuoles is an essential immune function and is necessary for efficient recognition of pathogens by inflammasome complexes in the cytosol.

  2. Cytoplasmic streaming in plant cells: the role of wall slip.

    Science.gov (United States)

    Wolff, K; Marenduzzo, D; Cates, M E

    2012-06-01

    We present a computer simulation study, via lattice Boltzmann simulations, of a microscopic model for cytoplasmic streaming in algal cells such as those of Chara corallina. We modelled myosin motors tracking along actin lanes as spheres undergoing directed motion along fixed lines. The sphere dimension takes into account the fact that motors drag vesicles or other organelles, and, unlike previous work, we model the boundary close to which the motors move as walls with a finite slip layer. By using realistic parameter values for actin lane and myosin density, as well as for endoplasmic and vacuole viscosity and the slip layer close to the wall, we find that this simplified view, which does not rely on any coupling between motors, cytoplasm and vacuole other than that provided by viscous Stokes flow, is enough to account for the observed magnitude of streaming velocities in intracellular fluid in living plant cells.

  3. Helicobacter pylori vacuolating toxin A and apoptosis

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    Rassow Joachim

    2011-11-01

    Full Text Available Abstract VacA, the vacuolating cytotoxin A of Helicobacter pylori, induces apoptosis in epithelial cells of the gastic mucosa and in leukocytes. VacA is released by the bacteria as a protein of 88 kDa. At the outer surface of host cells, it binds to the sphingomyelin of lipid rafts. At least partially, binding to the cells is facilitated by different receptor proteins. VacA is internalized by a clathrin-independent mechanism and initially accumulates in GPI-anchored proteins-enriched early endosomal compartments. Together with early endosomes, VacA is distributed inside the cells. Most of the VacA is eventually contained in the membranes of vacuoles. VacA assembles in hexameric oligomers forming an anion channel of low conductivity with a preference for chloride ions. In parallel, a significant fraction of VacA can be transferred from endosomes to mitochondria in a process involving direct endosome-mitochondria juxtaposition. Inside the mitochondria, VacA accumulates in the mitochondrial inner membrane, probably forming similar chloride channels as observed in the vacuoles. Import into mitochondria is mediated by the hydrophobic N-terminus of VacA. Apoptosis is triggered by loss of the mitochondrial membrane potential, recruitment of Bax and Bak, and release of cytochrome c.

  4. Large nuclear vacuoles in spermatozoa negatively affect pregnancy rate in IVF cycles.

    Science.gov (United States)

    Ghazali, Shahin; Talebi, Ali Reza; Khalili, Mohammad Ali; Aflatoonian, Abbas; Esfandiari, Navid

    2015-07-01

    Recently, motile sperm organelle morphology examination (MSOME) criteria as a new real time tool for evaluation of spermatozoa in intracytoplasmic sperm injection (ICSI) cycles has been considered. The aim was to investigate the predictive value of MSOME in in vitro fertilization (IVF) in comparison to ICSI cycles and evaluation of the association between MSOME parameters and traditional sperm parameters in both groups. This is a cross sectional prospective analysis of MSOME parameters in IVF (n=31) and ICSI cycles (n=35). MSOME parameters were also evaluated as the presence of vacuole (none, small, medium, large or mix); head size (normal, small or large); cytoplasmic droplet; head shape and acrosome normality. In sub-analysis, MSOME parameters were compared between two groups with successful or failed clinical pregnancy in each group. In IVF group, the rate of large nuclear vacuole showed significant increase in failed as compared to successful pregnancies (13.81±9.7vs7.38±4.4, respectively, p=0.045) while MSOME parameters were the same between successful and failed pregnancies in ICSI group. Moreover, a negative correlation was noticed between LNV and sperm shape normalcy. In ICSI group, a negative correlation was established between cytoplasmic droplet and sperm shape normalcy. In addition, there was a positive correlation between sperm shape normalcy and non-vacuolated spermatozoa. The high rate of large nuclear vacuoles in sperm used in IVF cycles with failed pregnancies confirms that MSOME, is a helpful tool for fine sperm morphology assessment, and its application may enhance the assisted reproduction technology success rates.

  5. Large nuclear vacuoles in spermatozoa negatively affect pregnancy rate in IVF cycles

    Directory of Open Access Journals (Sweden)

    Shahin Ghazali

    2015-07-01

    Full Text Available Background: Recently, motile sperm organelle morphology examination (MSOME criteria as a new real time tool for evaluation of spermatozoa in intracytoplasmic sperm injection (ICSI cycles has been considered. Objective: The aim was to investigate the predictive value of MSOME in in vitro fertilization (IVF in comparison to ICSI cycles and evaluation of the association between MSOME parameters and traditional sperm parameters in both groups. Materials and Methods: This is a cross sectional prospective analysis of MSOME parameters in IVF (n=31 and ICSI cycles (n=35. MSOME parameters were also evaluated as the presence of vacuole (none, small, medium, large or mix; head size (normal, small or large; cytoplasmic droplet; head shape and acrosome normality. In sub-analysis, MSOME parameters were compared between two groups with successful or failed clinical pregnancy in each group. Results: In IVF group, the rate of large nuclear vacuole showed significant increase in failed as compared to successful pregnancies (13.81±9.7vs7.38±4.4, respectively, p=0.045 while MSOME parameters were the same between successful and failed pregnancies in ICSI group. Moreover, a negative correlation was noticed between LNV and sperm shape normalcy. In ICSI group, a negative correlation was established between cytoplasmic droplet and sperm shape normalcy. In addition, there was a positive correlation between sperm shape normalcy and non-vacuolated spermatozoa. Conclusion: The high rate of large nuclear vacuoles in sperm used in IVF cycles with failed pregnancies confirms that MSOME, is a helpful tool for fine sperm morphology assessment, and its application may enhance the assisted reproduction technology success rates.

  6. Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

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    Glushakova Svetlana

    2013-01-01

    Full Text Available Abstract Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress

  7. Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration.

    Science.gov (United States)

    Markham, K R; Gould, K S; Ryan, K G

    2001-10-01

    It is widely accepted that the mix of flavonoids in the cell vacuole is the source of flavonoid based petal colour, and that analysis of the petal extract reveals the nature and relative levels of vacuolar flavonoid pigments. However, it has recently been established with lisianthus flowers that some petal flavonoids can be excluded from the vacuolar mix through deposition in the cell wall or through complexation with proteins inside the vacuole, and that these flavonoids are not readily extractable. The present work demonstrates that flavonoids can also be compartmented within the cell cytoplasm. Using adaxial epidermal peels from the petals of lisianthus (Eustoma grandiflorum), Lathyrus chrysanthus and Dianthus caryophyllus, light and laser scanning confocal microscopy studies revealed a significant concentration of petal flavonoids in the cell cytoplasm of some tissues. With lisianthus, flavonoid analyses of isolated protoplasts and vacuoles were used to establish that ca 14% of petal flavonoids are located in the cytoplasm (cf. 30% in the cell wall and 56% in the vacuole). The cytoplasmic flavonoids are predominantly acylated glycosides (cf. non-acylated in the cell wall). Flavonoid aggregation on a cytoplasmic protein substrate provides a rational mechanism to account for how colourless flavonoid glycosides can produce yellow colouration in petals, and perhaps also in other plant parts. High vacuolar concentrations of such flavonoids are shown to be insufficient.

  8. Cytoplasmic dynein nomenclature

    Science.gov (United States)

    Pfister, K. Kevin; Fisher, Elizabeth M.C.; Gibbons, Ian R.; Hays, Thomas S.; Holzbaur, Erika L.F.; McIntosh, J. Richard; Porter, Mary E.; Schroer, Trina A.; Vaughan, Kevin T.; Witman, George B.; King, Stephen M.; Vallee, Richard B.

    2005-01-01

    A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms. PMID:16260502

  9. The influence of osmotic stress on the content of calcium ions in the red beet vacuoles and on the transport activity of tonoplast proton pumps

    Directory of Open Access Journals (Sweden)

    Ozolina N.V.

    2012-05-01

    Full Text Available The contents of calcium ions in the isolated vacuoles and in intact red beets under the conditions of dormancy and osmotic stress was determined. It is demonstrated that the content of calcium ions in the red beet vacuoles not exposed to osmotic stress makes 13.3% of the total content these ions in intact red beets. Under the conditions of osmotic stress, this indicator increases substantially. Furthermore, under the conditions of hyperosmotic stress, the content of calcium ions in the vacuoles was 30%, while under hypoosmotic stress it was 49% of the total content of these ions in the intact red beet. The transition of calcium ions from the cytoplasm and other compartments into the vacuole under the conditions of osmotic stress is, probably, one of forms of participation of the vacuole in adaptation processes of the plant cell under this kind of abiotic stress. It has been demonstrated for the first time that tonoplast proton pumps, which actively participate in provision of calcium homeostasis in cytoplasm, substantially activate their transport activity under osmotic stress, what allows one to speak about their important role in the cell’s protective programs. Under normal (no stress conditions, artificial elevation of the content of calcium ions led to inhibition of activity of the tonoplast proton pumps, while under gipoosmotic stress the activity of tonoplast proton pumps increased, what might aid to restoring homeoctasis with respect to calcium ions in cytoplasm.

  10. Cytoplasmic and nuclear localizations are important for the hypersensitive response conferred by maize autoactive Rp1-D21 protein

    Science.gov (United States)

    Disease resistance (R-) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich-repeat (NLR) proteins that trigger a rapid localized programmed cell death termed the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, d...

  11. Involvement of the cytoplasmic cysteine-238 of CD40 in its up-regulation of CD23 expression and its enhancement of TLR4-triggered responses.

    Science.gov (United States)

    Nadiri, Amal; Jundi, Malek; El Akoum, Souhad; Hassan, Ghada S; Yacoub, Daniel; Mourad, Walid

    2015-11-01

    CD40, a member of the tumor necrosis factor receptor superfamily, plays a key role in both adaptive and innate immunity. Engagement of CD40 with its natural trimeric ligand or with cross-linked antibodies results in disulfide-linked CD40 (dl-CD40) homodimer formation, a process mediated by the cysteine-238 residues of the cytoplasmic tail of CD40. The present study was designed to elucidate the biological relevance of cysteine-238-mediated dl-CD40 homodimers to the expression of CD23 on B cells and to investigate its possible involvement in the innate response. Our results indicate that cysteine-238-mediated dl-CD40 homodimerization is required for CD40-induced activation of PI3-kinase/Akt signaling and the subsequent CD23 expression, as inhibition of dl-CD40 homodimer formation through a point mutation-approach specifically impairs these responses. Interestingly, cysteine-238-mediated dl-CD40 homodimers are also shown to play a crucial role in Toll-like receptor 4-induced CD23 expression, further validating the importance of this system in bridging innate and adaptive immune responses. This process also necessitates the activation of the PI3-kinase/Akt cascade. Thus, our results highlight new roles for CD40 and cysteine-238-mediated CD40 homodimers in cell biology and identify a potential new target for therapeutic strategies against CD40-associated chronic inflammatory diseases.

  12. H1-antihistamines induce vacuolation in astrocytes through macroautophagy

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Wei-Wei; Yang, Ying; Wang, Zhe; Shen, Zhe; Zhang, Xiang-Nan [Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058 (China); Wang, Guang-Hui [College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123 (China); Chen, Zhong, E-mail: chenzhong@zju.edu.cn [Department of Pharmacology, Key Laboratory of Medical Neurobiology of the Ministry of Health of China, Zhejiang Province Key Laboratory of Neurobiology, School of Basic Medical Sciences, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058 (China)

    2012-04-15

    H1-antihistamines induce vacuolation in vascular smooth muscle cells, which may contribute to their cardiovascular toxicity. The CNS toxicity of H1-antihistamines may also be related to their non-receptor-mediated activity. The aim of this study was to investigate whether H1-antihistamines induce vacuolation in astrocytes and the mechanism involved. The H1-antihistamines induced large numbers of giant vacuoles in astrocytes. Such vacuoles were marked with both the lysosome marker Lysotracker Red and the alkalescent fluorescence dye monodansylcadaverine, which indicated that these vacuoles were lysosome-like acidic vesicles. Quantitative analysis of monodansylcadaverine fluorescence showed that the effect of H1-antihistamines on vacuolation in astrocytes was dose-dependent, and was alleviated by extracellular acidification, but aggravated by extracellular alkalization. The order of potency to induce vacuolation at high concentrations of H1-antihistamines (diphenhydramine > pyrilamine > astemizole > triprolidine) corresponded to their pKa ranking. Co-treatment with histamine and the histamine receptor-1 agonist trifluoromethyl toluidide did not inhibit the vacuolation. Bafilomycin A1, a vacuolar (V)-ATPase inhibitor, which inhibits intracellular vacuole or vesicle acidification, clearly reversed the vacuolation and intracellular accumulation of diphenhydramine. The macroautophagy inhibitor 3-methyladenine largely reversed the percentage of LC3-positive astrocytes induced by diphenhydramine, while only partly reversing the number of monodansylcadaverine-labeled vesicles. In Atg5{sup −/−} mouse embryonic fibroblasts, which cannot form autophagosomes, the number of vacuoles induced by diphenhydramine was less than that in wild-type cells. These results indicated that H1-antihistamines induce V-ATPase-dependent acidic vacuole formation in astrocytes, and this is partly mediated by macroautophagy. The pKa and alkalescent characteristic of H1-antihistamines may be the

  13. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

  14. Response of the cytoplasmic and membrane proteome of Corynebacterium glutamicum ATCC 13032 to pH changes

    Directory of Open Access Journals (Sweden)

    Poetsch Ansgar

    2008-12-01

    Full Text Available Abstract Background C. glutamicum has traditionally been grown in neutral-pH media for amino acid production, but in a previous article we reported that this microorganism is a moderate alkaliphile since it grows optimally at pH 7.0–9.0, as shown in fermentor studies under tightly controlled pH conditions. We determined the best pH values to study differential expression of several genes after acidic or basic pH conditions (pH 6.0 for acidic expression and pH 9.0 for alkaline expression. Thus, it was interesting to perform a detailed analysis of the pH-adaptation response of the proteome of C. glutamicum ATCC 13032 to clarify the circuits involved in stress responses in this bacterium. In this paper we used the above indicated pH conditions, based on transcriptional studies, to confirm that pH adaptation results in significant changes in cytoplasmatic and membrane proteins. Results The cytoplasmatic and membrane proteome of Corynebacterium glutamicum ATCC 13032 at different pH conditions (6.0, 7.0 and 9.0 was analyzed by classical 2D-electrophoresis, and by anion exchange chromatography followed by SDS-PAGE (AIEC/SDS-PAGE. A few cytoplasmatic proteins showed differential expression at the three pH values with the classical 2D-technique including a hypothetical protein cg2797, L-2.3-butanediol dehydrogenase (ButA, and catalase (KatA. The AIEC/SDS-PAGE technique revealed several membrane proteins that respond to pH changes, including the succinate dehydrogenase complex (SdhABCD, F0F1-ATP synthase complex subunits b, α and δ (AtpF, AtpH and AtpA, the nitrate reductase II α subunit (NarG, and a hypothetical secreted/membrane protein cg0752. Induction of the F0F1-ATP synthase complex β subunit (AtpD at pH 9.0 was evidenced by Western analysis. By contrast, L-2.3-butanediol dehydrogenase (ButA, an ATPase with chaperone activity, the ATP-binding subunit (ClpC of an ATP-dependent protease complex, a 7 TMHs hypothetical protein cg0896, a conserved

  15. Vacuolation induced by unfavorable pH in cyanobacteria

    Institute of Scientific and Technical Information of China (English)

    赵以军; 吴红艳; 郭厚良; 许敏; 程凯; 祝海燕

    2001-01-01

    Six species or strains of cyanobacteria, Anabaena sp. 595, Plectonema boryanum 246, Scytonema hofmanni 248, Nostoc sp. 96, Oscillatoria animlis 284 and Spirulina maxima 438, were cultured in unfavorable pH conditions for vacuole induction. At pH 5.0, 6.5, or 7.0, vacuoles were observed to form in both Anabaena sp. 595 and Plectonema boryanum 246, especially in the former. The vacuolation took place with some morphological changes, such as the cells being inflated, spherical and vacuolated, and with unequalized division. The induced vacuoles in An- abaena sp. 595 and Plectonema boryanum 246 were in spherical shape and in rather transparent appearance under a phase microscope. For Scytonema hofmanni 248, it was less sensitive to pH, its vacuole formation was found only at pH 6.5. No vacuolization occurred in the cells of Nostoc sp. 96, Oscillatoria animlis 284 and Spirulina maxima 438 at all pH conditions we used. Vacuolization under unfavorable pH provides a new proof for the existence of vacuole in cells of cyanobacteria and reflects the prokaryote's function in ecological environment.

  16. 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...

  17. 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.

  18. Toxoplasma exports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression.

    Science.gov (United States)

    Bougdour, Alexandre; Tardieux, Isabelle; Hakimi, Mohamed-Ali

    2014-03-01

    Toxoplasma gondii is the most widespread apicomplexan parasite and occupies a large spectrum of niches by infecting virtually any warm-blooded animals. As an obligate intracellular parasite, Toxoplasma has evolved a repertoire of strategies to fine-tune the cellular environment in an optimal way to promote growth and persistence in host tissues hence increasing the chance to be transmitted to new hosts. Short and long-term intracellular survival is associated with Toxoplasma ability to both evade the host deleterious immune defences and to stimulate a beneficial immune balance by governing host cell gene expression. It is only recently that parasite proteins responsible for driving these transcriptional changes have been identified. While proteins contained in the apical secretory Rhoptry organelle have already been identified as bona fide secreted effectors that divert host signalling pathways, recent findings revealed that dense granule proteins should be added to the growing list of effectors as they reach the host cell cytoplasm and nucleus and target various host cell pathways in the course of cell infection. Herein, we emphasize on a novel subfamily of dense granule residentproteins, exemplified with the GRA16 and GRA24 members we recently discovered as both are exported beyond the vacuole-containing parasites and reach the host cell nucleus to reshape the host genome expression.

  19. Measurement of Cytoplasmic Streaming in Chara Corallina by Magnetic Resonance Velocimetry

    CERN Document Server

    van de Meent, Jan-Willem; Gladden, Lynn F; Goldstein, Raymond E

    2009-01-01

    In aquatic plants such as the Characean algae, the force generation that drives cyclosis is localized within the cytoplasm, yet produces fluid flows throughout the vacuole. For this to occur the tonoplast must transmit hydrodynamic shear efficiently. Here, using magnetic resonance velocimetry, we present the first whole-cell measurements of the cross-sectional longitudinal velocity field in Chara corallina and show that it is in quantitative agreement with a recent theoretical analysis of rotational cytoplasmic streaming driven by bidirectional helical forcing in the cytoplasm, with direct shear transmission by the tonoplast.

  20. Development of an Innovative Intradermal siRNA Delivery System Using a Combination of a Functional Stearylated Cytoplasm-Responsive Peptide and a Tight Junction-Opening Peptide

    Directory of Open Access Journals (Sweden)

    Hisako Ibaraki

    2016-09-01

    Full Text Available As a new category of therapeutics for skin diseases including atopic dermatitis (AD, nucleic acids are gaining importance in the clinical setting. Intradermal administration is noninvasive and improves patients′ quality of life. However, intradermal small interfering RNA (siRNA delivery is difficult because of two barriers encountered in the skin: intercellular lipids in the stratum corneum and tight junctions in the stratum granulosum. Tight junctions are the major barrier in AD; therefore, we focused on functional peptides to devise an intradermal siRNA delivery system for topical skin application. In this study, we examined intradermal siRNA permeability in the tape-stripped (20 times back skin of mice or AD-like skin of auricles treated with 6-carboxyfluorescein-aminohexyl phosphoramidite (FAM-labeled siRNA, the tight junction modulator AT1002, and the functional cytoplasm-responsive stearylated peptide STR-CH2R4H2C by using confocal laser microscopy. We found that strong fluorescence was observed deep and wide in the epidermis and dermis of back skin and AD-like ears after siRNA with STR-CH2R4H2C and AT1002 treatment. After 10 h from administration, brightness of FAM-siRNA was significantly higher for STR-CH2R4H2C + AT1002, compared to other groups. In addition, we confirmed the nontoxicity of STR-CH2R4H2C as a siRNA carrier using PAM212 cells. Thus, our results demonstrate the applicability of the combination of STR-CH2R4H2C and AT1002 for effective intradermal siRNA delivery.

  1. Active vacuolar H+ ATPase and functional cycle of Rab5 are required for the vacuolation defect triggered by PtdIns(3,5)P2 loss under PIKfyve or Vps34 deficiency.

    Science.gov (United States)

    Compton, Lauren M; Ikonomov, Ognian C; Sbrissa, Diego; Garg, Puneet; Shisheva, Assia

    2016-09-01

    The two evolutionarily conserved mammalian lipid kinases Vps34 and PIKfyve are involved in an important physiological relationship, whereby the former produces phosphatidylinositol (PtdIns) 3P that is used as a substrate for PtdIns(3,5)P2 synthesis by the latter. Reduced production of PtdIns(3,5)P2 in proliferating mammalian cells is phenotypically manifested by the formation of multiple translucent cytoplasmic vacuoles, readily rescued upon exogenous delivery of PtdIns(3,5)P2 or overproduction of PIKfyve. Although the aberrant vacuolation phenomenon has been frequently used as a sensitive functional measure of localized PtdIns(3,5)P2 reduction, cellular factors governing the appearance of cytoplasmic vacuoles under PtdIns3P-PtdIns(3,5)P2 loss remain elusive. To gain further mechanistic insight about the vacuolation process following PtdIns(3,5)P2 reduction, in this study we sought for cellular mechanisms required for manifestation of the aberrant endomembrane vacuoles triggered by PIKfyve or Vps34 dysfunction. The latter was achieved by various means such as pharmacological inhibition, gene disruption, or dominant-interference in several proliferating mammalian cell types. We report here that inhibition of V-ATPase with bafilomycin A1 as well as inactivation of the GTP-GDP cycle of Rab5a GTPase phenotypically rescued or completely precluded the cytoplasmic vacuolization despite the continued presence of inactivated PIKfyve or Vps34. Bafilomycin A1 also restored the aberrant EEA1-positive endosomes, enlarged upon short PIKfyve inhibition with YM201636. Together, our work identifies for the first time that factors such as active V-ATPase or functional Rab5a cycle are acting coincidentally with the PtdIns(3,5)P2 reduction in triggering formation of aberrant cytoplasmic vacuoles under PIKfyve or Vps34 dysfunction.

  2. Rimmed vacuoles and the added value of SMI-31 staining in diagnosing sporadic inclusion body myositis.

    Science.gov (United States)

    van der Meulen, M F; Hoogendijk, J E; Moons, K G; Veldman, H; Badrising, U A; Wokke, J H

    2001-07-01

    Problems in diagnosing sporadic inclusion body myositis may arise if all clinical features fit a diagnosis of polymyositis, but the muscle biopsy shows some rimmed vacuoles. Recently, immunohistochemistry with an antibody directed against phosphorylated neurofilament (SMI-31) has been advocated as a diagnostic test for sporadic inclusion body myositis. The aims of the present study were to define a quantitative criterion to differentiate sporadic inclusion body myositis from polymyositis based on the detection of rimmed vacuoles in the haematoxylin-eosin staining and to evaluate the additional diagnostic value of the SMI-31 staining. Based on clinical criteria and creatine kinase levels in patients with endomysial infiltrates, 18 patients complied with the diagnosis of sporadic inclusion body myositis, and 17 with the diagnosis of polymyositis. A blinded observer counted the abnormal fibres in haematoxylin-eosin-stained sections and in SMI-31-stained sections. The optimal cut-off in the haematoxylin-eosin test was 0.3% vacuolated fibres. Adding the SMI-31 staining significantly increased the positive predictive value from 87 to 100%, but increased the negative predictive value only to small extent. We conclude that (1) patients with clinical and laboratory features of polymyositis, including response to treatment, may show rimmed vacuoles in their muscle biopsy and that (2) adding the SMI-31 stain can be helpful in differentiating patients who respond to treatment from patients who do not.

  3. V-ATPase, ScNhxlp and Yeast Vacuole Fusion

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos.It is a central cellular reaction that plays important roles in signal transduction,protein sorting and subcellular compartmentation.Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summanzed in this article.It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast.Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH.V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast.Fission defects are epistatic to fusion defects.Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast,the fusion reaction does not need the transport activity but requires the physical presence of the proton pump.Vo,the membrane-integral sector of the V-ATPase,forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the Vo trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

  4. Emergence of the Terrestrial Ciliate Colpoda cucullus from a Resting Cyst: Rupture of the Cyst Wall by Active Expansion of an Excystment Vacuole

    Science.gov (United States)

    Funadani, Ryoji; Suetomo, Yasutaka; Matsuoka, Tatsuomi

    2013-01-01

    The first sign of excysting Colpoda cucullus cells is the initiation of the pulsation of a contractile vacuole, which is then replaced by a non-pulsating vacuole (excystment vacuole) that continues to expand and finally ruptures the outermost cyst wall (ectocyst) due to inner pressure. A ciliate surrounded by flexible membranes (endocyst) thus emerges. The osmolarity of the excysting cells is estimated to be 140 mOsm L−1 from the relationship between the frequency of contractile vacuole pulsation and the external sucrose concentration. Both the expansion of the excystment vacuole and the emergence of ciliates occurred even when the cysts were immersed in hypertonic medium. In hypotonic medium containing sodium azide (NaN3, a cytochrome c oxidase inhibitor), the contractile vacuole of vegetative cells stopped pulsating and gradually expanded, causing cells to burst. When C. cucullus was induced to encyst in a hypotonic medium containing NaN3, the expansion of the excystment vacuoles was inhibited. These results suggest that the active uptake of water may be responsible for the expansion of the excystment vacuole required for the ectocyst to rupture. PMID:23268793

  5. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45......% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied...... by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite...

  6. Cytoplasm Affects Embryonic Development

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    @@ Recent studies by CAS researchers furnish strong evidence that a fertilized egg's nucleus isn't the sole site of control for an embryo's development. A research team headed by Prof. Zhu Zuoyan from the CAS Institute of Hydrobiology in Wuhan discovered that cytoplasm affects the number of vertebrae in cloned offspring created when nuclei from one fish genus were transplanted to enucleated eggs of another.

  7. Cytoplasmic Z-RNA

    Energy Technology Data Exchange (ETDEWEB)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-09-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation.

  8. Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae

    Science.gov (United States)

    Michaillat, Lydie; Mayer, Andreas

    2013-01-01

    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. PMID:23383298

  9. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses

    OpenAIRE

    2015-01-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomona...

  10. Inborn errors of cytoplasmic triglyceride metabolism.

    Science.gov (United States)

    Wu, Jiang Wei; Yang, Hao; Wang, Shu Pei; Soni, Krishnakant G; Brunel-Guitton, Catherine; Mitchell, Grant A

    2015-01-01

    Triglyceride (TG) synthesis, storage, and degradation together constitute cytoplasmic TG metabolism (CTGM). CTGM is mostly studied in adipocytes, where starting from glycerol-3-phosphate and fatty acyl (FA)-coenzyme A (CoA), TGs are synthesized then stored in cytoplasmic lipid droplets. TG hydrolysis proceeds sequentially, producing FAs and glycerol. Several reactions of CTGM can be catalyzed by more than one enzyme, creating great potential for complex tissue-specific physiology. In adipose tissue, CTGM provides FA as a systemic energy source during fasting and is related to obesity. Inborn errors and mouse models have demonstrated the importance of CTGM for non-adipose tissues, including skeletal muscle, myocardium and liver, because steatosis and dysfunction can occur. We discuss known inborn errors of CTGM, including deficiencies of: AGPAT2 (a form of generalized lipodystrophy), LPIN1 (childhood rhabdomyolysis), LPIN2 (an inflammatory condition, Majeed syndrome, described elsewhere in this issue), DGAT1 (protein loosing enteropathy), perilipin 1 (partial lipodystrophy), CGI-58 (gene ABHD5, neutral lipid storage disease (NLSD) with ichthyosis and "Jordan's anomaly" of vacuolated polymorphonuclear leukocytes), adipose triglyceride lipase (ATGL, gene PNPLA2, NLSD with myopathy, cardiomyopathy and Jordan's anomaly), hormone-sensitive lipase (HSL, gene LIPE, hypertriglyceridemia, and insulin resistance). Two inborn errors of glycerol metabolism are known: glycerol kinase (GK, causing pseudohypertriglyceridemia) and glycerol-3-phosphate dehydrogenase (GPD1, childhood hepatic steatosis). Mouse models often resemble human phenotypes but may diverge markedly. Inborn errors have been described for less than one-third of CTGM enzymes, and new phenotypes may yet be identified.

  11. Parasitophorous vacuole membrane of Plasmodium knowlesi

    Energy Technology Data Exchange (ETDEWEB)

    Nillni, E.A.; Wallach, D.F.H.

    1986-05-01

    The authors have evaluated the occurrence of host cell membrane protein and parasite protein in the vacuolar membrane (VM) of isolated parasites. Parasites were labeled by incorporation of (/sup 35/S)methionine and by lactoperoxidase-catalyzed /sup 125/I iodination. Of the two prominent /sup 125/I-labeled components, one, not detected by metabolic labeling corresponded in M/sub r/ to erythrocyte band 3 (90 kDa). Trypsinization of radioiodinated parasites for 5' or 20' yield a 35 kDa fragment, not seen in untreated samples and compatible with the trypsin degradation of band 3 from the cytoplasmic side. Tryptic peptide maps of the 35 kDa revealed a very acidic peptide corresponding to the highly anionic tryptic peptide of band 3 showed by others. The second prominent /sup 125/I-labeled VM protein had an M/sub r/ 74,000 corresponding to a protein metabolically labeled with (/sup 35/S)methionine, suggesting it is inserted into the VM by the parasites. Several less prominent proteins labeling with both (/sup 35/S)methionine and /sup 125/I were also detected (140 kDa, 55 kDa, 45 kDa). A faint /sup 125/I-labeled triple (220-230 kDa) is compatible with a trace amounts of spectrin, usually a prominent component of red cell membrane. The results indicate that host cell band 3 is a prominent component of the VM, but that this membrane also contains several parasite-synthesized proteins.

  12. Effect of Helicobacter pylori's vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells.

    Science.gov (United States)

    Terebiznik, Mauricio R; Raju, Deepa; Vázquez, Cristina L; Torbricki, Karl; Kulkarni, Reshma; Blanke, Steven R; Yoshimori, Tamotsu; Colombo, María I; Jones, Nicola L

    2009-04-01

    Host cell responses to Helicobacter pylori infection are complex and incompletely understood. Here, we report that autophagy is induced within human-derived gastric epithelial cells (AGS) in response to H. pylori infection. These autophagosomes were distinct and different from the large vacuoles induced during H. pylori infection. Autophagosomes were detected by transmission electron microscopy, conversion of LC3-I to LC3-II, GFP-LC3 recruitment to autophagosomes, and depended on Atg5 and Atg12. The induction of autophagy depended on the vacuolating cytotoxin (VacA) and, moreover, VacA was sufficient to induce autophagosome formation. The channel-forming activity of VacA was necessary for inducing autophagy. Intracellular VacA partially co-localized with GFP-LC3, indicating that the toxin associates with autophagosomes. The inhibition of autophagy increased the stability of intracellular VacA, which in turn resulted in enhanced toxin-mediated cellular vacuolation. These findings suggest that the induction of autophagy by VacA may represent a host mechanism to limit toxin-induced cellular damage.

  13. 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...... Vps41. This accumulation is not due to a phosphorylation defect, but to reduced release of Vps41 from vma16Delta vacuoles. One reason could be a connection to vacuole fission, which is blocked in V-ATPase mutants. Vacuole fusion is not impaired between vacuoles lacking the V0-subunits Vma16 or Vma6...... and wild-type vacuoles, whereas fusion between mutant vacuoles is reduced. Our data suggest a connection between vacuole biogenesis and membrane fusion....

  14. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    Science.gov (United States)

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  15. PIKfyve Regulates Vacuole Maturation and Nutrient Recovery following Engulfment.

    Science.gov (United States)

    Krishna, Shefali; Palm, Wilhelm; Lee, Yongchan; Yang, Wendy; Bandyopadhyay, Urmi; Xu, Haoxing; Florey, Oliver; Thompson, Craig B; Overholtzer, Michael

    2016-09-12

    The scavenging of extracellular macromolecules by engulfment can sustain cell growth in a nutrient-depleted environment. Engulfed macromolecules are contained within vacuoles that are targeted for lysosome fusion to initiate degradation and nutrient export. We have shown that vacuoles containing engulfed material undergo mTORC1-dependent fission that redistributes degraded cargo back into the endosomal network. Here we identify the lipid kinase PIKfyve as a regulator of an alternative pathway that distributes engulfed contents in support of intracellular macromolecular synthesis during macropinocytosis, entosis, and phagocytosis. We find that PIKfyve regulates vacuole size in part through its downstream effector, the cationic transporter TRPML1. Furthermore, PIKfyve promotes recovery of nutrients from vacuoles, suggesting a potential link between PIKfyve activity and lysosomal nutrient export. During nutrient depletion, PIKfyve activity protects Ras-mutant cells from starvation-induced cell death and supports their proliferation. These data identify PIKfyve as a critical regulator of vacuole maturation and nutrient recovery during engulfment.

  16. Beyond Rab GTPases Legionella activates the small GTPase Ran to promote microtubule polymerization, pathogen vacuole motility, and infection.

    Science.gov (United States)

    Hilbi, Hubert; Rothmeier, Eva; Hoffmann, Christine; Harrison, Christopher F

    2014-01-01

    Legionella spp. are amoebae-resistant environmental bacteria that replicate in free-living protozoa in a distinct compartment, the Legionella-containing vacuole (LCV). Upon transmission of Legionella pneumophila to the lung, the pathogens employ an evolutionarily conserved mechanism to grow in LCVs within alveolar macrophages, thus triggering a severe pneumonia termed Legionnaires' disease. LCV formation is a complex and robust process, which requires the bacterial Icm/Dot type IV secretion system and involves the amazing number of 300 different translocated effector proteins. LCVs interact with the host cell's endosomal and secretory vesicle trafficking pathway. Accordingly, in a proteomics approach as many as 12 small Rab GTPases implicated in endosomal and secretory vesicle trafficking were identified and validated as LCV components. Moreover, the small GTPase Ran and its effector protein RanBP1 have been found to decorate the pathogen vacuole. Ran regulates nucleo-cytoplasmic transport, spindle assembly, and cytokinesis, as well as the organization of non-centrosomal microtubules. In L. pneumophila-infected amoebae or macrophages, Ran and RanBP1 localize to LCVs, and the small GTPase is activated by the Icm/Dot substrate LegG1. Ran activation by LegG1 leads to microtubule stabilization and promotes intracellular pathogen vacuole motility and bacterial growth, as well as chemotaxis and migration of Legionella-infected cells.

  17. Sat, the secreted autotransporter toxin of uropathogenic Escherichia coli, is a vacuolating cytotoxin for bladder and kidney epithelial cells.

    Science.gov (United States)

    Guyer, Debra M; Radulovic, Suzana; Jones, Faye-Ellen; Mobley, Harry L T

    2002-08-01

    The secreted autotransporter toxin (Sat) of uropathogenic Escherichia coli exhibits cytopathic activity upon incubation with HEp-2 cells. We further investigated the effects of Sat on cell lines more relevant to the urinary tract, namely, those derived from bladder and kidney epithelium. Sat elicited elongation of cells and apparent loosening of cellular junctions upon incubation with Vero kidney cells. Additionally, incubation with Sat triggered significant vacuolation within the cytoplasm of both human bladder (CRL-1749) and kidney (CRL-1573) cell lines. This activity has been associated with only a few other known toxins. Following transurethral infection of CBA mice with a sat mutant, no reduction of CFU in urine, bladder, or kidney tissue was seen compared to that in mice infected with wild-type E. coli CFT073. However, significant histological changes were observed within the kidneys of mice infected with wild-type E. coli CFT073, including dissolution of the glomerular membrane and vacuolation of proximal tubule cells. Such damage was not observed in kidney sections of mice infected with a Sat-deficient mutant. These results indicate that Sat, a vacuolating cytotoxin expressed by uropathogenic E. coli CFT073, elicits defined damage to kidney epithelium during upper urinary tract infection and thus contributes to pathogenesis of urinary tract infection.

  18. Electron tomography characterization of hemoglobin uptake in Plasmodium chabaudi reveals a stage-dependent mechanism for food vacuole morphogenesis.

    Science.gov (United States)

    Wendt, Camila; Rachid, Rachel; de Souza, Wanderley; Miranda, Kildare

    2016-05-01

    In the course of their intraerythrocytic development, malaria parasites incorporate and degrade massive amounts of the host cell cytoplasm. This mechanism is essential for parasite development and represents a physiological step used as target for many antimalarial drugs; nevertheless, the fine mechanisms underlying these processes in Plasmodium species are still under discussion. Here, we studied the events of hemoglobin uptake and hemozoin nucleation in the different stages of the intraerythrocytic cycle of the murine malaria parasite Plasmodium chabaudi using transmission electron tomography of cryofixed and freeze-substituted cells. The results showed that hemoglobin uptake in P. chabaudi starts at the early ring stage and is present in all developmental stages, including the schizont stage. Hemozoin nucleation occurs near the membrane of small food vacuoles. At the trophozoite stage, food vacuoles are found closely localized to cytostomal tubes and mitochondria, whereas in the schizont stage, we observed a large food vacuole located in the central portion of the parasite. Taken together, these results provide new insights into the mechanisms of hemoglobin uptake and degradation in rodent malaria parasites.

  19. Rotavirus disrupts cytoplasmic P bodies during infection.

    Science.gov (United States)

    Bhowmick, Rahul; Mukherjee, Arpita; Patra, Upayan; Chawla-Sarkar, Mamta

    2015-12-02

    Cytoplasmic Processing bodies (P bodies), the RNA-protein aggregation foci of translationally stalled and potentially decaying mRNA, have been reported to be differentially modulated by viruses. Rotavirus, the causative agent of acute infantile gastroenteritis is a double stranded RNA virus which completes its entire life-cycle exclusively in host cell cytoplasm. In this study, the fate of P bodies was investigated upon rotavirus infection. It was found that P bodies get disrupted during rotavirus infection. The disruption occurred by more than one different mechanism where deadenylating P body component Pan3 was degraded by rotavirus NSP1 and exonuclease XRN1 along with the decapping enzyme hDCP1a were relocalized from cytoplasm to nucleus. Overall the study highlights decay and subcellular relocalization of P body components as novel mechanisms by which rotavirus subverts cellular antiviral responses.

  20. Cytoplasmic localized infected cell protein 0 (bICP0) encoded by bovine herpesvirus 1 inhibits beta interferon promoter activity and reduces IRF3 (interferon response factor 3) protein levels

    Science.gov (United States)

    da Silva, Leticia Frizzo; Gaudreault, Natasha; Jones, Clinton

    2011-01-01

    Bovine herpesvirus 1 (BHV-1), an alpha-herpesvirinae subfamily member, establishes a life-long latent infection in sensory neurons. Periodically, BHV-1 reactivates from latency, infectious virus is spread, and consequently virus transmission occurs. BHV-1 acute infection causes upper respiratory track infections and conjunctivitis in infected cattle. As a result of transient immunesuppression, BHV-1 infections can also lead to life-threatening secondary bacterial pneumonia that is referred to as bovine respiratory disease. The infected cell protein 0 (bICP0) encoded by BHV-1 reduces human beta-interferon (IFN-β) promoter activity, in part, by inducing degradation of interferon response factor 3 (IRF3) and interacting with IRF7. In contrast to humans, cattle contain three IFN-β genes. All three bovine IFN-β proteins have anti-viral activity: but each IFN-β gene has a distinct transcriptional promoter. We have recently cloned and characterized the three bovine IFN-β promoters. Relative to the human IFN-β promoter, each of the three IFN-β promoters contain differences in the four positive regulatory domains that are required for virus-induced activity. In this study, we demonstrate that bICP0 effectively inhibits bovine IFN-β promoter activity following transfection of low passage bovine cells with interferon response factor 3 (IRF3) or IRF7. A bICP0 mutant that localizes to the cytoplasm inhibits bovine IFN-β promoter activity as efficiently as wt bICP0. The cytoplasmic localized bICP0 protein also induced IRF3 degradation with similar efficiency as wt bICP0. In summary, these studies suggested that cytoplasmic localized bICP0 plays a role in inhibiting the IFN-β response during productive infection. PMID:21689696

  1. Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1

    Science.gov (United States)

    Michaillat, Lydie; Baars, Tonie Luise; Mayer, Andreas

    2012-01-01

    Size and copy number of organelles are influenced by an equilibrium of membrane fusion and fission. We studied this equilibrium on vacuoles—the lysosomes of yeast. Vacuole fusion can readily be reconstituted and quantified in vitro, but it had not been possible to study fission of the organelle in a similar way. Here we present a cell-free system that reconstitutes fragmentation of purified yeast vacuoles (lysosomes) into smaller vesicles. Fragmentation in vitro reproduces physiological aspects. It requires the dynamin-like GTPase Vps1p, V-ATPase pump activity, cytosolic proteins, and ATP and GTP hydrolysis. We used the in vitro system to show that the vacuole-associated TOR complex 1 (TORC1) stimulates vacuole fragmentation but not the opposing reaction of vacuole fusion. Under nutrient restriction, TORC1 is inactivated, and the continuing fusion activity then dominates the fusion/fission equilibrium, decreasing the copy number and increasing the volume of the vacuolar compartment. This result can explain why nutrient restriction not only induces autophagy and a massive buildup of vacuolar/lysosomal hydrolases, but also leads to a concomitant increase in volume of the vacuolar compartment by coalescence of the organelles into a single large compartment. PMID:22238359

  2. Mutations that affect vacuole biogenesis inhibit proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae.

    Science.gov (United States)

    Koning, Ann J; Larson, Lynnelle L; Cadera, Emily J; Parrish, Mark L; Wright, Robin L

    2002-04-01

    In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.

  3. Vacuole import and degradation pathway:Insights into a specialized autophagy pathway

    Institute of Scientific and Technical Information of China (English)

    Abbas; A; Alibhoy; Hui-Ling; Chiang

    2011-01-01

    Glucose deprivation induces the synthesis of pivotagluconeogenic enzymes such as fructose-1,6-bisphos-phatase, malate dehydrogenase, phosphoenolpyruvatecarboxykinase and isocitrate lyase in Saccharomycescerevisiae. However, following glucose replenishment,these gluconeogenic enzymes are inactivated and de-graded. Studies have characterized the mechanismsby which these enzymes are inactivated in response toglucose. The site of degradation of these proteins hasalso been ascertained to be dependent on the dura-tion of starvation. Glucose replenishment of short-termstarved cells results in these proteins being degradedin the proteasome. In contrast, addition of glucose tocells starved for a prolonged period results in theseproteins being degraded in the vacuole. In the vacuoledependent pathway, these proteins are sequestered inspecialized vesicles termed vacuole import and degra-dation (Vid). These vesicles converge with the endo-cytic pathway and deliver their cargo to the vacuolefor degradation. Recent studies have identified thatinternalization, as mediated by actin polymerization, isessential for delivery of cargo proteins to the vacuolefor degradation. In addition, components of the targetof rapamycin complex 1 interact with cargo proteins during glucose starvation. Furthermore, Tor1p dissoci-ates from cargo proteins following glucose replenish-ment. Future studies will be needed to elaborate on the importance of internalization at the plasma membrane and the subsequent import of cargo proteins into Vid vesicles in the vacuole dependent degradation pathway.

  4. Methods to monitor autophagy in H. pylori vacuolating cytotoxin A (VacA)-treated cells.

    Science.gov (United States)

    Raju, Deepa; Jones, Nicola L

    2010-01-01

    Helicobacter pylori is a gram negative pathogen that infects at least half of the world's population and is associated not only with gastric cancer but also with other diseases such as gastritis and peptic ulcers. Indeed, H. pylori is considered the single most important risk factor for the development of gastric cancer. The vacuolating cytotoxin VacA, secreted by H. pylori, promotes intracellular survival of the bacterium and modulates host immune responses. In a recent study, we reported that VacA induces autophagy. Multilamellar autophagosomes are detected in gastric epithelial cells that are distinct from the large vacuoles formed by VacA. Furthermore, inhibition of autophagy stabilizes VacA and reduces vacuolation in the cells indicating that the toxin is being degraded by autophagy, thus limiting toxin-induced host cell damage. Many of the methods that were used for this study are commonly employed techniques that were adapted for H. pylori infection and VacA intoxication. In this paper, we describe the various methods and specific protocols used for the assessment and monitoring of autophagy during H. pylori infection.

  5. Characterization of the Vacuolating Cytotoxin in Helicobacter pylori Strains Isolated from Iran

    Directory of Open Access Journals (Sweden)

    Akbar Oghalaie

    2010-01-01

    Full Text Available Objective: Helicobacter pylori (H. pylori cytotoxin and its heterogeneity amongst strains hasbeen closely linked to the varying infection-associated clinical outcomes. In order to determinethe decisive role of the vacuolating cytotoxin (vacA gene mosaicism in its corresponding geneexpression and phenotype, we aimed to characterize vacA alleles of different H. pylori strainsin addition to the resulting protein and its vacuolating activity in epithelial cell culture.Materials and Methods: vacA gene polymorphism was determined for 80 H. pylori strainsisolated from dyspeptic patients, using multiplex gene-specific polymerase chain reaction(PCR. VacA protein was detected by immuno-blotting assay using a polyclonal anti-VacAantibody. In vitro cytotoxicity assay was conducted on HeLa cells in order to evaluate thevacuolating cytotoxin activity.Results: Genotyping revealed the following strain distribution: 26 (32.5% s1m1, 35(43.8% s1m2, and 19 (23.8% s2m2 subtypes. Infection with s1m1 type strain was significantlyassociated with gastric cancer as compared to non-ulcer dyspepsia (p=0.005and peptic ulcer disease (p=0.008. A 95-kDa immuno-reactive band that represented thevacuolating toxin was demonstrated in SDS-PAGE analysis of concentrated culture filtrate(CCF of H. pylori strains. H. pylori CCFs induced HeLa cell vacuolation which correlatedwith the strain genotype; s1m1 strains demonstrated higher levels of vacuolation as comparedto s1m2 strains, whereas s2m2 strains showed no detectable cytotoxic activity.Conclusion: The current study confirmed the relatively high cytotoxic activity of s1m1type H. pylori strains which infect the majority of patients suffering from gastric cancer andmay be partly responsible for the pathogenesis of this mortal disease.

  6. 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.

  7. 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.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. The Capsicum annuum class IV chitinase ChitIV interacts with receptor-like cytoplasmic protein kinase PIK1 to accelerate PIK1-triggered cell death and defence responses.

    Science.gov (United States)

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-04-01

    The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1.

  11. Genes controlling the development and function of plant vacuoles

    NARCIS (Netherlands)

    Li, Y.

    2017-01-01

    All plant cells contain numerous organelles, like mitochondria chloroplasts, with specific functions that are generally very similar among cell types and species. However, vacuoles, which are by far the largest compartments in plant cells, show a broad diversification in shape, dimensions, content

  12. Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome

    Science.gov (United States)

    Necchi, Vittorio; Minelli, Antonella; Sommi, Patrizia; Vitali, Agostina; Caruso, Roberta; Longoni, Daniela; Frau, Maria Rita; Nasi, Cristina; De Gregorio, Fabiola; Zecca, Marco; Ricci, Vittorio; Danesino, Cesare; Solcia, Enrico

    2012-01-01

    Background Shwachman–Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms. Design and Methods Blood neutrophils from 13 cases of Shwachman–Diamond syndrome – ten with and three without SBDS gene mutation – and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins. Results Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78±5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman–Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman–Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74±0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman–Diamond syndrome patients). Neutrophils from non-mutated Shwachman–Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and

  13. IFNγ inhibits the cytosolic replication of Shigella flexneri via the cytoplasmic RNA sensor RIG-I.

    Directory of Open Access Journals (Sweden)

    Stephanie P Jehl

    Full Text Available The activation of host cells by interferon gamma (IFNγ is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1, an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR retanoic acid-inducible gene I (RIG-I, a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS--but not cytosolic Nod-like receptors (NLRs--are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5'-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing

  14. Protein delivery to vacuole requires SAND protein-dependent Rab GTPase conversion for MVB-vacuole fusion

    NARCIS (Netherlands)

    Singh, M.K.; Krüger, F.; Beckmann, H.; Brumm, S.; Vermeer, J.E.M.; Munnik, T.; Mayer, U.; Stierhof, Y.D.; Grefen, C.; Schumacher, K.; Jürgens, G.

    2014-01-01

    Plasma-membrane proteins such as ligand-binding receptor kinases, ion channels, or nutrient transporters are turned over by targeting to a lytic compartment--lysosome or vacuole--for degradation. After their internalization, these proteins arrive at an early endosome, which then matures into a late

  15. Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells.

    Science.gov (United States)

    Taguchi; Fujikawa; Yazawa; Kodaira; Hayashida; Shimosaka; Okazaki

    2000-02-21

    Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.

  16. Cytoplasmic permeation pathway of neurotransmitter transporters.

    Science.gov (United States)

    Rudnick, Gary

    2011-09-06

    Ion-coupled solute transporters are responsible for transporting nutrients, ions, and signaling molecules across a variety of biological membranes. Recent high-resolution crystal structures of several transporters from protein families that were previously thought to be unrelated show common structural features indicating a large structural family representing transporters from all kingdoms of life. This review describes studies that led to an understanding of the conformational changes required for solute transport in this family. The first structure in this family showed the bacterial amino acid transporter LeuT, which is homologous to neurotransmitter transporters, in an extracellularly oriented conformation with a molecule of leucine occluded at the substrate site. Studies with the mammalian serotonin transporter identified positions, buried in the LeuT structure, that defined a potential pathway leading from the cytoplasm to the substrate binding site. Modeling studies utilized an inverted structural repeat within the LeuT crystal structure to predict the conformation of LeuT in which the cytoplasmic permeation pathway, consisting of positions identified in SERT, was open for diffusion of the substrate to the cytoplasm. From the difference between the model and the crystal structures, a simple "rocking bundle" mechanism was proposed, in which a four-helix bundle changed its orientation with respect to the rest of the protein to close the extracellular pathway and open the cytoplasmic one. Subsequent crystal structures from structurally related proteins provide evidence supporting this model for transport.

  17. In vivo accumulation of Helicobacter pylori products, NOD1, ubiquitinated proteins and proteasome in a novel cytoplasmic structure.

    Directory of Open Access Journals (Sweden)

    Vittorio Necchi

    Full Text Available Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer, are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance.

  18. In vivo accumulation of Helicobacter pylori products, NOD1, ubiquitinated proteins and proteasome in a novel cytoplasmic structure.

    Science.gov (United States)

    Necchi, Vittorio; Sommi, Patrizia; Ricci, Vittorio; Solcia, Enrico

    2010-03-16

    Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS) we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance.

  19. In Vivo Accumulation of Helicobacter pylori Products, NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure

    Science.gov (United States)

    Necchi, Vittorio; Sommi, Patrizia; Ricci, Vittorio; Solcia, Enrico

    2010-01-01

    Cell internalization and intracellular fate of H. pylori products/virulence factors in vivo by human gastric epithelium, the main target of H. pylori-induced pathologies (i.e., peptic ulcer and cancer), are still largely unknown. Investigating gastric endoscopic biopsies from dyspeptic patients by means of ultrastructural immunocytochemistry, here we show that, in human superficial-foveolar epithelium and its metaplastic or dysplastic foci, H. pylori virulence factors accumulated in a discrete cytoplasmic structure characterized by 13-nm-thick cylindrical particles of regular punctate-linear substructure resembling the proteasome complex in size and structure. Inside this particle-rich cytoplasmic structure (PaCS) we observed colocalization of VacA, CagA, urease and outer membrane proteins with NOD1 receptor, ubiquitin-activating enzyme E1, polyubiquitinated proteins, proteasome components and potentially oncogenic proteins like SHP2 and ERKs in human gastric epithelium. By means of electron and confocal microscopy, we demonstrate that the in vivo findings were reproduced in vitro by incubating human epithelial cell lines with H. pylori products/virulence factors. PaCSs differed from VacA-induced vacuoles, phagosomes, aggresomes or related bodies. Our data suggest that PaCS is a novel, proteasome-enriched structure arising in ribosome-rich cytoplasm at sites of H. pylori products accumulation. As a site of selective concentration of bacterial virulence factors, the ubiquitin-proteasome system and interactive proteins, PaCS is likely to modulate immune-inflammatory and proliferative responses of the gastric epithelium of potential pathologic relevance. PMID:20300534

  20. Cellular Subcompartments through Cytoplasmic Streaming.

    Science.gov (United States)

    Pieuchot, Laurent; Lai, Julian; Loh, Rachel Ann; Leong, Fong Yew; Chiam, Keng-Hwee; Stajich, Jason; Jedd, Gregory

    2015-08-24

    Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming.

  1. Vitrification Increased Vacuolization of Human Spematozoa: Application of MSOME Technology.

    Science.gov (United States)

    Taherzadeh, Sara; Khalili, Mohammad Ali; Agha-Rahimi, Azam; Anbari, Fateme; Ghazali, Shahin; Macchiarelli, Guido

    2017-01-01

    Sperm vitrification is a technique of ice and cryoprotectant free cryopreservation by direct plunging of sperm suspension into liquid nitrogen (LN2). The aim of this study was to investigate the influence of cryoprotectant free-vitrification on human sperm fine structure by MSOME technology and the fertility potential by zona binding assay (ZBA). 20 normo-ejaculates were prepared by swim up technique, and supernatants were divided into two parts of fresh and vitrified groups. For vitrification, sperm was dropped into LN2. Sperm motility, morphology, viability and MSOME were evaluated for each sample. In MSOM morphologically normal sperm (class 1), ≤2 small vacuoles (class 2), and one large vacuole or >2 small vacuoles (class 3) were evaluated. Also, fertility potential was evaluated by zona binding assay. Data was analyzed using paired t-test or Willcoxon's test and p-value <0.05 was considered significant. Vitrification significantly reduced both progressive motility, viability and morphology. Also, normal morphology of spermatozoa decreased significantly after vitrification. In MSOME evaluation, normal motile spermatozoa (Class 1) decreased from 23.00±12.44 to 16.00.56±10.79 after vitrification (p=0.008). Although spermatozoa classes 2 and 3 were increased, the difference was not significant. Moreover, fertility potential of motile spermatozoa was reduced after vitrification (9.0±13.87 vs. 13.40±22.73; p=0.07). Vitrification increased the rate of vacuolization in motile sperm head. Therefore, MSOME technology is recommended for assessment of sperm fine morphology in ICSI program used cryopreserved spermatozoa.

  2. 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.

  3. Immune complex stimulation of human neutrophils involves a novel Ca2+/H+ exchanger that participates in the regulation of cytoplasmic pH: flow cytometric analysis of Ca2+/pH responses by subpopulations.

    Science.gov (United States)

    Bernardo, John; Hartlaub, Hilary; Yu, Xin; Long, Heidi; Simons, Elizabeth R

    2002-12-01

    The activation of human phagocytic leukocytes by immune complexes (IC) or opsonized microbes via their Fc and complement receptors has been well-described. The mechanisms involved in this process are complex and depend on the receptors involved. The biochemical events that lead to the destruction of invading organisms in turn display varying degrees of interdependence, but the controlling elements that lead to the ultimate killing of ingested organisms within phagosomes by lysosomal enzymes and reactive oxygen intermediates are still not completely understood. We have addressed these mechanisms by following and correlating the kinetics of responses by individual cells, using multiparameter flow cytometry. Using nonopsonized IC as stimuli, we document here the presence of a novel Ca(2)(+)/H(+) voltage-independent channel in human neutrophils, which helps to control their cytoplasmic pH.

  4. Delivery of prolamins to the protein storage vacuole in maize aleurone cells.

    Science.gov (United States)

    Reyes, Francisca C; Chung, Taijoon; Holding, David; Jung, Rudolf; Vierstra, Richard; Otegui, Marisa S

    2011-02-01

    Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.

  5. Inhibitory monoclonal antibody against a (myristylated) small-molecular-weight antigen from Plasmodium falciparum associated with the parasitophorous vacuole membrane.

    Science.gov (United States)

    Kara, U A; Stenzel, D J; Ingram, L T; Bushell, G R; Lopez, J A; Kidson, C

    1988-04-01

    A small-molecular-weight antigen that occurs in asexual blood stages in synchronized cultures of Plasmodium falciparum was detected by a monoclonal antibody which inhibits parasite growth in vitro. This antigen, QF116, showed a molecular weight of 15,000 in parasite strain FCR-3K+ from The Gambia and 19,000 in strain FCQ-27 from Papua New Guinea. The protein did not show significant glycosylation by galactose or glucosamine labeling but was found to be acylated by myristic acid. By using immunogold labeling and electron microscopy, the location of the antigen could be attributed to the parasitophorous vacuole membrane and to inclusions and vesicles residing within the cytoplasm of the erythrocyte host cell.

  6. 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 honeycomb-type ar- rangement within the cytoplasm (see review by Walsby, 1994). These vesicles are cylindrical membrane tubes com- posed entirely of proteins and capped on each end with a half-cone. In Microcystis, individual vesicles have width and height... layers were assigned to the vacuole and chromatoplasm, respectively. By assigning the layer with the higher refractive index to be the outer layer, the effect of the cell wall membrane which is known to have a great impact on scattering (Quinby-Hunt et al...

  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. CTP synthase forms cytoophidia in the cytoplasm and nucleus

    Energy Technology Data Exchange (ETDEWEB)

    Gou, Ke-Mian [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); State Key Laboratory for Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193 (China); Chang, Chia-Chun [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Shen, Qing-Ji [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom); Sung, Li-Ying, E-mail: liyingsung@ntu.edu.tw [Institute of Biotechnology, National Taiwan University, Taipei, Taiwan, ROC (China); Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan, ROC (China); Liu, Ji-Long, E-mail: jilong.liu@dpag.ox.ac.uk [MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT (United Kingdom)

    2014-04-15

    CTP synthase is an essential metabolic enzyme responsible for the de novo synthesis of CTP. Multiple studies have recently showed that CTP synthase protein molecules form filamentous structures termed cytoophidia or CTP synthase filaments in the cytoplasm of eukaryotic cells, as well as in bacteria. Here we report that CTP synthase can form cytoophidia not only in the cytoplasm, but also in the nucleus of eukaryotic cells. Both glutamine deprivation and glutamine analog treatment promote formation of cytoplasmic cytoophidia (C-cytoophidia) and nuclear cytoophidia (N-cytoophidia). N-cytoophidia are generally shorter and thinner than their cytoplasmic counterparts. In mammalian cells, both CTP synthase 1 and CTP synthase 2 can form cytoophidia. Using live imaging, we have observed that both C-cytoophidia and N-cytoophidia undergo multiple rounds of fusion upon glutamine analog treatment. Our study reveals the coexistence of cytoophidia in the cytoplasm and nucleus, therefore providing a good opportunity to investigate the intracellular compartmentation of CTP synthase. - Highlights: • CTP synthase forms cytoophidia not only in the cytoplasm but also in the nucleus. • Glutamine deprivation and Glutamine analogs promotes cytoophidium formation. • N-cytoophidia exhibit distinct morphology when compared to C-cytoophidia. • Both CTP synthase 1 and CTP synthase 2 form cytoophidia in mammalian cells. • Fusions of cytoophidia occur in the cytoplasm and nucleus.

  9. Observation of sperm-head vacuoles and sperm morphology under light microscope.

    Science.gov (United States)

    Park, Yong-Seog; Park, Sol; Ko, Duck Sung; Park, Dong Wook; Seo, Ju Tae; Yang, Kwang Moon

    2014-09-01

    The presence of sperm-head vacuoles has been suspected to be deleterious to the outcomes of assisted reproductive technology (ART). It is difficult to accurately distinguish morphologically abnormal sperm with vacuoles under a light microscope. This study was performed to analyze the result of the observation of sperm-head vacuoles using Papanicolaou staining under a light microscope and whether the male partner's age affects these vacuoles. Sperm morphology with vacuoles was evaluated using Papanicolaou staining and observed under a light microscope (400×) in 980 men. The normal morphology was divided into three categories (group A, 14% of normal morphology). The criteria for the sperm-head vacuoles were those given in the World Health Organization manual. For the analysis of the age factor, the participants were divided into the following groups: 26-30 years, 31-35 years, 36-40 years, 41-45 years, and 46-50 years. The percentage of sperm-head vacuoles increased with normal sperm morphology (group A vs. groups B, C) (p<0.05). In the case of the age factor, a statistically significant difference was not observed across any of the age groups. A majority of the sperm-head vacuoles showed a statistically significant difference among normal morphology groups. Therefore, we should consider the probability of the percentage of sperm-head vacuoles not increasing with age but with abnormal sperm morphology. A further study is required to clarify the effect of the sperm-head vacuoles on ART outcomes.

  10. Redox Enzymes of Red Beetroot Vacuoles (Beta vulgaris L.

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2014-12-01

    Full Text Available Years of research have shown that some of the redox elements (enzymes, coenzymes, and co-substrate are isolated from each other kinetic and spatial manner (compartmentalization in the eukaryotic cells. The redox elements forming the "highly" and "widely" specialized redox system are found in all cell structures: mitochondria, plastids, peroxisomes, apoplast, nucleus etc. In recent years the active involvement of the central vacuole in the maintenance of the plant cell redox homeostasis is discussed, actually the information about the vacuolar redox system is very small. The high-priority redox processes and "redox-specialization" of the vacuolar compartment are not known. We have begun a study of red beet-root vacuole redox systems (Beta vulgaris L. and have identified redox enzymes such as: phenol peroxidase (EC 1.11.1.7, superoxide dismutase (EC 1.15.1.1 and glutathione reductase (EC 1.8.1.7. This paper presents some of the characteristics of these enzymes and considers the probable ways of their functioning in vacuolar redox chains.

  11. Enzymic and protein character of tonoplast from Hippeastrum vacuoles

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, G.J.

    1981-01-01

    The membrane of anthocyanin containing Hippeastrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg/sup 2 +/ -dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, ..cap alpha..-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K/sub 2/HPO/sub 4/ or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF,. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.

  12. The role of Plasmodium falciparum food vacuole plasmepsins.

    Science.gov (United States)

    Liu, Jun; Gluzman, Ilya Y; Drew, Mark E; Goldberg, Daniel E

    2005-01-14

    Plasmepsins (PMs) are thought to have an important function in hemoglobin degradation in the malarial parasite Plasmodium falciparum and have generated interest as antimalarial drug targets. Four paralogous plasmepsins reside in the food vacuole of P. falciparum. Targeted gene disruption by double crossover homologous recombination has been employed to study food vacuole plasmepsin function in cultured parasites. Parasite clones with deletions in each of the individual PM I, PM II, and HAP genes as well as clones with a double PM IV/PM I disruption have been generated. All of these clones lack the corresponding PMs, are viable, and appear morphologically normal. PM II and PM IV/I disruptions have longer doubling times than the 3D7 parental line in rich RPMI medium. This appears to be because of a decreased level of productive progeny rather than an increased cell cycle time. In amino acid-limited medium, all four knockouts exhibit slower growth than the parental strain. Compared with 3D7, knock-out clone sensitivity to aspartic and cysteine protease inhibitors is changed minimally. These results suggest substantial functional redundancy and have important implications for the design of antimalarial drugs. The slow growth phenotype may explain why P. falciparum has maintained four plasmepsin genes with overlapping functions.

  13. Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

    Directory of Open Access Journals (Sweden)

    Aurélie Fougère

    2016-11-01

    Full Text Available Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC. P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

  14. Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora

    Institute of Scientific and Technical Information of China (English)

    Yumi Hirakawa; Toshihisa Nomura; Seiichiro Hasezawa; Takumi Higaki

    2015-01-01

    Vacuoles are suggested to play crucial roles in plant defense-related cel death. During programmed cel death, previous live cel imaging studies have observed vacuoles to become simpler in structure and have implicated this simplification as a prelude to the vacuole’s rupture and consequent lysis of the plasma membrane. Here, we examined dynamics of the vacuole in cel cycle-synchronized tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yel ow 2) cel s during cel death induced by application of culture filtrates of Erwinia carotovora. The filtrate induced death in about 90%of the cel s by 24 h. Prior to cel death, vacuole shape simplified and endoplasmic actin filaments disassembled;however, the vacuoles did not rupture until after plasma membrane integrity was lost. Instead of facilitating rupture, the simplification of vacuole structure might play a role in the retrieval of membrane components needed for defense-related cel death.

  15. Immunofluorescent localization of ubiquitin and proteasomes in nucleolar vacuoles of soybean root meristematic cells

    OpenAIRE

    Stępiński, D.

    2012-01-01

    In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provi...

  16. Co-occurrence of tannin and tannin-less vacuoles in sensitive plants.

    Science.gov (United States)

    Fleurat-Lessard, Pierrette; Béré, Emile; Lallemand, Magali; Dédaldéchamp, Fabienne; Roblin, Gabriel

    2016-05-01

    Vacuoles of different types frequently coexist in the same plant cell, but the duality of the tannin/tannin-less vacuoles observed in Mimosa pudica L. is rare. In this plant, which is characterized by highly motile leaves, the development and original features of the double vacuolar compartment were detailed in primary pulvini from the young to the mature leaf stage. In young pulvini, the differentiation of tannin vacuoles first occurred in the epidermis and progressively spread toward the inner cortex. In motor cells of nonmotile pulvini, tannin deposits first lined the membranes of small vacuole profiles and then formed opaque clusters that joined together to form a large tannin vacuole (TV), the proportion of which in the cell was approximately 45%. At this stage, transparent vacuole profiles were rare and small, but as the parenchyma cells enlarged, these profiles coalesced to form a transparent vacuole with a convexity toward the larger-sized tannin vacuole. When leaf motility began to occur, the two vacuole types reached the same relative proportion (approximately 30%). Finally, in mature cells displaying maximum motility, the large transparent colloidal vacuole (CV) showed a relative proportion increasing to approximately 50%. At this stage, the proportion of the tannin vacuole, occurring in the vicinity of the nucleus, decreased to approximately 10%. The presence of the condensed type of tannins (proanthocyanidins) was proven by detecting their fluorescence under UV light and by specific chemical staining. This dual vacuolar profile was also observed in nonmotile parts of M. pudica (e.g., the petiole and the stem). Additional observations of leaflet pulvini showing more or less rapid movements showed that this double vacuolar structure was present in certain plants (Mimosa spegazzinii and Desmodium gyrans), but absent in others (Albizzia julibrissin, Biophytum sensitivum, and Cassia fasciculata). Taken together, these observations strongly suggest that a

  17. Fe homeostasis in plant cells: does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

    Science.gov (United States)

    Pich, A; Manteuffel, R; Hillmer, S; Scholz, G; Schmidt, W

    2001-10-01

    The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.

  18. The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

    Science.gov (United States)

    Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

    2014-07-01

    Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

  19. Glucose alleviates cadmium toxicity by increasing cadmium fixation in root cell wall and sequestration into vacuole in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Yuan-Zhi Shi; Xiao-Fang Zhu; Jiang-Xue Wan; Gui-Xin Li; Shao-Jian Zheng

    2015-01-01

    Glucose (Glu) is involved in not only plant physiological and developmental events but also plant responses to abiotic stresses. Here, we found that the exogenous Glu improved root and shoot growth, reduced shoot cadmium (Cd) concentration, and rescued Cd-induced chlorosis in Arabidopsis thaliana (Columbia ecotype, Col-0) under Cd stressed conditions. Glucose increased Cd retained in the roots, thus reducing its translocation from root to shoot significantly. The most Cd retained in the roots was found in the hemicellulose 1. Glucose combined with Cd (Glu þ Cd) treatment did not affect the content of pectin and its binding capacity of Cd while it increased the content of hemicelluloses 1 and the amount of Cd retained in it significantly. Furthermore, Leadmium Green staining indicated that more Cd was compartmented into vacuoles in Glu þ Cd treatment compared with Cd treatment alone, which was in accordance with the significant upregulation of the expression of tonoplast-localized metal transporter genes, suggesting that com-partmentation of Cd into vacuoles also contributes to the Glu-alleviated Cd toxicity. Taken together, we demonstrated that Glu-alleviated Cd toxicity is mediated through increas-ing Cd fixation in the root cell wall and sequestration into the vacuoles.

  20. Pathogen vacuole purification from legionella-infected amoeba and macrophages.

    Science.gov (United States)

    Hoffmann, Christine; Finsel, Ivo; Hilbi, Hubert

    2013-01-01

    Legionella pneumophila replicates intracellularly in environmental and immune phagocytes within a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). Formation of LCVs is strictly dependent on the Icm/Dot type IV secretion system and the translocation of "effector" proteins into the cell. Some effector proteins decorate the LCV membrane and subvert host cell vesicle trafficking pathways. Here we describe a method to purify intact LCVs from Dictyostelium discoideum amoebae and RAW 264.7 murine macrophages. The method comprises a two-step protocol: first, LCVs are enriched by immuno-magnetic separation using an antibody against a bacterial effector protein specifically localizing to the LCV membrane, and second, the LCVs are further purified by density gradient centrifugation. The purified LCVs can be characterized by proteomics and other biochemical approaches.

  1. Pleiotropic actions of Helicobacter pylori vacuolating cytotoxin, VacA.

    Science.gov (United States)

    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), RPTPalpha and RPTPbeta, on the surface of host cells. VacA bound to RPTPbeta, 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 into p33 and p55 fragments. The p55 domain not only plays a role in binding to target cells but also in the formation of oligomeric structures and anionic membrane channels. Oral administration of VacA to wild-type mice, but not to RPTPbeta knockout mice, resulted in gastric ulcers, in agreement with the clinical effect of VacA. VacA with s1/m1 allele has more potent cytotoxic activity in relation to peptic ulcer disease and appears to be associated with human gastric cancer. VacA activates pro-apoptotic Bcl-2 family proteins, and induces apoptosis via a mitochondria-dependent pathway. VacA can disrupt other signal transduction pathways; VacA activates p38 MAPK, enhancing production of IL-8 and PGE(2), and PI3K/Akt, suppressing GSK-3beta activity. VacA has immunomodulatory actions on T cells and other immune cells, possibly contributing to the chronic infection seen with this organism. H. pylori virulence factors including VacA and CagA, which is encoded by cytotoxin-associated gene A, along with host genetic and environmental factors, constitute a complex network to regulate chronic gastric injury and inflammation, which is involved in a multistep process leading to gastric carcinogenesis.

  2. Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it.

    Directory of Open Access Journals (Sweden)

    Sebastian Virreira Winter

    Full Text Available IFN-γ is a major cytokine that mediates resistance against the intracellular parasite Toxoplasma gondii. The p65 guanylate-binding proteins (GBPs are strongly induced by IFN-γ. We studied the behavior of murine GBP1 (mGBP1 upon infection with T. gondii in vitro and confirmed that IFN-γ-dependent re-localization of mGBP1 to the parasitophorous vacuole (PV correlates with the virulence type of the parasite. We identified three parasitic factors, ROP16, ROP18, and GRA15 that determine strain-specific accumulation of mGBP1 on the PV. These highly polymorphic proteins are held responsible for a large part of the strain-specific differences in virulence. Therefore, our data suggest that virulence of T. gondii in animals may rely in part on recognition by GBPs. However, phagosomes or vacuoles containing Trypanosoma cruzi did not recruit mGBP1. Co-immunoprecipitation revealed mGBP2, mGBP4, and mGBP5 as binding partners of mGBP1. Indeed, mGBP2 and mGBP5 co-localize with mGBP1 in T. gondii-infected cells. T. gondii thus elicits a cell-autonomous immune response in mice with GBPs involved. Three parasitic virulence factors and unknown IFN-γ-dependent host factors regulate this complex process. Depending on the virulence of the strains involved, numerous GBPs are brought to the PV as part of a large, multimeric structure to combat T. gondii.

  3. New insights into the tonoplast architecture of plant vacuoles and vacuolar dynamics during osmotic stress

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    Marty Francis

    2005-08-01

    Full Text Available Abstract Background The vegetative plant vacuole occupies >90% of the volume in mature plant cells. Vacuoles play fundamental roles in adjusting cellular homeostasis and allowing cell growth. The composition of the vacuole and the regulation of its volume depend on the coordinated activities of the transporters and channels localized in the membrane (named tonoplast surrounding the vacuole. While the tonoplast protein complexes are well studied, the tonoplast itself is less well described. To extend our knowledge of how the vacuole folds inside the plant cell, we present three-dimensional reconstructions of vacuoles from tobacco suspension cells expressing the tonoplast aquaporin fusion gene BobTIP26-1::gfp. Results 3-D reconstruction of the cell vacuole made possible an accurate analysis of large spanning folds of the vacuolar membrane under both normal and stressed conditions, and suggested interactions between surrounding plastids. Dynamic, high resolution 3-D pictures of the vacuole in tobacco suspension cells monitored under different growth conditions provide additional details about vacuolar architecture. The GFP-decorated vacuole is a single continuous compartment transected by tubular-like transvacuolar strands and large membrane surfaces. Cell culture under osmotic stress led to a complex vacuolar network with an increased tonoplast surface area. In-depth 3-D realistic inspections showed that the unity of the vacuole is maintained during acclimation to osmotic stress. Vacuolar unity exhibited during stress adaptation, coupled with the intimate associations of vacuoles with other organelles, suggests a physiological role for the vacuole in metabolism, and communication between the vacuole and organelles, respectively, in plant cells. Desiccation stress ensuing from PEG treatment generates "double" membrane structures closely linked to the tonoplast within the vacuole. These membrane structures may serve as membrane reservoirs for

  4. Effect of NaCl and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability

    Institute of Scientific and Technical Information of China (English)

    Juan Sun; Kazuo Aoki; Jin-Xu Zheng; Bing-Zhong Su; Xiao-Hui Ouyang; Junichi Misumi

    2006-01-01

    AIM: To determine whether Helicobacter pylori (H pylori) vacuolating cytotoxin (VacA) regulates release of proinflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6)or alters gastric epithelial cell viability and to determine whether NaCl affects these VacA-induced changes.METHODS: Vacuolating activity was determined by measuring the uptake of neutral red into vacuoles of VacA-treated human gastric epithelial (AGS) cells. AGS cell viability was assessed by direct cell counting. Specific enzyme-linked immunosorbent assays (ELISA) and reverse transcriptase-polymerase chain reaction(RT-PCR)were performed to examine the effects of Hpylori VacA and NaCl on cell pro-inflammatory cytokine production in AGS cells. Immunohistochemical staining of gastric tissue from Mongolian gerbils was used to confirm VacA-induced pro-inflammatory cytokine production and the effects of NaCl on this VacA-induced response.RESULTS: Addition of VacA alone reduced AGS cell viability (P< 0.05), and this reduction was enhanced by high doses of NaCl (P< 0.05). VacA alone induced expression of TNF-α, IL-8 and IL-1β, while NaCl alone induced expression of TNF-α and IL-1β. Changes in mRNA levels in the presence of both VacA and NaCl were more complicated. For the case of TNF-a, expression was dosedependent on NaCl. IL-6 mRNA was not detected. However, low levels of IL-6 were detected by ELISA. Positive immunohistochemical staining of IL- 1, IL-6, and TNF-αwas found in gastric tissue of H pylori-infected gerbils fed with either a normal diet or a high salt diet. However,the staining of these three cytokines was stronger in H pylori-infected animals fed with a 5g/kg NaCl diet.CONCLUSION: VacA decreases the viability of AGS cells, and this effect can be enhanced by NaCl. NaCl also affects the production of pro-inflammatory cytokines induced by Vac A, suggesting that NaCl plays an important role in Hpylori-induced gastric epithelial cell cytotoxicity.

  5. Trypanosoma cruzi Differentiates and Multiplies within Chimeric Parasitophorous Vacuoles in Macrophages Coinfected with Leishmania amazonensis.

    Science.gov (United States)

    Pessoa, Carina Carraro; Ferreira, Éden Ramalho; Bayer-Santos, Ethel; Rabinovitch, Michel; Mortara, Renato Arruda; Real, Fernando

    2016-05-01

    The trypanosomatids Leishmania amazonensis and Trypanosoma cruzi are excellent models for the study of the cell biology of intracellular protozoan infections. After their uptake by mammalian cells, the parasitic protozoan flagellates L. amazonensis and T. cruzi lodge within acidified parasitophorous vacuoles (PVs). However, whereas L. amazonensis develops in spacious, phagolysosome-like PVs that may enclose numerous parasites, T. cruzi is transiently hosted within smaller vacuoles from which it soon escapes to the host cell cytosol. To investigate if parasite-specific vacuoles are required for the survival and differentiation of T. cruzi, we constructed chimeric vacuoles by infection of L. amazonensis amastigote-infected macrophages with T. cruzi epimastigotes (EPIs) or metacyclic trypomastigotes (MTs). These chimeric vacuoles, easily observed by microscopy, allowed the entry and fate of T. cruzi in L. amazonensis PVs to be dynamically recorded by multidimensional imaging of coinfected cells. We found that although T. cruzi EPIs remained motile and conserved their morphology in chimeric vacuoles, T. cruzi MTs differentiated into amastigote-like forms capable of multiplying. These results demonstrate that the large adaptive vacuoles of L. amazonensis are permissive to T. cruzi survival and differentiation and that noninfective EPIs are spared from destruction within the chimeric PVs. We conclude that T. cruzi differentiation can take place in Leishmania-containing vacuoles, suggesting this occurs prior to their escape into the host cell cytosol.

  6. Preparative Procedures Markedly Influence the Appearance and Structural Integrity of Protein Storage Vacuoles in Soybean Seeds

    Science.gov (United States)

    In legumes, vacuoles serve as the final depository for storage proteins. The protein storage vacuoles (PSVs) of soybean contain electron-transparent globoid regions in which phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) is sequestered. Here, I report the effect of preparative procedures o...

  7. An investigation of the potential effect of sperm nuclear vacuoles in human spermatozoa on DNA fragmentation using a neutral and alkaline Comet assay.

    Science.gov (United States)

    Pastuszek, E; Kiewisz, J; Skowronska, P; Liss, J; Lukaszuk, M; Bruszczynska, A; Jakiel, G; Lukaszuk, K

    2017-03-01

    Presence of vacuoles and degree of sperm DNA damage are considered to be the basic factors used for the assessment of sperm fertilization capacity. We aimed to investigate the link between these two parameters. According to our knowledge, this is the first study where the Comet assay was used to assess the degree of DNA fragmentation of sperm categorized by Motile Sperm Organelle Morphology Examination (MSOME) Grades. Semen samples from 10 patients were assessed. Spermatozoa were graded into four MSOME groups according to the Vanderzwalmen's criteria. A total of 3930 motile spermatozoa were selected one-by-one using an inverted microscope and transferred onto two different slides. The degree of DNA fragmentation was analyzed by alkaline and neutral Comet assay. Results of the neutral Comet assay showed that Grade I spermatozoa (absence of vacuoles) presented significantly lower dsDNA fragmentation level (mean: 3.13 ± 1.17%) than Grade II (maximum of two small vacuoles; mean: 10.34 ± 2.65%), Grade III (more than two small vacuoles or at least one large vacuole; mean: 23.88 ± 8.37%), and Grade IV (large vacuoles associated with abnormal head shapes or other abnormalities; mean: 36.94 ± 7.78%; p fragmentation level (mean: 8.33 ± 3.62%) than Grade III (mean: 25.64 ± 9.15%) and Grade IV (mean: 40.10 ± 9.10%, p  0.05). Probably, the vacuoles may be responsible for double strand DNA breaks rather than single strand DNA breaks (only 2.39% spermatozoa in MSOME Grade II, 1.76% in III, and 3.16% in IV has single strand breaks). The results demonstrate that lower MSOME grading correlates with lower sperm DNA fragmentation. Therefore, the observation of sperm nuclear vacuoles using real-time optical microscopy without precise DNA fragmentation examination is not sufficient for optimal sperm selection for intracytoplasmic sperm injection. © 2017 American Society of Andrology and European Academy of Andrology.

  8. The nature of human sperm head vacuoles: a systematic literature review.

    Science.gov (United States)

    Boitrelle, Florence; Guthauser, Bruno; Alter, Laura; Bailly, Marc; Wainer, Robert; Vialard, François; Albert, Martine; Selva, Jacqueline

    2013-01-01

    Motile sperm organelle morphology examination (MSOME) involves the use of differential interference contrast microscopy (also called Nomarski contrast) at high magnification (at least 6300x) to improve the observation of live human spermatozoa. In fact, this technique evidences sperm head vacuoles that are not necessarily seen at lower magnifications - particularly if the vacuoles are small (i.e. occupying nature. In an attempt to clarify this debate, we performed a systematic literature review in accordance with the PRISMA guidelines. The PubMed database was searched from 2001 onwards with the terms "MSOME", "human sperm vacuoles", "high-magnification, sperm". Out of 180 search results, 21 relevant English-language publications on the nature of human sperm head vacuoles were finally selected and reviewed. Our review of the literature prompted us to conclude that sperm-head vacuoles are nuclear in nature and are related to chromatin condensation failure and (in some cases) sperm DNA damage.

  9. Motile tubular vacuoles in extramatrical mycelium and sheath hyphae of ectomycorrhizal systems.

    Science.gov (United States)

    Allaway, W G; Ashford, A E

    2001-01-01

    Extramatrical mycelium and outer hyphae of the sheath of Eucalyptus pilularis-Pisolithus tinctorius mycorrhizas contain abundant motile tubular vacuoles which accumulate the carboxyfluorescein analogue Oregon Green 488 carboxylic acid. The fluorochrome accumulates in a system of small vacuoles, tubules, and larger vacuoles, which are interlinked, motile, and pleiomorphic, in external hyphae, cords, and hyphae of the outer sheath. There is often a difference in fluorescence between two neighbouring cells, indicating that the dolipore septum exercises control on the movement of material between cells. Generally the motile tubular vacuole system in mycorrhizas resembles that previously found in isolated mycelium. The majority of fungal cells in the sheath contain no fluorochrome even after long exposure of the mycorrhiza to the solution, but with differential interference optics the cells are clearly seen to be alive and to contain vacuoles resembling those in the outer hyphae. In translocation experiments, long-distance transport of the fluorochrome is slow and slight, or even nonexistent in some cases.

  10. Gravity-dependent polarity of cytoplasmic streaming in Nitellopsis

    Science.gov (United States)

    Wayne, R.; Staves, M. P.; Leopold, A. C.

    1990-01-01

    The internodal cells of the characean alga Nitellopsis obtusa were chosen to investigate the effect of gravity on cytoplasmic streaming. Horizontal cells exhibit streaming with equal velocities in both directions, whereas in vertically oriented cells, the downward-streaming cytoplasm flows ca. 10% faster than the upward-streaming cytoplasm. These results are independent of the orientation of the morphological top and bottom of the cell. We define the ratio of the velocity of the downward- to the upward-streaming cytoplasm as the polar ratio (PR). The normal polarity of a cell can be reversed (PR polarity of cytoplasmic streaming. Less than 1 micromole Ca2+ resulted in a PR < 1 while greater than 1 micromole Ca2+ resulted in the normal gravity response. The voltage-dependent Ca(2+)-channel blocker, nifedipine, inhibited the gravity response in a reversible manner, while treatment with LaCl3 resulted in a PR < 1, indicating the presence of two types of Ca2+ channels. A new model for graviperception is presented in which the whole cell acts as the gravity sensor, and the plasma membrane acts as the gravireceptor. This is supported by ligation and UV irradiation experiments which indicate that the membranes at both ends of the cell are required for graviperception. The density of the external medium also affects the PR of Nitellopsis. Calculations are presented that indicate that the weight of the protoplasm may provide enough potential energy to open ion channels.

  11. Food vacuole associated enolase in plasmodium undergoes multiple post-translational modifications: evidence for atypical ubiquitination.

    Directory of Open Access Journals (Sweden)

    Saudamini Shevade

    Full Text Available Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of ~50 kDa and are likely to arise due to changes in pI, food vacuole (FV associated enolase showed three forms with MW~50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS, definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1 and the C-terminal G76 of the second (Ub2. Ub2 and third ubiquitin (Ub3 were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported.

  12. Ultrastructural transformations in the cytoplasm of differentiating Hyacinthus orientalis L. pollen cells

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    Elżbieta Bednarska

    2014-01-01

    Full Text Available The sequence of ultrastructural changes in the cytoplasm during the successive stages of pollen grain development in Hyacinthus orientulis pollen cells was studied. The cytoplasmic transformations of the generative cell included the elimination of plastids, increase in the number of mitochondria, assumption of a spindle shape with the aid of microtubules and the characteristic development of the vacuole system with the formation of so-called colored bodies. The cytoplasmic transformations of the generative cell encompassed changes in the plastids, which began to accumulate starch soon after the cell was formed, then released it shortly before anthesis, an increase in the number of mitochondria and an increase in the number of highly active dictyosomes just before anthesis. Changes in the structure of the border region between the differentiating pollen cells were associated mainly with the periodical appearance of a callose wall and the presence of lysosome-like bodies in the cytoplasm of the vegetative cell surrounding the generative cell. They arose soon after the disappearance of the callose wall and disappeared shortly before anthesis.

  13. Role of endodermal cell vacuoles in shoot gravitropism.

    Science.gov (United States)

    Kato, Takehide; Morita, Miyo Terao; Tasaka, Masao

    2002-06-01

    In higher plants, shoots and roots show negative and positive gravitropism, respectively. Data from surgical ablation experiments and analysis of starch deficient mutants have led to the suggestion that columella cells in the root cap function as gravity perception cells. On the other hand, endodermal cells are believed to be the statocytes (that is, gravity perceiving cells) of shoots. Statocytes in shoots and roots commonly contain amyloplasts which sediment under gravity. Through genetic research with Arabidopsis shoot gravitropism mutants, sgr1/scr and sgr7/shr, it was determined that endodermal cells are essential for shoot gravitropism. Moreover, some starch biosynthesis genes and EAL1 are important for the formation and maturation of amyloplasts in shoot endodermis. Thus, amyloplasts in the shoot endodermis would function as statoliths, just as in roots. The study of the sgr2 and zig/sgr4 mutants provides new insights into the early steps of shoot gravitropism, which still remains unclear. SGR2 and ZIG/SGR4 genes encode a phospholipase-like and a v-SNARE protein, respectively. Moreover, these genes are involved in vacuolar formation or function. Thus, the vacuole must play an important role in amyloplast sedimentation because the sgr2 and zig/sgr4 mutants display abnormal amyloplast sedimentation.

  14. The Tale of Protein Lysine Acetylation in the Cytoplasm

    Directory of Open Access Journals (Sweden)

    Karin Sadoul

    2011-01-01

    Full Text Available Reversible posttranslational modification of internal lysines in many cellular or viral proteins is now emerging as part of critical signalling processes controlling a variety of cellular functions beyond chromatin and transcription. This paper aims at demonstrating the role of lysine acetylation in the cytoplasm driving and coordinating key events such as cytoskeleton dynamics, intracellular trafficking, vesicle fusion, metabolism, and stress response.

  15. Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole

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    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. Phytochelatin-metal(loid) transport into vacuoles shows different substrate preferences in barley and Arabidopsis.

    Science.gov (United States)

    Song, Won-Yong; Mendoza-Cózatl, David G; Lee, Youngsook; Schroeder, Julian I; Ahn, Sang-Nag; Lee, Hyun-Sook; Wicker, Thomas; Martinoia, Enrico

    2014-05-01

    Cadmium (Cd) and arsenic (As) are toxic to all living organisms, including plants and humans. In plants, Cd and As are detoxified by phytochelatins (PCs) and metal(loid)-chelating peptides and by sequestering PC-metal(loid) complexes in vacuoles. Consistent differences have been observed between As and Cd detoxification. Whereas chelation of Cd by PCs is largely sufficient to detoxify Cd, As-PC complexes must be sequestered into vacuoles to be fully detoxified. It is not clear whether this difference in detoxification pathways is ubiquitous among plants or varies across species. Here, we have conducted a PC transport study using vacuoles isolated from Arabidopsis and barley. Arabidopsis vacuoles accumulated low levels of PC2 -Cd, and vesicles from yeast cells expressing either AtABCC1 or AtABCC2 exhibited negligible PC2 -Cd transport activity compared with PC2 -As. In contrast, barley vacuoles readily accumulated comparable levels of PC2 -Cd and PC2 -As. PC transport in barley vacuoles was inhibited by vanadate, but not by ammonium, suggesting the involvement of ABC-type transporters. Interestingly, barley vacuoles exhibited enhanced PC2 transport activity when essential metal ions, such as Zn(II), Cu(II) and Mn(II), were added to the transport assay, suggesting that PCs might contribute to the homeostasis of essential metals and detoxification of non-essential toxic metal(loid)s.

  17. Actin and myosin regulate cytoplasm stiffness in plant cells: a study using optical tweezers.

    Science.gov (United States)

    van der Honing, Hannie S; de Ruijter, Norbert C A; Emons, Anne Mie C; Ketelaar, Tijs

    2010-01-01

    Here, we produced cytoplasmic protrusions with optical tweezers in mature BY-2 suspension cultured cells to study the parameters involved in the movement of actin filaments during changes in cytoplasmic organization and to determine whether stiffness is an actin-related property of plant cytoplasm. Optical tweezers were used to create cytoplasmic protrusions resembling cytoplasmic strands. Simultaneously, the behavior of the actin cytoskeleton was imaged. After actin filament depolymerization, less force was needed to create cytoplasmic protrusions. During treatment with the myosin ATPase inhibitor 2,3-butanedione monoxime, more trapping force was needed to create and maintain cytoplasmic protrusions. Thus, the presence of actin filaments and, even more so, the deactivation of a 2,3-butanedione monoxime-sensitive factor, probably myosin, stiffens the cytoplasm. During 2,3-butanedione monoxime treatment, none of the tweezer-formed protrusions contained filamentous actin, showing that a 2,3-butanedione monoxime-sensitive factor, probably myosin, is responsible for the movement of actin filaments, and implying that myosin serves as a static cross-linker of actin filaments when its motor function is inhibited. The presence of actin filaments does not delay the collapse of cytoplasmic protrusions after tweezer release. Myosin-based reorganization of the existing actin cytoskeleton could be the basis for new cytoplasmic strand formation, and thus the production of an organized cytoarchitecture.

  18. Content and vacuole extravacuole distribution of neutral sugars, free amino acids, and anthocyanin in protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, G.J.

    1979-07-01

    Neutral sugar, free amino acid, and anthocyanin levels and vacuole/extravacuole distribution were determined for Hippeastrum and Tulipa petal and Tulipa leaf protoplasts. Glucose and fructose, the predominant neutral monosaccharides observed, were primarily vacuolar in location. Glutamine, the predominant free amino acid found, was primarily extravacuolar. ..gamma..-methyleneglutamate was identified as a major constituent of Tulipa protoplasts. Qualitative characterization of Hippeastrum petal and vacuole organic acids indicated the presence of oxalic, malic, citric, and isocitric acids. Data are presented which indicate that vacuoles obtained by gentle osmotic shock of protoplasts in dibasic phosphate have good purity and retain their contents.

  19. An AFM Observation on Fossil Cytoplasm

    Institute of Scientific and Technical Information of China (English)

    WANG Xin; YU Junping; FANG Xiaohong

    2008-01-01

    Fossil cytoplasm is a new research topic of interest in paleobotany. Atomic force microscope (AFM) is a new technology applied widely in physics and biology; however, it is rarely used in paleontology. Here we applied AFM for the first time to study fossil cytoplasm. The results indicate that the fossil cytoplasm is heterogeneous and full of ultrastructures, just like extant cytoplasm, and that the application of AFM, especially in combination with other techniques, can reveal the subcellular details of fossil plants with more confidence.

  20. A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

    Science.gov (United States)

    Rae, Anne L; Casu, Rosanne E; Perroux, Jai M; Jackson, Mark A; Grof, Christopher P L

    2011-06-15

    Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form.

  1. 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.

  2. 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.

  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. TATA-binding protein-related factor 2 is localized in the cytoplasm of mammalian cells and much of it migrates to the nucleus in response to genotoxic agents.

    Science.gov (United States)

    Park, Kyoung-ae; Tanaka, Yuji; Suenaga, Yusuke; Tamura, Taka-aki

    2006-10-31

    TBP (TATA-binding protein)-related factor 2 (TRF2) regulates transcription during a nuber of cellular processes. We previously demonstrated that it is localized in the cytoplasm and is translocated to the nucleus by DNA-damaging agents. However, the cytoplasmic localization of TRF2 is controversial. In this study, we reconfirmed its cytoplasmic localization in various ways and examined its nuclear migration. Stresses such as heat shock, redox agents, heavy metals, and osmotic shock did not affect localization whereas genotoxins such as methyl methanesulfonate (MMS), cisplatin, etoposide, and hydroxyurea caused it to migrate to the nucleus. Adriamycin, mitomycin C and gamma-rays had no obvious effect. We determined optimal conditions for the nuclear migration. The proportions of cells with nuclei enriched for TRF2 were 25-60% and 5-10% for stressed cells and control cells, respectively. Nuclear translocation was observed after 1 h, 4 h and 12 h for cisplatin, etoposide and MMS and hydroxyurea, respectively. The association of TRF2 with the chromatin and promoter region of the proliferating cell nuclear antigen (PCNA) gene, a putative target of TRF2, was increased by MMS treatment. Thus TRF2 may be involved in genotoxin-induced transcriptional regulation.

  5. Abscisic acid prevents the coalescence of protein storage vacuoles by upregulating expression of a tonoplast intrinsic protein gene in barley aleurone.

    Science.gov (United States)

    Lee, Sung-eun; Yim, Hui-kyung; Lim, Mi-na; Yoon, In sun; Kim, Jeong hoe; Hwang, Yong-sic

    2015-03-01

    Tonoplast intrinsic proteins (TIPs) are integral membrane proteins that are known to function in plants as aquaporins. Here, we propose another role for TIPs during the fusion of protein storage vacuoles (PSVs) in aleurone cells, a process that is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Studies of the expression of barley (Hordeum vulgare) TIP genes (HvTIP) showed that GA specifically decreased the abundance of HvTIP1;2 and HvTIP3;1 transcripts, while ABA strongly increased expression of HvTIP3;1. Increased or decreased expression of HvTIP3;1 interfered with the hormonal effects on vacuolation in aleurone protoplasts. HvTIP3;1 gain-of-function experiments delayed GA-induced vacuolation, whereas HvTIP3;1 loss-of-function experiments promoted vacuolation in ABA-treated aleurone cells. These results indicate that TIP plays a key role in preventing the coalescence of small PSVs in aleurone cells. Hormonal regulation of the HvTIP3;1 promoter is similar to the regulation of the endogenous gene, indicating that induction of the transcription of HvTIP3;1 by ABA is a critical factor in the prevention of PSV coalescence in response to ABA. Promoter analysis using deletions and site-directed mutagenesis of sequences identified three cis-acting elements that are responsible for ABA responsiveness in the HvTIP3;1 promoter. Promoter analysis also showed that ABA responsiveness of the HvTIP3;1 promoter is likely to occur via a unique regulatory system distinct from that involving the ABA-response promoter complexes.

  6. Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p RequirementD⃞

    OpenAIRE

    Tedrick, Kelly; Trischuk, Tim; Lehner, Richard; Eitzen, Gary

    2004-01-01

    Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Δ growth defect selective for vacuol...

  7. Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p RequirementD⃞

    OpenAIRE

    Tedrick, Kelly; Trischuk, Tim; Lehner, Richard; Eitzen, Gary

    2004-01-01

    Organization of lipids into membrane microdomains is a vital mechanism of protein processing. Here we show that overexpression of ERG6, a gene involved in ergosterol synthesis, elevates sterol levels 1.5-fold on the vacuole membrane and enhances their homotypic fusion. The mechanism of sterol-enhanced fusion is not via more efficient sorting, but instead promotes increased kinetics of fusion subreactions. We initially isolated ERG6 as a suppressor of a vrp1Δ growth defect selective for vacuol...

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W.; Wagner, G.J.; Siegelman, H.W.; Hind, G.

    1977-01-01

    Intact vacuoles were isolated from petals of Hippeastrum and Tulipa (Wagner, G. J. and Siegelman, H. W. (1975) Science 190, 1298 to 1299). The ATPase activity of fresh vacuole suspensions was found to be 2 to 3 times that of protoplasts from the same tissue. 70 to 80% of the ATPase activity of intact vacuoles was recovered in tonoplast preparations. The antibiotic Dio-9 at 6 ..mu..g/10/sup 6/ vacuoles or protoplasts causes 40% inhibition. However, only the protoplast ATPase is sensitive to oligomycin. N,N'-dicyclohexylcarbondiimide (DCCD) slightly stimulates ATPase activity in both vacuole and protoplast suspensions, whereas ethyl-3-(3-dimethylaminopropyl carbodiimide) (EDAC) strongly inhibits. Spectrophotometric studies show that in the petal the vacuolar contents have a pH of 4.0 for Tulipa and 4.3 for Hippeastrum, whereas the intact isolated vacuole has an internal pH of 7.0 (in pH 8.0 buffer) for Tulipa and about 7.3 for Hippeastrum. Internal ion concentrations of 150, 46, 30, 30 and 6 mM were found for K/sup +/, Na/sup +/, Mg/sup 2 +/, Cl/sup -/, and Ca/sup 2 +/ respectively, which are about the same as those in protoplasts.

  10. Cytoplasmic Streaming - Skylab Student Experiment ED-63

    Science.gov (United States)

    1973-01-01

    This chart describes the Skylab student experiment (ED-63), Cytoplasmic Streaming, proposed by Cheryl A. Peitz of Arapahoe High School, Littleton, Colorado. Experiment ED-63 was to observe the effect of zero-gravity on cytoplasmic streaming in the aquatic plant named Elodea, commonly called water weed or water thyme. The phenomenon of cytoplasmic streaming is not well understood, but it is recognized as the circulation mechanism of the internal materials or cytoplasm of a cell. Cytoplasm is a gelatinous substance that has the ability to change its viscosity and flow, carrying various cell materials with it. The activity can be stimulated by sunlight or heat. In March 1972, NASA and the National Science Teachers Association selected 25 experiment proposals for flight on Skylab. Science advisors from the Marshall Space Flight Center aided and assisted the students in developing the proposals for flight on Skylab.

  11. Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabiditis elegans embryo

    Science.gov (United States)

    Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

    2011-01-01

    Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex (“cortical flow”) is oriented toward the anterior, whereas the flow in the central region (“cytoplasmic flow”) is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos. PMID:21730185

  12. Hydrodynamic property of the cytoplasm is sufficient to mediate cytoplasmic streaming in the Caenorhabditis elegans embryo.

    Science.gov (United States)

    Niwayama, Ritsuya; Shinohara, Kyosuke; Kimura, Akatsuki

    2011-07-19

    Cytoplasmic streaming is a type of intracellular transport widely seen in nature. Cytoplasmic streaming in Caenorhabditis elegans at the one-cell stage is bidirectional; the flow near the cortex ("cortical flow") is oriented toward the anterior, whereas the flow in the central region ("cytoplasmic flow") is oriented toward the posterior. Both cortical flow and cytoplasmic flow depend on non-muscle-myosin II (NMY-2), which primarily localizes in the cortex. The manner in which NMY-2 proteins drive cytoplasmic flow in the opposite direction from remote locations has not been fully understood. In this study, we demonstrated that the hydrodynamic properties of the cytoplasm are sufficient to mediate the forces generated by the cortical myosin to drive bidirectional streaming throughout the cytoplasm. We quantified the flow velocities of cytoplasmic streaming using particle image velocimetry (PIV) and conducted a three-dimensional hydrodynamic simulation using the moving particle semiimplicit method. Our simulation quantitatively reconstructed the quantified flow velocity distribution resolved through PIV analysis. Furthermore, our PIV analyses detected microtubule-dependent flows during the pronuclear migration stage. These flows were reproduced via hydrodynamic interactions between moving pronuclei and the cytoplasm. The agreement of flow dynamics in vivo and in simulation indicates that the hydrodynamic properties of the cytoplasm are sufficient to mediate cytoplasmic streaming in C. elegans embryos.

  13. Vibrio cholerae hemolysin is required for lethality, developmental delay, and intestinal vacuolation in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Hediye Nese Cinar

    Full Text Available BACKGROUND: Cholera toxin (CT and toxin-co-regulated pili (TCP are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations. CONCLUSIONS/SIGNIFICANCE: Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.

  14. 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.

  15. New Challenges for the Design of High Value Plant Products: Stabilization of Anthocyanins in Plant Vacuoles.

    Science.gov (United States)

    Passeri, Valentina; Koes, Ronald; Quattrocchio, Francesca M

    2016-01-01

    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.

  16. Abortive Process of a Novel Rapeseed Cytoplasmic Male Sterility Line Derived from Somatic Hybrids Between Brassica napus and Sinapis alba

    Institute of Scientific and Technical Information of China (English)

    WANG Juan; GAO Ya-nan; KONG Yue-qin; JIANG Jin-jin; LI Ai-min; ZHANG Yong-tai; WANG You-ping

    2014-01-01

    Somatic hybridization is performed to obtain significant cytoplasmic male sterility (CMS) lines, whose CMS genes are derived either from the transfer of sterile genes from the mitochondrial genome of donor parent to the counterpart of receptor or production of new sterile genes caused by mitochondrial genome recombination of the biparent during protoplast fusion. In this study, a novel male sterile line, SaNa-1A, was obtained from the somatic hybridization between Brassica napus and Sinapis alba. The normal anther development of the maintainer line, SaNa-1B, and the abortive process of SaNa-1A were described through phenotypic observations and microtome sections. The lforal organ of the sterile line SaNa-1A was sterile with a shortened iflament and delfated anther. No detectable pollen grains were found on the surface of the sterile anthers. Semi-thin sections indicated that SaNa-1A aborted in the pollen mother cell (PMC) stage when vacuolization of the tapetum and PMCs began. The tapetum radically elongated and became highly vacuolated, occupying the entire locule together with the vacuolated microspores. Therefore, SaNa-1A is different from other CMS lines, such as ogu CMS, pol CMS and nap CMS as shown by the abortive process of the anther.

  17. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Emanuela Mari

    2016-11-01

    Full Text Available Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2 and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS, mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  18. Regulation of autophagy by cytoplasmic p53.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, M Chiara; Galluzzi, Lorenzo; Vitale, Ilio; Djavaheri-Mergny, Mojgan; D'Amelio, Marcello; Criollo, Alfredo; Morselli, Eugenia; Zhu, Changlian; Harper, Francis; Nannmark, Ulf; Samara, Chrysanthi; Pinton, Paolo; Vicencio, José Miguel; Carnuccio, Rosa; Moll, Ute M; Madeo, Frank; Paterlini-Brechot, Patrizia; Rizzuto, Rosario; Szabadkai, Gyorgy; Pierron, Gérard; Blomgren, Klas; Tavernarakis, Nektarios; Codogno, Patrice; Cecconi, Francesco; Kroemer, Guido

    2008-06-01

    Multiple cellular stressors, including activation of the tumour suppressor p53, can stimulate autophagy. Here we show that deletion, depletion or inhibition of p53 can induce autophagy in human, mouse and nematode cells subjected to knockout, knockdown or pharmacological inhibition of p53. Enhanced autophagy improved the survival of p53-deficient cancer cells under conditions of hypoxia and nutrient depletion, allowing them to maintain high ATP levels. Inhibition of p53 led to autophagy in enucleated cells, and cytoplasmic, not nuclear, p53 was able to repress the enhanced autophagy of p53(-/-) cells. Many different inducers of autophagy (for example, starvation, rapamycin and toxins affecting the endoplasmic reticulum) stimulated proteasome-mediated degradation of p53 through a pathway relying on the E3 ubiquitin ligase HDM2. Inhibition of p53 degradation prevented the activation of autophagy in several cell lines, in response to several distinct stimuli. These results provide evidence of a key signalling pathway that links autophagy to the cancer-associated dysregulation of p53.

  19. Fractal organization of feline oocyte cytoplasm.

    Science.gov (United States)

    De Vico, G; Peretti, V; Losa, G A

    2005-01-01

    The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display self-similar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400x with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD). The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

  20. Fractal organization of feline oocyte cytoplasm

    Directory of Open Access Journals (Sweden)

    G De Vico

    2009-06-01

    Full Text Available The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display selfsimilar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400 X with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD. The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

  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. Quantitative trait locus mapping of genes associated with vacuolation in the adrenal X-zone of the DDD/Sgn inbred mouse

    Directory of Open Access Journals (Sweden)

    Suto Jun-ichi

    2012-11-01

    Full Text Available Abstract Background Adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse B6 strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors. Identification of the genetic factors controlling such strain differences should help analyze the X-zone function. In this study, a quantitative trait locus (QTL analysis for the extent of X-zone vacuolation was performed for two types of F2 female mice: F2Ay mice (F2 mice with the Ay allele and F2 non-Ay mice (F2 mice without the Ay allele. These were produced by crossing B6 females and DDD.Cg-Ay males. DDD.Cg-Ay is a congenic mouse strain for the Ay allele at the agouti locus and is used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The Ay allele is dominant and homozygous lethal; therefore, living Ay mice are invariably heterozygotes. Results Single QTL scans identified significant QTLs on chromosomes 1, 2, 6, and X for F2 non-Ay mice, and on chromosomes 2, 6, and 12 for F2Ay mice. The QTL on chromosome 2 was considered to be because of the agouti locus, which has been suggested to be associated with X-zone vacuolation. A significant QTL that interacted with the agouti locus was identified on chromosome 8. Conclusions The extent of X-zone vacuolation in DDD females was controlled by multiple genes with complex interactions. The murine X-zone is considered analogous structure to the human fetal zone. Therefore, the results of this study will aid in understanding function of not only of the X-zone but also of the human fetal zone

  3. Quantitative trait locus mapping of genes associated with vacuolation in the adrenal X-zone of the DDD/Sgn inbred mouse.

    Science.gov (United States)

    Suto, Jun-Ichi

    2012-11-06

    Adrenal gland of mice contains a transient zone between the adrenal cortex and the adrenal medulla: the X-zone. There are clear strain differences in terms of X-zone morphology. Nulliparous females of the inbred mouse DDD strain develop adrenal X-zones containing exclusively vacuolated cells, whereas females of the inbred mouse B6 strain develop X-zones containing only non-vacuolated cells. The X-zone vacuolation is a physiologic process associated with the X-zone degeneration and is tightly regulated by genetic factors. Identification of the genetic factors controlling such strain differences should help analyze the X-zone function. In this study, a quantitative trait locus (QTL) analysis for the extent of X-zone vacuolation was performed for two types of F2 female mice: F2Ay mice (F2 mice with the Ay allele) and F2 non-Ay mice (F2 mice without the Ay allele). These were produced by crossing B6 females and DDD.Cg-Ay males. DDD.Cg-Ay is a congenic mouse strain for the Ay allele at the agouti locus and is used for this study because a close association between the X-zone morphology and the agouti locus genotype has been suggested. The Ay allele is dominant and homozygous lethal; therefore, living Ay mice are invariably heterozygotes. Single QTL scans identified significant QTLs on chromosomes 1, 2, 6, and X for F2 non-Ay mice, and on chromosomes 2, 6, and 12 for F2Ay mice. The QTL on chromosome 2 was considered to be because of the agouti locus, which has been suggested to be associated with X-zone vacuolation. A significant QTL that interacted with the agouti locus was identified on chromosome 8. The extent of X-zone vacuolation in DDD females was controlled by multiple genes with complex interactions. The murine X-zone is considered analogous structure to the human fetal zone. Therefore, the results of this study will aid in understanding function of not only of the X-zone but also of the human fetal zone. Identifying the genes responsible for the QTLs will be

  4. Disruption of Toxoplasma gondii parasitophorous vacuoles by the mouse p47-resistance GTPases.

    Directory of Open Access Journals (Sweden)

    Sascha Martens

    2005-11-01

    Full Text Available The p47 GTPases are essential for interferon-gamma-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-gamma-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-gamma-mediated T. gondii growth restriction in mouse astrocytes.

  5. Disruption of Toxoplasma gondii Parasitophorous Vacuoles by the Mouse p47-Resistance GTPases.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available The p47 GTPases are essential for interferon-gamma-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-gamma-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-gamma-mediated T. gondii growth restriction in mouse astrocytes.

  6. The Expression of VacA in BCF of Helicobacter Pylori and Its Relationship to Vacuolated Effect

    Institute of Scientific and Technical Information of China (English)

    施理; 侯晓华; 易粹琼; 张锦坤

    2002-01-01

    Summary: The vacuolated effect of Helicobacter (H. Pylori) and its relationship to vacuolated cyto toxin antigen (VacA) were investigated by the method of cytotoxic test and SDS-pobyacrylamide gel electrophoresis (SDS-PAGE). Of the 62 clinical isolates, the broth culture filter (BCF) of 43 strains causecl the Vero cell intracytoplasmically vacuolated. H. Pylori strains were divided into H. Pylori (Toxin+) group with vacuolated effect and H. Pylori (Toxin-) group without vacuolated effect. The analysis of the BCF of H. Pylori (Toxin+) and that of H. Pylori (Toxin-) was studied by SDS-PAGE and Scan reader. A kind of protein with 87 ku molecular weight was recognized in the BCF of 30.23 % (13/43) H. Pylori (Toxin+) strains but in none of that of H. Pylori (Toxin-) strains, the difference was statistically significant (P<0. 05). There was a significant and concordant relation ship between OD of the protein band with 87 ku molecular weight and titer of vacuolated activity of H. Pylori(Toxin+) (r=0. 67 and P<0. 05 by linear regression analysis). H. Pylori strains were di-vided into H. Pylori (Toxin+) group with vacuolated effect and H. Pylori (Toxin-) group without vacuolated effect. The vacuolated effect of H. Pylori (Toxin+) was caused by the protein with 87 ku molecular weight (VacA).

  7. Imaging of calcium dynamics in pollen tube cytoplasm.

    Science.gov (United States)

    Barberini, María Laura; Muschietti, Jorge

    2015-01-01

    Cytoplasmic calcium [(Ca(2+))cyt] is a central component of cellular signal transduction pathways. In plants, many external and internal stimuli transiently elevate (Ca(2+))cyt, initiating downstream responses that control different features of plant development. In pollen tubes the establishment of an oscillatory gradient of calcium at the tip is essential for polarized growth. Disruption of the cytosolic Ca(2+) gradient by chelators or channel blockers inhibits pollen tube growth. To quantify the physiological role of (Ca(2+))cyt in cellular systems, genetically encoded Ca(2+) indicators such as Yellow Cameleons (YCs) have been developed. The Cameleons are based on a fluorescence resonance energy transfer (FRET) process. Here, we describe a method for imaging cytoplasmic Ca(2+) dynamics in growing pollen tubes that express the fluorescent calcium indicator Yellow Cameleon 3.6 (YC 3.6), using laser-scanning confocal microscopy.

  8. Ultrastructural and autoradiographic studies of the role of nucleolar vacuoles in soybean root meristem.

    OpenAIRE

    Dariusz Stepiński

    2004-01-01

    Ultrastructural and autoradiographic studies of nucleoli in soybean root meristematic cells in seedlings: (1) grown for 3 days at 25 degrees C (control), (2) grown for three days at 25 degrees C and for 4 days at 10 degrees C, and (3) grown as in (2) and recovered for 1 day at 25 degrees C were carried out. Control nucleoli had dense structure and a few small nucleolar vacuoles. Chilled plant nucleoli had less dense structure and no vacuoles. Nucleoli of plants recovered at 25 degrees C had b...

  9. CD248 facilitates tumor growth via its cytoplasmic domain

    Directory of Open Access Journals (Sweden)

    Janssens Tom

    2011-05-01

    Full Text Available Abstract Background Stromal fibroblasts participate in the development of a permissive environment for tumor growth, yet molecular pathways to therapeutically target fibroblasts are poorly defined. CD248, also known as endosialin or tumor endothelial marker 1 (TEM1, is a transmembrane glycoprotein expressed on activated fibroblasts. We recently showed that the cytoplasmic domain of CD248 is important in facilitating an inflammatory response in a mouse model of arthritis. Others have reported that CD248 gene inactivation in mice results in dampened tumor growth. We hypothesized that the conserved cytoplasmic domain of CD248 is important in regulating tumor growth. Methods Mice lacking the cytoplasmic domain of CD248 (CD248CyD/CyD were generated and evaluated in tumor models, comparing the findings with wild-type mice (CD248WT/WT. Results As compared to the response in CD248WT/WT mice, growth of T241 fibrosarcomas and Lewis lung carcinomas was significantly reduced in CD248CyD/CyD mice. Tumor size was similar to that seen with CD248-deficient mice. Conditioned media from CD248CyD/CyD fibroblasts were less effective at supporting T241 fibrosarcoma cell survival. In addition to our previous observation of reduced release of activated matrix metalloproteinase (MMP-9, CD248CyD/CyD fibroblasts also had impaired PDGF-BB-induced migration and expressed higher transcripts of tumor suppressor factors, transgelin (SM22α, Hes and Hey1. Conclusions The multiple pathways regulated by the cytoplasmic domain of CD248 highlight its potential as a therapeutic target to treat cancer.

  10. Plasmodium falciparum PfA-M1 aminopeptidase is trafficked via the parasitophorous vacuole and marginally delivered to the food vacuole

    Directory of Open Access Journals (Sweden)

    Nyalwidhe Julius

    2010-06-01

    Full Text Available Abstract Background The Plasmodium falciparum PfA-M1 aminopeptidase, encoded by a single copy gene, displays a neutral optimal activity at pH 7.4. It is thought to be involved in haemoglobin degradation and/or invasion of the host cells. Although a series of inhibitors developed against PfA-M1 suggest that this enzyme is a promising target for therapeutic intervention, the biological function(s of the three different forms of the enzyme (p120, p96 and p68 are not fully understood. Two recent studies using PfA-M1 transfections have also provided conflicting results on PfA-M1 localization within or outside the food vacuole. Alternative destinations, such as the nucleus, have also been proposed. Methods By using a combination of techniques, such as cellular and biochemical fractionations, biochemical analysis, mass-spectrometry, immunofluorescence assays and live imaging of GFP fusions to various PfA-M1 domains, evidence is provided for differential localization and behaviour of the three different forms of PfA-M1 in the infected red blood cell which had not been established before. Results The high molecular weight p120 form of PfA-M1, the only version of the protein with a hydrophobic transmembrane domain, is detected both inside the parasite and in the parasitophorous vacuole while the processed p68 form is strictly soluble and localized within the parasite. The transient intermediate and soluble p96 form is localized at the border of parasitophorous vacuole and within the parasite in a compartment sensitive to high concentrations of saponin. Upon treatment with brefeldin A, the PfA-M1 maturation is blocked and the enzyme remains in a compartment close to the nucleus. Conclusions The PfA-M1 trafficking/maturation scenario that emerges from this data indicates that PfA-M1, synthesized as the precursor p120 form, is targeted to the parasitophorous vacuole via the parasite endoplasmic reticulum/Golgi, where it is converted into the transient p96

  11. Xenopus egg cytoplasm with intact actin.

    Science.gov (United States)

    Field, Christine M; Nguyen, Phuong A; Ishihara, Keisuke; Groen, Aaron C; Mitchison, Timothy J

    2014-01-01

    We report optimized methods for preparing Xenopus egg extracts without cytochalasin D, that we term "actin-intact egg extract." These are undiluted egg cytoplasm that contains abundant organelles, and glycogen which supplies energy, and represents the least perturbed cell-free cytoplasm preparation we know of. We used this system to probe cell cycle regulation of actin and myosin-II dynamics (Field et al., 2011), and to reconstitute the large, interphase asters that organize early Xenopus embryos (Mitchison et al., 2012; Wühr, Tan, Parker, Detrich, & Mitchison, 2010). Actin-intact Xenopus egg extracts are useful for analysis of actin dynamics, and interaction of actin with other cytoplasmic systems, in a cell-free system that closely mimics egg physiology, and more generally for probing the biochemistry and biophysics of the egg, zygote, and early embryo. Detailed protocols are provided along with assays used to check cell cycle state and tips for handling and storing undiluted egg extracts.

  12. Cytoplasmic Streaming in the Drosophila Oocyte.

    Science.gov (United States)

    Quinlan, Margot E

    2016-10-06

    Objects are commonly moved within the cell by either passive diffusion or active directed transport. A third possibility is advection, in which objects within the cytoplasm are moved with the flow of the cytoplasm. Bulk movement of the cytoplasm, or streaming, as required for advection, is more common in large cells than in small cells. For example, streaming is observed in elongated plant cells and the oocytes of several species. In the Drosophila oocyte, two stages of streaming are observed: relatively slow streaming during mid-oogenesis and streaming that is approximately ten times faster during late oogenesis. These flows are implicated in two processes: polarity establishment and mixing. In this review, I discuss the underlying mechanism of streaming, how slow and fast streaming are differentiated, and what we know about the physiological roles of the two types of streaming.

  13. How crowded is the prokaryotic cytoplasm?

    Science.gov (United States)

    Spitzer, Jan; Poolman, Bert

    2013-07-11

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded cytogel extends the vectorial character of the plasma membrane deeper into the cytoplasm by about 20-70 nm. We discuss useful physiological insights that this model gives into the functioning of a prokaryotic cell on the micrometer scale. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole

    DEFF Research Database (Denmark)

    Payne, Richard; Xu, Deyang; Foureau, Emilien

    2017-01-01

    /peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding...

  15. PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for Pompe disease.

    Science.gov (United States)

    Hagemans, Marloes L C; Stigter, Rolinda L; van Capelle, Carine I; van der Beek, Nadine A M E; Winkel, Leon P F; van Vliet, Laura; Hop, Wim C J; Reuser, Arnold J J; Beishuizen, Auke; van der Ploeg, Ans T

    2010-04-01

    Screening of blood films for the presence of periodic acid-Schiff (PAS)-positive lymphocyte vacuoles is sometimes used to support the diagnosis of Pompe disease, but the actual diagnostic value is still unknown. We collected peripheral blood films from 65 untreated Pompe patients and 51 controls. Lymphocyte vacuolization was quantified using three methods: percentage vacuolated lymphocytes, percentage PAS-positive lymphocytes, and a PAS score depending on staining intensity. Diagnostic accuracy of the tests was assessed using receiver operating characteristic (ROC) curves. All three methods fully discerned classic infantile patients from controls. The mean values of patients with milder forms of Pompe disease were significantly higher than those of controls, but full separation was not obtained. The area under the ROC curve was 0.98 for the percentage vacuolated lymphocytes (optimal cutoff value 3; sensitivity 91%, specificity 96%) and 0.99 for the percentage PAS-positive lymphocytes and PAS score (optimal cutoff value 9; sensitivity 100%, specificity 98%). Our data indicate that PAS-stained blood films can be used as a reliable screening tool to support a diagnosis of Pompe disease. The percentage of PAS-positive lymphocytes is convenient for use in clinical practice but should always be interpreted in combination with other clinical and laboratory parameters.

  16. Retromer and the dynamin Vps1 cooperate in the retrieval of transmembrane proteins from vacuoles

    NARCIS (Netherlands)

    Arlt, Henning; Reggiori, Fulvio; Ungermann, Christian

    2015-01-01

    Endosomes are dynamic organelles that need to combine the ability to successfully deliver proteins and lipids to the lysosome-like vacuole, and recycle others to the Golgi or the plasma membrane. We now show that retromer, which is implicated in retrieval of proteins from endosomes to the Golgi or t

  17. Effects of lysosomal membrane protein depletion on the Salmonella-containing vacuole.

    Directory of Open Access Journals (Sweden)

    Everett A Roark

    Full Text Available Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2 are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane.

  18. Retromer and the dynamin Vps1 cooperate in the retrieval of transmembrane proteins from vacuoles

    NARCIS (Netherlands)

    Arlt, Henning; Reggiori, Fulvio; Ungermann, Christian

    2015-01-01

    Endosomes are dynamic organelles that need to combine the ability to successfully deliver proteins and lipids to the lysosome-like vacuole, and recycle others to the Golgi or the plasma membrane. We now show that retromer, which is implicated in retrieval of proteins from endosomes to the Golgi or t

  19. Vacuoles in sperm head are not associated with head morphology, DNA damage and reproductive success.

    Science.gov (United States)

    Fortunato, Adriana; Boni, Raffaele; Leo, Rita; Nacchia, Giuseppina; Liguori, Francesca; Casale, Sofia; Bonassisa, Paolo; Tosti, Elisabetta

    2016-02-01

    In this retrospective study of 873 men enrolled for assisted reproduction techniques, relationships between sperm quality parameters, motile sperm organelle morphology examination (MSOME), DNA damage and live birth rate were evaluated. The presence of vacuoles in the sperm heads was detected by MSOME. Either chromatin decondensation or DNA fragmentation was used to study DNA damage. Results show that age significantly affected some of the examined parameters. In particular, sperm concentration was positively correlated (R = 0.088; P = 0.01) and chromatin decondensation was negatively correlated (R = -0.102; P = 0.003) with age. Furthermore, live birth rate was significantly lower in men aged 40 years or older (P fragmentation and live birth rate. Considering sperm heads in relation to the shape (normal/abnormal) and vacuoles (presence/absence), no significant variations in the occurrence of vacuoles in either normal or abnormal heads were found. These data suggest that vacuoles are physiological features that do not alter sperm functionality, and it seems that MSOME is not necessary for increasing the success of assisted reproduction techniques.

  20. PAS-positive lymphocyte vacuoles can be used as diagnostic screening test for Pompe disease

    NARCIS (Netherlands)

    M.L.C. Hagemans (Marloes); R.L. Stigter; C.I. van Capelle (Carine); N.A.M.E. van der Beek (Nadine); L.P.F. Winkel (Léon); L. van Vliet (Laura); W.C.J. Hop (Wim); A.J.J. Reuser (Arnold); A. Beishuizen (Auke); A.T. van der Ploeg (Ans)

    2010-01-01

    textabstractScreening of blood films for the presence of periodic acid-Schiff (PAS)-positive lymphocyte vacuoles is sometimes used to support the diagnosis of Pompe disease, but the actual diagnostic value is still unknown. We collected peripheral blood films from 65 untreated Pompe patients and 51

  1. Ionizing Radiation Induces HMGB1 Cytoplasmic Translocation and Extracellular Release

    Institute of Scientific and Technical Information of China (English)

    Lili Wang; Li He; Guoqiang Bao; Xin He; Saijun Fan; Haichao Wang

    2016-01-01

    Objective A nucleosomal protein,HMGBI,can be secreted by activated immune cells or passively released by dying cells,thereby amplifying rigorous inflammatory responses.In this study we aimed to test the possibility that radiation similarly induces cytoplasmic HMGB1 translocation and release.Methods Human skin fibroblast (GM0639) and bronchial epithelial (16HBE) cells and rats were exposed to X-ray radiation,and HMGB1 translocation and release were then assessed by immunocytochemistry and immunoassay,respectively.Results At a wide dose range(4.0-12.0 Gy),X-ray radiation induced a dramatic cytoplasmic HMGB1 translocation,and triggered a time-and dose-dependent HMGB1 release both in vitro and in vivo.The radiation-mediated HMGB1 release was also associated with noticeable chromosomal DNA damage and loss of cell viability.Conclusions Radiation induces HMGB1 cytoplasmic translocation and extracellular release through active secretion and passive leakage processes.

  2. 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

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

    Science.gov (United States)

    Gavrin, Aleksandr; Kaiser, Brent N; Geiger, Dietmar; Tyerman, Stephen D; Wen, Zhengyu; Bisseling, Ton; Fedorova, Elena E

    2014-09-01

    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 cells and thus hypothesized that microsymbionts may cause modifications in vacuole formation or function. To examine this, we quantified the volumes and surface areas of plant cells, vacuoles, and symbiosomes in root nodules of Medicago truncatula and analyzed the expression and localization of VPS11 and VPS39, members of the HOPS vacuole-tethering complex. During the maturation of symbiosomes to become N2-fixing organelles, a developmental switch occurs and changes in vacuole features are induced. For example, we found that expression of VPS11 and VPS39 in infected cells is suppressed and host cell vacuoles contract, permitting the expansion of symbiosomes. Trafficking of tonoplast-targeted proteins in infected symbiotic cells is also altered, as shown by retargeting of the aquaporin TIP1g from the tonoplast membrane to the symbiosome membrane. This retargeting appears to be essential for the maturation of symbiosomes. We propose that these alterations in the function of the vacuole are key events in the adaptation of the plant cell to host intracellular symbiotic bacteria. © 2014 American Society of Plant Biologists. All rights reserved.

  4. Saccharomyces cerevisiae depend on vesicular traffic between Golgi and vacuole when Inositolphosphorylceramide synthase Aur1 is inactivated

    DEFF Research Database (Denmark)

    Voynova, Natalia S; Roubaty, Carole; Vazquez, Hector M

    2015-01-01

    that vesicle mediated transport between Golgi, endosomes and vacuole becomes crucial for survival when Aur1 is repressed, irrespective of the mode of repression. In addition, vacuolar acidification becomes essential when cells are acutely stressed by AbA, and Quinacrine uptake into vacuoles shows that Ab...

  5. 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 cell

  6. Natural Diversity in the N Terminus of the Mature Vacuolating Cytotoxin of Helicobacter pylori Determines Cytotoxin Activity

    OpenAIRE

    Letley, D. P.; Atherton, J C

    2000-01-01

    Naturally occurring noncytotoxic vacA type s2 strains of Helicobacter pylori have a 12-residue extension to the vacuolating cytotoxin (VacA) compared with cytotoxic type s1 strains. We show that adding the region encoding this extension to type s1 vacA completely abolishes vacuolating cytotoxin activity but has no effect on VacA production.

  7. Characterization of Helicobacter pylori VacA-containing vacuoles (VCVs), VacA intracellular trafficking and interference with calcium signalling in T lymphocytes.

    Science.gov (United States)

    Kern, Beate; Jain, Utkarsh; Utsch, Ciara; Otto, Andreas; Busch, Benjamin; Jiménez-Soto, Luisa; Becher, Dörte; Haas, Rainer

    2015-12-01

    The human pathogen Helicobacter pylori colonizes half of the global population. Residing at the stomach epithelium, it contributes to the development of diseases such as gastritis, duodenal and gastric ulcers, and gastric cancer. A major factor is the secreted vacuolating toxin VacA, which forms anion-selective channels in the endosome membrane that cause the compartment to swell, but the composition and purpose of the resulting VacA-containing vacuoles (VCVs) are still unknown. VacA exerts influence on the host immune response in various ways, including inhibition of T-cell activation and proliferation and suppression of the host immune response. In this study, for the first time the composition of VCVs from T cells was comprehensively analysed to investigate VCV function. VCVs were successfully isolated via immunomagnetic separation, and the purified vacuoles were analysed by mass spectrometry. We detected a set of 122 VCV-specific proteins implicated among others in immune response, cell death and cellular signalling processes, all of which VacA is known to influence. One of the individual proteins studied further was stromal interaction molecule (STIM1), a calcium sensor residing in the endoplasmic reticulum (ER) that is important in store-operated calcium entry. Live cell imaging microscopy data demonstrated colocalization of VacA with STIM1 in the ER and indicated that VacA may interfere with the movement of STIM1 towards the plasma membrane-localized calcium release activated calcium channel protein ORAI1 in response to Ca(2+) store depletion. Furthermore, VacA inhibited the increase of cytosolic-free Ca(2+) in the Jurkat E6-1 T-cell line and human CD4(+) T cells. The presence of VacA in the ER and its trafficking to the Golgi apparatus was confirmed in HeLa cells, identifying these two cellular compartments as novel VacA target structures.

  8. Retargeting a maize β-glucosidase to the vacuole--evidence from intact plants that zeatin-O-glucoside is stored in the vacuole.

    Science.gov (United States)

    Kiran, Nagavalli S; Benková, Eva; Reková, Alena; Dubová, Jaroslava; Malbeck, Jiří; Palme, Klaus; Brzobohatý, Břetislav

    2012-07-01

    Cytokinin (CK) activity is regulated by the complex interplay of their metabolism, transport, stability and cellular/tissue localization. O-glucosides of zeatin-type CKs are postulated to be storage and/or transport forms. Active CK levels are determined in part by their differential distribution of CK metabolites across different subcellular compartments. We have previously shown that overexpressing chloroplast-localized Zm-p60.1, a maize β-glucosidase capable of releasing active cytokinins from their O- and N3-glucosides, perturbs CK homeostasis in transgenic tobacco. We obtained tobacco (Nicotiana tabacum L., cv Petit Havana SR1) plants overexpressing a recombinant Zm-p60.1 that is targeted to the vacuole. The protein is correctly processed and localized to the vacuole. When grown on medium containing exogenous zeatin, transgenic seedlings rapidly accumulate fresh weight due to ectopic growths at the base of the hypocotyl. The presence of the enzyme in these ectopic structures is shown by histochemical staining. CK quantification reveals that these transgenic seedlings are unable to accumulate zeatin-O-glucoside to levels similar to those observed in the wild type. When crossed with tobacco overexpressing the zeatin-O-glucosyltransferase gene from Phaseolus, the vacuolar variant shows an almost complete reversion in the root elongation assay. This is the first evidence from intact plants that the vacuole is the storage organelle for CK O-glucosides and that they are available to attack by Zm-p60.1. We propose the use of Zm-p60.1 as a robust molecular tool that exploits the reversibility of O-glucosylation and enables delicate manipulations of active CK content at the cellular level.

  9. Subunit organization in cytoplasmic dynein subcomplexes

    Science.gov (United States)

    King, Stephen J.; Bonilla, Myriam; Rodgers, Michael E.; Schroer, Trina A.

    2002-01-01

    Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain–binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo. PMID:11967380

  10. Antineutrophil cytoplasmic antibodies in juvenile chronic arthritis

    NARCIS (Netherlands)

    Mulder, L; Horst, G; Limburg, P; deGraeffMeeder, ER; Kuis, W; Kallenberg, C

    1997-01-01

    Objective, To evaluate the diagnostic significance of antineutrophil cytoplasmic antibodies (ANCA) by assessing the prevalence of ANCA in juvenile chronic arthritis (JCA) (n = 93) of either oligoarticular, polyarticular, or systemic onset. To investigate the prevalence of ANCA in other diseases of c

  11. How crowded is the prokaryotic cytoplasm?

    NARCIS (Netherlands)

    Spitzer, Jan; Poolman, Bert; Ferguson, Stuart

    2013-01-01

    We consider biomacromolecular crowding within the cytoplasm of prokaryotic cells as a two-phase system of 'supercrowded' cytogel and 'dilute' cytosol; we simplify and quantify this model for a coccoid cell over a wide range of biomacromolecular crowding. The key result shows that the supercrowded

  12. Detection of antineutrophil cytoplasmic antibodies (ANCAs)

    DEFF Research Database (Denmark)

    Damoiseaux, Jan; Csernok, Elena; Rasmussen, Niels

    2017-01-01

    of diagnosis) from 251 patients with ANCA-associated vasculitis (AAV), including granulomatosis with polyangiitis and microscopic polyangiitis, and from 924 disease controls were tested for the presence of cytoplasmic pattern/perinuclear pattern and atypical ANCA (A-ANCA) by indirect immunofluorescence (IIF...

  13. Haemocytes of the cockle Cerastoderma glaucum: morphological characterisation and involvement in immune responses.

    Science.gov (United States)

    Matozzo, Valerio; Rova, Giulio; Marin, Maria Gabriella

    2007-10-01

    For the first time, morpho-functional characterisation of haemocytes from the cockle Cerastoderma glaucum was performed to identify circulating cell types and to study their involvement in immune responses. Haemocyte mean number was 5.5 (x 10(5)) cells/mL haemolymph. Two main haemocyte types were found in haemolymph: granulocytes (85%), about 10 microm in diameter and with evident cytoplasmic granules, and hyalinocytes (15%), 8 to 14 microm in diameter, with a few or no granules. Most of the cytoplasmic granules stained in vivo with Neutral Red, indicating that they were lysosomes. On the basis of haemocyte staining properties, granulocytes and hyalinocytes were further classified as basophils and acidophils. Acidophil hyalinocytes were the largest haemocyte type (about 14 microm in diameter) and had an eccentric nucleus and a large cytoplasmic vacuole. Both granulocytes and hyalinocytes (except acidophils) were able to phagocytise yeast cells, although the basal phagocytic index was very low (about 2%). It increased significantly (up to 26%) after pre-incubation of yeast in cell-free haemolymph, suggesting that haemolymph has opsonising properties. Haemocytes also produced superoxide anion. Moreover, both granulocytes and hyalinocytes (except acidophils) were positive for some important hydrolytic and oxidative enzymes. Lysozyme-like activity was recorded in both cell-free haemolymph and haemocyte lysate, although enzyme activity in cell lysate was significantly higher. Results indicate that haemocytes from C. glaucum are effective cells in immune responses.

  14. Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

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    Toshiyuki Takahashi

    Full Text Available A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

  15. Myelin vacuolation, optic neuropathy and retinal degeneration after closantel overdosage in sheep and in a goat.

    Science.gov (United States)

    van der Lugt, J J; Venter, I

    2007-01-01

    Toxicity of closantel, a halogenated salicylanilide anthelmintic, is described in 11 sheep and a goat, humanely killed 4-70 days after accidental overdosage. Status spongiosis of the cerebrum and cerebellum was present, its severity decreasing with time after treatment. Ultrastructurally, vacuoles in the cerebral white matter were seen to be intramyelinic due to splitting of myelin lamellae at the intraperiod lines, indicating myelin oedema. In the optic nerves, Wallerian degeneration and eventual fibrosis and atrophy of the nerves followed myelin vacuolation. Lesions in the optic nerves were particularly advanced in the intracanalicular portion, indicating a compressive neuropathy within the optic canal. Acute retinal lesions consisted of papilloedema, necrosis of the outer retinal layers (especially the photoreceptor layer), and retinal separation in tapetal and non-tapetal areas. In more chronic cases, the outer nuclear layer was diffusely attenuated and generally reduced to a single row of cells.

  16. Vacuole/extravacuole distribution of soluble protease in Hippeastrum petal and Triticum leaf protoplasts

    Energy Technology Data Exchange (ETDEWEB)

    Wagner, G.J.; Mulready, P.; Cutt, J.

    1981-11-01

    The subcellular distribution of soluble protease in anthesis-stage, anthocyanin-containing Hippeastrum cv. Dutch Red Hybrid petal protoplasts has been reevaluated and that of Triticum aestivum L. var. Red Coat leaf protoplasts determined using /sup 125/I-fibrin as a protease substrate and improved methods for protoplast and vacuole volume estimation. Results indicate that about 20% of the Hippeastrum petal-soluble protease and about 90% of the wheat leaf-soluble protease can be assigned to the vacuole. Protoplast isolation enzyme labeled with /sup 125/I has been used to assess the efficiency of removing isolation enzyme from protoplasts by repeated washing and by separation of protoplasts from debris using density centrifugation. Results of these studies suggest that protoplasts prepared by both methods retain low levels of isolation enzyme. However, when protoplasts prepared by either method were lysed with washing medium lacking osmoticum, little isolation enzyme contaminated the lysates.

  17. An Acanthamoeba castellanii metacaspase associates with the contractile vacuole and functions in osmoregulation.

    Science.gov (United States)

    Saheb, Entsar; Trzyna, Wendy; Bush, John

    2013-03-01

    Acanthamoeba castellanii is a free-living protozoan. Some strains are opportunistic pathogens. A type-I metacaspase was identified in A. castellanii (Acmcp) and was shown to be expressed through the encystation process. The model organism, Dictyostelium discoideum, has been used here as a model for studying these caspase-like proteins. Separate cell lines expressing a GFP-tagged version of the full length Acmcp protein, as well as a deletion proline region mutant of Acmcp protein (GFP-Acmcp-dpr), have been introduced into D. discoideum. Both mutants affect the cellular metabolism, characterized by an increase in the growth rate. Microscopic imaging revealed an association between Acmcp and the contractile vacuole system in D. discoideum. The treatment of cells with selected inhibitors in different environments added additional support to these findings. This evidence shows that Acmcp plays an important role in contractile vacuole regulation and mediated membrane trafficking in D. discoideum. Additionally, the severe defect in contractile vacuole function in GFP-Acmcp-dpr mutant cells suggests that the proline-rich region in Acmcp has an essential role in binding this protein with other partners to maintain this process. Furthermore, Yeast two-hybrid system identified there are weak interactions of the Dictyostelium contractile vacuolar proteins, including Calmodulin, RabD, Rab11 and vacuolar proton ATPase, with Acmcp protein. Taken together, our findings suggest that A. castellanii metacaspase associate with the contractile vacuole and have an essential role in cell osmoregulation, which contributes to its attractiveness as a possible target for treatment therapies against A. castellanii infection.

  18. Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles.

    Science.gov (United States)

    Nolan, Sabrina J; Romano, Julia D; Luechtefeld, Thomas; Coppens, Isabelle

    2015-05-01

    Toxoplasma gondii and Neospora caninum, which cause the diseases toxoplasmosis and neosporosis, respectively, are two closely related apicomplexan parasites. They have similar heteroxenous life cycles and conserved genomes and share many metabolic features. Despite these similarities, T. gondii and N. caninum differ in their transmission strategies and zoonotic potential. Comparative analyses of the two parasites are important to identify the unique biological features that underlie the basis of host preference and pathogenicity. T. gondii and N. caninum are obligate intravacuolar parasites; in contrast to T. gondii, events that occur during N. caninum infection remain largely uncharacterized. We examined the capability of N. caninum (Liverpool isolate) to interact with host organelles and scavenge nutrients in comparison to that of T. gondii (RH strain). N. caninum reorganizes the host microtubular cytoskeleton and attracts endoplasmic reticulum (ER), mitochondria, lysosomes, multivesicular bodies, and Golgi vesicles to its vacuole though with some notable differences from T. gondii. For example, the host ER gathers around the N. caninum parasitophorous vacuole (PV) but does not physically associate with the vacuolar membrane; the host Golgi apparatus surrounds the N. caninum PV but does not fragment into ministacks. N. caninum relies on plasma lipoproteins and scavenges cholesterol from NPC1-containing endocytic organelles. This parasite salvages sphingolipids from host Golgi Rab14 vesicles that it sequesters into its vacuole. Our data highlight a remarkable degree of conservation in the intracellular infection program of N. caninum and T. gondii. The minor differences between the two parasites related to the recruitment and rearrangement of host organelles around their vacuoles likely reflect divergent evolutionary paths.

  19. Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis.

    Science.gov (United States)

    Tsai, I-Ting; Lin, Jyun-Liang; Chiang, Yi-Hsuan; Chuang, Yu-Chien; Liang, Shu-Shan; Chuang, Chi-Ning; Huang, Tzyy-Nan; Wang, Ting-Fang

    2014-02-01

    Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

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

    Institute of Scientific and Technical Information of China (English)

    Ting Chen; Xiang-Hui Lu; Hui-Fang Wang; Rui Ban; Hua-Xu Liu; Qiang Shi; Qian Wang

    2016-01-01

    Background:Myopathies with rimmed vacuoles are a heterogeneous group of muscle disorders with progressive muscle weakness and varied clinical manifestations but similar features in muscle biopsies.Here,we describe a novel autosomal dominant myopathy with rimmed vacuoles in a large family with 11 patients of three generations affected.Methods:A clinical study including family history,obstetric,pediatric,and development history was recorded.Clinical examinations including physical examination,electromyography (EMG),serum creatine kinase (CK),bone X-rays,and brain magnetic resonance imaging (MRI) were performed in this family.Open muscle biopsies were performed on the proband and his mother.To find the causative gene,the whole-exome sequencing was carried out.Results:Disease onset was from adolescence to adulthood,but the affected patients of the third generation presented an earlier onset and more severe clinical manifestations than the older generations.Clinical features were characterized as dysarthria,dysphagia,external ophthalmoplegia,limb weakness,hypophrenia,deafness,and impaired vision.However,not every patient manifested all symptoms.Serum CK was mildly elevated and EMG indicated a myopathic pattern.Brain MRI showed cerebellum and brain stem mildly atrophy.Rimmed vacuoles and inclusion bodies were observed in muscle biopsy.The whole-exome sequencing was performed,but the causative gene has not been found.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.

  2. Protein diffusion in mammalian cell cytoplasm.

    Science.gov (United States)

    Kühn, Thomas; Ihalainen, Teemu O; Hyväluoma, Jari; Dross, Nicolas; Willman, Sami F; Langowski, Jörg; Vihinen-Ranta, Maija; Timonen, Jussi

    2011-01-01

    We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol) and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP) experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS). A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

  3. Protein diffusion in mammalian cell cytoplasm.

    Directory of Open Access Journals (Sweden)

    Thomas Kühn

    Full Text Available We introduce a new method for mesoscopic modeling of protein diffusion in an entire cell. This method is based on the construction of a three-dimensional digital model cell from confocal microscopy data. The model cell is segmented into the cytoplasm, nucleus, plasma membrane, and nuclear envelope, in which environment protein motion is modeled by fully numerical mesoscopic methods. Finer cellular structures that cannot be resolved with the imaging technique, which significantly affect protein motion, are accounted for in this method by assigning an effective, position-dependent porosity to the cell. This porosity can also be determined by confocal microscopy using the equilibrium distribution of a non-binding fluorescent protein. Distinction can now be made within this method between diffusion in the liquid phase of the cell (cytosol/nucleosol and the cytoplasm/nucleoplasm. Here we applied the method to analyze fluorescence recovery after photobleach (FRAP experiments in which the diffusion coefficient of a freely-diffusing model protein was determined for two different cell lines, and to explain the clear difference typically observed between conventional FRAP results and those of fluorescence correlation spectroscopy (FCS. A large difference was found in the FRAP experiments between diffusion in the cytoplasm/nucleoplasm and in the cytosol/nucleosol, for all of which the diffusion coefficients were determined. The cytosol results were found to be in very good agreement with those by FCS.

  4. [Effect of acetylcholine and acetylcholinesterase on the activity of contractile vacuole of Amoeba proteus].

    Science.gov (United States)

    Bagrov, Ia Iu; Manusova, N B

    2011-01-01

    Acetylcholine (ACh, 1 microM) stimulates activity of the contractile vacuole of proteus. The effect of ACh is not mimicked by its analogs which are not hydrolyzed by acetylcholinesterase (AChE), i. e., carbacholine and 5-methylfurmethide. The effect of ACh is not sensitive to the blocking action of M-cholinolytics, atropine and mytolone, but is suppressed by N-cholinolytic, tubocurarine. The inhibitors of AChE, eserine (0.01 microM) and armine (0.1 microM), suppress the effect of ACh on amoeba contractile vacuole. ACh does not affect activation of contractile vacuole induced by arginine-vasopressin (1 microM), but it blocks such effect of opiate receptors agonist, dynorphin A1-13 (0.01 microM). This effect of ACh is also suppressed by the inhibitors of AChE. These results suggest that, in the above-described effects of ACh, AChE acts not as an antagonist, but rather as a synergist.

  5. Vacuolization and apoptosis induced by nano-selenium in HeLa cell line

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Selenium(Se),a potential drug candidate for cancer prevention,has a special property:Its nutritional dosage and tolerable upper intake level appear in a narrow range,while the therapeutic use of this mineral may depend on a higher body intake level.Nano-selenium(nano-Se) particles,however,preserve the selenium element’s low toxicity characteristic but give a high biochemical activity effect of selenium compounds.In the present study different morphologies of synthesized nano-Se were evaluated concerning its anti-proliferation and apoptosis-inducing effect.Then nano-Se(sphere) were picked out to investigate its influence on two significant events involved in apoptosis,cell cycle arrest and mitochondrial membrane potential disruption.Furthermore,massive vacuolization of HeLa cells treated by nano-Se(sphere) was observed and more methods were used to measure the level of vacuolization.Such vacuolization needs energy supply and has been demonstrated to be related to Se endocytosis.These results suggest a possible mechanism to trigger apoptosis initiation.

  6. The vacuole model: new terms in the second order deflection of light

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Amrita; Nandi, Kamal K. [Department of Mathematics, University of North Bengal, Raja Rammohunpur, Siliguri 734 013 (India); Garipova, Guzel M. [Department of Theoretical Physics, Sterlitamak State Pedagogical Academy, 49, Lenin Street, Sterlitamak 453103 (Russian Federation); Laserra, Ettore [DMI, Università di Salerno, Via Ponte Don Melillo, 84084 Fisciano, Salerno (Italy); Bhadra, Arunava, E-mail: amrita852003@yahoo.co.in, E-mail: goldberg144@gmail.com, E-mail: elaserra@unisa.it, E-mail: aru_bhadra@yahoo.com, E-mail: kamalnandi1952@yahoo.co.in [High Energy and Cosmic Ray Research Center, University of North Bengal, Raja Rammohunpur, Siliguri 734 013 (India)

    2011-02-01

    The present paper is an extension of a recent work (Bhattacharya et al. 2010) to the Einstein-Strauss vacuole model with a cosmological constant, where we work out the light deflection by considering perturbations up to order M{sup 3} and confirm the light bending obtained previously in their vacuole model by Ishak et al. (2008). We also obtain another local coupling term −5πM{sup 2}Λ/8 related to Λ, in addition to the one obtained by Sereno (2008, 2009). We argue that the vacuole method for light deflection is exclusively suited to cases where the cosmological constant Λ disappears from the path equation. However, the original Rindler-Ishak method (2007) still applies even if a certain parameter γ of Weyl gravity does not disappear. Here, using an alternative prescription, we obtain the known term −γR/2, as well as another new local term 3πγM/2 between M and γ. Physical implications are compared, where we argue that the repulsive term −γR/2 can be masked by the Schwarzschild term 2M/R in the halo regime supporting attractive property of the dark matter.

  7. Ergosteryl-β-glucosidase (Egh1) involved in sterylglucoside catabolism and vacuole formation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Watanabe, Takashi; Tani, Motohiro; Ishibashi, Yohei; Endo, Ikumi; Okino, Nozomu; Ito, Makoto

    2015-10-01

    Sterylglucosides (SGs) are composed of a glucose and sterol derivatives, and are distributed in fungi, plants and mammals. We recently identified EGCrP1 and EGCrP2 (endoglycoceramidase-related proteins 1 and 2) as a β-glucocerebrosidase and steryl-β-glucosidase, respectively, in Cryptococcus neoformans. We herein describe an EGCrP2 homologue (Egh1; ORF name, Yir007w) involved in SG catabolism in Saccharomyces cerevisiae. The purified recombinant Egh1 hydrolyzed various β-glucosides including ergosteryl β-glucoside (EG), cholesteryl β-glucoside, sitosteryl β-glucoside, para-nitrophenyl β-glucoside, 4-methylumberifellyl β-glucoside and glucosylceramide. The disruption of EGH1 in S. cerevisiae BY4741 (egh1Δ) resulted in the accumulation of EG and fragmentation of vacuoles. The expression of EGH1 in egh1Δ (revertant) reduced the accumulation of EG, and restored the morphology of vacuoles. The accumulation of EG was not detected in EGH1 and UGT51(ATG26) double-disrupted mutants (ugt51Δegh1Δ), indicating that EG was synthesized by Ugt51(Atg26) and degraded by Egh1 in vivo. These results clearly demonstrated that Egh1 is an ergosteryl-β-glucosidase that is functionally involved in the EG catabolic pathway and vacuole formation in S. cerevisiae.

  8. Anti-neutrophil cytoplasm autoantibodies (ANCA) in autoimmune liver diseases

    NARCIS (Netherlands)

    Roozendaal, C.; Kallenberg, Cees

    1999-01-01

    Anti-neutrophil cytoplasm antibodies (ANCA) are autoantibodies directed against cytoplasmic constituents of neutrophil granulocytes and monocytes. ANCA have been detected in serum from patients with inflammatory bowel diseases (mainly ulcerative colitis) and autoimmune mediated liver diseases (mainl

  9. Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis

    NARCIS (Netherlands)

    Kallenberg, Cees G. M.

    2007-01-01

    Purpose of reviews This review focuses on recent advance in the diagnosis pathogenesis and treatment of antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Recent findings Antineutrophil cytoplasmic autoantibodies are closely associated with Wegener's granulomatosis and micro

  10. The Pathogen-Occupied Vacuoles of Anaplasma phagocytophilum and Anaplasma marginale Interact with the Endoplasmic Reticulum.

    Science.gov (United States)

    Truchan, Hilary K; Cockburn, Chelsea L; Hebert, Kathryn S; Magunda, Forgivemore; Noh, Susan M; Carlyon, Jason A

    2016-01-01

    The genus Anaplasma consists of tick-transmitted obligate intracellular bacteria that invade white or red blood cells to cause debilitating and potentially fatal infections. A. phagocytophilum, a human and veterinary pathogen, infects neutrophils to cause granulocytic anaplasmosis. A. marginale invades bovine erythrocytes. Evidence suggests that both species may also infect endothelial cells in vivo. In mammalian and arthropod host cells, A. phagocytophilum and A. marginale reside in host cell derived pathogen-occupied vacuoles (POVs). While it was recently demonstrated that the A. phagocytophilum-occupied vacuole (ApV) intercepts membrane traffic from the trans-Golgi network, it is unclear if it or the A. marginale-occupied vacuole (AmV) interacts with other secretory organelles. Here, we demonstrate that the ApV and AmV extensively interact with the host endoplasmic reticulum (ER) in endothelial, myeloid, and/or tick cells. ER lumen markers, calreticulin, and protein disulfide isomerase, and the ER membrane marker, derlin-1, were pronouncedly recruited to the peripheries of both POVs. ApV association with the ER initiated early and continued throughout the infection cycle. Both the ApV and AmV interacted with the rough ER and smooth ER. However, only derlin-1-positive rough ER derived vesicles were delivered into the ApV lumen where they localized with intravacuolar bacteria. Transmission electron microscopy identified multiple ER-POV membrane contact sites on the cytosolic faces of both species' vacuoles that corresponded to areas on the vacuoles' lumenal faces where intravacuolar Anaplasma organisms closely associated. A. phagocytophilum is known to hijack Rab10, a GTPase that regulates ER dynamics and morphology. Yet, ApV-ER interactions were unhindered in cells in which Rab10 had been knocked down, demonstrating that the GTPase is dispensable for the bacterium to parasitize the ER. These data establish the ApV and AmV as pathogen-host interfaces that directly

  11. Cytoplasmic TRADD Confers a Worse Prognosis in Glioblastoma

    Directory of Open Access Journals (Sweden)

    Sharmistha Chakraborty

    2013-08-01

    Full Text Available Tumor necrosis factor receptor 1 (TNFR1-associated death domain protein (TRADD is an important adaptor in TNFR1 signaling and has an essential role in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB activation and survival signaling. Increased expression of TRADD is sufficient to activate NF-κB. Recent studies have highlighted the importance of NF-κB activation as a key pathogenic mechanism in glioblastoma multiforme (GBM, the most common primary malignant brain tumor in adults.We examined the expression of TRADD by immunohistochemistry (IHC and find that TRADD is commonly expressed at high levels in GBM and is detected in both cytoplasmic and nuclear distribution. Cytoplasmic IHC TRADD scoring is significantly associated with worse progression-free survival (PFS both in univariate and multivariate analysis but is not associated with overall survival (n = 43 GBMs. PFS is a marker for responsiveness to treatment. We propose that TRADD-mediated NF-κB activation confers chemoresistance and thus a worse PFS in GBM. Consistent with the effect on PFS, silencing TRADD in glioma cells results in decreased NF-κB activity, decreased proliferation of cells, and increased sensitivity to temozolomide. TRADD expression is common in glioma-initiating cells. Importantly, silencing TRADD in GBM-initiating stem cell cultures results in decreased viability of stem cells, suggesting that TRADD may be required for maintenance of GBM stem cell populations. Thus, our study suggests that increased expression of cytoplasmic TRADD is both an important biomarker and a key driver of NF-κB activation in GBM and supports an oncogenic role for TRADD in GBM.

  12. Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells in the hippocampal neurogenesis involving myelin vacuolation of cholinergic and glutamatergic inputs in mice.

    Science.gov (United States)

    Kato, Mizuho; Abe, Hajime; Itahashi, Megu; Kikuchihara, Yoh; Kimura, Masayuki; Mizukami, Sayaka; Yoshida, Toshinori; Shibutani, Makoto

    2016-02-01

    Hexachlorophene (HCP) has been shown to induce myelin vacuolation due to intramyelinic edema of the nerve fibers in animal neural tissue. We investigated the maternal exposure effect of HCP on hippocampal neurogenesis in the offspring of pregnant mice supplemented with 0 (control), 33 or 100 ppm HCP in diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, offspring as examined in males exhibited decreased granule cell lineage populations expressing paired box 6, sex-determining region Y-box 2 and eomesodermin in the hippocampal subgranular zone (SGZ) accompanied by myelin vacuolation involving white matter tracts of the hippocampal fimbria at ≥ 33 ppm. However, SGZ cellular populations expressing brain lipid binding protein and doublecortin were unchanged at any dose. Transcript expression of cholinergic receptor genes, Chrna4 and Chrnb2, and glutamate receptor genes, Grm1 and Grin2d, examined at 100 ppm, decreased in the dentate gyrus. HCP exposure did not alter the number of proliferating or apoptotic cells in the SGZ, or reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)ergic interneurons in the dentate hilus, on PND 21 and PND 77. All neurogenesis-related changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77, suggesting that maternal HCP exposure at ≥ 33 ppm reversibly decreased type 2 intermediate-stage progenitor cells in the hippocampal neurogenesis. Myelin vacuolation might be responsible for changes in neurogenesis possibly by reducing nerve conduction velocity of cholinergic inputs from the septal-hippocampal pathway to granule cell lineages and/or GABAergic interneurons, and of glutamatergic inputs to granule cell lineages.

  13. Anaplasma phagocytophilum APH0032 Is Exposed on the Cytosolic Face of the Pathogen-Occupied Vacuole and Co-opts Host Cell SUMOylation

    Science.gov (United States)

    Oki, Aminat T.; Huang, Bernice; Beyer, Andrea R.; May, Levi J.; Truchan, Hilary K.; Walker, Naomi J.; Galloway, Nathan L.; Borjesson, Dori L.; Carlyon, Jason A.

    2016-01-01

    Anaplasma phagocytophilum, a member of the family Anaplasmataceae and the obligate intracellular bacterium that causes granulocytic anaplasmosis, resides in a host cell-derived vacuole. Bacterial proteins that localize to the A. phagocytophilum-occupied vacuole membrane (AVM) are critical host-pathogen interfaces. Of the few bacterial AVM proteins that have been identified, the domains responsible for AVM localization and the host cell pathways that they co-opt are poorly defined. APH0032 is an effector that is expressed and localizes to the AVM late during the infection cycle. Herein, the APH0032 domain that is essential for associating with host cell membranes was mapped. Immunofluorescent labeling of infected cells that had been differentially permeabilized confirmed that APH0032 is exposed on the AVM's cytosolic face, signifying its potential to interface with host cell processes. SUMOylation is the covalent attachment of a member of the small ubiquitin-like modifier (SUMO) family of proteins to lysines in target substrates. Previous work from our laboratory determined that SUMOylation is important for A. phagocytophilum survival and that SUMOylated proteins decorate the AVM. Algorithmic prediction analyses identified APH0032 as a candidate for SUMOylation. Endogenous APH0032 was precipitated from infected cells using a SUMO affinity matrix, confirming that the effector co-opts SUMOylation during infection. APH0032 pronouncedly colocalized with SUMO1, but not SUMO2/3 moieties on the AVM. Ectopic expression of APH0032 in A. phagocytophilum infected host cells significantly boosted the bacterial load. This study delineates the first domain of any Anaplasmataceae protein that is essential for associating with the pathogen-occupied vacuole membrane, demonstrates the importance of APH0032 to infection, and identifies it as the second A. phagocytophilum effector that co-opts SUMOylation, thus underscoring the relevance of this post-translational modification to

  14. Molecular cloning, immunochemical localization to the vacuole, and expression in transgenic yeast and tobacco of a putative sugar transporter from sugar beet.

    Science.gov (United States)

    Chiou, T J; Bush, D R

    1996-02-01

    Several plant genes have been cloned that encode members of the sugar transporter subgroup of the major facilitator superfamily of transporters. Here we report the cloning, expression, and membrane localization of one of these porters found in sugar beet (Beta vulgaris L.). This clone, cDNA-1, codes for a protein with 490 amino acids and an estimated molecular mass of 54 kD. The predicted membrane topology and sequence homology suggest that cDNA-1 is a member of the sugar transporter family. RNA gel blot analysis revealed that this putative sugar transporter is expressed in all vegetative tissues and expression increases with development in leaves. DNA gel blot analysis indicated that multiple gene copies exist for this putative sugar transporter in the sugar beet genome. Antibodies directed against small peptides representing the N- and C-terminal domains of the cDNA1 protein identified a 40-kD polypeptide in microsomes isolated from cDNA-1-transformed yeast (Saccharomyces cerevisiae). Moreover, the same protein was identified in sugar beet and transgenic tobacco (Nicotaina tobacum L.) membrane fractions. Detailed analysis of the transporter's distribution across linear sucrose gradients and flotation centrifugations showed that it co-migrates with tonoplast membrane markers. We conclude that this carrier is located on the tonoplast membrane and that it may mediate sugar partitioning between the vacuole and cytoplasmic compartments.

  15. A functional connection of Dictyostelium paracaspase with the contractile vacuole and a possible partner of the vacuolar proton ATPase

    Indian Academy of Sciences (India)

    Entsar Saheb; Ithay Biton; Katherine Maringer; John Bush

    2013-09-01

    Dictyostelium discoideum possesses only one caspase family member, paracaspase (pcp). Two separate mutant cell lines were first analysed: one cell line was an over-expressed GFP-tagged Pcp (GFP-Pcp), while the other cell line was a pcp-null (pcp-). Microscopic analysis of cells expressing GFP-Pcp revealed that Pcp was associated with the contractile vacuole membrane consisting of bladder-like vacuoles. This association was disrupted when cells were exposed to osmotic stress conditions. Compared with wild-type cells, the GFP-Pcp-over-expressing cells were susceptible to osmotic stress and were seen to be very rounded in hypo-osmotic conditions and contained more abnormally swollen contractile vacuole. Cells with pcp- were also rounded but had few, if any, contractile vacuoles. These observations suggest that Pcp is essential for Dictyostelium osmotic regulation via its functioning in the contractile vacuole system. Subjecting these cells to selected contractile vacuole inhibitor provided additional support for these findings. Furthermore, yeast two-hybrid system identified vacuolar proton ATPase (VatM) as the protein interacting with Pcp. Taken together, this work gives evidence for an eukaryotic paracaspase to be associated with both localization in and regulation of the contractile vacuolar system, an organelle critical for maintaining the normal morphology of the cell.

  16. Endogenous Mouse Dicer Is an Exclusively Cytoplasmic Protein.

    Directory of Open Access Journals (Sweden)

    Christian Much

    2016-06-01

    Full Text Available Dicer is a large multi-domain protein responsible for the ultimate step of microRNA and short-interfering RNA biogenesis. In human and mouse cell lines, Dicer has been shown to be important in the nuclear clearance of dsRNA as well as the establishment of chromatin modifications. Here we set out to unambiguously define the cellular localization of Dicer in mice to understand if this is a conserved feature of mammalian Dicer in vivo. To this end, we utilized an endogenously epitope tagged Dicer knock-in mouse allele. From primary mouse cell lines and adult tissues, we determined with certainty by biochemical fractionation and confocal immunofluorescence microscopy that endogenous Dicer is exclusively cytoplasmic. We ruled out the possibility that a fraction of Dicer shuttles to and from the nucleus as well as that FGF or DNA damage signaling induce Dicer nuclear translocation. We also explored Dicer localization during the dynamic and developmental context of embryogenesis, where Dicer is ubiquitously expressed and strictly cytoplasmic in all three germ layers as well as extraembryonic tissues. Our data exclude a direct role for Dicer in the nuclear RNA processing in the mouse.

  17. Antineutrophil cytoplasmic autoantibodies in ulcerative colitis

    Directory of Open Access Journals (Sweden)

    Vodjgani M

    2000-10-01

    Full Text Available Antineutrophil cytoplasmic autoanibodies (ANCA were detecte in patients with certain autoimmune vascular disease such as Wegner’s granulomatosis, polyarthrits nodosa and systemic luuc erythematous. Indirect immunofluorescence (IIF technique was employed to detec these autoantibodies.ANCA have been recently detected in some forms of inflammatory bowel disease (IBD, ulcerative colitis (U.C. Crohn’s disease (C.D and primary sclerosing cholangitis (PSC. By IIF method, two general patterns of ANCA were seen: a cytoplasmic (C-ANCA and perinuclear form (P-ANCA. In this study we evaluated the presece of ANCA in 52 U.C. patients and 69 matched normal control group by IIF technique, and it’s relationship with disease activity. Site of colon involvement and, lesion extent. The results showed that all control group were ANCA negative, but 58% of patients had ANCA, and most cases (70% had C- ANCA. The obtained results also revealed that there was no relationship between ANCA and disease activity

  18. Molecular classification of Maize cytoplasms in a breeding program

    Directory of Open Access Journals (Sweden)

    Colombo. N * , Presello, D.A. , Kandus M. , G.E. Eyherabide and J.C. Salerno

    2012-06-01

    Full Text Available Cytoplasmic male sterility (CMS is maternally inherited in most of higher plants species. Together with nuclear restorer genes (Rf, CMS cytoplasms contribute significantly to the efficient production of hybrid seed. Three main types of male sterile cytoplasms are known in maize: T, S and C, which can be distinguished by crossing with specific restorer lines. Recently, PCR markers have been developed allowing the identification of different cytoplasms quickly and accurately. Our objective was to classify the cytoplasm type of maize inbred lines used in our breeding program and F1s obtained from crosses between CMS lines and elite maize lines using PCR multiplex. A multiplex PCR protocol was optimized for our conditions. We obtained the molecular classification of the analyzed cytoplasms. The optimized protocol is a valuable tool to trace male sterile cytoplasms and determine hybrid seed purity in our maize breeding program.

  19. Changes in vacuolation in the root apex cells of soybean seedlings in microgravity

    Science.gov (United States)

    Klymchuk, D. O.; Kordyum, E. L.; Vorobyova, T. V.; Chapman, D. K.; Brown, C. S.

    2003-05-01

    Changes in the vacuolation in root apex cells of soybean ( Glycine max L. [Merr.]) seedlings grown in microgravity were investigated. Spaceflight and ground control seedlings were grown in the absence or presence of KMnO 4 (to remove ethylene) for 6 days. After landing, in order to study of cell ultrastructure and subcellular free calcium ion distribution, seedling root apices were fixed in 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer and 2% (w/v) glutaraldehyde, 2.5% (w/v) formaldehyde, 2% (w/v) potassium antimonate K[Sb(OH) 6] in 0.1 M K 2HPO 4 buffer with an osmolarity (calculated theoretically) of 0.45 and 1.26 osmol. The concentrations of ethylene in all spaceflight canisters were significantly higher than in the ground control canisters. Seedling growth was reduced in the spaceflight-exposed plants. Additionally, the spaceflight-exposed plants exhibited progressive vacuolation in the root apex cells, particularly in the columella cells, to a greater degree than the ground controls. Plasmolysis was observed in columella cells of spaceflight roots fixed in solutions with relatively high osmolarity (1.26 osmol). The appearance of plasmolysis permitted the evaluation of the water status of cells. The water potential of the spaceflight cells was higher than the surrounding fixative solution. A decrease in osmotic potential and/or an increase in turgor potential may have induced increases in cell water potential. However, the plasmolysed (i.e. nonturgid) cells implied that increases in water potential were accompanied with a decrease in osmotic potential. In such cells changes in vacuolation may have been involved to maintain turgor pressure or may have been a result of intensification of other vacuolar functions like digestion and storage

  20. Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Liu Ronghua

    2011-09-01

    Full Text Available Abstract Background P21(WAF1/Cip1 binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. Methods RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. Results p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. Conclusions Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors

  1. The Vtc proteins in vacuole fusion: coupling NSF activity to V(0) trans-complex formation

    DEFF Research Database (Denmark)

    Müller, Oliver; Bayer, Martin J; Peters, Christopher

    2002-01-01

    vacuole system has revealed two subsequent molecular events: trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events......, LMA1 release, but dispensible for all preceding steps, including V(0) trans-complex formation. This suggests that Vtc3p might act close to or at fusion pore opening. We propose that Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0) sectors in order to activate them for V(0) trans...

  2. Atypical, cytoplasmic and perinuclear anti-neutrophil cytoplasmic antibodies in patients with inflammatory bowel disease.

    Science.gov (United States)

    Frenzer, A; Fierz, W; Rundler, E; Hammer, B; Binek, J

    1998-09-01

    Atypical, cytoplasmic and perinuclear anti-neutrophil cytoplasmic antibodies (x-, c- and pANCA, respectively) are associated with a variety of inflammatory diseases, including inflammatory bowel disease (IBD). Anti-neutrophil cytoplasmic antibodies are more common in patients with ulcerative colitis (UC) than in patients with Crohn's disease (CD). Most publications only refer to p- and cANCA in relation to IBD. We have prospectively evaluated the reactivity of sera from 58 patients with IBD and 10 healthy controls against human neutrophils with emphasis on the distinction of the ANCA types. The sera were incubated with ethanol- and formaldehyde-fixed granulocytes to differentiate between c-, p- and xANCA. The results showed that 10 of 24 patients with UC were positive for ANCA, whereas only one of 34 patients with CD was ANCA positive. These results correspond to a sensitivity of 42%, a specificity of 97%, a negative predictive value of 91% and a positive predictive value of 75% in UC. Of the 11 ANCA-positive sera, two showed a cytoplasmic staining pattern, three showed a perinuclear and six an atypical staining pattern. The disease activity was not correlated to either the ANCA titre or to the presence of ANCA in the serum. In conclusion, ANCA are of limited value in differentiating between UC and CD. Because the majority of ANCA in patients with IBD are xANCA, these ANCA should be explored by not only incubating on ethanol-fixed granulocytes, but also on formaldehyde-fixed granulocytes.

  3. Autophagy is induced by anti-neutrophil cytoplasmic Abs and promotes neutrophil extracellular traps formation.

    Science.gov (United States)

    Sha, Li-Li; Wang, Huan; Wang, Chen; Peng, Hong-Ying; Chen, Min; Zhao, Ming-Hui

    2016-11-01

    Dysregulated neutrophil extracellular traps (NETs) formation contributes to the pathogenesis of anti-neutrophil cytoplasmic Ab (ANCA)-associated vasculitis (AAV). Increasing evidence indicates that autophagy is involved in the process of NETs formation. In this study, we aimed to investigate whether ANCA could induce autophagy in the process of NETs formation. Autophagy was detected using live cell imaging, microtubule-associated protein light chain 3B (LC3B) accumulation and Western blotting. The results showed that autophagy vacuolization was detected in neutrophils treated with ANCA-positive IgG by live cell imaging. This effect was enhanced by rapamycin, the autophagy inducer, and weakened by 3-methyladenine (3-MA), the autophagy inhibitor. In line with these results, the autophagy marker, LC3B, showed a punctate distribution pattern in the neutrophils stimulated with ANCA-positive IgG. In the presence of rapamycin, LC3B accumulation was further increased; however, this effect was attenuated by 3-MA. Moreover, incubated with ANCA-positive IgG, the NETosis rate significantly increased compared with the unstimulated group. And, the rate significantly increased or decreased in the neutrophils pretreated with rapamycin or 3-MA, respectively, as compared with the cells incubated with ANCA-positive IgG. Overall, this study demonstrates that autophagy is induced by ANCA and promotes ANCA-induced NETs formation.

  4. Development of cytoplasmic-nuclear male sterility, its inheritance, and potential use in hybrid pigeonpea breeding.

    Science.gov (United States)

    Saxena, Kul B; Ravikoti, V Kumar; Dalvi, Vijay A; Pandey, Lalji B; Gaddikeri, Guruprasad

    2010-01-01

    Pigeonpea [Cajanus cajan (L.) Millsp.] is a unique food legume because of its partial (20-30%) outcrossing nature, which provides an opportunity to breed commercial hybrids. To achieve this, it is essential to have a stable male-sterility system. This paper reports the selection of a cytoplasmic-nuclear male-sterility (CMS) system derived from an interspecific cross between a wild relative of pigeonpea (Cajanus sericeus Benth. ex. Bak.) and a cultivar. This male-sterility source was used to breed agronomically superior CMS lines in early (ICPA 2068), medium (ICPA 2032), and late (ICPA 2030) maturity durations. Twenty-three fertility restorers and 30 male-sterility maintainers were selected to develop genetically diverse hybrid combinations. Histological studies revealed that vacuolation of growing tetrads and persistence of tetrad wall were primary causes of the manifestation of male sterility. Genetic studies showed that 2 dominant genes, of which one had inhibitory gene action, controlled fertility restoration in the hybrids. The experimental hybrids such as TK 030003 and TK 030009 in early, ICPH 2307 and TK 030625 in medium, and TK 030861 and TK 030851 in late maturity groups exhibited 30-88% standard heterosis in multilocation trials.

  5. Model for bidirectional movement of cytoplasmic dynein

    CERN Document Server

    Sumathy, S

    2014-01-01

    Cytoplasmic dynein exhibits a directional processive movement on microtubule filaments and is known to move in steps of varying length based on the number of ATP molecules bound to it and the load that it carries. It is experimentally observed that dynein takes occasional backward steps and the frequency of such backward steps increases as the load approaches the stall force. Using a stochastic process model, we investigate the bidirectional movement of single head of a dynein motor. The probability for backward step is implemented based on Crook's fluctuation theorem of non-equilibrium statistical mechanics. We find that the movement of dynein motor is characterized with negative velocity implying backward motion beyond stall force. We observe that the motor moves backward for super stall forces by hydrolyzing the ATP exactly the same way as it does while moving forward for sub stall forces.

  6. The epididymis, cytoplasmic droplets and male fertility

    Institute of Scientific and Technical Information of China (English)

    Trevor G Cooper

    2011-01-01

    The potential of spermatozoa to become motile during post-testicular maturation,and the relationship between the cytoplasmic droplet and fertilizing capacity are reviewed.Post-testicular maturation of spermatozoa involves the autonomous induction of motility,which can occur in vivo in testes with occluded excurrent ducts and in vitro in testicular explants,and artefactual changes in morphology that appear to occur in the testis in vitro.Both modifications may reflect time-dependent oxidation of disulphide bonds of head and tail proteins.Regulatory volume decrease(RVD),which counters sperm swelling at ejaculation,is discussed in relation to loss of cytoplasmic droplets and consequences for fertility.It is postulated that:(i)fertile males possess spermatozoa with sufficient osmolytes to drive RVD at ejaculation,permitting the droplet to round up and pinch off without membrane rupture; and(ⅱ)infertile males possess spermatozoa with insufficient osmolytes so that RVD is inadequate,the droplet swells and the resulting flagellar angulation prevents droplet loss.Droplet retention at ejaculation is a harbinger of infertility caused by failure of the spermatozoon to negotiate the uterotubal junction or mucous and reach the egg.In this hypothesis,the epididymis regulates fertility indirectly by the extent of osmolyte provision to spermatozoa,which influences RVD and therefore droplet loss.Man is an exception,because ejaculated human spermatozoa retain their droplets.This may reflect their short midpiece,approximating head length,permitting a swollen droplet to extend along the entire midpiece; this not only obviates droplet migration and flagellar angulation but also hampers droplet loss.

  7. Hitchhiking vesicular transport routes to the vacuole: amyloid recruitment to the Insoluble Protein Deposit (IPOD).

    Science.gov (United States)

    Kumar, Rajesh; Neuser, Nicole; Tyedmers, Jens

    2017-03-09

    Sequestration of aggregates into specialized deposition sites occurs in many species across all kingdoms of life ranging from bacteria to mammals and is commonly believed to have a cytoprotective function. Yeast cells possess at least three different spatially separated deposition sites, one of which is termed "Insoluble Protein Deposit (IPOD)" and harbors amyloid aggregates. We have recently discovered that recruitment of amyloid aggregates to the IPOD employs an actin cable based recruitment machinery that also involves vesicular transport (1) . Here we discuss how different proteins known to be involved in vesicular transport processes to the vacuole might act to guide amyloid aggregates to the IPOD. These factors include the Myosin V motor protein Myo2 involved in transporting vacuolar vesicles along actin cables, the transmembrane protein Atg9 involved in the recruitment of large precursor hydrolase complexes to the vacuole, the phosphatidylinositol/ phosphatidylcholine (PI/PC) transfer protein Sec 14 and the SNARE chaperone Sec 18. Furthermore, we present new data suggesting that the yeast dynamin homolog Vps1 is also crucial for faithful delivery of the amyloid model protein PrD-GFP to the IPOD. This is in agreement with a previously identified role for Vps1 in recruitment of heat-denatured aggregates to a perivacuolar deposition site (2) .

  8. Campylobacter jejuni actively invades the amoeba Acanthamoeba polyphaga and survives within non digestive vacuoles.

    Directory of Open Access Journals (Sweden)

    Jenny Olofsson

    Full Text Available The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81-176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.

  9. Campylobacter jejuni actively invades the amoeba Acanthamoeba polyphaga and survives within non digestive vacuoles.

    Science.gov (United States)

    Olofsson, Jenny; Axelsson-Olsson, Diana; Brudin, Lars; Olsen, Björn; Ellström, Patrik

    2013-01-01

    The Gram-negative bacterium Campylobacter jejuni is able to enter, survive and multiply within the free living amoeba Acanthamoeba polyphaga, but the molecular mechanisms behind these events are still unclear. We have studied the uptake and intracellular trafficking of viable and heat killed bacterial cells of the C. jejuni strain 81-176 in A. polyphaga. We found that viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat killed bacteria. Furthermore, the kinetics of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were dramatically influenced by bacterial viability. Viable bacteria were internalized at a high rate already after 1 h of co-incubation and were observed in small vacuoles tightly surrounding the bacteria. In contrast, internalization of heat killed C. jejuni was low at early time points and did not peak until 96 h. These cells were gathered in large spacious vacuoles that were part of the degradative pathway as determined by the uptake of fluorescently labeled dextran. The amount of heat killed bacteria internalized by A. polyphaga did never reach the maximal amount of internalized viable bacteria. These results suggest that the uptake and intracellular survival of C. jejuni in A. polyphaga is bacterially induced.

  10. The Rice RMR1 Associates with a Distinct Prevacuolar Compartment for the Protein Storage Vacuole Pathway

    Institute of Scientific and Technical Information of China (English)

    Yun Shen; Junqi Wang; Yu Ding; SzeWan Lo; Guillaume Gouzerh; Jean-Marc Neuhaus; Liwen Jiang

    2011-01-01

    Transport of vacuolar proteins from Golgi apparatus or trans-Golgi network (TGN) to vacuoles is a receptormediated process via an intermediate membrane-bound prevacuolar compartment (PVC) in plant cells.Both vacuolar sorting receptor (VSR) and receptor homology region-transmembrane domain-RING-H2 (RMR) proteins have been shown to function in transporting storage proteins to protein storage vacuole (PSV),but little is known about the nature of the PVC for the PSV pathway.Here,we use the rice RMR1 (OsRMR1) as a probe to study the PSV pathway in plants.Immunogold electron microscopy (EM) with specific OsRMR1 antibodies showed that OsRMR1 proteins were found in the Golgi apparatus,TGN,and a distinct organelle with characteristics of PVC in both rice culture cells and developing rice seeds,as well as the protein body type Ⅱ (PBII) or PSV in developing rice seeds.This organelle,also found in both tobacco BY-2 and Arabidopsis suspension cultured cells,is morphologically distinct from the VSR-positive multivesicular lytic PVC or multivesicular body (MVB) and thus represent a PVC for the PSV pathway that we name storage PVC (sPVC).Further in vivo and in vitro interaction studies using truncated OsRMR1 proteins secreted into the culture media of transgenic BY-2 suspension cells demonstrated that OsRMR1 functions as a sorting receptor in transporting vicilin-like storage proteins.

  11. Identification and characterization of receptors for vacuolating activity of subtilase cytotoxin.

    Science.gov (United States)

    Yahiro, Kinnosuke; Morinaga, Naoko; Satoh, Mamoru; Matsuura, Gen; Tomonaga, Takeshi; Nomura, Fumio; Moss, Joel; Noda, Masatoshi

    2006-10-01

    Some shiga toxin-producing Escherichia coli secrete a novel AB5 cytotoxin, named subtilase cytotoxin (SubAB), which induces vacuole formation in addition to cytotoxicity in susceptible cells. By immunoprecipitation with SubAB from Vero cells, we discovered proteins of 100 kDa, 135 kDa and 155 kDa as potential candidates for its receptor. These proteins were N-glycosylated in their extracellular domains, a modification that was necessary for interaction with SubAB. Biotinylated receptors were partially purified by Datura stramonium agglutinin affinity chromatography and avidin-agarose and analysed by TOF mass spectroscopy. The peptide sequences of p135 were identical to beta1 integrin, and its identification was confirmed with anti-integrin beta1 antibody. The p155 protein was identified as alpha2 integrin using anti-integrin alpha2 antibody. In addition, treatment of Vero cells with beta1 integrin RNAi before exposure to SubAB prevented vacuolating activity. These results suggested that SubAB recognizes alpha2beta1 integrin as a functional receptor; this first interaction may be an important key step leading to the SubAB-induced morphological changes in Vero cells.

  12. Preparative procedures markedly influence the appearance and structural integrity of protein storage vacuoles in soybean seeds.

    Science.gov (United States)

    Krishnan, Hari B

    2008-05-14

    In legumes, vacuoles serve as the final depository for storage proteins. The protein storage vacuoles (PSVs) of soybean contain electron-transparent globoid regions in which phytic acid ( myo-inositol-1,2,3,4,5,6-hexakisphosphate) is sequestered. This paper reports the effect of preparative procedures on the appearance and ultrastructural integrity of PSVs in soybeans. Electron microscopy examination of both developing and mature soybean seeds that were postfixed with osmium tetroxide revealed PSVs that had a homogeneous appearance with very few globoid crystals dispersed in them. Numerous electron-dense lipid bodies were readily seen in these cells. Omission of osmium tetroxide strikingly altered the appearance of PSVs and aided the visualization of the location of the globoids in the PSVs. In contrast to the osmicated tissue, lipid bodies appeared as electron-transparent spheres. The choice of dehydration reagent or staining procedure had little influence on the appearance of the PSVs. The results of this study demonstrate the profound effect of osmium tetroxide on the appearance and structural integrity of PSVs in soybean.

  13. LegC3, an effector protein from Legionella pneumophila, inhibits homotypic yeast vacuole fusion in vivo and in vitro.

    Directory of Open Access Journals (Sweden)

    Terry L Bennett

    Full Text Available During infection, the intracellular pathogenic bacterium Legionella pneumophila causes an extensive remodeling of host membrane trafficking pathways, both in the construction of a replication-competent vacuole comprised of ER-derived vesicles and plasma membrane components, and in the inhibition of normal phagosome:endosome/lysosome fusion pathways. Here, we identify the LegC3 secreted effector protein from L. pneumophila as able to inhibit a SNARE- and Rab GTPase-dependent membrane fusion pathway in vitro, the homotypic fusion of yeast vacuoles (lysosomes. This vacuole fusion inhibition appeared to be specific, as similar secreted coiled-coiled domain containing proteins from L. pneumophila, LegC7/YlfA and LegC2/YlfB, did not inhibit vacuole fusion. The LegC3-mediated fusion inhibition was reversible by a yeast cytosolic extract, as well as by a purified soluble SNARE, Vam7p. LegC3 blocked the formation of trans-SNARE complexes during vacuole fusion, although we did not detect a direct interaction of LegC3 with the vacuolar SNARE protein complexes required for fusion. Additionally, LegC3 was incapable of inhibiting a defined synthetic model of vacuolar SNARE-driven membrane fusion, further suggesting that LegC3 does not directly inhibit the activity of vacuolar SNAREs, HOPS complex, or Sec17p/18p during membrane fusion. LegC3 is likely utilized by Legionella to modulate eukaryotic membrane fusion events during pathogenesis.

  14. Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Logan, Michael R.; Jones, Lynden [Department of Cell Biology, University of Alberta, Edmonton, Alta., Canada T6G 2H7 (Canada); Eitzen, Gary, E-mail: gary.eitzen@ualberta.ca [Department of Cell Biology, University of Alberta, Edmonton, Alta., Canada T6G 2H7 (Canada)

    2010-03-26

    Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P{sub 2} specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.

  15. The molecular mechanism and physiological role of cytoplasmic streaming.

    Science.gov (United States)

    Tominaga, Motoki; Ito, Kohji

    2015-10-01

    Cytoplasmic streaming occurs widely in plants ranging from algae to angiosperms. However, the molecular mechanism and physiological role of cytoplasmic streaming have long remained unelucidated. Recent molecular genetic approaches have identified specific myosin members (XI-2 and XI-K as major and XI-1, XI-B, and XI-I as minor motive forces) for the generation of cytoplasmic streaming among 13 myosin XIs in Arabidopsis thaliana. Simultaneous knockout of these myosin XI members led to a reduced velocity of cytoplasmic streaming and marked defects of plant development. Furthermore, the artificial modifications of myosin XI-2 velocity changed plant and cell sizes along with the velocity of cytoplasmic streaming. Therefore, we assume that cytoplasmic streaming is one of the key regulators in determining plant size.

  16. cPrG-HCl a potential H+/Cl- symporter prevents acidification of storage vacuoles in aleurone cells and inhibits GA-dependent hydrolysis of storage protein and phytate.

    Science.gov (United States)

    Hwang, Yong-sic; Bethke, Paul C; Gubler, Frank; Jones, Russell L

    2003-07-01

    The putative H+/Cl- symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when aleurone cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in aleurone layers and prevents synthesis of secretory proteins such as alpha-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the aleurone layer. Vacuolation of the aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl- symporter in aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of alpha-amylase when Cl- ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated cells.

  17. Distinct cytoplasmic and nuclear functions of the stress induced protein DDIT3/CHOP/GADD153.

    Directory of Open Access Journals (Sweden)

    Alexandra Jauhiainen

    Full Text Available DDIT3, also known as GADD153 or CHOP, encodes a basic leucine zipper transcription factor of the dimer forming C/EBP family. DDIT3 is known as a key regulator of cellular stress response, but its target genes and functions are not well characterized. Here, we applied a genome wide microarray based expression analysis to identify DDIT3 target genes and functions. By analyzing cells carrying tamoxifen inducible DDIT3 expression constructs we show distinct gene expression profiles for cells with cytoplasmic and nuclear localized DDIT3. Of 175 target genes identified only 3 were regulated by DDIT3 in both cellular localizations. More than two thirds of the genes were downregulated, supporting a role for DDIT3 as a dominant negative factor that could act by either cytoplasmic or nuclear sequestration of dimer forming transcription factor partners. Functional annotation of target genes showed cell migration, proliferation and apoptosis/survival as the most affected categories. Cytoplasmic DDIT3 affected more migration associated genes, while nuclear DDIT3 regulated more cell cycle controlling genes. Cell culture experiments confirmed that cytoplasmic DDIT3 inhibited migration, while nuclear DDIT3 caused a G1 cell cycle arrest. Promoters of target genes showed no common sequence motifs, reflecting that DDIT3 forms heterodimers with several alternative transcription factors that bind to different motifs. We conclude that expression of cytoplasmic DDIT3 regulated 94 genes. Nuclear translocation of DDIT3 regulated 81 additional genes linked to functions already affected by cytoplasmic DDIT3. Characterization of DDIT3 regulated functions helps understanding its role in stress response and involvement in cancer and degenerative disorders.

  18. Effects of Nuclear Genomes on Anther Development in Cytoplasmic Male Sterile Chicories (Cichorium intybus L.: Morphological Analysis

    Directory of Open Access Journals (Sweden)

    Ildephonse Habarugira

    2015-01-01

    Full Text Available The Cichorium intybus flower development in fertile, cytoplasmic male sterility (CMS 524 and various phenotypes carrying the 524 male sterile cytoplasm was investigated macroscopically and by light microscopy. The development was similar in fertile and in male sterile florets up to meiosis, and then it was affected in anther wall structure and pollen grain development in male sterile floret. In the male sterile plants, the tapetum intrusion after meiosis was less remarkable, the microspores started to abort at vacuolate stage, the connective tissue collapsed, and endothecium failed to expand normally and did not undergo cell wall lignification, which prevented anther opening since the septum and stomium were not disrupted. Crosses undertaken in order to introduce the CMS 524 into two different nuclear backgrounds gave rise to morphologically diversified progenies due to different nuclear-mitochondrial interactions. Macroscopic and cytological investigations showed that pollen-donor plants belonging to Jupiter population had potential capacity to restore fertility while the CC line could be considered as a sterility maintainer.

  19. Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Færgeman, Nils J.; Feddersen, Søren; Christiansen, Janne K

    2004-01-01

    In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide......-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I...... be compartmentalized. We suggest that the reduced ceramide synthesis in Acb1p-depleted cells leads to severely altered vacuole morphology, perturbed vacuole assembly and strong inhibition of homotypic vacuole fusion....

  20. Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration

    Directory of Open Access Journals (Sweden)

    Rajendra Rai

    2016-05-01

    Full Text Available A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR, long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc13 sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477–493 region required for effective cytoplasmic Gln3-Myc13 sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc13 to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc13 sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477–493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration.

  1. Applying the genetic theories of ageing to the cytoplasm: cytoplasmic genetic covariation for fitness and lifespan.

    Science.gov (United States)

    Dowling, D K; Maklakov, A A; Friberg, U; Hailer, F

    2009-04-01

    Two genetic models exist to explain the evolution of ageing - mutation accumulation (MA) and antagonistic pleiotropy (AP). Under MA, a reduced intensity of selection with age results in accumulation of late-acting deleterious mutations. Under AP, late-acting deleterious mutations accumulate because they confer beneficial effects early in life. Recent studies suggest that the mitochondrial genome is a major player in ageing. It therefore seems plausible that the MA and AP models will be relevant to genomes within the cytoplasm. This possibility has not been considered previously. We explore whether patterns of covariation between fitness and ageing across 25 cytoplasmic lines, sampled from a population of Drosophila melanogaster, are consistent with the genetic associations predicted under MA or AP. We find negative covariation for fitness and the rate of ageing, and positive covariation for fitness and lifespan. Notably, the direction of these associations is opposite to that typically predicted under AP.

  2. Vps13-Mcp1 interact at vacuole-mitochondria interfaces and bypass ER-mitochondria contact sites.

    Science.gov (United States)

    John Peter, Arun T; Herrmann, Beatrice; Antunes, Diana; Rapaport, Doron; Dimmer, Kai Stefan; Kornmann, Benoît

    2017-10-02

    Membrane contact sites between endoplasmic reticulum (ER) and mitochondria, mediated by the ER-mitochondria encounter structure (ERMES) complex, are critical for mitochondrial homeostasis and cell growth. Defects in ERMES can, however, be bypassed by point mutations in the endosomal protein Vps13 or by overexpression of the mitochondrial protein Mcp1. How this bypass operates remains unclear. Here we show that the mitochondrial outer membrane protein Mcp1 functions in the same pathway as Vps13 by recruiting it to mitochondria and promoting its association to vacuole-mitochondria contacts. Our findings support a model in which Mcp1 and Vps13 work as functional effectors of vacuole-mitochondria contact sites, while tethering is mediated by other factors, including Vps39. Tethered and functionally active vacuole-mitochondria interfaces then compensate for the loss of ERMES-mediated ER-mitochondria contact sites. © 2017 John Peter et al.

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

    Science.gov (United States)

    Remis, Natalie N; Wiwatpanit, Teerawat; Castiglioni, Andrew J; Flores, Emma N; Cantú, Jorge A; García-Añoveros, Jaime

    2014-12-01

    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 with lysosomal

  4. 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

  5. A physical perspective on cytoplasmic streaming (invited)

    CERN Document Server

    Goldstein, Raymond E

    2015-01-01

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed $100$ $\\mu$m. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant $Chara$, whose cells can exceed $10$ cm in length and $1$ mm in diameter. Two spiraling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to $100$ $\\mu$m/s, motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as "cytoplasmic streaming", found in a wide range of eukaryotic organisms - algae, plants, amoebae, nematodes, and flies - often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size, and discuss the possible role of self-organi...

  6. Molecular analysis of cytoplasmic male sterility

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, M.R.

    1990-01-01

    The ultimate aims of the project are to understand the molecular mechanism of the disruption in pollen development which occurs in cytoplasmic male sterile plants and to understand the control of respiratory energy flow in the higher plant cell. A mitochondrial locus termed S-pcf segregates with sterility and with an alteration in respiration in Petunia. This cloned locus contains three genes, an abnormal fused gene termed pcf, a gene for a subunit of an NADH dehydrogenase complex, and a small ribosomal subunit protein. The pcf gene is comprised of partial sequences of ATPase subunit 9, cytochrome oxidase subunit II, and an unidentified reading frame. Components of the S-Pcf locus will be introduced into the nuclear of a fertile genotype under the control of appropriate regulatory signals, and polypeptide products of introduced genes will be directed to the mitochondrion with a transit peptide. By examining transgenic plants, we can determine what elements of the locus are critical for altered respiration or sterility. Such knowledge could explain how mitochondrial DNA affects pollen development in the large number of plant species which exhibit the agronomically important trait of male sterility. 10 refs., 3 figs.

  7. A physical perspective on cytoplasmic streaming.

    Science.gov (United States)

    Goldstein, Raymond E; van de Meent, Jan-Willem

    2015-08-06

    Organisms show a remarkable range of sizes, yet the dimensions of a single cell rarely exceed 100 µm. While the physical and biological origins of this constraint remain poorly understood, exceptions to this rule give valuable insights. A well-known counterexample is the aquatic plant Chara, whose cells can exceed 10 cm in length and 1 mm in diameter. Two spiralling bands of molecular motors at the cell periphery drive the cellular fluid up and down at speeds up to 100 µm s(-1), motion that has been hypothesized to mitigate the slowness of metabolite transport on these scales and to aid in homeostasis. This is the most organized instance of a broad class of continuous motions known as 'cytoplasmic streaming', found in a wide range of eukaryotic organisms-algae, plants, amoebae, nematodes and flies-often in unusually large cells. In this overview of the physics of this phenomenon, we examine the interplay between streaming, transport and cell size and discuss the possible role of self-organization phenomena in establishing the observed patterns of streaming.

  8. Cytoplasmic Dynein Promotes HIV-1 Uncoating

    Directory of Open Access Journals (Sweden)

    Paulina Pawlica

    2014-11-01

    Full Text Available Retroviral capsid (CA cores undergo uncoating during their retrograde transport (toward the nucleus, and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.

  9. Optimal cytoplasmic transport in viral infections.

    Directory of Open Access Journals (Sweden)

    Maria R D'Orsogna

    Full Text Available For many viruses, the ability to infect eukaryotic cells depends on their transport through the cytoplasm and across the nuclear membrane of the host cell. During this journey, viral contents are biochemically processed into complexes capable of both nuclear penetration and genomic integration. We develop a stochastic model of viral entry that incorporates all relevant aspects of transport, including convection along microtubules, biochemical conversion, degradation, and nuclear entry. Analysis of the nuclear infection probabilities in terms of the transport velocity, degradation, and biochemical conversion rates shows how certain values of key parameters can maximize the nuclear entry probability of the viral material. The existence of such "optimal" infection scenarios depends on the details of the biochemical conversion process and implies potentially counterintuitive effects in viral infection, suggesting new avenues for antiviral treatment. Such optimal parameter values provide a plausible transport-based explanation of the action of restriction factors and of experimentally observed optimal capsid stability. Finally, we propose a new interpretation of how genetic mutations unrelated to the mechanism of drug action may nonetheless confer novel types of overall drug resistance.

  10. An NPF transporter exports a central monoterpene indole alkaloid intermediate from the vacuole.

    Science.gov (United States)

    Payne, Richard M E; Xu, Deyang; Foureau, Emilien; Teto Carqueijeiro, Marta Ines Soares; Oudin, Audrey; Bernonville, Thomas Dugé de; Novak, Vlastimil; Burow, Meike; Olsen, Carl-Erik; Jones, D Marc; Tatsis, Evangelos C; Pendle, Ali; Ann Halkier, Barbara; Geu-Flores, Fernando; Courdavault, Vincent; Nour-Eldin, Hussam Hassan; O'Connor, Sarah E

    2017-01-13

    Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.

  11. Analysis of cytoplasmic effects and fine-mapping of a genic male sterile line in rice.

    Directory of Open Access Journals (Sweden)

    Peng Qin

    Full Text Available Cytoplasm has substantial genetic effects on progeny and is important for yield improvement in rice breeding. Studies on the cytoplasmic effects of cytoplasmic male sterility (CMS show that most types of CMS have negative effects on yield-related traits and that these negative effects vary among CMS. Some types of genic male sterility (GMS, including photo-thermo sensitive male sterility (PTMS, have been widely used in rice breeding, but the cytoplasmic effects of GMS remain unknown. Here, we identified a GMS mutant line, h2s, which exhibited small, white anthers and failed to produce mature pollen. Unlike CMS, the h2s had significant positive cytoplasmic effects on the seed set rate, weight per panicle, yield, and general combining ability (GCA for plant height, seed set rate, weight per panicle, and yield. These effects indicated that h2s cytoplasm may show promise for the improvement of rice yield. Genetic analysis suggested that the phenotype of h2s was controlled by a single recessive locus. We mapped h2s to a 152 kb region on chromosome 6, where 22 candidate genes were predicted. None of the 22 genes had previously been reported to be responsible for the phenotypes of h2s. Sequencing analysis showed a 12 bp deletion in the sixth exon of Loc_Os06g40550 in h2s in comparison to wild type, suggesting that Loc_Os06g40550 is the best candidate gene. These results lay a strong foundation for cloning of the H2S gene to elucidate the molecular mechanism of male reproduction.

  12. Cell-to-cell spread and massive vacuole formation after Cryptococcus neoformans infection of murine macrophages

    Directory of Open Access Journals (Sweden)

    Casadevall Arturo

    2007-08-01

    Full Text Available Abstract Background The interaction between macrophages and Cryptococcus neoformans (Cn is critical for containing dissemination of this pathogenic yeast. However, Cn can either lyse macrophages or escape from within them through a process known as phagosomal extrusion. Both events result in live extracellular yeasts capable of reproducing and disseminating in the extracellular milieu. Another method of exiting the intracellular confines of cells is through host cell-to-cell transfer of the pathogen, and this commonly occurs with the human immuno-deficiency virus (HIV and CD4+ T cells and macrophages. In this report we have used time-lapse imaging to determine if this occurs with Cn. Results Live imaging of Cryptococcus neoformans interactions with murine macrophages revealed cell-to-cell spread of yeast cells from infected donor cells to uninfected cells. Although this phenomenon was relatively rare its occurrence documents a new capacity for this pathogen to infect adjacent cells without exiting the intracellular space. Cell-to-cell spread appeared to be an actin-dependent process. In addition, we noted that cryptococcal phagosomal extrusion was followed by the formation of massive vacuoles suggesting that intracellular residence is accompanied by long lasting damage to host cells. Conclusion C. neoformans can escape the intracellular confines of macrophages in an actin dependent manner by cell-to-cell transfer of the yeast leading to infection of adjacent cells. In addition, complete extrusion of internalized Cn cells can lead to the formation of a massive vacuole which may be a sign of damage to the host macrophage. These observations document new outcomes for the interaction of C. neoformans with host cells that provide precedents for cell biological effects that may contribute to the pathogenesis of cryptococcal infections.

  13. Trypanosoma cruzi: Entry Into Mammalian Host Cells and Parasitophorous Vacuole Formation

    Directory of Open Access Journals (Sweden)

    Emile Santos Barrias

    2013-08-01

    Full Text Available Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are the metacyclic trypomastigotes, amastigotes and bloodstream trypomastigotes. The recognition between the parasite and mammalian host cell, involves numerous molecules present in both cell types, and similar to several intracellular pathogens, T.cruzi is internalized by host cells via multiple endocytic pathways. Morphological studies demonstrated that after the interaction of the infective forms of T.cruzi with phagocytic or non-phagocytic cell types, plasma membrane protrusions can form, showing similarity with those observed during canonical phagocytosis or macropinocytic events. Additionally, several molecules known to be molecular markers of membrane rafts, macropinocytosis and phagocytosis have been demonstrated to be present at the invasion site. These events may or may not depend on the host cell lysosomes and cytoskeleton. In addition, after penetration, components of the host endosomal-lysosomal system, such as early endosomes, late endosomes and lysosomes, participate in the formation of the nascent parasithophorous vacuole (VP. Dynamin, a molecule involved in vesicle formation, has been shown to be involved in the parasitophorous vacuole release from the host cell plasma membrane. This review focuses on the multiple pathways that T.cruzi can use to enter the host cells until complete VP formation. We will describe different endocytic processes, such as phagocytosis, macropinocytosis, endocytosis using membrane microdomains and clathrin-dependent endocytosis and show results that are consistent with their use by this smart parasite. We will also discuss other mechanisms that have been described, such as active penetration and the process that takes advantage of cell membrane wound repair.

  14. Heterozygous alleles restore male fertility to cytoplasmic male-sterile radish (Raphanus sativus L.): a case of overdominance.

    Science.gov (United States)

    Wang, Zhi Wei; De Wang, Chuan; Wang, Chuan; Gao, Lei; Mei, Shi Yong; Zhou, Yuan; Xiang, Chang Ping; Wang, Ting

    2013-04-01

    The practice of hybridization has greatly contributed to the increase in crop productivity. A major component that exploits heterosis in crops is the cytoplasmic male sterility (CMS)/nucleus-controlled fertility restoration (Rf) system. Through positional cloning, it is shown that heterozygous alleles (RsRf3-1/RsRf3-2) encoding pentatricopeptide repeat (PPR) proteins are responsible for restoring fertility to cytoplasmic male-sterile radish (Raphanus sativus L.). Furthermore, it was found that heterozygous alleles (RsRf3-1/RsRf3-2) show higher expression and RNA polymerase II occupancy in the CMS cytoplasmic background compared with their homozygous alleles (RsRf3-1/RsRf3-1 or RsRf3-2/RsRf3-2). These data provide new insights into the molecular mechanism of fertility restoration to cytoplasmic male-sterile plants and illustrate a case of overdominance.

  15. ROS-activated ATM-dependent phosphorylation of cytoplasmic substrates identified by large scale phosphoproteomics screen

    DEFF Research Database (Denmark)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper

    2016-01-01

    ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoi......ATM (ataxia-telangiectasia, mutated) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signalling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle...... to identify cytoplasmic proteins altered in their phosphorylation state in control and A-T (ataxia-telangiectasia) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites...

  16. Long Non-coding RNAs in the Cytoplasm

    Institute of Scientific and Technical Information of China (English)

    Farooq Rashid; Abdullah Shah; Ge Shan

    2016-01-01

    An enormous amount of long non-coding RNAs (lncRNAs) transcribed from eukaryotic genome are important regulators in different aspects of cellular events. Cytoplasm is the residence and the site of action for many lncRNAs. The cytoplasmic lncRNAs play indispensable roles with multiple molecular mechanisms in animal and human cells. In this review, we mainly talk about functions and the underlying mechanisms of lncRNAs in the cytoplasm. We highlight relatively well-studied examples of cytoplasmic lncRNAs for their roles in modulating mRNA stability, regulating mRNA translation, serving as competing endogenous RNAs, functioning as precursors of microRNAs, and mediating protein modifications. We also elaborate the perspectives of cytoplasmic lncRNA studies.

  17. Cytoplasmic streaming velocity as a plant size determinant.

    Science.gov (United States)

    Tominaga, Motoki; Kimura, Atsushi; Yokota, Etsuo; Haraguchi, Takeshi; Shimmen, Teruo; Yamamoto, Keiichi; Nakano, Akihiko; Ito, Kohji

    2013-11-11

    Cytoplasmic streaming is active transport widely occurring in plant cells ranging from algae to angiosperms. Although it has been revealed that cytoplasmic streaming is generated by organelle-associated myosin XI moving along actin bundles, the fundamental function in plants remains unclear. We generated high- and low-speed chimeric myosin XI by replacing the motor domains of Arabidopsis thaliana myosin XI-2 with those of Chara corallina myosin XI and Homo sapiens myosin Vb, respectively. Surprisingly, the plant sizes of the transgenic Arabidopsis expressing high- and low-speed chimeric myosin XI-2 were larger and smaller, respectively, than that of the wild-type plant. This size change correlated with acceleration and deceleration, respectively, of cytoplasmic streaming. Our results strongly suggest that cytoplasmic streaming is a key determinant of plant size. Furthermore, because cytoplasmic streaming is a common system for intracellular transport in plants, our system could have applications in artificial size control in plants.

  18. Antineutrophil Cytoplasmic Antibodies Associated With Infective Endocarditis

    Science.gov (United States)

    Langlois, Vincent; Lesourd, Anais; Girszyn, Nicolas; Ménard, Jean-Francois; Levesque, Hervé; Caron, Francois; Marie, Isabelle

    2016-01-01

    Abstract To determine the prevalence of antineutrophil cytoplasmic antibodies (ANCA) in patients with infective endocarditis (IE) in internal medicine; and to compare clinical and biochemical features and outcome between patients exhibiting IE with and without ANCA. Fifty consecutive patients with IE underwent ANCA testing. The medical records of these patients were reviewed. Of the 50 patients with IE, 12 exhibited ANCA (24%). ANCA-positive patients with IE exhibited: longer duration between the onset of first symptoms and IE diagnosis (P = 0.02); and more frequently: weight loss (P = 0.017) and renal impairment (P = 0.08), lower levels of C-reactive protein (P = 0.0009) and serum albumin (P = 0.0032), involvement of both aortic and mitral valves (P = 0.009), and longer hospital stay (P = 0.016). Under multivariate analysis, significant factors for ANCA-associated IE were: longer hospital stay (P = 0.004), lower level of serum albumin (P = 0.02), and multiple valve involvement (P = 0.04). Mortality rate was 25% in ANCA patients; death was because of IE complications in all these patients. Our study identifies a high prevalence of ANCA in unselected patients with IE in internal medicine (24%). Our findings further underscore that ANCA may be associated with a subacute form of IE leading to multiple valve involvement and more frequent renal impairment. Because death was due to IE complications in all patients, our data suggest that aggressive therapy may be required to improve such patients’ outcome. PMID:26817911

  19. Antineutrophil Cytoplasmic Antibodies, Autoimmune Neutropenia, and Vasculitis

    Science.gov (United States)

    Grayson, Peter C.; Sloan, J. Mark; Niles, John L.; Monach, Paul A.; Merkel, Peter A.

    2011-01-01

    Objectives Reports of an association between antineutrophil cytoplasmic antibodies (ANCA) and autoimmune neutropenia have rarely included cases of proven vasculitis. A case of ANCA-associated vasculitis (AAV) with recurrent neutropenia is described and relevant literature on the association between ANCA, neutropenia, and vasculitis is reviewed. Methods Longitudinal clinical assessments and laboratory findings are described in a patient with AAV and recurrent episodes of profound neutropenia from December 2008 – October 2010. A PubMed database search of the medical literature was performed for papers published from 1960 through October 2010 to identify all reported cases of ANCA and neutropenia. Results A 49 year-old man developed recurrent neutropenia, periodic fevers, arthritis, biopsy-proven cutaneous vasculitis, sensorineural hearing loss, epididymitis, and positive tests for ANCA with specificity for antibodies to both proteinase 3 and myeloperoxidase. Antineutrophil membrane antibodies were detected during an acute neutropenic phase and were not detectable in a post-recovery sample, whereas ANCA titers did not seem to correlate with neutropenia. An association between ANCA and neutropenia has been reported in 74 cases from 24 studies in the context of drug/toxin exposure, underlying autoimmune disease, or chronic neutropenia without underlying autoimmune disease. In these cases, the presence of atypical ANCA patterns and other antibodies were common; however, vasculitis was uncommon and when it occurred was usually limited to the skin and in cases of underlying toxin exposure. Conclusions ANCA is associated with autoimmune neutropenia, but systemic vasculitis rarely occurs in association with ANCA and neutropenia. The interaction between neutrophils and ANCA may provide insight into understanding both autoimmune neutropenia and AAV. PMID:21507463

  20. Nuclear reprogramming by interphase cytoplasm of 2-cell mouse embryos

    Science.gov (United States)

    Kang, Enugu; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P.; Schöler, Hans; Mitalipov, Shoukhrat

    2014-01-01

    Summary Successful mammalian cloning employing somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II-arrested (MII) oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing pluripotency in somatic cell nuclei1-3. However, these poorly defined maternal factors presumably decline sharply after fertilization since cytoplasm of pronuclear stage zygotes is reportedly inactive4, 5. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase (M-phase) can also support derivation of embryonic stem cells (ESCs) following SCNT6-8, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in M-phase but not in interphase cytoplasm are “trapped” inside the nucleus during interphase and effectively removed during enucleation9. Here, we investigated the presence of reprogramming activity in the interphase cytoplasm of 2-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated M-phase and interphase zygotes and 2-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Then, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ESC, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ESCs capable of contributing to traditional germline and tetraploid chimeras. In addition, direct transfer of cloned embryos, reconstructed with ESC nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to utilize interphase cytoplasm in SCNT could impact efforts to generate autologous human ESCs for

  1. LAMP proteins account for the maturation delay during the establishment of the Coxiella burnetii-containing vacuole.

    Science.gov (United States)

    Schulze-Luehrmann, Jan; Eckart, Rita A; Ölke, Martha; Saftig, Paul; Liebler-Tenorio, Elisabeth; Lührmann, Anja

    2016-02-01

    The obligate intracellular pathogen Coxiella burnetii replicates in a large phagolysosomal-like vacuole. Currently, both host and bacterial factors required for creating this replicative parasitophorous C. burnetii-containing vacuole (PV) are poorly defined. Here, we assessed the contributions of the most abundant proteins of the lysosomal membrane, LAMP-1 and LAMP-2, to the establishment and maintenance of the PV. Whereas these proteins were not critical for uptake of C. burnetii, they influenced the intracellular replication of C. burnetii. In LAMP-1/2 double-deficient fibroblasts as well as in LAMP-1/2 knock-down cells, C. burnetii establishes a significantly smaller, yet faster maturing vacuole, which harboured more bacteria. The accelerated maturation of PVs in LAMP double-deficient fibroblasts, which was partially or fully reversed by ectopic expression of LAMP-1 or LAMP-2, respectively, was characterized by an increased fusion rate with endosomes, lysosomes and bead-containing phagosomes, but not by different fusion kinetics with autophagy vesicles. These findings establish that LAMP proteins are critical for the maturation delay of PVs. Unexpectedly, neither the creation of the spacious vacuole nor the delay in maturation was found to be prerequisites for the intracellular replication of C. burnetii.

  2. 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.

    2008-01-01

    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: 296-30

  3. Differential Compartmentation of Gibberellin A1 and Its Metabolites in Vacuoles of Cowpea and Barley Leaves 1

    Science.gov (United States)

    Garcia-Martinez, Jose L.; Ohlrogge, John B.; Rappaport, Lawrence

    1981-01-01

    The metabolism and efflux of gibberellin A1 (GA1) taken up by leaves of cowpea (Vigna sinensis cv. Blackeye pea No. 5), as well as the distribution of GA1 metabolites in the protoplasts and vacuoles of cowpea and barley (Hordeum vulgare L. cv. Numar), were studied. GA1 is metabolized rapidly in cowpea leaf discs to products tentatively identified as gibberellin A8 (GA8) and gibberellin A8 glucoside (GA8-glu). After labeling leaf discs with [3H]GA1 for 1 hour, the release of radioactivity from the leaf was followed. Over a 12-hour period, the level of radioisotope in the tissue declined to about 35% of the original, after which no further release was observed. At this time, almost all of the radioactivity remaining in the leaf was GA8-glu, while most of the radioactivity which had been released was unmetabolized GA1. Mesophyll protoplasts and vacuoles were isolated from cowpea and barley leaves previously fed [3H]GA1. These protoplasts retain the ability to metabolize GA1, indicating that neither the leaf structure nor the cell wall is necessary for this metabolism. A higher proportion of GA8-glu was found in the vacuoles relative to the entire protoplasts. The results obtained suggest that GA1 metabolites are preferentially compartmentalized in the vacuoles relative to GA1. PMID:16662014

  4. The vacuolating cytotoxin of Helicobacter pylori%幽门螺杆菌空泡毒素研究进展

    Institute of Scientific and Technical Information of China (English)

    刘纯杰; 陶好霞; 张兆山

    2001-01-01

    幽门螺杆菌空泡毒素是该菌产生的与已知其它细菌毒素无明显同源性的唯一蛋白毒素。该毒素是幽门螺杆菌重要的毒力致病因子,它的产生与感染者胃肠上皮损伤和溃疡形成密切相关。本文就幽门螺杆菌空泡毒素的结构与功能研究进展以及在未来免疫预防与免疫治疗中的作用进行了简述。%The vacuolating cytotoxin is a unique proteinous cytotoxin producted by H. pylori that showed no striking primary sequence homology with other known baterial toxins. The cytotoxin is an important fator in the pathogenesis of H. pylori, which induces vacuolation of epithelial cells and plays an important role in gastric epithelial necrosis and peptic ulceration. In the paper, the progress on structure and function of the vacuolating cytotoxin of H. pylori and the roles of the H. pylori vacuolating cytotoxin in the future immunoprophylaxis and immunotherapy were reviewed.

  5. Hyperacidification of Vacuoles by the Combined Action of Two Different P-ATPases in the Tonoplast Determines Flower Color

    Directory of Open Access Journals (Sweden)

    Marianna Faraco

    2014-01-01

    Full Text Available The acidification of endomembrane compartments is essential for enzyme activities, sorting, trafficking, and trans-membrane transport of various compounds. Vacuoles are mildly acidic in most plant cells because of the action of V-ATPase and/or pyrophosphatase proton pumps but are hyperacidified in specific cells by mechanisms that remained unclear. Here, we show that the blue petal color of petunia ph mutants is due to a failure to hyperacidify vacuoles. We report that PH1 encodes a P3B-ATPase, hitherto known as Mg2+ transporters in bacteria only, that resides in the vacuolar membrane (tonoplast. In vivo nuclear magnetic resonance and genetic data show that PH1 is required and, together with the tonoplast H+ P3A-ATPase PH5, sufficient to hyperacidify vacuoles. PH1 has no H+ transport activity on its own but can physically interact with PH5 and boost PH5 H+ transport activity. Hence, the hyperacidification of vacuoles in petals, and possibly other tissues, relies on a heteromeric P-ATPase pump.

  6. The I-BAR protein Ivy1 is an effector of the Rab7 GTPase Ypt7 involved in vacuole membrane homeostasis

    NARCIS (Netherlands)

    Numrich, Johannes; Péli-Gulli, Marie-Pierre; Arlt, Henning; Sardu, Alessandro; Griffith, Janice; Levine, Tim; Engelbrecht-Vandré, Siegfried; Reggiori, Fulvio; De Virgilio, Claudio; Ungermann, Christian

    2015-01-01

    Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show

  7. Pediatric Inflammatory Bowel Disease with Cytoplasmic Staining of Antineutrophil Cytoplasmic Antibodies

    Directory of Open Access Journals (Sweden)

    Omar I. Saadah

    2013-01-01

    Full Text Available Background. It is unusual for the antineutrophil cytoplasmic antibody with cytoplasmic pattern (cANCA to present in patients with inflammatory bowel disease (IBD without vasculitis. The purpose of this study was to describe the occurrence and characteristics of pediatrics IBD with cANCA. Methods. A retrospective review of pediatric IBD associated with cANCA serology in patients from King Abdulaziz University Hospital, Saudi Arabia, between September 2002 and February 2012. Results. Out of 131 patients with IBD screened for cANCAs, cANCA was positive in 7 (5.3% patients of whom 4 had ulcerative colitis and 3 had Crohn's disease. The median age was 8.8 years (2–14.8 years. Six (86% were males. Of the 7 patients, 5 (71% were Saudi Arabians and 2 were of Indian ethnicity. The most common symptoms were diarrhea, abdominal pain, weight loss, and rectal bleeding. None had family history or clinical features suggestive of vasculitis involving renal and respiratory systems. No difference in the disease location or severity was observed between cANCA positive and cANCA negative patients apart from male preponderance in cANCA positive patients. Conclusion. The occurrence of cANCA in pediatric IBD is rare. Apart from male preponderance, there were no peculiar characteristics for the cANCA positive patients.

  8. Uniparental Inheritance Promotes Adaptive Evolution in Cytoplasmic Genomes.

    Science.gov (United States)

    Christie, Joshua R; Beekman, Madeleine

    2017-03-01

    Eukaryotes carry numerous asexual cytoplasmic genomes (mitochondria and plastids). Lacking recombination, asexual genomes should theoretically suffer from impaired adaptive evolution. Yet, empirical evidence indicates that cytoplasmic genomes experience higher levels of adaptive evolution than predicted by theory. In this study, we use a computational model to show that the unique biology of cytoplasmic genomes-specifically their organization into host cells and their uniparental (maternal) inheritance-enable them to undergo effective adaptive evolution. Uniparental inheritance of cytoplasmic genomes decreases competition between different beneficial substitutions (clonal interference), promoting the accumulation of beneficial substitutions. Uniparental inheritance also facilitates selection against deleterious cytoplasmic substitutions, slowing Muller's ratchet. In addition, uniparental inheritance generally reduces genetic hitchhiking of deleterious substitutions during selective sweeps. Overall, uniparental inheritance promotes adaptive evolution by increasing the level of beneficial substitutions relative to deleterious substitutions. When we assume that cytoplasmic genome inheritance is biparental, decreasing the number of genomes transmitted during gametogenesis (bottleneck) aids adaptive evolution. Nevertheless, adaptive evolution is always more efficient when inheritance is uniparental. Our findings explain empirical observations that cytoplasmic genomes-despite their asexual mode of reproduction-can readily undergo adaptive evolution. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  9. Differences in catalytic properties between cerebral cytoplasmic and mitochondrial hexokinases.

    Science.gov (United States)

    Thompson, M F; Bachelard, H S

    1977-03-01

    1. Clear kinetic differences between cytoplasmic and mitochondrial forms of type-I cerebral hexokinase were demonstrated from experiments performed under identical conditions on three (cytoplasmic, bound mitochondrial and solubilized mitochondrial) preparations of the enzyme. 2. Whereas the Michaelis constant for glucose (KmGlc) was consistent, that for MgATP2- (KmATP) was lower in the cytoplasmic than in the two mitochondrial preparations. The substrate dissociation constants (KsGlc and KsATP) were both higher in the cytoplasmic than in the mitochondrial preparations. A further difference in the substrate kinetic patterns was that KmATP=KmATP for the cytoplasmic enzyme, in contrast with the mitochondrial enzyme, where KmATP was clearly not equal to KsATP [Bachelard et al. (1971) Biochem. J. 123, 707-715]. 3. Dead-end inhibition produced by N-acetyl-glucosamine and by AMP also exhibited different quantitative kinetic patterns for the two enzyme sources. Both inhibitions gave Ki values similar or equal to those of Ki' for the cytoplasmic activity, whereas Ki was clearly not equal to Ki' for the mitochondrial activity. 4. All of these studies demonstrated the similarity of the two mitochondrial activities (particulate and solubilized), which were both clearly different from the cytoplasmic activity. 5. The analysis gives a practical example of our previous theoretical treatment on the derivation of true inhibition constants. 6. The results are discussed in terms of the function of cerebral hexokinases.

  10. Enhanced electroporation in plant tissues via low frequency pulsed electric fields: influence of cytoplasmic streaming.

    Science.gov (United States)

    Asavasanti, Suvaluk; Stroeve, Pieter; Barrett, Diane M; Jernstedt, Judith A; Ristenpart, William D

    2012-01-01

    Pulsed electric fields (PEF) are known to be effective at permeabilizing plant tissues. Prior research has demonstrated that lower pulse frequencies induce higher rates of permeabilization, but the underlying reason for this response is unclear. Intriguingly, recent microscopic observations with onion tissues have also revealed a correlation between PEF frequency and the subsequent speed of intracellular convective motion, i.e., cytoplasmic streaming. In this paper, we investigate the effect of cytoplasmic streaming on the efficacy of plant tissue permeabilization via PEF. Onion tissue samples were treated with Cytochalasin B, a known inhibitor of cytoplasmic streaming, and changes in cellular integrity and viability were measured over a wide range of frequencies and field strengths. We find that at low frequencies (f streaming results in a 19% decrease in the conductivity disintegration index compared with control samples. Qualitatively, similar results were observed using a microscopic cell viability assay. The results suggest that at low frequencies convection plays a statistically significant role in distributing more conductive fluid throughout the tissue, making subsequent pulses more efficacious. The key practical implication is that PEF pretreatment at low frequency can increase the rate of tissue permeabilization in dehydration or extraction processes, and that the treatment will be most effective when cytoplasmic streaming is most active, i.e., with freshly prepared plant tissues.

  11. Gravitational effects on the rearrangement of cytoplasmic components during axial formation in amphibian development

    Science.gov (United States)

    Phillips, C. R.; Whalon, B.; Moore, J.; Danilchik, M.

    The spatial positioning of the dorsal-ventral axis in the amphibian, Xenopus laevis, can be experimentally manipulated either by tipping the embryo relative to Earth's gravitational force vector or by centrifugation. Experimental evidence suggests that certain cytoplasmic components are redistributed during the first cell cycle and that these components are, in part, responsible for the establishment of this axis. Further studies indicate that at least some of the cytoplasmic components responsible for establishing this axis may be RNA. Recombinant cDNA and PCR technology are utilized to isolate DNA clones for messenger RNA which becomes spatially localized to the dorsal side of the embryo. These clones are being used to study the mechanisms of spatial localization and the function of the localized RNA transcripts.

  12. Poorly differentiated angiosarcoma without vasoformative channels but with focal intracytoplastic vacuoles mimicking liposarcomas

    Directory of Open Access Journals (Sweden)

    Tadashi Terada, MD, PhD

    2016-03-01

    Full Text Available Angiosarcoma (AS showed diverse morphologies from well formed malignant vasculatures to poorly differentiated tumor with only a few clues of endothelial differentiation. Herein reported are two cases of AS without primitive vasoformative channels (VC. They showed, instead, a very few foci of intracytoplasmic vacuoles (ICV that mimicked liposarcoma. The two cases were found in 12 cases of AS in computer database. Both are men, 57 and 68 years. One is cutaneous (foot AS and another is soft tissue (thigh AS. The largest diameter of cutaneous AS was 5 cm, and that of soft tissue AS 9 cm. The prognosis of both patients was poor; both died of metastases 4 and 6 years after initial presentation. In both cases, hematoxylin and eosin (HE diagnosis was difficult because there were no VC, and most of the tumors were composed of primitive mesenchymal tissues. In both cases, however, a few very tiny foci consisting of ICV were seen. At first, the author considered them as mucins or fat, and suspected liposarcoma. In fact, they were pseudolipoblasts. Several mucin stains showed no mucins, and fat stains of frozen sections of formalin fixed tissue were negative for fat. Immunohistochemically, the vacuoles were positive for factor VIII-related antigen (F-VIII-RA, Ulex lectin, CD31, CD34, vimentin, p53 and Ki-67 (labeling index = 64% and 75%, but negative for various types of cytokeratins (CK, EMA, CEA, CA19-9, CD45, smooth muscle actins, S100 protein, myoglobin, HMB-45, Melan A, NCAM, and NSE. F-VIII-RA is specific and Ulex lectin and CD31 are relatively specific for endothelium. Therefore, the pathological diagnosis of AS could be made by the combined histologic features (ICV and Immunohistochemical positivity of F-VIII-RA, Ulex lectin, and CD31. Thus, it appeared that the ICV may be the only clue of poorly differentiated or undifferentiated AS. In such undifferentiated cases, combined observations of meticulous histologic observations (intracytoplasmic

  13. Refractory disease in antineutrophil cytoplasmic antibodies associated vasculitis

    NARCIS (Netherlands)

    Rutgers, Abraham; Kallenberg, Cornelis

    2012-01-01

    Purpose of review Induction treatment of antineutrophil cytoplasmic antibodies (ANCA) associated vasculitis (AAV) is not always successful and nonresponding patients are considered refractory. Recent findings Refractory disease should be subdefined to the treatment that was received. Cyclophosphamid

  14. On the evolution of cytoplasmic incompatibility in haplodiploid species

    NARCIS (Netherlands)

    Egas, C.J.M.; de Freitas Vala Salvador, F.; Breeuwer, J.A.J.

    2002-01-01

    The most enigmatic sexual manipulation by Wolbachia endosymbionts is cytoplasmic incompatibility (CI): infected mates are reproductively incompatible with uninfected females. In this paper, we extend the theory on population dynamics and evolution of CI, with emphasis on haplodiploid species. First,

  15. Cytoplasmic tail of coronavirus spike protein has intracellular targeting signals

    Indian Academy of Sciences (India)

    JIBIN SADASIVAN; MANMEET SINGH; JAYASRI DAS SARMA

    2017-06-01

    Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is localized in the ER or ERGIC compartment and OC43 spike protein is predominantly localized in thelysosome. Differential localization can be explained by signal sequence. The sequence alignment using Clustal Wshows that the signal sequence present at the cytoplasmic tail plays an important role in spike protein localization. Aunique GYQEL motif is identified at the cytoplasmic terminal of OC43 spike protein which helps in localization in thelysosome, and a novel KLHYT motif is identified in the cytoplasmic tail of SARS spike protein which helps in ER orERGIC localization. This study sheds some light on the role of cytoplasmic tail of spike protein in cell-to-cell fusion,coronavirus host cell fusion and subsequent pathogenicity.

  16. Critical amino acids in syndecan-4 cytoplasmic domain modulation of turkey satellite cell growth and development.

    Science.gov (United States)

    Song, Yan; McFarland, Douglas C; Velleman, Sandra G

    2012-02-01

    Syndecan-4 is composed of a core protein and covalently attached glycosaminoglycan (GAG) and N-linked glycosylated (N-glycosylated) chains. The core protein is divided into extracellular, transmembrane, and cytoplasmic domains. The cytoplasmic domain has two conserved regions and a variable region in the middle. The Ser residue in the conserved region 1 and the Tyr residue in the variable region are important in regulating protein kinase C alpha (PKCα) membrane localization and focal adhesion formation. The objective of the current study was to investigate the role of syndecan-4 Ser and Tyr residues in combination with the GAG and N-glycosylated chains in turkey satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) responsiveness, and PKCα membrane localization. Site-directed mutagenesis was used to generate Ser and Tyr mutants with or without GAG and N-glycosylated chains. The wild type and mutant syndecan-4 constructs were transfected into turkey satellite cells. The over-expression of Ser and Tyr mutants increased cell proliferation and differentiation and decreased membrane localization of PKCα. Furthermore, Ser mutants enhanced cellular responsiveness to FGF2. The results from this study are the first demonstration of a role of syndecan-4 cytoplasmic domain Ser and Tyr residues in regulating satellite cell proliferation, differentiation, and the modulation of cellular responsiveness to FGF2.

  17. [A single strand comformation polymorphism of vacuolating cytotoxin gene in H. pylori].

    Science.gov (United States)

    Peng, H; Pan, G; Chao, S

    1999-03-01

    To use PCR/SSCP analysis of the vacuolating cytotoxin gene (vacA) of H. pylori for differentiation of various strains of H. pylori. PCR was performed using the primers amplifing vacA gene of the bacteria embeded in the gastric mucosa of 159 patients with various gastric duodenal diseases. The products of PCR were further processed for SSCP analysis and southern blot hybridization. In the meantime, vacA genes of three different SSCP-patterns from three patients with duodenal ulcers were sequenced. The rate of detection of H. pylori with the method was 100%. vacA1 and vacA2, the two subtypes of vacA, were 76.5% (114/149) and 23.5%(35/149), respectively. Eight different SSCP-patterns were distributed in various gastroduodenal diseases, and that 80% of duodenal ulcers was predominated with B pattern. Sequencing of DNA indicated a diversity of vacA gene structure. PCR/SSCP can be used in the differentiation of different strains of H. pylori in epidemology, and in the follow up study after H. pylori eradication, especially in the differentiation between H. pylori recrudescence and reinfection.

  18. Transport of Arginine and Aspartic Acid into Isolated Barley Mesophyll Vacuoles 1

    Science.gov (United States)

    Martinoia, Enrico; Thume, Monika; Vogt, Esther; Rentsch, Doris; Dietz, Karl-Josef

    1991-01-01

    The transport of arginine into isolated barley (Hordeum vulgare L.) mesophyll vacuoles was investigated. In the absence of ATP, arginine uptake was saturable with a Km of 0.3 to 0.4 millimolar. Positively charged amino acids inhibited arginine uptake, lysine being most potent with a Ki of 1.2 millimolar. In the presence of free ATP, but not of its Mg-complex, uptake of arginine was drastically enhanced and a linear function of its concentration up to 16 millimolar. The nonhydrolyzable adenylyl imidodiphosphate, but no other nucleotide tested, could substitute for ATP. Therefore, it is suggested that this process does not require energy and does not involve the tonoplast ATPase. The ATP-dependent arginine uptake was strongly inhibited by p-chloromercuriphenylsulfonic acid. Furthermore, hydrophobic amino acids were inhibitory (I50 phenylalanine 1 millimolar). Similar characteristics were observed for the uptake of aspartic acid. However, rates of ATP-stimulated aspartic acid transport were 10-fold lower as compared to arginine transport. Uptake of aspartate in the absence of ATP was negligible. PMID:16668447

  19. Shape, shell, and vacuole formation during the drying of a single concentrated whey protein droplet.

    Science.gov (United States)

    Sadek, Céline; Tabuteau, Hervé; Schuck, Pierre; Fallourd, Yannick; Pradeau, Nicolas; Le Floch-Fouéré, Cécile; Jeantet, Romain

    2013-12-17

    The drying of milk concentrate droplets usually leads to specific particle morphology influencing their properties and their functionality. Understanding how the final shape of the particle is formed therefore represents a key issue for industrial applications. In this study, a new approach to the investigation of droplet-particle conversion is proposed. A single droplet of concentrated globular proteins extracted from milk was deposited onto a hydrophobic substrate and placed in a dry environment. Complementary methods (high-speed camera, confocal microscopy, and microbalance) were used to record the drying behavior of the concentrated protein droplets. Our results showed that whatever the initial concentration, particle formation included three dynamic stages clearly defined by the loss of mass and the evolution of the internal and external shapes of the droplet. A new and reproducible particle shape was related in this study. It was observed after drying a smooth, hemispherical cap-shaped particle, including a uniform protein shell and the nucleation of an internal vacuole. The particle morphology was strongly influenced by the drying environment, the contact angle, and the initial protein concentration, all of which governed the duration of the droplet shrinkage, the degree of buckling, and the shell thickness. These results are discussed in terms of specific protein behaviors in forming a predictable and a characteristic particle shape. The way the shell is formed may be the starting point in shaping particle distortion and thus represents a potential means of tuning the particle morphology.

  20. 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.

  1. Structure of CARDS toxin, a unique ADP-ribosylating and vacuolating cytotoxin from Mycoplasma pneumoniae.

    Science.gov (United States)

    Becker, Argentina; Kannan, T R; Taylor, Alexander B; Pakhomova, Olga N; Zhang, Yanfeng; Somarajan, Sudha R; Galaleldeen, Ahmad; Holloway, Stephen P; Baseman, Joel B; Hart, P John

    2015-04-21

    Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.

  2. 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.

  3. Mitochondrial targeted β-lapachone induces mitochondrial dysfunction and catastrophic vacuolization in cancer cells.

    Science.gov (United States)

    Ma, Jing; Lim, Chaemin; Sacher, Joshua R; Van Houten, Bennett; Qian, Wei; Wipf, Peter

    2015-11-01

    Mitochondria play important roles in tumor cell physiology and survival by providing energy and metabolites for proliferation and metastasis. As part of their oncogenic status, cancer cells frequently produce increased levels of mitochondrial-generated reactive oxygen species (ROS). However, extensive stimulation of ROS generation in mitochondria has been shown to be able to induce cancer cell death, and is one of the major mechanisms of action of many anticancer agents. We hypothesized that enhancing mitochondrial ROS generation through direct targeting of a ROS generator into mitochondria will exhibit tumor cell selectivity, as well as high efficacy in inducing cancer cell death. We thus synthesized a mitochondrial targeted version of β-lapachone (XJB-Lapachone) based on our XJB mitochondrial targeting platform. We found that the mitochondrial targeted β-lapachone is more efficient in inducing apoptosis compared to unconjugated β-lapachone, and the tumor cell selectivity is maintained. XJB-Lapachone also induced extensive cellular vacuolization and autophagy at a concentration not observed with unconjugated β-lapachone. Through characterization of mitochondrial function we revealed that XJB-Lapachone is indeed more capable of stimulating ROS generation in mitochondria, which led to a dramatic mitochondrial uncoupling and autophagic degradation of mitochondria. Taken together, we have demonstrated that targeting β-lapachone accomplishes higher efficacy through inducing ROS generation directly in mitochondria, resulting in extensive mitochondrial and cellular damage. XJB-Lapachone will thus help to establish a novel platform for the design of next generation mitochondrial targeted ROS generators for cancer therapy.

  4. Evolutionary conservation of sequence elements controlling cytoplasmic polyadenylylation.

    OpenAIRE

    1996-01-01

    Cytoplasmic polyadenylylation is an evolutionarily conserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilizati...

  5. Lung transplantation for severe antineutrophilic cytoplasmic antibody-associated vasculitis.

    Science.gov (United States)

    Weinkauf, J; Puttagunta, L; Stewart, K; Humar, A; Homik, J; Caldwell, S; Fenton, M; Nador, R; Lien, Dale

    2010-09-01

    Antineutrophil cytoplasmic antibody-associated vasculitis is a life-threatening disorder for which medical therapy has greatly improved survival. However, there is still significant mortality associated with antineutrophil cytoplasmic antibody-associated vasculitis. Little data exists on the utility of lung transplantation for patients, especially with an acute and severe form of this disease. Herein, we report successful lung transplantation for a patient with life-threatening pulmonary hemorrhage and respiratory failure as a consequence of this pulmonary renal syndrome.

  6. Rimmed vacuoles with beta-amyloid and ubiquitinated filamentous deposits in the muscles of patients with long-standing denervation (postpoliomyelitis muscular atrophy): similarities with inclusion body myositis.

    Science.gov (United States)

    Semino-Mora, C; Dalakas, M C

    1998-10-01

    In the chronically denervated muscles of patients with prior paralytic poliomyelitis, there are secondary myopathic features, including endomysial inflammation and rare vacuolated fibers. To assess the frequency and characteristics of the vacuoles and their similarities with those seen in inclusion body myositis (IBM), we examined 58 muscle biopsy specimens from patients with prior paralytic poliomyelitis for (1) the presence of rimmed vacuoles; (2) acid-phosphatase reactivity; (3) Congo-red-positive amyloid deposits; (4) electron microscopy, searching for tubulofilaments; and (5) immunoelectron microscopy, using antibodies against beta-amyloid and ubiquitin. We found vacuolated muscle fibers in 18 of 58 (31%) biopsies, with a mean frequency of 2.06 +/- 0.42 fibers per specimen. The vacuoles contained acid phosphatase-positive material in 6 of the 18 (33.30%) specimens and stained positive for Congo red in five (27.80%). By immunoelectron microscopy, the vacuoles contained 5.17 +/- 0.13 nm fibrils and 14.9 +/- 0.31 nm filaments that immunoreacted with antibodies to beta-amyloid and ubiquitin in a pattern identical to the one seen in IBM. We conclude that vacuolated muscle fibers containing filamentous inclusions positive for amyloid and ubiquitin are not unique to IBM and the other vacuolar myopathies but can also occur in a chronic neurogenic condition, such as postpoliomyelitis. The chronicity of the underlying disease, rather than the cause, may lead to vacuolar formation, amyloid deposition, and accumulation of ubiquitinated filaments.

  7. Expression of 87 kD protein in the broth culture filtrate of Helicobacter pylori and its association with the vacuolating effect

    Institute of Scientific and Technical Information of China (English)

    SHI Li; YIE Gui-an; NAN Qing-zhen; SUN Yong; ZHANG Ya-li; ZHANG Zhen-shu; ZHOU Dian-yuan

    2001-01-01

    To study the vacuolating effect of Helicobacter pylori(H.pylori). Method: The vacuolating effect and its relationship with vacuolating cytotoxin antigen (an 87 kD protein) were investigated by the method of cytotoxic test, SDS-PAGE and scanning. Result: Of the 62 clinical isolates, 43 strains were H.pylori (Toxin+) with vacuolating effect, while the others were H.pylori (Toxin-) without vacuolating effect. Altogether 78.26%(36/46) patients with peptic ulcer were infected with H.pylori (Toxin+) strains, and only 42.86%(6/14) who had gastritis were infected with H.pylori (Toxin+) strains, with significant difference between them(χ2=4.83,P<0.05). A protein with relativemolecular mass of 87 kD was identified in the broth culture filter(BCF) of 30.23% H. Pylori (Toxin+) strains (13/43) but in none of that of H.pylori (Toxin-) strains, and the difference was statistically significant(P<0.05). There was a significant and concordant relationship between the OD value of the protein band and the titer of vacuolating activity of H.pylori (Toxin+) (r=0.67 and P<0.05 by linear regression analysis). Conclusion: H.pylori (Toxin+) were more often associated with peptic ulcerous diseases than with gastritis diseases. The vacuolating effect of H.pylori (Toxin+) may be caused by the 87 kD protein.

  8. An ABC transporter B family protein, ABCB19, is required for cytoplasmic streaming and gravitropism of the inflorescence stems.

    Science.gov (United States)

    Okamoto, Keishi; Ueda, Haruko; Shimada, Tomoo; Tamura, Kentaro; Koumoto, Yasuko; Tasaka, Masao; Morita, Miyo Terao; Hara-Nishimura, Ikuko

    2016-01-01

    A significant feature of plant cells is the extensive motility of organelles and the cytosol, which was originally defined as cytoplasmic streaming. We suggested previously that a three-way interaction between plant-specific motor proteins myosin XIs, actin filaments, and the endoplasmic reticulum (ER) was responsible for cytoplasmic streaming. (1) Currently, however, there are no reports of molecular components for cytoplasmic streaming other than the actin-myosin-cytoskeleton and ER-related proteins. In the present study, we found that elongated cells of inflorescence stems of Arabidopsis thaliana exhibit vigorous cytoplasmic streaming. Statistical analysis showed that the maximal velocity of plastid movements is 7.26 µm/s, which is much faster than the previously reported velocities of organelles. Surprisingly, the maximal velocity of streaming in the inflorescence stem cells was significantly reduced to 1.11 µm/s in an Arabidopsis mutant, abcb19-101, which lacks ATP BINDING CASSETTE SUBFAMILY B19 (ABCB19) that mediates the polar transport of the phytohormone auxin together with PIN-FORMED (PIN) proteins. Polar auxin transport establishes the auxin concentration gradient essential for plant development and tropisms. Deficiency of ABCB19 activity eventually caused enhanced gravitropic responses of the inflorescence stems and abnormally flexed inflorescence stems. These results suggest that ABCB19-mediated auxin transport plays a role not only in tropism regulation, but also in cytoplasmic streaming.

  9. The Cytoplasmic Permeation Pathway of Neurotransmitter Transporters†

    OpenAIRE

    Rudnick, Gary

    2011-01-01

    Ion-coupled solute transporters are responsible for transporting nutrients, ions and signaling molecules across a variety of biological membranes. Recent high-resolution crystal structures of several transporters from protein families that were previously thought to be unrelated show common structural features indicating a large structural family representing transporters from all kingdoms of life. This review describes studies that led to an understanding of the conformational changes requir...

  10. In Vivo Biotinylation of the Toxoplasma Parasitophorous Vacuole Reveals Novel Dense Granule Proteins Important for Parasite Growth and Pathogenesis

    Science.gov (United States)

    Nadipuram, Santhosh M.; Kim, Elliot W.; Vashisht, Ajay A.; Lin, Andrew H.; Bell, Hannah N.; Coppens, Isabelle; Wohlschlegel, James A.

    2016-01-01

    ABSTRACT Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated. In this report, we utilize the promiscuous biotin ligase BirA* to biotinylate GRA proteins secreted into the vacuole and then identify those proteins by affinity purification and mass spectrometry. Using GRA-BirA* fusion proteins as bait, we have identified a large number of known and candidate GRAs and verified localization of 13 novel GRA proteins by endogenous gene tagging. We proceeded to functionally characterize three related GRAs from this group (GRA38, GRA39, and GRA40) by gene knockout. While Δgra38 and Δgra40 parasites showed no altered phenotype, disruption of GRA39 results in slow-growing parasites that contain striking lipid deposits in the parasitophorous vacuole, suggesting a role in lipid regulation that is important for parasite growth. In addition, parasites lacking GRA39 showed dramatically reduced virulence and a lower tissue cyst burden in vivo. Together, the findings from this work reveal a partial vacuolar proteome of T. gondii and identify a novel GRA that plays a key role in parasite replication and pathogenesis. PMID:27486190

  11. Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration.

    Science.gov (United States)

    Saera-Vila, Alfonso; Kish, Phillip E; Louie, Ke'ale W; Grzegorski, Steven J; Klionsky, Daniel J; Kahana, Alon

    2016-10-02

    Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity.

  12. Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR.

    Science.gov (United States)

    Kanno, Yuichiro; Suzuki, Motoyoshi; Nakahama, Takayuki; Inouye, Yoshio

    2005-09-10

    The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

  13. Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion

    DEFF Research Database (Denmark)

    Ungermann, C; von Mollard, G F; Jensen, Ole Nørregaard

    1999-01-01

    Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion...... cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p....

  14. Flow-induced channel formation in the cytoplasm of motile cells

    Science.gov (United States)

    Guy, Robert D.; Nakagaki, Toshiyuki; Wright, Grady B.

    2011-07-01

    A model is presented to explain the development of flow channels within the cytoplasm of the plasmodium of the giant amoeba Physarum polycephalum. The formation of channels is related to the development of a self-organizing tubular network in large cells. Experiments indicate that the flow of cytoplasm is involved in the development and organization of these networks, and the mathematical model proposed here is motivated by recent experiments involving the observation of development of flow channel in small cells. A model of pressure-driven flow through a polymer network is presented in which the rate of flow increases the rate of depolymerization. Numerical solutions and asymptotic analysis of the model in one spatial dimension show that under very general assumptions this model predicts the formation of channels in response to flow.

  15. Interactions between Salmonella typhimurium and Acanthamoeba polyphaga, and observation of a new mode of intracellular growth within contractile vacuoles.

    Science.gov (United States)

    Gaze, W H; Burroughs, N; Gallagher, M P; Wellington, E M H

    2003-10-01

    Acanthamoeba polyphaga feeding on Salmonella typhimurium in a simple model biofilm were observed by light microscopy and a detailed record of interactions kept by digital image capture and image analysis. A strain of S. typhimurium SL1344 carrying a fis: gfp reporter construct (pPDT105) was used to assess intracellular growth in A. polyphaga on non-nutrient agar (NNA) plates. Invasion of the contractile vacuole (CV) was observed at a frequency of 1:100-1000 acanthamoebae at 35 degrees C. The salmonellae contained in CVs illustrated significant up-regulation of fis relative to extracellular bacteria, indicating that they were in the early stages of logarithmic growth, and reached numbers of 100-200 cells per vacuole after 4 days. This is the first report of this mode of intracellular growth. Up-regulation of fis was also observed in a proportion of S. typhimurium cells contained within food vacuoles. Filamentation of S. typhimurium and E. coli cells was frequently observed in coculture with A. polyphaga on NNA plates, with bacterial cells reaching lengths of up to 500 microm after 10 days' incubation at 35 degrees C. A. polyphaga was also seen to mediate bacterial translocation over the agar surface; egested salmonellae subsequently formed microcolonies along amoebal tracks. This illustrated intracellular survival of a fraction of the S. typhimurium population. These phenomena suggest that protozoa such as A. polyhaga may play an important role in the ecology of S. typhimurium in soil and aquatic environments.

  16. Further characterization of particulate fractions from lysed cell envelopes of Halobacterium halobium and isolation of gas vacuole membranes.

    Science.gov (United States)

    Toeckenius, W; Kunau, W H

    1968-08-01

    Lysates of cell envelopes from Halobacterium halobium have been separated into four fractions. A soluble, colorless fraction (I) containing protein, hexosamines, and no lipid is apparently derived from the cell wall. A red fraction (II), containing approximately 40 per cent lipid, 60 per cent protein, and a small amount of hexosamines consists of cell membrane disaggregated into fragments of small size. A third fraction (III) of purple color consists of large membrane sheets and has a very similar composition to II, containing the same classes of lipids but no hexosamines; its buoyant density is 1.18 g/ml. The fourth fraction (IV) has a buoyant density of 1.23 g/ml and contains the "intracytoplasmic membranes." These consist mainly of protein, and no lipid can be extracted with chloroform-methanol. Fractions I and II, which result from disaggregation of cell wall and cell membrane during lysis, contain a high proportion of dicarboxyl amino acids; this is in good agreement with the assumption that disruption of the cell envelope upon removal of salt is due to the high charge density. The intracytoplasmic membranes (IV) represent the gas vacuole membranes in the collapsed state. In a number of mutants that have lost the ability to form gas vacuoles, no vacuole membranes or any structure that could be related to them has been found.

  17. Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, enlarge the parasite's food vacuole and alter drug sensitivities.

    Science.gov (United States)

    Pulcini, Serena; Staines, Henry M; Lee, Andrew H; Shafik, Sarah H; Bouyer, Guillaume; Moore, Catherine M; Daley, Daniel A; Hoke, Matthew J; Altenhofen, Lindsey M; Painter, Heather J; Mu, Jianbing; Ferguson, David J P; Llinás, Manuel; Martin, Rowena E; Fidock, David A; Cooper, Roland A; Krishna, Sanjeev

    2015-01-01

    Mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, are the major determinant of chloroquine resistance in this lethal human malaria parasite. Here, we describe P. falciparum lines subjected to selection by amantadine or blasticidin that carry PfCRT mutations (C101F or L272F), causing the development of enlarged food vacuoles. These parasites also have increased sensitivity to chloroquine and some other quinoline antimalarials, but exhibit no or minimal change in sensitivity to artemisinins, when compared with parental strains. A transgenic parasite line expressing the L272F variant of PfCRT confirmed this increased chloroquine sensitivity and enlarged food vacuole phenotype. Furthermore, the introduction of the C101F or L272F mutation into a chloroquine-resistant variant of PfCRT reduced the ability of this protein to transport chloroquine by approximately 93 and 82%, respectively, when expressed in Xenopus oocytes. These data provide, at least in part, a mechanistic explanation for the increased sensitivity of the mutant parasite lines to chloroquine. Taken together, these findings provide new insights into PfCRT function and PfCRT-mediated drug resistance, as well as the food vacuole, which is an important target of many antimalarial drugs.

  18. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Directory of Open Access Journals (Sweden)

    Noriaki Kadohama

    Full Text Available It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg' and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  19. Sudden collapse of vacuoles in Saintpaulia sp. palisade cells induced by a rapid temperature decrease.

    Science.gov (United States)

    Kadohama, Noriaki; Goh, Tatsuaki; Ohnishi, Miwa; Fukaki, Hidehiro; Mimura, Tetsuro; Suzuki, Yoshihiro

    2013-01-01

    It is well known that saintpaulia leaf is damaged by the rapid temperature decrease when cold water is irrigated onto the leaf surface. We investigated this temperature sensitivity and the mechanisms of leaf damage in saintpaulia (Saintpaulia sp. cv. 'Iceberg') and other Gesneriaceae plants. Saintpaulia leaves were damaged and discolored when subjected to a rapid decrease in temperature, but not when the temperature was decreased gradually. Sensitivity to rapid temperature decrease increased within 10 to 20 min during pre-incubation at higher temperature. Injury was restricted to the palisade mesophyll cells, where there was an obvious change in the color of the chloroplasts. During a rapid temperature decrease, chlorophyll fluorescence monitored by a pulse amplitude modulated fluorometer diminished and did not recover even after rewarming to the initial temperature. Isolated chloroplasts were not directly affected by the rapid temperature decrease. Intracellular pH was monitored with a pH-dependent fluorescent dye. In palisade mesophyll cells damaged by rapid temperature decrease, the cytosolic pH decreased and the vacuolar membrane collapsed soon after a temperature decrease. In isolated chloroplasts, chlorophyll fluorescence declined when the pH of the medium was lowered. These results suggest that a rapid temperature decrease directly or indirectly affects the vacuolar membrane, resulting in a pH change in the cytosol that subsequently affects the chloroplasts in palisade mesophyll cells. We further confirmed that the same physiological damage occurs in other Gesneriaceae plants. These results strongly suggested that the vacuoles of palisade mesophyll cells collapsed during the initial phase of leaf injury.

  20. Light-induced morphological alteration in anthocyanin-accumulating vacuoles of maize cells

    Directory of Open Access Journals (Sweden)

    Grotewold Erich

    2005-05-01

    Full Text Available Abstract Background Plant pigmentation is affected by a variety of factors. Light, an important plant developmental signal, influences the accumulation of anthocyanins primarily through the activation of the transcription factors that regulate the flavonoid biosynthetic pathway. In this study, we utilized maize Black Mexican Sweet (BMS cells expressing the R and C1 regulators of anthocyanin biosynthesis from a light-insensitive promoter as a means to investigate the existence of additional levels of control of pigmentation by light. Results BMS cells expressing the R and C1 regulators from the CaMV 35S constitutive promoter accumulate anthocyanins when grown in complete darkness, suggesting that the transcription factors R and C1 are sufficient for the transcription of the genes corresponding to the structural enzymes of the pathway, with no requirement for additional light-induced regulators. Interestingly, light induces a "darkening" in the color of the purple anthocyanin pigmentation of transgenic BMS cells expressing R and C1. This change in the pigment hue is not associated with a variation in the levels or types of anthocyanins present, or with an alteration of the transcript levels of several flavonoid biosynthetic genes. However, cytological observations show that light drives unexpected changes in the morphology and distribution of the anthocyanins-containing vacuolar compartments. Conclusion By uncoupling the effect of light on anthocyanin accumulation, we have found light to induce the fusion of anthocyanin-containing vacuoles, the coalescence of anthocyanic vacuolar inclusion (AVI-like structures contained, and the spread of anthocyanins from the inclusions into the vacuolar sap. Similar light-induced alterations in vacuolar morphology are also evident in the epidermal cells of maize floral whorls accumulating anthocyanins. Our findings suggest a novel mechanism for the action of light on the vacuolar storage of anthocyanin.

  1. Trypanosoma cruzi: Entry into Mammalian Host Cells and Parasitophorous Vacuole Formation

    Science.gov (United States)

    Barrias, Emile Santos; de Carvalho, Tecia Maria Ulisses; De Souza, Wanderley

    2013-01-01

    Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted to vertebrate hosts by blood-sucking insects. This protozoan is an obligate intracellular parasite. The infective forms of the parasite are the metacyclic trypomastigotes, amastigotes, and bloodstream trypomastigotes. The recognition between the parasite and mammalian host cell, involves numerous molecules present in both cell types, and similar to several intracellular pathogens, T. cruzi is internalized by host cells via multiple endocytic pathways. Morphological studies demonstrated that after the interaction of the infective forms of T. cruzi with phagocytic or non-phagocytic cell types, plasma membrane (PM) protrusions can form, showing similarity with those observed during canonical phagocytosis or macropinocytic events. Additionally, several molecules known to be molecular markers of membrane rafts, macropinocytosis, and phagocytosis have been demonstrated to be present at the invasion site. These events may or may not depend on the host cell lysosomes and cytoskeleton. In addition, after penetration, components of the host endosomal-lysosomal system, such as early endosomes, late endosomes, and lysosomes, participate in the formation of the nascent parasitophorous vacuole (PV). Dynamin, a molecule involved in vesicle formation, has been shown to be involved in the PV release from the host cell PM. This review focuses on the multiple pathways that T. cruzi can use to enter the host cells until complete PV formation. We will describe different endocytic processes, such as phagocytosis, macropinocytosis, and endocytosis using membrane microdomains and clathrin-dependent endocytosis and show results that are consistent with their use by this smart parasite. We will also discuss others mechanisms that have been described, such as active penetration and the process that takes advantage of cell membrane wound repair. PMID:23914186

  2. Enzymatic Characterization of Recombinant Food Vacuole Plasmepsin 4 from the Rodent Malaria Parasite Plasmodium berghei.

    Directory of Open Access Journals (Sweden)

    Peng Liu

    Full Text Available The rodent malaria parasite Plasmodium berghei is a practical model organism for experimental studies of human malaria. Plasmepsins are a class of aspartic proteinase isoforms that exert multiple pathological effects in malaria parasites. Plasmepsins residing in the food vacuole (FV of the parasite hydrolyze hemoglobin in red blood cells. In this study, we cloned PbPM4, the FV plasmepsin gene of P. berghei that encoded an N-terminally truncated pro-segment and the mature enzyme from genomic DNA. We over-expressed this PbPM4 zymogen as inclusion bodies (IB in Escherichia coli, and purified the protein following in vitro IB refolding. Auto-maturation of the PbPM4 zymogen to mature enzyme was carried out at pH 4.5, 5.0, and 5.5. Interestingly, we found that the PbPM4 zymogen exhibited catalytic activity regardless of the presence of the pro-segment. We determined the optimal catalytic conditions for PbPM4 and studied enzyme kinetics on substrates and inhibitors of aspartic proteinases. Using combinatorial chemistry-based peptide libraries, we studied the active site preferences of PbPM4 at subsites S1, S2, S3, S1', S2' and S3'. Based on these results, we designed and synthesized a selective peptidomimetic compound and tested its inhibition of PbPM4, seven FV plasmepsins from human malaria parasites, and human cathepsin D (hcatD. We showed that this compound exhibited a >10-fold selectivity to PbPM4 and human malaria parasite plasmepsin 4 orthologs versus hcatD. Data from this study furthesr our understanding of enzymatic characteristics of the plasmepsin family and provides leads for anti-malarial drug design.

  3. Evidence for a cytoplasmic microprocessor of pri-miRNAs.

    Science.gov (United States)

    Shapiro, Jillian S; Langlois, Ryan A; Pham, Alissa M; Tenoever, Benjamin R

    2012-07-01

    microRNAs (miRNAs) represent a class of noncoding RNAs that fine-tune gene expression through post-transcriptional silencing. While miRNA biogenesis occurs in a stepwise fashion, initiated by the nuclear microprocessor, rare noncanonical miRNAs have also been identified. Here we characterize the molecular components and unique attributes associated with the processing of virus-derived cytoplasmic primary miRNAs (c-pri-miRNAs). RNA in situ hybridization and inhibition of cellular division demonstrated a complete lack of nuclear involvement in c-pri-miRNA cleavage while genetic studies revealed that maturation still relied on the canonical nuclear RNase III enzyme, Drosha. The involvement of Drosha was mediated by a dramatic relocalization to the cytoplasm following virus infection. Deep sequencing analyses revealed that the cytoplasmic localization of Drosha does not impact the endogenous miRNA landscape during infection, despite allowing for robust synthesis of virus-derived miRNAs in the cytoplasm. Taken together, this research describes a unique function for Drosha in the processing of highly structured cytoplasmic RNAs in the context of virus infection.

  4. Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects

    Energy Technology Data Exchange (ETDEWEB)

    Rao, A.K.; Kowalska, M.A.; Disa, J. (Temple Univ. School of Medicine, Philadelphia, PA (USA))

    1989-08-01

    Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and {sup 14}C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, (Ca++)i, was normal; however, peak (Ca++)i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak (Ca++)i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.

  5. Thymic Nurse Cells Exhibit Epithelial Progenitor Phenotype and Create Unique Extra-Cytoplasmic Membrane Space for Thymocyte Selection

    Science.gov (United States)

    Hendrix, Tonya M.; Chilukuri, Rajendra V.E.; Martinez, Marcia; Olushoga, Zachariah; Blake, Andrew; Brohi, Moazzam; Walker, Christopher; Samms, Michael; Guyden, Jerry C.

    2010-01-01

    Thymic nurse cells (TNCs) are epithelial cells in the thymic cortex that contain as many as fifty thymocytes within specialized cytoplasmic vacuoles. The function of this cell-in-cell interaction has created controversy since their discovery in 1980. Further, some skepticism exists about the idea that apoptotic thymocytes within the TNC complex result from negative selection, a process believed to occur exclusively within the medulla. In this report, we have microscopic evidence that defines a unique membranous environment wherein lipid raft aggregates around the αβTCR expressed on captured thymocytes and class II MHC molecules expressed on TNCs. Further, immunohistological examination of thymic sections show TNCs located within the cortico-medullary junction to express cytokeratins five and eight (K5 and K8), and the transcription factor Trp-63, the phenotype defined elsewhere as the thymic epithelial progenitor subset. Our results suggest that the microenvironment provided by TNCs plays an important role in thymocyte selection as well as the potential for TNCs to be involved in the maintenance of thymic epithelia. PMID:20035931

  6. Cytoplasmic expression of C-MYC protein is associated with risk stratification of mantle cell lymphoma

    Directory of Open Access Journals (Sweden)

    Yi Gong

    2017-06-01

    increased positive cytoplasmic expression of C-MYC protein and decreased CD8+TIL appeared to be associated with a poor response to chemotherapy, but the correlation was not statistically significant. Conclusion Our study suggested that assessment of cytoplasmic C-MYC overexpression and cytotoxic T lymphocytes (CTLs by immunohistochemical staining might be helpful for MCL risk stratification and outcome prediction. However, large cohort studies of MCL patients with complete follow up are needed to validate our speculation.

  7. Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

    Science.gov (United States)

    Kintner, C

    1992-04-17

    Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of cell adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytoplasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytoplasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types.

  8. Evolutionary conservation of sequence elements controlling cytoplasmic polyadenylylation.

    Science.gov (United States)

    Verrotti, A C; Thompson, S R; Wreden, C; Strickland, S; Wickens, M

    1996-08-20

    Cytoplasmic polyadenylylation is an evolutionarily conserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilization development. These experiments demonstrate that the sequence elements that govern cytoplasmic polyadenylylation, and hence one form of translational activation, function across species. We infer that the requisite regulatory sequence elements, and likely the trans-acting components with which they interact, have been conserved since the divergence of vertebrates and arthropods.

  9. Cytoplasmic RNA: a case of the tail wagging the dog.

    Science.gov (United States)

    Norbury, Chris J

    2013-10-01

    The addition of poly(A) tails to eukaryotic nuclear mRNAs promotes their stability, export to the cytoplasm and translation. Subsequently, the balance between exonucleolytic deadenylation and selective re-establishment of translation-competent poly(A) tails by cytoplasmic poly(A) polymerases is essential for the appropriate regulation of gene expression from oocytes to neurons. In recent years, surprising roles for cytoplasmic poly(A) polymerase-related enzymes that add uridylyl, rather than adenylyl, residues to RNA 3' ends have also emerged. These terminal uridylyl transferases promote the turnover of certain mRNAs but also modify microRNAs, their precursors and other small RNAs to modulate their stability or biological functions.

  10. Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins.

    Science.gov (United States)

    Kim, Minsoo; Carman, Christopher V; Springer, Timothy A

    2003-09-19

    Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the alpha and beta cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane.

  11. Colicin S8 export: extracellular and cytoplasmic colicin are different.

    Science.gov (United States)

    Garcia Diaz, Maria-Elena; Concepción Curbelo, Juan Luis

    2003-12-01

    The properties of colicin S8 are different for the cytoplasmic, periplasmic and extracellular protein. Interactions with its specific receptors reflect this. Active cell extracts separate into a non-anionic along with an anionic fraction by DEAE-Sephacell chromatography. Previously, we have purified cell-associated colicin S8 as an aggregation of highly related polypeptides; cytoplasmic colicin S8 seems to be post-translationally processed into an aggregation of polypeptides of molecular mass ranging from 45,000 Da to 60,000 Da. We suggest that a conformational change to colicin S8 may occur related to the export process.

  12. Doppler OCT imaging of cytoplasm shuttle flow in Physarum polycephalum.

    Science.gov (United States)

    Bykov, Alexander V; Priezzhev, Alexander V; Lauri, Janne; Myllylä, Risto

    2009-09-01

    The Doppler optical coherence tomography technique was applied to image the oscillatory dynamics of protoplasm in the strands of the plasmodium of slime mould Physarum polycephalum. Radial contractions of the gel-like walls of the strands and the velocity distributions in the sol-like endoplasm streaming along the plasmodial strands are imaged. The motility inhibitor effect of carbon dioxide on the cytoplasm shuttle flow and strand-wall contraction is shown. The optical attenuation coefficient of cytoplasm is estimated. 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  13. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji, E-mail: tanaka-kj@igakuken.or.jp; Saeki, Yasushi, E-mail: saeki-ys@igakuken.or.jp

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

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

    Directory of Open Access Journals (Sweden)

    Anton R Dluzewski

    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.

  15. Histopathological comparison of Kearns-Sayre syndrome and PGC-1α-deficient mice suggests a novel concept for vacuole formation in mitochondrial encephalopathy.

    Science.gov (United States)

    Szalardy, L; Molnar, M; Torok, R; Zadori, D; Vecsei, L; Klivenyi, P; Liberski, P; Kovacs, G G

    2016-01-01

    Despite the current hypotheses about myelinic and astrocytic ion-dyshomeostasis underlying white (WM) and grey matter (GM) vacuolation in mitochondrial encephalopathies, there is a paucity of data on the exact mechanism of vacuole formation. To revisit the concepts of vacuole formation associated with mitochondrial dysfunction, we performed a comparative neuropathological analysis in Kearns-Sayre syndrome (KSS) and full-length peroxisome proliferator-activated receptor-g coactivator-1a (FL-PGC-1a)-deficient mice, a recently proposed morphological model of mitochondrial encephalopathies. Brain tissues from an individual with genetically proven KSS (22-year-old man) and aged FL-PGC-1a-deficient and wild-type (male, 70-75-week-old) mice were analysed using ultrastructural and immunohistochemical methods, with a specific focus on myelin-related, oligodendroglial, axonal and astrocytic pathologies. Besides demonstrating remarkable similarities in the lesion profile of KSS and FL-PGC-1a-deficient mice, this study first provides morphological evidence for the identical origin of WM and GM vacuolation as well as for the presence of intracytoplasmic oligodendroglial vacuoles in mitochondriopathies. Based on these observations, the paper proposes a theoretical model for the development of focal myelin vacuolation as opposed to the original concepts of intramyelin oedema. Placing oligodendrocytes in the centre of tissue lesioning in conditions related to defects in mitochondria, our observations support the rationale for cytoprotective targeting of oligodendrocytes in mitochondrial encephalopathies, and may also have implications in brain aging and multiple sclerosis, as discussed.

  16. Analysis of embryo, cytoplasm and maternal effects on fatty acid components in soybean (Glycine max Merill.)

    Institute of Scientific and Technical Information of China (English)

    NING Hailong; LI Wenxia; LI Wenbin

    2007-01-01

    The quality of oil determined by the constituents and proportion of fatty acid components,and the understanding of heredity of fatty acid components are of importance to breeding good quality soybean varieties.Embryo,cytoplasmic and maternal effects and genotype×environment interaction effects for quality traits of soybean [Glycine max (L.) Merrill.] seeds were analyzed using a general genetic model for quantitative traits of seeds with parents,F1 and F2,of 20 crosses from a diallel mating design of five parents planted in the field in 2003 and 2004 in Harbin,China.The interaction effects of palmitic,stearic,and linoleic acid contents were larger than the genetic main effects,while the genetic main effects were equal to interaction effects for linolenic and oleic acid content.Among all kinds of genetic main effects,the embryo effects were the largest for palmitic,stearic,and linoleic acids,while the cytoplasm effects were the largest for oleic and linolenic acids.Among all kinds of interaction effects,the embryo interaction effects were the largest for fatty acids.The sum of additive and additive× environment effects were larger than that of dominance and dominance×environment effects for the linolenic acid content,but not for other quality traits.The general heritabilities were the main parts of heritabilities for palmitic and oleic acid contents,but the interaction was more important for stearic,linoleic,and linolenic acid contents.For the general heritability,maternal and cytoplasm heritabilities were the main components for palmitic,oleic,and linolenic acid contents.It was shown for the interaction heritabilities that the embryo interaction heritabilities were more important for oleic and linolenic acid contents,while the maternal interaction heritabilities were more important for linoleic acid content.Among selection response components,the maternal and cytoplasm general responses and/or interaction responses were more important for palmitic

  17. Cytoplasmic translocation, aggregation, and cleavage of TDP-43 by enteroviral proteases modulate viral pathogenesis

    Science.gov (United States)

    Fung, G; Shi, J; Deng, H; Hou, J; Wang, C; Hong, A; Zhang, J; Jia, W; Luo, H

    2015-01-01

    We have previously demonstrated that infection by coxsackievirus B3 (CVB3), a positive-stranded RNA enterovirus, results in the accumulation of insoluble ubiquitin–protein aggregates, which resembles the common feature of neurodegenerative diseases. The importance of protein aggregation in viral pathogenesis has been recognized; however, the underlying regulatory mechanisms remain ill-defined. Transactive response DNA-binding protein-43 (TDP-43) is an RNA-binding protein that has an essential role in regulating RNA metabolism at multiple levels. Cleavage and cytoplasmic aggregation of TDP-43 serves as a major molecular marker for amyotrophic lateral sclerosis and frontotemporal lobar degeneration and contributes significantly to disease progression. In this study, we reported that TDP-43 is translocated from the nucleus to the cytoplasm during CVB3 infection through the activity of viral protease 2A, followed by the cleavage mediated by viral protease 3C. Cytoplasmic translocation of TDP-43 is accompanied by reduced solubility and increased formation of protein aggregates. The cleavage takes place at amino-acid 327 between glutamine and alanine, resulting in the generation of an N- and C-terminal cleavage fragment of ~35 and ~8 kDa, respectively. The C-terminal product of TDP-43 is unstable and quickly degraded through the proteasome degradation pathway, whereas the N-terminal truncation of TDP-43 acts as a dominant-negative mutant that inhibits the function of native TDP-43 in alternative RNA splicing. Lastly, we demonstrated that knockdown of TDP-43 results in an increase in viral titers, suggesting a protective role for TDP-43 in CVB3 infection. Taken together, our findings suggest a novel model by which cytoplasmic redistribution and cleavage of TDP-43 as a consequence of CVB3 infection disrupts the solubility and transcriptional activity of TDP-43. Our results also reveal a mechanism evolved by enteroviruses to support efficient viral infection. PMID

  18. Reprogramming of round spermatids by the germinal vesicle cytoplasm in mice.

    Directory of Open Access Journals (Sweden)

    Peng-Cheng Kong

    Full Text Available The birthrate following round spermatid injection (ROSI remains low in current and evidence suggests that factors in the germinal vesicle (GV cytoplasm and certain substances in the GV such as the nucleolus might be responsible for genomic reprogramming and embryonic development. However, little is known whether the reprogramming factors in GV oocyte cytoplasm and/or nucleolus in GV are beneficial to the reprogramming of round spermatids and development of ROSI embryos. Here, round spermatids were treated with GV cytolysates and injected this round spermatid alone or co-injected with GV oocyte nucleolus into mature metaphase II oocytes. Subsequent embryonic development was assessed morphologically and by Oct4 expression in blastocysts. There was no significant difference between experimental groups at the zygote to four-cell development stages. Blastocysts derived from oocytes which were injected with cytolysate treated-round spermatid alone or co-injected with nucleoli injection yielded 63.6% and 70.3% high quality embryos, respectively; comparable to blastocysts derived by intracytoplasmic sperm injection (ICSI, but higher than these oocytes which were co-injected with lysis buffer-treated round spermatids and nucleoli or injected with the lysis buffer-treated round spermatids alone. Furthermore, the proportion of live offspring resulting from oocytes which were co-injected with cytolysate treated-round spermatids and nucleoli or injected with cytolysate treated-round spermatids alone was higher than those were injected with lysis buffer treated-round spermaids, but comparable with the ICSI group. Our results demonstrate that factors from the GV cytoplasm improve round spermatid reprogramming, and while injection of the extra nucleolus does not obviously improve reprogramming its potential contribution, although which cannot be definitively excluded. Thus, some reprogramming factors are evidently present in GV oocyte cytoplasm and could

  19. The transmission of cytoplasmic genes in Aspergillus nidulans.

    NARCIS (Netherlands)

    Coenen, A.

    1997-01-01

    IntroductionThis manuscript concerns the spread of selfish cytoplasmic genes in the fungus Aspergillus nidulans. A.nidulans is a common soil fungus that grows vegetatively by forming a network (mycelium) of hyphae and reproduces via sexual ascospores and asexual conidiospores. Cytop

  20. Optomechatronic System For Automated Intra Cytoplasmic Sperm Injection*

    Directory of Open Access Journals (Sweden)

    Shulev Assen

    2015-12-01

    Full Text Available This paper presents a complex optomechatronic system for In-Vitro Fertilization (IVF, offering almost complete automation of the Intra Cytoplasmic Sperm Injection (ICSI procedure. The compound parts and sub-systems, as well as some of the computer vision algorithms, are described below. System capabilities for ICSI have been demonstrated on infertile oocyte cells.

  1. The transmission of cytoplasmic genes in Aspergillus nidulans

    NARCIS (Netherlands)

    Coenen, A.

    1997-01-01


    Introduction

    This manuscript concerns the spread of selfish cytoplasmic genes in the fungus Aspergillus nidulans. A.nidulans is a common soil fungus that grows vegetatively by forming a network (mycelium) of hyphae and reproduces

  2. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming.

    Science.gov (United States)

    Kimura, Kenji; Mamane, Alexandre; Sasaki, Tohru; Sato, Kohta; Takagi, Jun; Niwayama, Ritsuya; Hufnagel, Lars; Shimamoto, Yuta; Joanny, Jean-François; Uchida, Seiichi; Kimura, Akatsuki

    2017-04-01

    Cytoplasmic streaming refers to a collective movement of cytoplasm observed in many cell types. The mechanism of meiotic cytoplasmic streaming (MeiCS) in Caenorhabditis elegans zygotes is puzzling as the direction of the flow is not predefined by cell polarity and occasionally reverses. Here, we demonstrate that the endoplasmic reticulum (ER) network structure is required for the collective flow. Using a combination of RNAi, microscopy and image processing of C. elegans zygotes, we devise a theoretical model, which reproduces and predicts the emergence and reversal of the flow. We propose a positive-feedback mechanism, where a local flow generated along a microtubule is transmitted to neighbouring regions through the ER. This, in turn, aligns microtubules over a broader area to self-organize the collective flow. The proposed model could be applicable to various cytoplasmic streaming phenomena in the absence of predefined polarity. The increased mobility of cortical granules by MeiCS correlates with the efficient exocytosis of the granules to protect the zygotes from osmotic and mechanical stresses.

  3. Animal models of antineutrophil cytoplasm antibody-associated vasculitis.

    LENUS (Irish Health Repository)

    Salama, Alan D

    2012-01-01

    To provide an update on the experimental models that have been developed recapitulating clinical antineutrophil cytoplasm antibody (ANCA) associated vasculitis. The application of the models in the study of pathogenesis, and the therapeutic implications of this, are covered in the article by van Timmeren and Heeringa in this issue.

  4. Nuclear proteins hijacked by mammalian cytoplasmic plus strand RNA viruses

    Energy Technology Data Exchange (ETDEWEB)

    Lloyd, Richard E., E-mail: rlloyd@bcm.edu

    2015-05-15

    Plus strand RNA viruses that replicate in the cytoplasm face challenges in supporting the numerous biosynthetic functions required for replication and propagation. Most of these viruses are genetically simple and rely heavily on co-opting cellular proteins, particularly cellular RNA-binding proteins, into new roles for support of virus infection at the level of virus-specific translation, and building RNA replication complexes. In the course of infectious cycles many nuclear-cytoplasmic shuttling proteins of mostly nuclear distribution are detained in the cytoplasm by viruses and re-purposed for their own gain. Many mammalian viruses hijack a common group of the same factors. This review summarizes recent gains in our knowledge of how cytoplasmic RNA viruses use these co-opted host nuclear factors in new functional roles supporting virus translation and virus RNA replication and common themes employed between different virus groups. - Highlights: • Nuclear shuttling host proteins are commonly hijacked by RNA viruses to support replication. • A limited group of ubiquitous RNA binding proteins are commonly hijacked by a broad range of viruses. • Key virus proteins alter roles of RNA binding proteins in different stages of virus replication.

  5. Method for Confirming Cytoplasmic Delivery of RNA Aptamers

    Science.gov (United States)

    Dickey, David D; Dassie, Justin P; Giangrande, Paloma H

    2016-01-01

    RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity. PMID:26472453

  6. [Sexual reproduction of insects is regulated by cytoplasmic bacteria].

    Science.gov (United States)

    Markov, A V; Zakharov, I A

    2005-01-01

    The effects have been considered that the intracellular symbiotic alpha-proteobacteria Wolbachia pipientis induces in its hosts, such as insects and other arthropods: cytoplasmic incompatibility upon mating, feminization, parthenogenesis, and androcide. Specific features of the bacterium genome and possible mechanisms of its action on hosts are discussed.

  7. Experimental Analysis of Cell Function Using Cytoplasmic Streaming

    Science.gov (United States)

    Janssens, Peter; Waldhuber, Megan

    2012-01-01

    This laboratory exercise investigates the phenomenon of cytoplasmic streaming in the fresh water alga "Nitella". Students use the fungal toxin cytochalasin D, an inhibitor of actin polymerization, to investigate the mechanism of streaming. Students use simple statistical methods to analyze their data. Typical student data are provided. (Contains 3…

  8. Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain

    DEFF Research Database (Denmark)

    Oh, E S; Couchman, J R; Woods, A

    1997-01-01

    Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus seque...

  9. Pulp tissue vacuolization and necrosis after direct pulp capping with calcium hydroxide and transforming growth factor-β1

    Directory of Open Access Journals (Sweden)

    Sri Kunarti

    2008-03-01

    Full Text Available Mechanical pulp exposure by a rotary cutting instrument or a hand-cutting instrument often happens in deep caries. Application of protective dressing can protect the pulp from additional injury by facilitating healing and repair. Pulp capping has been suggested as one treatment of choice after pulp exposure to maintain pulp vitality. TGF-β1 is growth factor that has important rule in wound healing. The application of Ca(OH2 and exogenous TGF-β1 as direct pulp capping tr4eatment must be experimented in-vivo to see the vacuolization and necrosis in 7, 14, and 21 days after application. This research was done in vivo experiment from orthodontic patients indicated for premolar extraction, between ages 10–15 years. A class V cavity preparation was created in the buccal aspect 1 mm above gingival margin until pulp exposure. Cavity was irrigated slowly with saline solution and dried with a sterile small cotton pellet. Group 1 calcium hydroxide was applied as manufacture procedure. Group 2, the sterile absorbable collagen membrane used, as inert carrier of TGF-β1 was soaked with 5 ml. All groups were covered by a Teflon pledge to separate pulp capping agent from glass ionomer cement restoration. Teeth extracted in 7, 14 and 21 days after treatment. All samples were hystopathologically examined. There were significant difference of TGF-β1 (p < 0.05 in the vacuolization day 14th and 21th compared with 7th. there were not significant difference in necrosis for all variables. Vacuolization and necrosis decreased in the application of TGF-β1.

  10. Effect of Trimethyltin Chloride on Slow Vacuolar (SV) Channels in Vacuoles from Red Beet (Beta vulgaris L.) Taproots.

    Science.gov (United States)

    Trela, Zenon; Burdach, Zbigniew; Siemieniuk, Agnieszka; Przestalski, Stanisław; Karcz, Waldemar

    2015-01-01

    In the present study, patch-clamp techniques have been used to investigate the effect of trimethyltin chloride (Met3SnCl) on the slow vacuolar (SV) channels in vacuoles from red beet (Beta vulgaris L.) taproots. Activity of SV channels has been measured in whole-vacuole and cytosolic side-out patch configurations. It was found that addition of trimethyltin chloride to the bath solution suppressed, in a concentration-dependent manner, SV currents in red beet vacuoles. The time constant, τ, increased significantly in the presence of the organotin. When single channel activity was analyzed, only little channel activity could be recorded at 100 μM Met3SnCl. Trimethyltin chloride added to the bath medium significantly decreased (by ca. threefold at 100 μM Met3SnCl and at 100 mV voltage, as compared to the control medium) the open probability of single channels. Single channel recordings obtained in the presence and absence of trimethyltin chloride showed that the organotin only slightly (by <10%) decreased the unitary conductance of single channels. It was also found that Met3SnCl significantly diminished the number of SV channel openings, whereas it did not change the opening times of the channels. Taking into account the above and the fact that under the here applied experimental conditions (pH = 7.5) Met3SnCl is a non-dissociated (more lipophilic) compound, we suggest that the suppression of SV currents observed in the presence of the organotin results probably from its hydrophobic properties allowing this compound to translocate near the selectivity filter of the channel.

  11. Analysis of a β-helical region in the p55 domain of Helicobacter pylori vacuolating toxin

    Directory of Open Access Journals (Sweden)

    Algood Holly

    2010-02-01

    Full Text Available Abstract Background Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and contributes to the development of gastric cancer and peptic ulcer disease. VacA, a toxin secreted by H. pylori, is comprised of two domains, designated p33 and p55. Analysis of the crystal structure of the p55 domain indicated that its structure is predominantly a right-handed parallel β-helix, which is a characteristic of autotransporter passenger domains. Substitution mutations of specific amino acids within the p33 domain abrogate VacA activity, but thus far, it has been difficult to identify small inactivating mutations within the p55 domain. Therefore, we hypothesized that large portions of the p55 domain might be non-essential for vacuolating toxin activity. To test this hypothesis, we introduced eight deletion mutations (each corresponding to a single coil within a β-helical segment spanning VacA amino acids 433-628 into the H. pylori chromosomal vacA gene. Results All eight of the mutant VacA proteins were expressed by the corresponding H. pylori mutant strains and underwent proteolytic processing to yield ~85 kDa passenger domains. Three mutant proteins (VacA Δ484-504, Δ511-536, and Δ517-544 were secreted and induced vacuolation of mammalian cells, which indicated that these β-helical coils were dispensable for vacuolating toxin activity. One mutant protein (VacA Δ433-461 exhibited reduced vacuolating toxin activity compared to wild-type VacA. Other mutant proteins, including those containing deletions near the carboxy-terminal end of the β-helical region (amino acids Val559-Asn628, exhibited marked defects in secretion and increased susceptibility to proteolytic cleavage by trypsin, which suggested that these proteins were misfolded. Conclusions These results indicate that within the β-helical segment of the VacA p55 domain, there are regions of plasticity that tolerate alterations without detrimental effects on protein

  12. Cytoplasmic location of α1A voltage-gated calcium channel C-terminal fragment (Cav2.1-CTF aggregate is sufficient to cause cell death.

    Directory of Open Access Journals (Sweden)

    Makoto Takahashi

    Full Text Available The human α1A voltage-dependent calcium channel (Cav2.1 is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C-tail contains a small poly-glutamine (Q tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6. A recent study has shown that a 75-kDa C-terminal fragment (CTF containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (rCTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12 cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range than with Q13 (normal-length. Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB and phosphorylated-CREB (p-CREB in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Cav2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.

  13. Mechanodelivery of nanoparticles to the cytoplasm of living cells

    Science.gov (United States)

    Emerson, Nyssa T.; Hsia, Chih-Hao; Rafalska-Metcalf, Ilona U.; Yang, Haw

    2014-04-01

    Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials.Nanotechnology has opened up the opportunity to probe, sense, and manipulate the chemical environment of biological systems with an unprecedented level of spatiotemporal control. A major obstacle to the full realization of these novel technologies is the lack of a general, robust, and simple method for the delivery of arbitrary nanostructures to the cytoplasm of intact live cells. Here, we identify a new delivery modality, based on mechanical disruption of the plasma membrane, which efficiently mediates the delivery of nanoparticles to the cytoplasm of mammalian cells. We use two distinct execution modes, two adherent cell lines, and three sizes of semiconducting nanocrystals, or quantum dots, to demonstrate its applicability and effectiveness. As the underlying mechanism is purely physical, we anticipate that such ``mechanodelivery'' can be generalized to other modes of execution as well as to the cytoplasmic introduction of a structurally diverse array of functional nanomaterials

  14. Listeriolysin O Membrane Damaging Activity Involves Arc Formation and Lineaction -- Implication for Listeria monocytogenes Escape from Phagocytic Vacuole.

    Directory of Open Access Journals (Sweden)

    Yi Ruan

    2016-04-01

    Full Text Available Listeriolysin-O (LLO plays a crucial role during infection by Listeria monocytogenes. It enables escape of bacteria from phagocytic vacuole, which is the basis for its spread to other cells and tissues. It is not clear how LLO acts at phagosomal membranes to allow bacterial escape. The mechanism of action of LLO remains poorly understood, probably due to unavailability of suitable experimental tools that could monitor LLO membrane disruptive activity in real time. Here, we used high-speed atomic force microscopy (HS-AFM featuring high spatio-temporal resolution on model membranes and optical microscopy on giant unilamellar vesicles (GUVs to investigate LLO activity. We analyze the assembly kinetics of toxin oligomers, the prepore-to-pore transition dynamics and the membrane disruption in real time. We reveal that LLO toxin efficiency and mode of action as a membrane-disrupting agent varies strongly depending on the membrane cholesterol concentration and the environmental pH. We discovered that LLO is able to form arc pores as well as damage lipid membranes as a lineactant, and this leads to large-scale membrane defects. These results altogether provide a mechanistic basis of how large-scale membrane disruption leads to release of Listeria from the phagocytic vacuole in the cellular context.

  15. Effects of the disaggregation of high-polymerized particles in guard cell vacuoles on osmoregulation of stomatal aperture (stomata opening)

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Observation under an electron microscope reveals that in closed and open stomata of V. faba, the average volume of particles in guard cell vacuoles (GCV) reduces about 3 orders in magnitude, while the distribution density of the particles increases about 2 orders of magnitude. By using the method of the ratio of fluorescent emissions with laser scanning confocal microscopy, the monitoring to stomata opening shows that during 10 to 30 s before the first distinguishable aperture of stomata, there is a change of pH in GCV about-0.5 units. A quick stomatal opening immediately follows the changes of pH in GCV to reach a steady aperture about 12μm in 100-200 s. This work proposes a model for the osmoregulation in GCV for stomatal opening. The proposed osmoregulation is related to the disaggregation of some polymerized particles inside GCV, which is probably induced by a -(pH in the vacuole. This model describes a process of osmoregulation that avoids the massive energy consuming transportation across cell membranes, which is a foundation of the current chemiosmotic hypothesis. This model is a supplement to the multiple controlling hypothesis for the stomatal movement, which widens research principle ideas for other quick movements in plants.

  16. Ubiquilin/Dsk2 promotes inclusion body formation and vacuole (lysosome)-mediated disposal of mutated huntingtin.

    Science.gov (United States)

    Chuang, Kun-Han; Liang, Fengshan; Higgins, Ryan; Wang, Yanchang

    2016-07-01

    Ubiquilin proteins contain a ubiquitin-like domain (UBL) and ubiquitin-associated domain(s) that interact with the proteasome and ubiquitinated substrates, respectively. Previous work established the link between ubiquilin mutations and neurodegenerative diseases, but the function of ubiquilin proteins remains elusive. Here we used a misfolded huntingtin exon I containing a 103-polyglutamine expansion (Htt103QP) as a model substrate for the functional study of ubiquilin proteins. We found that yeast ubiquilin mutant (dsk2Δ) is sensitive to Htt103QP overexpression and has a defect in the formation of Htt103QP inclusion bodies. Our evidence further suggests that the UBL domain of Dsk2 is critical for inclusion body formation. Of interest, Dsk2 is dispensable for Htt103QP degradation when Htt103QP is induced for a short time before noticeable inclusion body formation. However, when the inclusion body forms after a long Htt103QP induction, Dsk2 is required for efficient Htt103QP clearance, as well as for autophagy-dependent delivery of Htt103QP into vacuoles (lysosomes). Therefore our data indicate that Dsk2 facilitates vacuole-mediated clearance of misfolded proteins by promoting inclusion body formation. Of importance, the defect of inclusion body formation in dsk2 mutants can be rescued by human ubiquilin 1 or 2, suggesting functional conservation of ubiquilin proteins.

  17. Virulence-related Mycobacterium avium subsp hominissuis MAV_2928 gene is associated with vacuole remodeling in macrophages

    Directory of Open Access Journals (Sweden)

    Vogt Steven

    2010-04-01

    Full Text Available Abstract Background Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928 homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion. Results MAV_2928 gene is primarily upregulated upon phagocytosis. The transcriptional profile of macrophages infected with the wild-type bacterium and the mutant were examined using DNA microarray, which showed that the two bacteria interact uniquely with mononuclear phagocytes. Based on the results, it was hypothesized that the phagosome environment and vacuole membrane of the wild-type bacterium might differ from the mutant. Wild-type bacterium phagosomes expressed a number of proteins different from those infected with the mutant. Proteins on the phagosomes were confirmed by fluorescence microscopy and Western blot. The environment in the phagosome of macrophages infected with the mutant differed from the environment of vacuoles with M. hominissuis wild-type in the concentration of zinc, manganese, calcium and potassium. Conclusion The results suggest that the MAV_2928 gene/operon might participate in the establishment of bacterial intracellular environment in macrophages.

  18. Genetic analysis of the cytoplasmic dynein subunit families.

    Directory of Open Access Journals (Sweden)

    K Kevin Pfister

    2006-01-01

    Full Text Available Cytoplasmic dyneins, the principal microtubule minus-end-directed motor proteins of the cell, are involved in many essential cellular processes. The major form of this enzyme is a complex of at least six protein subunits, and in mammals all but one of the subunits are encoded by at least two genes. Here we review current knowledge concerning the subunits, their interactions, and their functional roles as derived from biochemical and genetic analyses. We also carried out extensive database searches to look for new genes and to clarify anomalies in the databases. Our analysis documents evolutionary relationships among the dynein subunits of mammals and other model organisms, and sheds new light on the role of this diverse group of proteins, highlighting the existence of two cytoplasmic dynein complexes with distinct cellular roles.

  19. MRP2, a human conjugate export pump, is present and transports fluo 3 into apical vacuoles of Hep G2 cells.

    Science.gov (United States)

    Cantz, T; Nies, A T; Brom, M; Hofmann, A F; Keppler, D

    2000-04-01

    The multidrug resistance protein 2 (MRP2, symbol ABCC2) transports anionic conjugates and certain amphiphilic anions across the apical membrane of polarized cells. Human hepatoma Hep G2 cells retain hepatic polarity and form apical vacuoles into which cholephilic substances are secreted. Immunofluorescence microscopy showed that human MRP2 was expressed in the apical vacuole membrane of polarized Hep G2 cells, whereas the isoform MRP3 was localized to the lateral membrane. Expression of both MRP2 and MRP3 was confirmed by immunoblotting and reverse transcription PCR. Fluo 3 secretion into the apical vacuoles was inhibited by cyclosporin A but not by selective inhibitors of multidrug resistance 1 P-glycoprotein. In addition, carboxyfluorescein, rhodamine 123, and the fluorescent bile salt derivatives ursodeoxycholyl-(Nepsilon-nitrobenzoxadiazolyl)-lysine and cholylglycylamido-fluorescein were secreted into the apical vacuoles; the latter two probably via the bile salt export pump. We conclude that MRP2 mediates fluo 3 secretion into the apical vacuoles of polarized Hep G2 cells. Thus the function of human MRP2 and the action of inhibitors can be analyzed by the secretion of fluorescent anions such as fluo 3.

  20. NOX2-Mediated TFEB Activation and Vacuolization Regulate Lysosome-Associated Cell Death Induced by Gypenoside L, a Saponin Isolated from Gynostemma pentaphyllum.

    Science.gov (United States)

    Zheng, Kai; Jiang, Yingchun; Liao, Chenghui; Hu, Xiaopeng; Li, Yan; Zeng, Yong; Zhang, Jian; Wu, Xuli; Wu, Haiqiang; Liu, Lizhong; Wang, Yifei; He, Zhendan

    2017-08-09

    Downregulation of apoptotic signal pathway and activation of protective autophagy mainly contribute to the chemoresistance of tumor cells. Therefore, exploring efficient chemotherapeutic agents or isolating novel natural products that can trigger nonapoptotic and nonautophagic cell death such as lysosome-associated death is emergently required. We have recently extracted a saponin, gypenoside L (Gyp-L), from Gynostemma pentaphyllum and showed that Gyp-L was able to induce nonapoptotic cell death of esophageal cancer cells associated with lysosome swelling. However, contributions of vacuolization and lysosome to cell death remain unclear. Herein, we reveal a critical role for NADPH oxidase NOX2-mediated vacuolization and transcription factor EB (TFEB) activation in lysosome-associated cell death. We found that Gyp-L initially induced the abnormal enlarged and alkalized vacuoles, which were derived from lipid rafts dependent endocytosis. Besides, NOX2 was activated to promote vacuolization and mTORC1-independent TFEB-mediated lysosome biogenesis. Finally, raising lysosome pH could enhance Gyp-L induced cell death. These findings suggest a protective role of NOX2-TFEB-mediated lysosome biogenesis in cancer drug resistance and the tight interaction between lipid rafts and vacuolization. In addition, Gyp-L can be utilized as an alternative option to overcome drug-resistance though inducing lysosome associated cell death.

  1. Influenza a virus assembly intermediates fuse in the cytoplasm.

    Directory of Open Access Journals (Sweden)

    Seema S Lakdawala

    2014-03-01

    Full Text Available Reassortment of influenza viral RNA (vRNA segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA protein (WSN PA-GFP to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.

  2. Nucleoporin Nup98 mediates galectin-3 nuclear-cytoplasmic trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Funasaka, Tatsuyoshi, E-mail: funasaka@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Balan, Vitaly; Raz, Avraham [Department of Oncology and Pathology, Karmanos Cancer Institute, Wayne State University, School of Medicine, Detroit, MI (United States); Wong, Richard W., E-mail: rwong@staff.kanazawa-u.ac.jp [Laboratory of Molecular and Cellular Biology, Department of Biology, Faculty of Natural Systems, Institute of Science and Engineering, Kanazawa University, Ishikawa (Japan); Bio-AFM Frontier Research Center, Kanazawa Kanazawa University, Ishikawa (Japan)

    2013-04-26

    Highlights: •Nuclear pore protein Nup98 is a novel binding partner of galectin-3. •Nup98 transports galectin-3 into cytoplasm. •Nup98 depletion leads to galectin-3 nuclear transport and induces growth retardation. •Nup98 may involve in ß-catenin pathway through interaction with galectin-3. -- Abstract: Nucleoporin Nup98 is a component of the nuclear pore complex, and is important in transport across the nuclear pore. Many studies implicate nucleoporin in cancer progression, but no direct mechanistic studies of its effect in cancer have been reported. We show here that Nup98 specifically regulates nucleus–cytoplasm transport of galectin-3, which is a ß-galactoside-binding protein that affects adhesion, migration, and cancer progression, and controls cell growth through the ß-catenin signaling pathway in cancer cells. Nup98 interacted with galectin-3 on the nuclear membrane, and promoted galectin-3 cytoplasmic translocation whereas other nucleoporins did not show these functions. Inversely, silencing of Nup98 expression by siRNA technique localized galectin-3 to the nucleus and retarded cell growth, which was rescued by Nup98 transfection. In addition, Nup98 RNA interference significantly suppressed downstream mRNA expression in the ß-catenin pathway, such as cyclin D1 and FRA-1, while nuclear galectin-3 binds to ß-catenin to inhibit transcriptional activity. Reduced expression of ß-catenin target genes is consistent with the Nup98 reduction and the galectin-3–nucleus translocation rate. Overall, the results show Nup98’s involvement in nuclear–cytoplasm translocation of galectin-3 and ß-catenin signaling pathway in regulating cell proliferation, and the results depicted here suggest a novel therapeutic target/modality for cancers.

  3. Interaction of the growth hormone receptor cytoplasmic domain with the JAK2 tyrosine kinase.

    Science.gov (United States)

    Frank, S J; Gilliland, G; Kraft, A S; Arnold, C S

    1994-11-01

    An early step in GH action involves tyrosine phosphorylation of various cellular proteins. Recently, it has been shown in murine preadipocytes that GH promotes the association of its receptor (the GHR) with and the activation of the JAK2 tyrosine kinase. In this study, we confirmed the human (h) GH-induced association of JAK2 with hGHR in IM-9 cells by coimmunoprecipitation experiments using anti-hGHR serum. We further examined the interaction of JAK2 with the GHR cytoplasmic domain by two lines of investigation. For in vitro studies, we assayed by immunoblotting the ability of cell-derived JAK2 to interact with glutathione S-transferase fusion proteins containing elements of the hGHR cytoplasmic domain. A fusion protein containing the entire hGHR cytoplasmic domain (residues 271-620) specifically associated with JAK2 independent of prior stimulation of cells with hGH. This interaction was not dependent on tyrosine phosphorylation of either partner. Mutational analysis of the hGHR cytoplasmic domain component of the fusions indicated that a membrane-proximal 20-residue region that includes the proline-rich box 1 was necessary for the interaction. This region appeared to cooperate with another region(s), largely in the N-terminal one third of the cytoplasmic domain, to promote full interaction with JAK2. For in vivo reconstitution experiments, wild-type (WT) and mutant rabbit GHRs (rGHRs) along with murine JAK2 were expressed by transient transfection in COS-7 cells. rGHR mutations were confined to the cytoplasmic domain and included C-terminal truncations as well as internal deletions of residues 297-406 and 278-292 (the latter contains box 1). All mutant rGHRs were expressed at the cell surface and bound hGH to a degree similar to the WT rGHR. Receptors were tested for their ability to mediate the hGH-induced immunoprecipitability of JAK2 with phosphotyrosine (APT) antibodies. A rGHR truncated to residue 275 [rGHR-(1-275)], which contains only five cytoplasmic

  4. THE KINETICS OF CYTOPLASMIC GRANULE SECRETION IN NATURAL KILLER CYTOTOXICITY

    Institute of Scientific and Technical Information of China (English)

    龚伊红; R.R.Hcrberman; C.W.Reynolds

    1994-01-01

    Antisexum against purified cytoplasmic granules from rat LGL tumor cells, and protein A-gold inmmnoelec-tron microscopy were used to study the secretory events in lysis of YAC-1 tumor cells by rat LGL tumor cells or by isolated LGL from normal rats. After 30 min incubation of effector and target cells together, gold-labeled cyto-plasmic granules were often seen concentrated in the area of the LGL adjacent to the ~ YAC-1 Within 60min,the grantees were observed to move to the cell border near the conjugazed site. At this point, fine granules were fused with file cell membrane, and subsequently released file gold-labeled contents into the junction between the LGL and the target cell. Gold particles could be seen at the B-T interface, on the surface,or sometimes on the target cell surface.These data provide direct evidence for the hypothesis that under conditions of active cytotoxicity,natural killer cells secrete their cytoplasmic granule contents leading to the deposition of granule material on the target cell surface and the eventual lysis of the cell.

  5. Genetic studies on cytoplasmic male sterility in maize

    Energy Technology Data Exchange (ETDEWEB)

    Laughnan, J.R.

    1992-01-01

    Our research concerns the basic mechanisms of cytoplasmic male sterility (CMS) and fertility restoration in maize. The molecular determination of CMS is in the DNA of the mitochondria (mtDNA) but specific nuclear restorer-of-fertility (Rf) genes can overrule the male-sterile effect of the cytoplasm. Our approach to the study of the Rf genes is threefold. We are attempting to tag the cms-S Rf genes and the cms-T Rf2 gene with controlling elements (CEs). Since we have identified a number of spontaneous Rf genes for cms-S and have demonstrated that they are themselves transposable, we are also searching for cases in which an Rf gene is inserted into a wild-type gene. The other aspect of our research involves the nuclear control over the organization of the mitochondrial genome. We found that the changes in mtDNA organization upon cytoplasmic reversion to fertility were characteristic of the nuclear background in which the reversion event occurred. We have investigated whether these differences are a reflection of differences in the organization of the mtDNA genome before reversion.

  6. Mutant p53 protein localized in the cytoplasm inhibits autophagy.

    Science.gov (United States)

    Morselli, Eugenia; Tasdemir, Ezgi; Maiuri, Maria Chiara; Galluzzi, Lorenzo; Kepp, Oliver; Criollo, Alfredo; Vicencio, José Miguel; Soussi, Thierry; Kroemer, Guido

    2008-10-01

    The knockout, knockdown or chemical inhibition of p53 stimulates autophagy. Moreover, autophagy-inducing stimuli such as nutrient depletion, rapamycin or lithium cause the depletion of cytoplasmic p53, which in turn is required for the induction of autophagy. Here, we show that retransfection of p53(-/-) HCT 116 colon carcinoma cells with wild type p53 decreases autophagy down to baseline levels. Surprisingly, one third among a panel of 22 cancer-associated p53 single amino acid mutants also inhibited autophagy when transfected into p53(-/-) cells. Those variants of p53 that preferentially localize to the cytoplasm effectively repressed autophagy, whereas p53 mutants that display a prominently nuclear distribution failed to inhibit autophagy. The investigation of a series of deletion mutants revealed that removal of the DNA-binding domain from p53 fails to interfere with its role in the regulation of autophagy. Altogether, these results identify the cytoplasmic localization of p53 as the most important feature for p53-mediated autophagy inhibition. Moreover, the structural requirements for the two biological activities of extranuclear p53, namely induction of apoptosis and inhibition of autophagy, are manifestly different.

  7. Sequences determining the cytoplasmic localization of a chemoreceptor domain.

    Science.gov (United States)

    Seligman, L; Bailey, J; Manoil, C

    1995-01-01

    The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure. PMID:7730259

  8. PaCS is a novel cytoplasmic structure containing functional proteasome and inducible by cytokines/trophic factors.

    Directory of Open Access Journals (Sweden)

    Patrizia Sommi

    Full Text Available A variety of ubiquitinated protein-containing cytoplasmic structures has been reported, from aggresomes to aggresome-like induced structures/sequestosomes or particle-rich cytoplasmic structures (PaCSs that we recently observed in some human diseases. Nevertheless, the morphological and cytochemical patterns of the different structures remain largely unknown thus jeopardizing their univocal identification. Here, we show that PaCSs resulted from proteasome and polyubiquitinated protein accumulation into well-demarcated, membrane-free, cytoskeleton-poor areas enriched in glycogen and glycosaminoglycans. A major requirement for PaCS detection by either electron or confocal microscopy was the addition of osmium to aldehyde fixatives. However, by analyzing living cells, we found that proteasome chymotrypsin-like activity concentrated in well-defined cytoplasmic structures identified as PaCSs by ultrastructural morphology and immunocytochemistry of the same cells. PaCSs differed ultrastructurally and cytochemically from sequestosomes which may coexist with PaCSs. In human dendritic or natural killer cells, PaCSs were induced in vitro by cytokines/trophic factors during differentiation/activation from blood progenitors. Our results provide evidence that PaCS is indeed a novel distinctive cytoplasmic structure which may play a critical role in the ubiquitin-proteasome system response to immune, infectious or proneoplastic stimuli.

  9. Holophytochrome-Interacting Proteins in Physcomitrella: Putative Actors in Phytochrome Cytoplasmic Signaling.

    Science.gov (United States)

    Ermert, Anna Lena; Mailliet, Katharina; Hughes, Jon

    2016-01-01

    Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We also used database information to study their expression patterns relative to those of phy4. We discuss the likely functional characteristics of these holophytochrome-interacting proteins (HIP's) and their possible roles in signaling.

  10. Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

    Science.gov (United States)

    Lechuga, Susana; Baranwal, Somesh; Li, Chao; Naydenov, Nayden G; Kuemmerle, John F; Dugina, Vera; Chaponnier, Christine; Ivanov, Andrei I

    2014-10-15

    Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not β-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis.

  11. [Anti-neutrophil cytoplasmic antibodies (ANCA) in patients with symptomatic and asymptomatic HIV infection].

    Science.gov (United States)

    Habegger de Sorrentino, A; Motta, P; Iliovich, E; Sorrentino, A P

    1997-01-01

    The cytopathic effect of HIV on CD4 T cells, as well as the active autoimmune mechanism occurring during infection, have been documented. Of the cytokines involved in the pathogenesis of AIDS, the main one produced by the monocyte-macrophage series is tumor necrosis factor alfa (TNF alpha). This cytokine induces antigens such as proteinase 3 (Pr 3) or mieloperoxidase (MPO). Anti-neutrophil cytoplasmic antibodies (ANCA) are directed against this type of PMN antigens. In the present paper, the role of anti-neutrophil cytoplasmic antibodies (ANCA) in HIV infected patients as responsible for autoimmune phenomena in relation to opportunistic infections, was studied. A total of 88 serum samples belonging to 49 asymptomatic and 39 symptomatic HIV infected patients were tested for ANCA by an indirect immunofluorescence (IIF) test over a neutrophil substrate. ANCA were detected in 53.8% of symptomatic patients as compared to 4.1% in asymptomatic cases (p tuberculosis is a frequent finding in HIV infected patients from Northeastern Argentina. When the presence of ANCA in TB(+) HIV(+) and TB(+) HIV(-) patients was studied, it was seen that positive-ANCA significantly correlated with the first group (p pulmonar TB, could indicate that the virus may not be responsible for the induction of these antibodies.

  12. Holophytochrome-interacting proteins in Physcomitrella: putative actors in phytochrome cytoplasmic signaling

    Directory of Open Access Journals (Sweden)

    Anna Lena eErmert

    2016-05-01

    Full Text Available Phytochromes are the principle photoreceptors in light-regulated plant development, primarily acting via translocation of the light-activated photoreceptor into the nucleus and subsequent gene regulation. However, several independent lines of evidence indicate unambiguously that an additional cytoplasmic signaling mechanism must exist. Directional responses in filament tip cells of the moss Physcomitrella patens are steered by phy4 which has been shown to interact physically with the blue light receptor phototropin at the plasma membrane. This complex might perceive and transduce vectorial information leading to cytoskeleton reorganization and finally a directional growth response. We developed yeast two-hybrid procedures using photochemically-functional, full-length phy4 as bait in Physcomitrella cDNA library screens and growth assays under different light conditions, revealing Pfr-dependent interactions possibly associated with phytochrome cytoplasmic signaling. Candidate proteins were then expressed in planta with fluorescent protein tags to determine their intracellular localization in darkness and red light. Of 14 candidates, 12 were confirmed to interact with phy4 in planta using bimolecular fluorescence complementation. We discuss the roles these putative holophytochrome-interacting proteins (HIP's might have in signaling.

  13. Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non‐neuronal cells

    National Research Council Canada - National Science Library

    Grenier, Catherine; Bissonnette, Cyntia; Volkov, Leonid; Roucou, Xavier

    2006-01-01

    .... The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non‐neuronal cells...

  14. Reactive Oxygen Species (ROS)-Activated ATM-Dependent Phosphorylation of Cytoplasmic Substrates Identified by Large-Scale Phosphoproteomics Screen.

    Science.gov (United States)

    Kozlov, Sergei V; Waardenberg, Ashley J; Engholm-Keller, Kasper; Arthur, Jonathan W; Graham, Mark E; Lavin, Martin

    2016-03-01

    Ataxia-telangiectasia, mutated (ATM) protein plays a central role in phosphorylating a network of proteins in response to DNA damage. These proteins function in signaling pathways designed to maintain the stability of the genome and minimize the risk of disease by controlling cell cycle checkpoints, initiating DNA repair, and regulating gene expression. ATM kinase can be activated by a variety of stimuli, including oxidative stress. Here, we confirmed activation of cytoplasmic ATM by autophosphorylation at multiple sites. Then we employed a global quantitative phosphoproteomics approach to identify cytoplasmic proteins altered in their phosphorylation state in control and ataxia-telangiectasia (A-T) cells in response to oxidative damage. We demonstrated that ATM was activated by oxidative damage in the cytoplasm as well as in the nucleus and identified a total of 9,833 phosphorylation sites, including 6,686 high-confidence sites mapping to 2,536 unique proteins. A total of 62 differentially phosphorylated peptides were identified; of these, 43 were phosphorylated in control but not in A-T cells, and 19 varied in their level of phosphorylation. Motif enrichment analysis of phosphopeptides revealed that consensus ATM serine glutamine sites were overrepresented. When considering phosphorylation events, only observed in control cells (not observed in A-T cells), with predicted ATM sites phosphoSerine/phosphoThreonine glutamine, we narrowed this list to 11 candidate ATM-dependent cytoplasmic proteins. Two of these 11 were previously described as ATM substrates (HMGA1 and UIMCI/RAP80), another five were identified in a whole cell extract phosphoproteomic screens, and the remaining four proteins had not been identified previously in DNA damage response screens. We validated the phosphorylation of three of these proteins (oxidative stress responsive 1 (OSR1), HDGF, and ccdc82) as ATM dependent after H2O2 exposure, and another protein (S100A11) demonstrated ATM

  15. 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.

  16. Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires.

    Science.gov (United States)

    Wood, Rebecca E; Newton, Patrice; Latomanski, Eleanor A; Newton, Hayley J

    2015-10-01

    Legionella organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the Legionella-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of Legionella pneumophila. Here, we have characterized the Legionella longbeachae replicative vacuole (longbeachae-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of L. pneumophila (pneumophila-LCV). We constructed a Dot/Icm-deficient strain by deleting dotB and demonstrated the inability of this mutant to replicate inside THP-1 cells. L. longbeachae does not enter THP-1 cells as efficiently as L. pneumophila, and this is reflected in the observation that translocation of BlaM-RalFLLO (where RalFLLO is the L. longbeachae homologue of RalF) into THP-1 cells by the L. longbeachae Dot/Icm system is less efficient than that by L. pneumophila. This difference is negated in A549 cells where L. longbeachae and L. pneumophila infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the Rab-like effector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the longbeachae-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the longbeachae-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the L. longbeachae replicative vacuole, highlighting phenotypic similarities to the vacuole of L. pneumophila as well as unique aspects of LCV biology.

  17. Histopathological comparison of Kearns-Sayre syndrome and PGC-1α-deficient mice suggests a novel concept for vacuole formation in mitochondrial encephalopathy

    Directory of Open Access Journals (Sweden)

    Levente Szalardy

    2016-03-01

    Full Text Available Despite the current hypotheses about myelinic and astrocytic ion-dyshomeostasis underlying white (WM and grey matter (GM vacuolation in mitochondrial encephalopathies, there is a paucity of data on the exact mechanism of vacuole formation. To revisit the concepts of vacuole formation associated with mitochondrial dysfunction, we performed a comparative neuropathological analysis in Kearns-Sayre syndrome (KSS and full-length peroxisome proliferator-activated receptor-g coactivator-1a (FL-PGC-1a-deficient mice, a recently proposed morphological model of mitochondrial encephalopathies. Brain tissues from an individual with genetically proven KSS (22-year-old man and aged FL-PGC-1a-deficient and wild-type (male, 70-75-week-old mice were analysed using ultrastructural and immunohistochemical methods, with a specific focus on myelin-related, oligodendroglial, axonal and astrocytic pathologies. Besides demonstrating remarkable similarities in the lesion profile of KSS and FL-PGC-1a-deficient mice, this study first provides morphological evidence for the identical origin of WM and GM vacuolation as well as for the presence of intracytoplasmic oligodendroglial vacuoles in mitochondriopathies. Based on these observations, the paper proposes a theoretical model for the development of focal myelin vacuolation as opposed to the original concepts of intramyelin oedema. Placing oligodendrocytes in the centre of tissue lesioning in conditions related to defects in mitochondria, our observations support the rationale for cytoprotective targeting of oligodendrocytes in mitochondrial encephalopathies, and may also have implications in brain aging and multiple sclerosis, as discussed.

  18. Liver-specific Aquaporin 11 knockout mice show rapid vacuolization of the rough endoplasmic reticulum in periportal hepatocytes after feeding amino acids

    DEFF Research Database (Denmark)

    Rojek, Aleksandra; Füchtbauer, Ernst-Martin; Füchtbauer, Annette

    2013-01-01

    of the mice (24 h) induced modest dilatation of the rough endoplasmic reticulum (RER) in the periportal hepatocytes. Refeeding with standard mouse chow induced rapid generation of large RER-derived vacuoles in Aqp11 KO mice hepatocytes. Similar effects were observed following oral administration of pure...... protein or larger doses of various amino acids. The fasting/refeeding challenge is associated with increased expression of markers of ER stress Grp78 and GADD153 and decreased glutathione levels, suggesting that ER stress may play role in the development of vacuoles in the AQP11-deficient hepatocytes. NMR...

  19. Three v-SNAREs and two t-SNAREs, present in a pentameric cis-SNARE complex on isolated vacuoles, are essential for homotypic fusion

    DEFF Research Database (Denmark)

    Ungermann, C; von Mollard, G F; Jensen, Ole Nørregaard

    1999-01-01

    in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles...... cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p....

  20. Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain.

    Science.gov (United States)

    Montiel, Maria-Dolores; Krzewinski-Recchi, Marie-Ange; Delannoy, Philippe; Harduin-Lepers, Anne

    2003-01-01

    The nucleotide sequence of the short and long transcripts of beta1,4- N -acetylgalactosaminyltransferase have been submitted to the DDBJ, EMBL, GenBank(R) and GSDB Nucleotide Sequence Databases under accession nos AJ517770 and AJ517771 respectively. The human Sd(a) antigen is formed through the addition of an N -acetylgalactosamine residue via a beta1,4-linkage to a sub-terminal galactose residue substituted with an alpha2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4- N -acetylgalactosaminyltransferase (Sd(a) beta1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sd(a) beta1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1(S) and E1(L), leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to alpha2,3-sialylated acceptor

  1. Burkholderia cenocepacia type VI secretion system mediates escape of type II secreted proteins into the cytoplasm of infected macrophages.

    Directory of Open Access Journals (Sweden)

    Roberto Rosales-Reyes

    Full Text Available Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS. We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

  2. DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

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

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    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.

  4. 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.

  5. Complementary DNA display selection of high-affinity peptides binding the vacuolating toxin (VacA) of Helicobacter pylori.

    Science.gov (United States)

    Hayakawa, Yumiko; Matsuno, Mitsuhiro; Tanaka, Makoto; Wada, Akihiro; Kitamura, Koichiro; Takei, Osamu; Sasaki, Ryuzo; Mizukami, Tamio; Hasegawa, Makoto

    2015-09-01

    Artificial peptides designed for molecular recognition of a bacterial toxin have been developed. Vacuolating cytotoxin A protein (VacA) is a major virulence factor of Helicobacter pylori, a gram-negative microaerophilic bacterium inhabiting the upper gastrointestinal tract, particularly the stomach. This study attempted to identify specific peptide sequences with high affinity for VacA using systematic directed evolution in vitro, a cDNA display method. A surface plasmon resonance-based biosensor and fluorescence correlation spectroscopy to examine binding of peptides with VacA identified a peptide (GRVNQRL) with high affinity. Cyclization of the peptide by attaching cysteine residues to both termini improved its binding affinity to VacA, with a dissociation constant (Kd ) of 58 nm. This study describes a new strategy for the development of artificial functional peptides, which are promising materials in biochemical analyses and medical applications.

  6. Big Roles of Small Kinases:The Complex Functions of Receptor-Like Cytoplasmic Kinases in Plant Immunity and Development

    Institute of Scientific and Technical Information of China (English)

    Wenwei Lin; Xiyu Ma; Libo Shan; Ping He

    2013-01-01

    Plants have evolved a large number of receptor-like cytoplasmic kinases (RLCKs) that often functionally and physically associate with receptor-like kinases (RLKs) to modulate plant growth, development and immune responses. Without any apparent extracellular domain, RLCKs relay intracellular signaling often via RLK complex-mediated transphosphorylation events. Recent advances have suggested essential roles of diverse RLCKs in concert with RLKs in regulating various cellular and physiological responses. We summarize here the complex roles of RLCKs in mediating plant immune responses and growth regulation, and discuss specific and overlapping functions of RLCKs in transducing diverse signaling pathways.

  7. Vacuolating cytotoxin A (VacA) - A multi-talented pore-forming toxin from Helicobacter pylori.

    Science.gov (United States)

    Junaid, Muhammad; Linn, Aung Khine; Javadi, Mohammad Bagher; Al-Gubare, Sarbast; Ali, Niaz; Katzenmeier, Gerd

    2016-08-01

    Helicobacter pylori is associated with severe and chronic diseases of the stomach and duodenum such as peptic ulcer, non-cardial adenocarcinoma and gastric lymphoma, making Helicobacter pylori the only bacterial pathogen which is known to cause cancer. The worldwide rate of incidence for these diseases is extremely high and it is estimated that about half of the world's population is infected with H. pylori. Among the bacterial virulence factors is the vacuolating cytotoxin A (VacA), which represents an important determinant of pathogenicity. Intensive characterization of VacA over the past years has provided insight into an ample variety of mechanisms contributing to host-pathogen interactions. The toxin is considered as an important target for ongoing research for several reasons: i) VacA displays unique features and structural properties and its mechanism of action is unrelated to any other known bacterial toxin; ii) the toxin is involved in disease progress and colonization by H. pylori of the stomach; iii) VacA is a potential and promising candidate for the inclusion as antigen in a vaccine directed against H. pylori and iv) the vacA gene is characterized by a high allelic diversity, and allelic variants contribute differently to the pathogenicity of H. pylori. Despite the accumulation of substantial data related to VacA over the past years, several aspects of VacA-related activity have been characterized only to a limited extent. The biologically most significant effect of VacA activity on host cells is the formation of membrane pores and the induction of vacuole formation. This review discusses recent findings and advances on structure-function relations of the H. pylori VacA toxin, in particular with a view to membrane channel formation, oligomerization, receptor binding and apoptosis.

  8. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

    Science.gov (United States)

    Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

    2016-04-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

  9. Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

    Science.gov (United States)

    Blodgett, David M.; De Zutter, Julie K.; Levine, Kara B.; Karim, Pusha; Carruthers, Anthony

    2007-01-01

    Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7. PMID:17635959

  10. Corpos de inclusão citoplasmática: estudo em diversas doenças e revisão da literatura Inclusion cytoplasmic bodies: a study in several diseases and a literature review

    Directory of Open Access Journals (Sweden)

    Rosana Herminia Scola

    1996-06-01

    Full Text Available Estudamos 16 casos entre 1400 biópsias musculares que apresentavam vacúolos marginados, cujo aspecto histológico sugeria corpos de inclusão citoplasmáticos. Procuramos correlacionar os dados clínicos, laboratoriais e histopatológicos, a fim de determinar a especificidade dos corpos de inclusão citoplasmáticos para determinadas doenças. A creatinaquinase mostrou-se elevada em 10 casos. A eletromiografia foi anormal em todos os casos. A histoquímica muscular em 5 casos revelou uma miopatia, em 7 padrão misto, em dois desinervação e em 2 casos miopatia inflamatória. A microscopia eletrônica demonstrou a presença de filamentos em 8 casos (nucleares, dispersos no citoplasma ou na região subsarcolemal. Os pacientes foram classificados conforme a história clínica, hereditariedade, dados laboratoriais, eletrofisiológicos, histoquímicos e microscopia eletrônica Encontramos miosite com corpos de inclusão citoplasmática (4 casos, atrofia muscular espinhal juvenil (6 casos, miopatias distais (3 casos, distrofia de cinturas pélvica e escapular (2 casos e polineuropatia periférica (1 caso. Apresentamos revisão sobre a patogenia, formação e possível etiologia dos vacúolos marginados e sua relação com as diversas entidades em que foram detectados, sugerindo que não são específicos para uma única doença.Among 1400 muscle biopsies, we studied 16 cases with rimmed vacuoles, whose histology suggests cytoplasm inclusion bodies. We tried to correlate the clinical, laboratory and histopatological data in order to verify the specificity of cytoplasm inclusion bodies to certain diseases. The creatinekinase was increased in 10 cases. In all cases electromyography was abnormal. Muscle histochemistry revealed myopathy in 5 cases, mixed pattern in 7, denervation in 2 and in 2 cases, inflammatory myopathy. Electron microscopy showed the presence of filaments in 8 cases (nuclear, disseminated in cytoplasm or in the subsarcolemmal region

  11. Symmetrical infantile thalamic degeneration with focal cytoplasmic calcification.

    Science.gov (United States)

    Ambler, M; O'Neil, W

    1975-10-27

    Infantile thalamic degeneration is a rare clinico-pathological entity. Restricted location of the lesion and peculiar cytopathological changes serve to distinguish this disorder from other common encephalopathies. Optical and ultrastructural studies demonstrate cytoplasmic calcopherules in previously viable cells. According to current concepts of acute cellular reactions to injury and mechanism of intracellular calcification, the cytological changes cannot be attributed to either hypoxic ischemic cell change or dystrophic calcification. By analogy to other human and pathological material, the most likely basis for nondystrophic calcopherule formation is toxic or infectious injury with local synthesis, or autophagic or phagolysosomal degradation of cellular debris of specific chemical composition favoring calcium deposition.

  12. The Role of MUC1 Cytoplasmic Domain in Tumorigenesis

    Science.gov (United States)

    2005-05-01

    5 AD Award Number: DAMD17-02-1-0476 TITLE: The Role of MUCI Cytoplasmic Domain in Tumorigenesis PRINCIPAL INVESTIGATOR: Assah Al-Masri CONTRACTING ...The overall aim of the research is to develop a better understanding of the role of MUCI in breast cancer. Loss of Mucl (mouse homologue of MUC1...significant delay in tumor progression that is observed in the absence of Mucl . We suggest that the interaction of Mucl with c-Src, a key player in PyV MT

  13. Expression of a vacuole-localized BURP-domain protein from soybean (SALI3-2 enhances tolerance to cadmium and copper stresses.

    Directory of Open Access Journals (Sweden)

    Yulin Tang

    Full Text Available The plant-specific BURP family proteins play diverse roles in plant development and stress responses, but the function mechanism of these proteins is still poorly understood. Proteins in this family are characterized by a highly conserved BURP domain with four conserved Cys-His repeats and two other Cys, indicating that these proteins potentially interacts with metal ions. In this paper, an immobilized metal affinity chromatography (IMAC assay showed that the soybean BURP protein SALI3-2 could bind soft transition metal ions (Cd(2+, Co(2+, Ni(2+, Zn(2+ and Cu(2+ but not hard metal ions (Ca(2+ and Mg(2+ in vitro. A subcellular localization analysis by confocal laser scanning microscopy revealed that the SALI3-2-GFP fusion protein was localized to the vacuoles. Physiological indexes assay showed that Sali3-2-transgenic Arabidopsis thaliana seedlings were more tolerant to Cu(2+ or Cd(2+ stresses than the wild type. An inductively coupled plasma optical emission spectrometry (ICP-OES analysis illustrated that, compared to the wild type seedlings the Sali3-2-transgenic seedlings accumulated more cadmium or copper in the roots but less in the upper ground tissues when the seedlings were exposed to excessive CuCl2 or CdCl2 stress. Therefore, our findings suggest that the SALI3-2 protein may confer cadmium (Cd(2+ and copper (Cu(2+ tolerance to plants by helping plants to sequester Cd(2+ or Cu(2+ in the root and reduce the amount of heavy metals transported to the shoots.

  14. The neutral lipid composition present in the digestive vacuole of Plasmodium falciparum concentrates heme and mediates β-hematin formation with an unusually low activation energy.

    Science.gov (United States)

    Hoang, Anh N; Sandlin, Rebecca D; Omar, Aneesa; Egan, Timothy J; Wright, David W

    2010-11-30

    In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono- and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets, partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of β-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.

  15. A Small GTPase Activator Protein Interacts with Cytoplasmic Phytochromes in Regulating Root Development*

    Science.gov (United States)

    Shin, Dong Ho; Cho, Man-Ho; Kim, Tae-Lim; Yoo, Jihye; Kim, Jeong-Il; Han, Yun-Jeong; Song, Pill-Soon; Jeon, Jong-Seong; Bhoo, Seong Hee; Hahn, Tae-Ryong

    2010-01-01

    Phytochromes enable plants to sense light information and regulate developmental responses. Phytochromes interact with partner proteins to transmit light signals to downstream components for plant development. PIRF1 (phytochrome-interacting ROP guanine-nucleotide exchange factor (RopGEF 1)) functions as a light-signaling switch regulating root development through the activation of ROPs (Rho-like GTPase of plant) in the cytoplasm. In vitro pulldown and yeast two-hybrid assays confirmed the interaction between PIRF1 and phytochromes. PIRF1 interacted with the N-terminal domain of phytochromes through its conserved PRONE (plant-specific ROP nucleotide exchanger) region. PIRF1 also interacted with ROPs and activated them in a phytochrome-dependent manner. The Pr form of phytochrome A enhanced the RopGEF activity of PIRF1, whereas the Pfr form inhibited it. A bimolecular fluorescence complementation analysis demonstrated that PIRF1 was localized in the cytoplasm and bound to the phytochromes in darkness but not in light. PIRF1 loss of function mutants (pirf1) of Arabidopsis thaliana showed a longer root phenotype in the dark. In addition, both PIRF1 overexpression mutants (PIRF1-OX) and phytochrome-null mutants (phyA-211 and phyB-9) showed retarded root elongation and irregular root hair formation, suggesting that PIRF1 is a negative regulator of phytochrome-mediated primary root development. We propose that phytochrome and ROP signaling are interconnected through PIRF1 in regulating the root growth and development in Arabidopsis. PMID:20551316

  16. Melatonin biosynthesis in plants: multiple pathways catalyze tryptophan to melatonin in the cytoplasm or chloroplasts.

    Science.gov (United States)

    Back, Kyoungwhan; Tan, Dun-Xian; Reiter, Russel J

    2016-11-01

    Melatonin is an animal hormone as well as a signaling molecule in plants. It was first identified in plants in 1995, and almost all enzymes responsible for melatonin biosynthesis had already been characterized in these species. Melatonin biosynthesis from tryptophan requires four-step reactions. However, six genes, that is, TDC, TPH, T5H, SNAT, ASMT, and COMT, have been implicated in the synthesis of melatonin in plants, suggesting the presence of multiple pathways. Two major pathways have been proposed based on the enzyme kinetics: One is the tryptophan/tryptamine/serotonin/N-acetylserotonin/melatonin pathway, which may occur under normal growth conditions; the other is the tryptophan/tryptamine/serotonin/5-methoxytryptamine/melatonin pathway, which may occur when plants produce large amounts of serotonin, for example, upon senescence. The melatonin biosynthetic capacity associated with conversion of tryptophan to serotonin is much higher than that associated with conversion of serotonin to melatonin, which yields a low level of melatonin synthesis in plants. Many melatonin intermediates are produced in various subcellular compartments, such as the cytoplasm, endoplasmic reticulum, and chloroplasts, which either facilitates or impedes the subsequent enzymatic steps. Depending on the pathways, the final subcellular sites of melatonin synthesis vary at either the cytoplasm or chloroplasts, which may differentially affect the mode of action of melatonin in plants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Calcium wave for cytoplasmic streaming of Physarum polycephalum.

    Science.gov (United States)

    Yoshiyama, Shinji; Ishigami, Mitsuo; Nakamura, Akio; Kohama, Kazuhiro

    2009-12-16

    The plasmodium Physarum polycephalum exhibits periodic cycles of cytoplasmic streaming in association with those of contraction and relaxation movement. In the present study, we injected Calcium Green dextran as a fluorescent Ca2+ indicator into the thin-spread living plasmodium. We found changes in the [Ca2+]i (intracellular concentration of Ca2+), which propagated in a wave-like form in its cytoplasm. The Ca2+ waves were also detected when we used Fura dextran which detected [Ca2+]i by the ratio of two wavelengths. We prepared the plasmodial fragment from the thin-spread and found that the cycles of the contraction-relaxation movement was so synchronized that the measurement of its area provided an indication of the movement. We observed that [Ca2+]i also synchronized in the entire fragment and that the relaxation ensued upon the reduction in [Ca2+]i. We suggest that the Ca2+ wave generated periodically is one of the major factors playing a crucial role in the relaxation of P. polycephalum.

  18. Effects of cytoplasmic inheritance on preweaning traits of Hereford cattle

    Directory of Open Access Journals (Sweden)

    Mezzadra Carlos Alberto

    2005-01-01

    Full Text Available The influence of cytoplasmic inheritance on birth and weaning weight was evaluated in an experimental Hereford herd. Data on 1,720 records for birth and weaning weights from calves born between 1963 and 2002 were studied. Variance components were estimated using MTDFREML procedures and an animal model was fitted for each trait. Direct and maternal additive effects and permanent environment and maternal lineage effects were treated as random, while year and month of birth, age of dam and sex of the calf were treated as fixed. Identification of maternal lineages was based on pedigree information. The contribution to phenotypic variance of cytoplasmic lineages defined by pedigree information was negligible for both traits. Mitochondrial genotypes of cows present in the herd in 2002 were analyzed by single strand conformation polymorphism (SSCP analysis. Only five different genotypes were identified among 23 maternal lineages. All the animals with records were assigned to maternal genotypes based on pedigree information. The statistical analysis was repeated, removing maternal lineage from the model and including mitochondrial genotype as a fixed effect. No evidence of genotype effects was detected. These results suggest a negligible effect of the mitochondrial genome on the preweaning traits of this Hereford herd.

  19. Formation of hydrogen sulfide from cysteine in Saccharomyces cerevisiae BY4742: genome wide screen reveals a central role of the vacuole.

    Directory of Open Access Journals (Sweden)

    Gal Winter

    Full Text Available Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S, are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes.

  20. Combination of acid phosphatase positivity and rimmed vacuoles as useful markers in the diagnosis of adult-onset Pompe disease lacking specific clinical and pathological features

    Directory of Open Access Journals (Sweden)

    Claire Dolfus

    2016-10-01

    Full Text Available Introduction: The clinical and histological presentations of the adult form of Pompe disease may be atypical. In such cases, identifying histological signs that point to the diagnosis would be crucial to avoid a delay in care. The aim of our study was to investigate the presence of rimmed vacuoles and acid phosphatase positivity in muscle biopsies of patients with late-onset Pompe disease. Material and methods: We retrospectively studied muscle biopsies of all cases of the adult form of Pompe disease diagnosed at the University Hospital of Caen. Three of these four cases showed atypical clinical signs, and diagnosis was established tardily based on family history or systematic analysis of acid maltase activity. Results: All biopsies showed some rimmed vacuoles. The acid phosphatase reaction showed positive inclusions and labelled vacuoles in biopsies of all patients. Conclusions: The presence of rimmed vacuoles and acid phosphatase positivity in muscle biopsy should suggest the diagnosis of the adult form of Pompe disease, this is decisive since effective therapy is available.

  1. A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2.

    Science.gov (United States)

    Rivera-Serrano, Efraín E; Fritch, Ethan J; Scholl, Elizabeth H; Sherry, Barbara

    2017-04-01

    To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein μ2 from strain T1L, but not strain T3D, represses one antiviral response: alpha/beta interferon signaling. We report here that T1L, but not T3D, μ2 localizes to nuclear speckles, where it forms a complex with the mRNA splicing factor SRSF2 and alters its subnuclear localization. Reovirus replicates in cytoplasmic viral factories, and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L μ2 might target splicing of cell RNAs. Indeed, RNA sequencing revealed that reovirus T1L, but not T3D, infection alters the splicing of transcripts for host genes involved in mRNA posttranscriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L μ2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing.IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. We demonstrate here that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles, where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Our results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus. Copyright © 2017 American Society for Microbiology.

  2. A comparative toxicity study between small and large size zinc oxide nanoparticles in tilapia (Oreochromis niloticus): Organ pathologies, osmoregulatory responses and immunological parameters.

    Science.gov (United States)

    Kaya, Hasan; Aydın, Fatih; Gürkan, Mert; Yılmaz, Sevdan; Ates, Mehmet; Demir, Veysel; Arslan, Zikri

    2016-02-01

    Tilapia (Oreochromis niloticus) was exposed to different sizes of zinc oxide nanoparticles (ZnO-NPs) to evaluate their organ pathologies (kidney, liver, gill, and intestine), osmoregulatory responses and immunological parameters. Sub-chronic exposure was conducted in fresh water with 1 and 10 mg/L concentrations of the small (10-30 nm) and large-size ZnO (100 nm) particles for 7 and 14 days. In this study, it is found that small and large forms of ZnO-NPs cause various pathologic findings in the target organs at all concentrations. These findings are increased of melanomacrophage aggregates, tubular deformations, necrosis and cytoplasmic vacuolations in the kidney, oedema, mononuclear cell infiltrations, fatty changes, pyknotic nuclei and hepatocellular vacuolations in the liver, hyperplasia, aneurysms, and epithelial liftings in the gills, and hyperplasia, swelled of goblet cells, villus deformations in the intestine. Results showed that respiratory burst and potential killing activity at the small-size ZnO concentration significantly increased compared to the control group (p < 0.05) but significant reductions of these parameters at the large-size ZnO concentrations compared to control (p < 0.05) were measured. These findings demonstrate the potential of each particle size to cause significant damage to the immune system. Moreover, because ZnO NPs inhibit the Na(+), K(+)-ATPase activity at all concentrations and increase serum Ca(2+) and Cl(-) levels especially in gill, these particles are osmoregulatory and toxicant for tilapia fish. As a summary, both sizes of the particles have led to organ damage, osmoregulatory changes and immune disorder in tilapia fish.

  3. Cellular Response of the Amoeba Acanthamoeba castellanii to Chlorine, Chlorine Dioxide, and Monochloramine Treatments ▿

    Science.gov (United States)

    Mogoa, Emerancienne; Bodet, Charles; Morel, Franck; Rodier, Marie-Hélène; Legube, Bernard; Héchard, Yann

    2011-01-01

    Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii. PMID:21602398

  4. Cellular response of the amoeba Acanthamoeba castellanii to chlorine, chlorine dioxide, and monochloramine treatments.

    Science.gov (United States)

    Mogoa, Emerancienne; Bodet, Charles; Morel, Franck; Rodier, Marie-Hélène; Legube, Bernard; Héchard, Yann

    2011-07-01

    Acanthamoeba castellanii is a free-living amoebae commonly found in water systems. Free-living amoebae might be pathogenic but are also known to bear phagocytosis-resistant bacteria, protecting these bacteria from water treatments. The mode of action of these treatments is poorly understood, particularly on amoebae. It is important to examine the action of these treatments on amoebae in order to improve them. The cellular response to chlorine, chlorine dioxide, and monochloramine was tested on A. castellanii trophozoites. Doses of disinfectants leading to up to a 3-log reduction were compared by flow cytometry and electron microscopy. Chlorine treatment led to size reduction, permeabilization, and retraction of pseudopods. In addition, treatment with chlorine dioxide led to a vacuolization of the cytoplasm. Monochloramine had a dose-dependent effect. At the highest doses monochloramine treatment resulted in almost no changes in cell size and permeability, as shown by flow cytometry, but the cell surface became smooth and dense, as seen by electron microscopy. We show that these disinfectants globally induced size reduction, membrane permeabilization, and morphological modifications but that they have a different mode of action on A. castellanii.

  5. Constructing and Analyzing Fusion Promoter of Partial Sericin 1 and Bombyx A3 Cytoplasmic Actin

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Previous report showed that the 209 bp DNA sequence upstream of the sericin 1 transcriptional start site (-586 to -378 bp) is involved in promoting transcription and responsible for the tissue specificity of sericin 1 promoter in silkworm Bombyx mori. In the present study, this 209 bp sequence exhibited enhancive effect by assembling in two different locations of ubiquitous Bombyx A3 cytoplasmic actin promoter. Sf-9 cells were transfected with recombinant plasmids using Cellfectin reagent. Firefly luciferase gene located downstream of fusion promoter was considered as a reporter, whereas the activity of the co-transfected Renilla luciferase gene (pGL2-SV40) provides an internal control. This 209 bp region up-regulates the strength of A3 promoter significantly (P < 0.01) when it enters into A3 promoter with respect to the position in sericin 1 gene promoter. This 209-bp fragment was almost functionless when being located upstream of A3 promoter.

  6. Developing Seeds of Arabidopsis Store Different Minerals in Two Types of Vacuoles and in the Endoplasmic Reticulum

    Science.gov (United States)

    Otegui, Marisa S.; Capp, Roberta; Staehelin, L. Andrew

    2002-01-01

    Mineral-accumulating compartments in developing seeds of Arabidopsis were studied using high-pressure-frozen/freeze-substituted samples. Developing seeds store minerals in three locations: in the protein storage vacuoles of the embryo, and transiently in the endoplasmic reticulum (ER) and vacuolar compartments of the chalazal endosperm. Energy dispersive x-ray spectroscopy and enzyme treatments suggest that the minerals are stored as phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) salts in all three compartments, although they differ in cation composition. Whereas embryo globoids contain Mg, K, and Ca as cations, the chalazal ER deposits show high levels of Mn, and the chalazal vacuolar deposits show high levels of Zn. The appearance of the first Zn-phytate crystals coincides with the formation of network-like extensions of the chalazal vacuoles. The core of these networks consists of a branched network of tubular ER membranes, which are separated from the delineating tonoplast membranes by a layer of cytosolic material. Degradation of the networks starts with the loss of the cytosol and is followed by the retraction of the ER, generating a network of collapsed tonoplast membranes that are resorbed. Studies of fertilized fis2 seeds, which hyperaccumulate Zn-phytate crystals in the chalazal vacuolar compartments, suggest that only the intact network is active in mineral sequestration. Mineral determination analysis and structural observations showed that Zn and Mn are mobilized from the endosperm to the embryo at different developmental stages. Thus, Zn appears to be removed from the endosperm at the late globular stage, and Mn stores appear to be removed at the late bent-cotyledon stage of embryo development. The disappearance of the Mn-phytate from the endosperm coincides with the accumulation of two major Mn binding proteins in the embryo, the 33-kD protein from the oxygen-evolving complex of photosystem II and the Mn superoxide dismutase. The possible

  7. Sequestration of p53 in the Cytoplasm by Adenovirus Type 12 E1B 55-Kilodalton Oncoprotein Is Required for Inhibition of p53-Mediated Apoptosis

    OpenAIRE

    2003-01-01

    The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in b...

  8. Cytoplasmic flows as signatures for the mechanics of mitotic positioning

    CERN Document Server

    Nazockdast, Ehssan; Needleman, Daniel; Shelley, Michael

    2015-01-01

    The proper positioning of the mitotic spindle is crucial for asymmetric cell division and generating cell diversity during development. Proper position in the single-cell embryo of Caenorhabditis elegans is achieved initially by the migration and rotation of the pronuclear complex (PNC) and its two associated centrosomal arrays of microtubules (MTs). We present here the first systematic theoretical study of how these $O(1000)$ centrosomal microtubules (MTs) interact through the immersing cytoplasm, the cell periphery and PNC, and with each other, to achieve proper position. This study is made possible through our development of a highly efficient and parallelized computational framework that accounts explicitly for long-ranged hydrodynamic interactions (HIs) between the MTs, while also capturing their flexibility, dynamic instability, and interactions with molecular motors and boundaries. First, we show through direct simulation that previous estimates of the PNC drag coefficient, based on either ignoring or ...

  9. Antineutrophil cytoplasmic antibodies crescentic allograft glomerulonephritis after sofosbuvir therapy

    Science.gov (United States)

    Gadde, Shilpa; Lee, Belinda; Kidd, Laura; Zhang, Rubin

    2016-01-01

    Antineutrophil cytoplasmic antibodies (ANCA) are well known to be associated with several types of vasculitis, including pauci-immune crescentic glomerulonephritis, a form of rapid progressive glomerular nephritis (RPGN). ANCA vasculitis has also been reported after administration of propylthiouracil, hydralazine, cocaine (adulterated with levimasole), allopurinol, penicillamine and few other drugs. All previously reported cases of drug-associated ANCA glomerulonephritis were in native kidneys. Sofosbuvir is a new and effective drug for hepatitis C virus infection. Here, we report a case of ANCA vasculitis and RPGN following sofosbuvir administration in a kidney transplant recipient. It also represents the first case of drug-associated ANCA vasculitis in a transplanted kidney. Further drug monitoring is necessary to elucidate the degree of association and possible causal effect of sofosbuvir and perinuclear ANCA vasculitis. PMID:27872837

  10. Exporting RNA from the nucleus to the cytoplasm.

    Science.gov (United States)

    Köhler, Alwin; Hurt, Ed

    2007-10-01

    The transport of RNA molecules from the nucleus to the cytoplasm is fundamental for gene expression. The different RNA species that are produced in the nucleus are exported through the nuclear pore complexes via mobile export receptors. Small RNAs (such as tRNAs and microRNAs) follow relatively simple export routes by binding directly to export receptors. Large RNAs (such as ribosomal RNAs and mRNAs) assemble into complicated ribonucleoprotein (RNP) particles and recruit their exporters via class-specific adaptor proteins. Export of mRNAs is unique as it is extensively coupled to transcription (in yeast) and splicing (in metazoa). Understanding the mechanisms that connect RNP formation with export is a major challenge in the field.

  11. Nucleo-cytoplasmic transport of proteins and RNA in plants.

    Science.gov (United States)

    Merkle, Thomas

    2011-02-01

    Transport of macromolecules between the nucleus and the cytoplasm is an essential necessity in eukaryotic cells, since the nuclear envelope separates transcription from translation. In the past few years, an increasing number of components of the plant nuclear transport machinery have been characterised. This progress, although far from being completed, confirmed that the general characteristics of nuclear transport are conserved between plants and other organisms. However, plant-specific components were also identified. Interestingly, several mutants in genes encoding components of the plant nuclear transport machinery were investigated, revealing differential sensitivity of plant-specific pathways to impaired nuclear transport. These findings attracted attention towards plant-specific cargoes that are transported over the nuclear envelope, unravelling connections between nuclear transport and components of signalling and developmental pathways. The current state of research in plants is summarised in comparison to yeast and vertebrate systems, and special emphasis is given to plant nuclear transport mutants.

  12. Propylthiouracil-Induced Vasculitis With Antineutrophil Cytoplasmic Antibody.

    Science.gov (United States)

    Criado, Paulo Ricardo; Grizzo Peres Martins, Ana Claudia; Gaviolli, Camila Fatima; Alavi, Afsaneh

    2015-06-01

    Propylthiouracil (PTU)-associated vasculitis is a potentially life-threatening disease with a recent increase in the reported cases in the medical literature. This increase may suggest that some earlier cases have been unrecognized or assigned to an alternative nosology category. Although the skin can be the only organ affected by PTU-associated vasculitis, there are many reports with multiple-system involvement. Classically, the symptoms appear under a tetrad of fever, sore throat, arthralgia, and skin lesions. Cutaneous lesions in reported cases of PTU vasculitis have most commonly consisted of retiform acral, purpuric plaques, or nodules. We report a case of perinuclear antineutrophil cytoplasmic antibody-associated vasculitis developed during treatment with PTU for Grave's disease. © The Author(s) 2014.

  13. Cytoplasmic sulfur trafficking in sulfur-oxidizing prokaryotes.

    Science.gov (United States)

    Dahl, Christiane

    2015-04-01

    Persulfide groups are chemically versatile and participate in a wide array of biochemical pathways. Although it is well documented that persulfurated proteins supply a number of important and elaborate biosynthetic pathways with sulfane sulfur, it is far less acknowledged that the enzymatic generation of persulfidic sulfur, the successive transfer of sulfur as a persulfide between multiple proteins, and the oxidation of sulfane sulfur in protein-bound form are also essential steps during dissimilatory sulfur oxidation in bacteria and archaea. Here, the currently available information on sulfur trafficking in sulfur oxidizing prokaryotes is reviewed, and the idea is discussed that sulfur is always presented to cytoplasmic oxidizing enzymes in a protein-bound form, thus preventing the occurrence of free sulfide inside of the prokaryotic cell. Thus, sulfur trafficking emerges as a central element in sulfur-oxidizing pathways, and TusA homologous proteins appear to be central and common elements in these processes.

  14. Regulation of Cytoplasmic Dynein ATPase by Lis1

    Science.gov (United States)

    Mesngon, Mariano T.; Tarricone, Cataldo; Hebbar, Sachin; Guillotte, Aimee M.; Schmitt, E. William; Lanier, Lorene; Musacchio, Andrea; King, Stephen J.; Smith, Deanna S.

    2015-01-01

    Mutations in Lis1 cause classical lissencephaly, a developmental brain abnormality characterized by defects in neuronal positioning. Over the last decade, a clear link has been forged between Lis1 and the microtubule motor cytoplasmic dynein. Substantial evidence indicates that Lis1 functions in a highly conserved pathway with dynein to regulate neuronal migration and other motile events. Yeast two-hybrid studies predict that Lis1 binds directly to dynein heavy chains (Sasaki et al., 2000; Tai et al., 2002), but the mechanistic significance of this interaction is not well understood. We now report that recombinant Lis1 binds to native brain dynein and significantly increases the microtubule-stimulated enzymatic activity of dynein in vitro. Lis1 does this without increasing the proportion of dynein that binds to microtubules, indicating that Lis1 influences enzymatic activity rather than microtubule association. Dynein stimulation in vitro is not a generic feature of microtubule-associated proteins, because tau did not stimulate dynein. To our knowledge, this is the first indication that Lis1 or any other factor directly modulates the enzymatic activity of cytoplasmic dynein. Lis1 must be able to homodimerize to stimulate dynein, because a C-terminal fragment (containing the dynein interaction site but missing the self-association domain) was unable to stimulate dynein. Binding and colocalization studies indicate that Lis1 does not interact with all dynein complexes found in the brain. We propose a model in which Lis1 stimulates the activity of a subset of motors, which could be particularly important during neuronal migration and long-distance axonal transport. PMID:16481446

  15. Genetics of Fertility Restoration in Cytoplasmic Male Sterile Pepper

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Pepper hybrid seeds production using male sterility could lower cost by reducing time and labour, and increase the genetic purity of the F1 seeds. To investigate the genetics of fertility restoration of the Peterson cytoplasmic sterility in pepper, a doubled haploid population of 115 pepper lines obtained from anther culture of the F1 hybrid between Yolo Wonder (sterility maintainer line) and Perennial (fertility restorer line) and the parental lines were test-crossed by 77013A (a strict cytoplasmic-genic male sterile line). The fertility of the test-crossed lines was assessed in greenhouse and open field with the following three criteria: pollen index (PI, visual estimation of pollen amount per flower), pollen number (PN, pollen counting under microscope), and seed number (SN, the number of seeds per fruit in open pollination). Correlations between the each couple of criteria within, as well as between the cultivation methods ranged from 0.55 to 0.84. Analysis of variance showed that the genotype (DH line) and environment were the significant sources of variation of the fertility.Narrow sense of heritance of fertility restoration ranged from 0.38 to 0.92, depending on the criteria and environment. The distribution of the progeny was continuous between the parental genotypes indicating the quantitative inheritance of fertility restoration. Inferred from segregation according to Snape et al.(1984), the number of segregating genes was estimated to be that three to four genetic factors were involved in pollen traits (PI and PN) and five to eight genetic factors in seed production (SN). The heredity analysis of the CMS will be helpful for understanding of the genetic mechanism of the fertility restoration and the exploitation of the CMS in hybrid seed production.

  16. Stringent control of cytoplasmic Ca2+ in guard cells of intact plants compared to their counterparts in epidermal strips or guard cell protoplasts.

    Science.gov (United States)

    Levchenko, V; Guinot, D R; Klein, M; Roelfsema, M R G; Hedrich, R; Dietrich, P

    2008-01-01

    Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions. We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts. Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM. Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips. Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.

  17. Proteolytic Activation of the Essential Parasitophorous Vacuole Cysteine Protease SERA6 Accompanies Malaria Parasite Egress from Its Host Erythrocyte*

    Science.gov (United States)

    Ruecker, Andrea; Shea, Michael; Hackett, Fiona; Suarez, Catherine; Hirst, Elizabeth M. A.; Milutinovic, Katarina; Withers-Martinez, Chrislaine; Blackman, Michael J.

    2012-01-01

    The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV). The PV and host cell membranes eventually rupture, releasing merozoites in a process called egress. Certain inhibitors of serine and cysteine proteases block egress, indicating a crucial role for proteases. The Plasmodium falciparum genome encodes nine serine-repeat antigens (SERAs), each of which contains a central domain homologous to the papain-like (clan CA, family C1) protease family. SERA5 and SERA6 are indispensable in blood-stage parasites, but the function of neither is known. Here we show that SERA6 localizes to the PV where it is precisely cleaved just prior to egress by an essential serine protease called PfSUB1. Mutations that replace the predicted catalytic Cys of SERA6, or that block SERA6 processing by PfSUB1, could not be stably introduced into the parasite genomic sera6 locus, indicating that SERA6 is an essential enzyme and that processing is important for its function. We demonstrate that cleavage of SERA6 by PfSUB1 converts it to an active cysteine protease. Our observations reveal a proteolytic activation step in the malarial PV that may be required for release of the parasite from its host erythrocyte. PMID:22984267

  18. 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.

  19. Dictyostelium discoideum RabS and Rab2 colocalize with the Golgi and contractile vacuole system and regulate osmoregulation

    Indian Academy of Sciences (India)

    Katherine Maringer; Azure Yarbrough; Sunder Sims-Lucas; Entsar Saheb; Sanaa Jawed; John Bush

    2016-06-01

    Small-molecular-weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and is required for protein transport from the ER to the Golgi complex; however, Rab2 has yet to be characterized in Dictyostelium discoideum. DdRabS is a Dictyostelium Rab that is 80% homologous to DdRab1 which is required for protein transport between the ER and Golgi. Expression of GFP-tagged DdRab2 and DdRabS proteins showed localization to Golgi membranes and to the contractile vacuole system (CV) in Dictyostelium. Microscopic imaging indicates that the DdRab2 and DdRabS proteins localize at, and are essential for, the proper structure of Golgi membranes and the CV system. Dominant negative (DN) forms show fractionation of Golgi membranes, supporting their role in the structure and function of it. DdRab2 and DdRabS proteins, and their dominant negative and constitutively active (CA) forms, affect osmoregulation of the cells, possibly by the influx and discharge of fluids, which suggests a role in the function of the CV system. This is the first evidence of GTPases being localized to both Golgi membranes and the CV system in Dictyostelium.

  20. 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.

  1. Functional analysis of Plasmodium falciparum parasitophorous vacuole membrane protein (Pfs16) during gametocytogenesis and gametogenesis by targeted gene disruption.

    Science.gov (United States)

    Kongkasuriyachai, Darin; Fujioka, Hisashi; Kumar, Nirbhay

    2004-02-01

    Gametocytogenesis is a tightly regulated process marked by differentiation through distinct morphological forms and coordinated expression of sexual stage gene products. The earliest known gene product expressed at the onset of Plasmodium falciparum gametocytogenesis is Pfs16 localized on the parasitophorous vacuole membrane (PVM). Targeted gene disruption was undertaken to disrupt expression of Pfs16 and examine its potential role during sexual development. Three independent clones were demonstrated to have the coding sequence of Ps16 gene disrupted by the targeting plasmid by homologous recombination. No full-length transcripts and PVM localized 16 kDa protein were detected. Instead, all three "16ko" clones expressed a protein of 14 kDa recognized by Pfs16 specific antibodies that was mislocalized to an unidentified double membrane compartment in the parasites. Disruption of Pfs16 gene resulted in a significant reduction in gametocyte production, although the small number of gametocytes produced appeared to be normal by molecular and phenotypic evidences. Preliminary observation also suggested impaired ability of male gametocytes to exflagellate in vitro. Pfs16 does not appear to be essential for sexual development, instead may be required for optimal production of sexual parasites. Understanding mechanisms involved in the development of sexual stages of P. falciparum may identify novel targets for drugs and vaccines effective in reducing malaria transmission.

  2. Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

    Science.gov (United States)

    Shevchuk, Olga; Pägelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel; Steinert, Michael

    2014-11-01

    L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.

  3. Formation of a pathogen vacuole according to Legionella pneumophila: how to kill one bird with many stones.

    Science.gov (United States)

    Finsel, Ivo; Hilbi, Hubert

    2015-07-01

    Legionella species are ubiquitous, waterborne bacteria that thrive in numerous ecological niches. Yet, in contrast to many other environmental bacteria, Legionella spp. are also able to grow intracellularly in predatory protozoa. This feature mainly accounts for the pathogenicity of Legionella pneumophila, which causes the majority of clinical cases of a severe pneumonia termed Legionnaires' disease. The pathomechanism underlying L. pneumophila infection is based on macrophage resistance, which in turn is largely defined by the opportunistic pathogen's resistance towards amoebae. L. pneumophila replicates in macrophages or amoebae in a unique membrane-bound compartment, the Legionella-containing vacuole (LCV). LCV formation requires the bacterial intracellular multiplication/defective for organelle trafficking (Icm/Dot) type IV secretion system and involves a plethora of translocated effector proteins, which subvert pivotal processes in the host cell. Of the ca. 300 different experimentally validated Icm/Dot substrates, about 50 have been studied and attributed a cellular function to date. The versatility and ingenuity of these effectors' mode of actions is striking. In this review, we summarize insight into the cellular functions and biochemical activities of well-characterized L. pneumophila effector proteins and the host pathways they target. Recent studies not only substantially increased our knowledge about pathogen-host interactions, but also shed light on novel biological mechanisms.

  4. TcPho91 is a contractile vacuole phosphate sodium symporter that regulates phosphate and polyphosphate metabolism in Trypanosoma cruzi.

    Science.gov (United States)

    Jimenez, Veronica; Docampo, Roberto

    2015-09-01

    We have identified a phosphate transporter (TcPho91) localized to the bladder of the contractile vacuole complex (CVC) of Trypanosoma cruzi, the etiologic agent of Chagas disease. TcPho91 has 12 transmembrane domains, an N-terminal regulatory SPX (named after SYG1, Pho81 and XPR1) domain and an anion permease domain. Functional expression in Xenopus laevis oocytes followed by two-electrode voltage clamp showed that TcPho91 is a low-affinity transporter with a Km for Pi in the millimolar range, and sodium-dependency. Epimastigotes overexpressing TcPho91-green fluorescent protein have significantly higher levels of pyrophosphate (PPi ) and short-chain polyphosphate (polyP), suggesting accumulation of Pi in these cells. Moreover, when overexpressing parasites were maintained in a medium with low Pi , they grew at higher rates than control parasites. Only one allele of TcPho91 in the CL strain encodes for the complete open reading frame, while the other one is truncated encoding for only the N-terminal domain. Taking advantage of this characteristic, knockdown experiments were performed resulting in cells with reduced growth rate as well as a reduction in PPi and short-chain polyP levels. Our results indicate that TcPho91 is a phosphate sodium symporter involved in Pi homeostasis in T. cruzi.

  5. The infectious intracellular lifestyle of Salmonella enterica relies on the adaptation to nutritional conditions within the Salmonella-containing vacuole.

    Science.gov (United States)

    Diacovich, Lautaro; Lorenzi, Lucía; Tomassetti, Mauro; Méresse, Stéphane; Gramajo, Hugo

    2016-12-09

    Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative pathogen that causes various host-specific diseases. During their life cycle, Salmonellae survive frequent exposures to a variety of environmental stresses, e.g. carbon-source starvation. The virulence of this pathogen relies on its ability to establish a replicative niche, named Salmonella-containing vacuole, inside host cells. However, the microenvironment of the SCV and the bacterial metabolic pathways required during infection are largely undefined. In this work we developed different biological probes whose expression is modulated by the environment and the physiological state of the bacterium. We constructed transcriptional reporters by fusing promoter regions to the gfpmut3a gene to monitor the expression profile of genes involved in glucose utilization and lipid catabolism. The induction of these probes by a specific metabolic change was first tested in vitro, and then during different conditions of infection in macrophages. We were able to determine that Entner-Doudoroff is the main metabolic pathway utilized by Salmonella during infection in mouse macrophages. Furthermore, we found sub-populations of bacteria expressing genes involved in pathways for the utilization of different sources of carbon. These populations are modified in presence of different metabolizable substrates, suggesting the coexistence of Salmonella with diverse metabolic states during the infection.

  6. Live Cell Imaging During Germination Reveals Dynamic Tubular Structures Derived from Protein Storage Vacuoles of Barley Aleurone Cells.

    Science.gov (United States)

    Ibl, Verena; Stoger, Eva

    2014-01-01

    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.

  7. Targeting host syntaxin-5 preferentially blocks Leishmania parasitophorous vacuole development in infected cells and limits experimental Leishmania infections.

    Science.gov (United States)

    Canton, Johnathan; Kima, Peter E

    2012-10-01

    Our previous observations established a role for syntaxin-5 in the development of Leishmania parasitophorous vacuoles (LPVs). In this study, we took advantage of the recent identification of Retro-2, a small organic molecule that can cause the redistribution of syntaxin-5; we show herein that Retro-2 blocks LPV development within 2 hours of adding it to cells infected with Leishmania amazonensis. In infected cells incubated for 48 hours with Retro-2, LPV development was significantly limited; furthermore, infected cells harbored four to five times fewer parasites than infected cells incubated in vehicle alone. In vivo studies revealed that Retro-2 curbed experimental L. amazonensis infections in a dose-dependent manner. Retro-2 did not have any appreciable effect on the host cell physiological characteristics; furthermore, it had no apparent toxicity in experimental animals. An unexpected, but welcome, finding was that Retro-2 inhibited the replication of Leishmania parasites in axenic cultures. This study is significant because it identifies an endoplasmic reticulum/Golgi SNARE as a potential target for the control of Leishmania infections; moreover, it suggests that small organic molecules can be identified that can selectively disrupt the vesicle fusion machinery that promotes the development of pathogen-containing compartments without exerting toxic effects on the host.

  8. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

    Science.gov (United States)

    Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Cheng, Lailiang; Hao, Yu-Jin

    2016-03-01

    Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants.

  9. Novel origin of lamin-derived cytoplasmic intermediate filaments in tardigrades.

    Science.gov (United States)

    Hering, Lars; Bouameur, Jamal-Eddine; Reichelt, Julian; Magin, Thomas M; Mayer, Georg

    2016-02-03

    Intermediate filament (IF) proteins, including nuclear lamins and cytoplasmic IF proteins, are essential cytoskeletal components of bilaterian cells. Despite their important role in protecting tissues against mechanical force, no cytoplasmic IF proteins have been convincingly identified in arthropods. Here we show that the ancestral cytoplasmic IF protein gene was lost in the entire panarthropod (onychophoran + tardigrade + arthropod) rather than arthropod lineage and that nuclear, lamin-derived proteins instead acquired new cytoplasmic roles at least three times independently in collembolans, copepods, and tardigrades. Transcriptomic and genomic data revealed three IF protein genes in the tardigrade Hypsibius dujardini, one of which (cytotardin) occurs exclusively in the cytoplasm of epidermal and foregut epithelia, where it forms belt-like filaments around each epithelial cell. These results suggest that a lamin derivative has been co-opted to enhance tissue stability in tardigrades, a function otherwise served by cytoplasmic IF proteins in all other bilaterians.

  10. EhVps32 Is a Vacuole-Associated Protein Involved in Pinocytosis and Phagocytosis of Entamoeaba histolytica.

    Directory of Open Access Journals (Sweden)

    Yunuen Avalos-Padilla

    2015-07-01

    Full Text Available Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member, Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.

  11. EhVps32 Is a Vacuole-Associated Protein Involved in Pinocytosis and Phagocytosis of Entamoeaba histolytica.

    Science.gov (United States)

    Avalos-Padilla, Yunuen; Betanzos, Abigail; Javier-Reyna, Rosario; García-Rivera, Guillermina; Chávez-Munguía, Bibiana; Lagunes-Guillén, Anel; Ortega, Jaime; Orozco, Esther

    2015-07-01

    Here, we investigated the role of EhVps32 protein (a member of the endosomal-sorting complex required for transport) in endocytosis of Entamoeba histolytica, a professional phagocyte. Confocal microscopy, TEM and cell fractionation revealed EhVps32 in cytoplasmic vesicles and also located adjacent to the plasma membrane. Between 5 to 30 min of phagocytosis, EhVps32 was detected on some erythrocytes-containing phagosomes of acidic nature, and at 60 min it returned to cytoplasmic vesicles and also appeared adjacent to the plasma membrane. TEM images revealed it in membranous structures in the vicinity of ingested erythrocytes. EhVps32, EhADH (an ALIX family member), Gal/GalNac lectin and actin co-localized in the phagocytic cup and in some erythrocytes-containing phagosomes, but EhVps32 was scarcely detected in late phagosomes. During dextran uptake, EhVps32, EhADH and Gal/GalNac lectin, but not actin, co-localized in pinosomes. EhVps32 recombinant protein formed oligomers composed by rings and filaments. Antibodies against EhVps32 monomers stained cytoplasmic vesicles but not erythrocytes-containing phagosomes, suggesting that in vivo oligomers are formed on phagosome membranes. The involvement of EhVps32 in phagocytosis was further study in pNeoEhvps32-HA-transfected trophozoites, which augmented almost twice their rate of erythrophagocytosis as well as the membranous concentric arrays built by filaments, spirals and tunnel-like structures. Some of these structures apparently connected phagosomes with the phagocytic cup. In concordance, the EhVps32-silenced G3 trophozoites ingested 80% less erythrocytes than the G3 strain. Our results suggest that EhVps32 participates in E. histolytica phagocytosis and pinocytosis. It forms oligomers on erythrocytes-containing phagosomes, probably as a part of the scission machinery involved in membrane invagination and intraluminal vesicles formation.

  12. Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin.

    Science.gov (United States)

    Quentmeier, H; Martelli, M P; Dirks, W G; Bolli, N; Liso, A; Macleod, R A F; Nicoletti, I; Mannucci, R; Pucciarini, A; Bigerna, B; Martelli, M F; Mecucci, C; Drexler, H G; Falini, B

    2005-10-01

    We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.

  13. Astrocytic exportin-7 responds to ischemia through mediating LKB1 translocation from the nucleus to the cytoplasm.

    Science.gov (United States)

    Liang, Hai Jie; Chai, Rui Chao; Li, Xi; Kong, Jin Ge; Jiang, Jiao Hua; Ma, Ju; Vatcher, Greg; Yu, Albert Cheung Hoi

    2015-02-01

    The superfamily of importin-β-related proteins is the largest class of nuclear transport receptors and can be generally divided into importins and exportins according to their transport directions. Eleven importins and seven exportins have been identified, and the expression patterns of both classes are important for their functions in nucleocytoplasmic transport activities. This study demonstrates that all of the importins (importin-β; transportin-1, -2, and -3; and importin-4, -5, -7, -8, -9, -11, and -13) and all the exportins (exportin-1, -2, -4, -5, -6, -7, and -t) are differentially expressed in the cerebral cortex, cerebellum, hippocampus, and brainstem and in primary cultures of cerebral cortical astrocytes and neurons. For astrocytes, we observed that different importins and exportins displayed different expression changes during 0-6 hr of ischemia treatment, especially an increase of both the mRNA and the protein of exportin-7. Immunostaining showed that exportin-7 accumulated inside the nucleus and around the nuclear envelope. In addition, we noticed an increased cytoplasmic distribution of one of the cargo proteins of exportin-7, LKB1, an important element in maintaining energy homeostasis. This increased cytoplasmic distribution was accompanied by an increased expression of exportin-7 under ischemia in astrocytes. We demonstrate that exportin-7 responds to ischemia in astrocytes and that this response involves translocation of LKB1, a protein that plays important roles during metabolic stress, from the nucleus to the cytoplasm.

  14. A protein allergen microarray detects specific IgE to pollen surface, cytoplasmic, and commercial allergen extracts.

    Directory of Open Access Journals (Sweden)

    Katinka A Vigh-Conrad

    Full Text Available Current diagnostics for allergies, such as skin prick and radioallergosorbent tests, do not allow for inexpensive, high-throughput screening of patients. Additionally, extracts used in these methods are made from washed pollen that lacks pollen surface materials that may contain allergens.We sought to develop a high-throughput assay to rapidly measure allergen-specific IgE in sera and to explore the relative allergenicity of different pollen fractions (i.e. surface, cytoplasmic, commercial extracts. To do this, we generated a protein microarray containing surface, cytoplasmic, and commercial extracts from 22 pollen species, commercial extracts from nine non-pollen allergens, and five recombinant allergenic proteins. Pollen surface and cytoplasmic fractions were prepared by extraction into organic solvents and aqueous buffers, respectively. Arrays were incubated with <25 uL of serum from 176 individuals and bound IgE was detected by indirect immunofluorescence, providing a high-throughput measurement of IgE. We demonstrated that the allergen microarray is a reproducible method to measure allergen-specific IgE in small amounts of sera. Using this tool, we demonstrated that specific IgE clusters according to the phylogeny of the allergen source. We also showed that the pollen surface, which has been largely overlooked in the past, contained potent allergens. Although, as a class, cytoplasmic fractions obtained by our pulverization/precipitation method were comparable to commercial extracts, many individual allergens showed significant differences.These results support the hypothesis that protein microarray technology is a useful tool for both research and in the clinic. It could provide a more efficient and less painful alternative to traditionally used skin prick tests, making it economically feasible to compare allergen sensitivity of different populations, monitor individual responses over time, and facilitate genetic studies on pollen allergy.

  15. Poliovirus infection induces the co-localization of cellular protein SRp20 with TIA-1, a cytoplasmic stress granule protein.

    Science.gov (United States)

    Fitzgerald, Kerry D; Semler, Bert L

    2013-09-01

    Different types of environmental stress cause mammalian cells to form cytoplasmic foci, termed stress granules, which contain mRNPs that are translationally silenced. These foci are transient and dynamic, and contain components of the cellular translation machinery as well as certain mRNAs and RNA binding proteins. Stress granules are known to be induced by conditions such as hypoxia, nutrient deprivation, and oxidative stress, and a number of cellular factors have been identified that are commonly associated with these foci. More recently it was discovered that poliovirus infection also induces the formation of stress granules, although these cytoplasmic foci appear to be somewhat compositionally unique. Work described here examined the punctate pattern of SRp20 (a host cell mRNA splicing protein) localization in the cytoplasm of poliovirus-infected cells, demonstrating the partial co-localization of SRp20 with the stress granule marker protein TIA-1. We determined that SRp20 does not co-localize with TIA-1, however, under conditions of oxidative stress, indicating that the close association of these two proteins during poliovirus infection is not representative of a general response to cellular stress. We confirmed that the expression of a dominant negative version of TIA-1 (TIA-1-PRD) results in the dissociation of stress granules. Finally, we demonstrated that expression of wild type TIA-1 or dominant negative TIA-1-PRD in cells during poliovirus infection does not dramatically affect viral translation. Taken together, these studies provide a new example of the unique cytoplasmic foci that form during poliovirus infection.

  16. Selective eicosanoid-generating capacity of cytoplasmic phospholipase A2 in Pseudomonas aeruginosa-infected epithelial cells.

    Science.gov (United States)

    Hurley, Bryan P; Pirzai, Waheed; Mumy, Karen L; Gronert, Karsten; McCormick, Beth A

    2011-02-01

    Airway neutrophil infiltration is a pathological hallmark observed in multiple lung diseases including pneumonia and cystic fibrosis. Bacterial pathogens such as Pseudomonas aeruginosa instigate neutrophil recruitment to the air space. Excessive accumulation of neutrophils in the lung often contributes to tissue destruction. Previous studies have unveiled hepoxilin A(3) as the key molecular signal driving neutrophils across epithelial barriers. The eicosanoid hepoxilin A(3) is a potent neutrophil chemoattractant produced by epithelial cells in response to infection with P. aeruginosa. The enzyme phospholipase A(2) liberates arachidonic acid from membrane phospholipids, the rate-limiting step in the synthesis of all eicosanoids, including hepoxilin A(3). Once generated, aracidonic acid is acted upon by multiple cyclooxygenases and lipoxygenases producing an array of functionally diverse eicosanoids. Although there are numerous phospholipase A(2) isoforms capable of generating arachidonic acid, the isoform most often associated with eicosanoid generation is cytoplasmic phospholipase A(2)α. In the current study, we observed that the cytoplasmic phospholipase A(2)α isoform is required for mediating P. aeruginosa-induced production of certain eicosanoids such as prostaglandin E(2). However, we found that neutrophil transepithelial migration induced by P. aeruginosa does not require cytoplasmic phospholipase A(2)α. Furthermore, P. aeruginosa-induced hepoxilin A(3) production persists despite cytoplasmic phospholipase A(2)α suppression and generation of the 12-lipoxygenase metabolite 12-HETE is actually enhanced in this context. These results suggest that alterative phospholipase A(2) isoforms are utilized to synthesize 12-lipoxygenase metabolites. The therapeutic implications of these findings are significant when considering anti-inflammatory therapies based on targeting eicosanoid synthesis pathways.

  17. Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Gestl, Erin E., E-mail: egestl@wcupa.edu [Department of Biology, West Chester University, 750 S Church Street, West Chester, PA 19383 (United States); Anne Boettger, S., E-mail: aboettger@wcupa.edu [Department of Biology, West Chester University, 750 S Church Street, West Chester, PA 19383 (United States)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer Eight human colorectal cell lines were evaluated for p53 and mortalin localization. Black-Right-Pointing-Pointer Six cell lines displayed cytoplasmic sequestration of the tumor suppressor p53. Black-Right-Pointing-Pointer Direct interaction between mortalin and p53 was shown in five cell lines. Black-Right-Pointing-Pointer Cell lines positive for p53 sequestration yielded elevated p53 expression levels. Black-Right-Pointing-Pointer This study yields the first evidence of cytoplasmic sequestration p53 by mortalin. -- Abstract: While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53

  18. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

    Energy Technology Data Exchange (ETDEWEB)

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, Andre; Cocquerel, Laurence [INSERM U1019, CNRS UMR 8204, CIIL, F-59021 Lille (France); Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Allet, Cecile [INSERM U837-JPARC, 59045 Lille (France); Dumont, Patrick [Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); CNRS UMR 8161, F-59021 Lille (France); Loyens, Anne [INSERM U837-JPARC, 59045 Lille (France); Leteurtre, Emmanuelle [Service d' Anatomie et de Cytologie Pathologiques, Centre de Biologie Pathologie, CHRU de Lille, Avenue Oscar-Lambret, Lille cedex (France); Omary, M. Bishr [Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America (United States); Dubuisson, Jean; Rouille, Yves [INSERM U1019, CNRS UMR 8204, CIIL, F-59021 Lille (France); Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Wychowski, Czeslaw, E-mail: czeslaw.wychowski@ibl.fr [INSERM U1019, CNRS UMR 8204, CIIL, F-59021 Lille (France); Universite Lille Nord de France, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  19. Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies.

    Science.gov (United States)

    Delavalle, Pierre-Yves; Alsaleh, Khaled; Pillez, André; Cocquerel, Laurence; Allet, Cécile; Dumont, Patrick; Loyens, Anne; Leteurtre, Emmanuelle; Omary, M Bishr; Dubuisson, Jean; Rouillé, Yves; Wychowski, Czeslaw

    2011-11-01

    Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which

  20. Studies on the Utilization Potentiality of the Nucleo-Cytoplasmic Hybrid in Wheat

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    A series of comparative studies was carried out on the genetic effects of 25 alien cytoplasms of wheat on the growth potential, heading stage , fertility, resistance against diseases, important agronomic traits and its heterosis of 125 nuclei-cytoplasmic hybrids of wheat. The results indicated that there were clearly effects of alien cytoplasms on some characteristics, but the nucleus still exerted main effect on other characteristics. The effect of interactions between nucleus and cytoplasm was comparative obvious in some combination. Consequently, when we utilize the effects of alien cytoplasms, we should pay full attention to the facts such as the characteristic to be improved, the effects of cytoplasm ,nucleus, the nucleus-cytoplasm interactions on that characteristics . From the preliminary studies, we believed that the cytoplasmic types of M°, S1, Sv, D2, D and B, and the nucleo-cytoplasmic hybrids of (Ae. sharonensis) -Bl74, (Ae. squarrosa)352-35, (Ae. cylindrica) -352-35, (Ae. cylindrica)-E EN-1, (Ae. cylindrica)- NPFP, and (Ae. speltoides)352-35 would have some utilization potentiality in cultivar improvement.

  1. Heterogeneous anomalous diffusion of virus in cytoplasm of a living cell

    CERN Document Server

    Itto, Yuichi

    2010-01-01

    The infection pathway of virus in cytoplasm of a living cell is studied from the viewpoint of diffusion theory. The cytoplasm plays a role of a medium for stochastic motion of the virus contained in the endosome as well as the free virus. It is experimentally known that the exponent of anomalous diffusion fluctuates in localized areas of the cytoplasm. Here, generalizing fractional kinetic theory, such fluctuations are described in terms of the exponent locally distributed over the cytoplasm, and a theoretical proposition is presented for its statistical form. The proposed fluctuations may be examined in an experiment of heterogeneous diffusion in the infection pathway.

  2. Identification of cytoplasm types in rapeseed (Brassica napus L.) accessions by a multiplex PCR assay.

    Science.gov (United States)

    Zhao, H X; Li, Z J; Hu, S W; Sun, G L; Chang, J J; Zhang, Z H

    2010-08-01

    Cytoplasmic male sterility (CMS) has widely been used as an efficient pollination control system in rapeseed hybrid production. Identification of cytoplasm type of rapeseed accessions is becoming the most important basic work for hybrid-rapeseed breeding. In this study, we report a simple multiplex PCR method to distinguish the existing common cytoplasm resources, Pol, Nap, Cam, Ogu and Ogu-NWSUAF cytoplasm, in rapeseed. Cytoplasm type of 35 F(1) hybrids and 140 rapeseed open pollinated varieties or breeding lines in our rapeseed breeding programme were tested by this method. The results indicated that 10 of 35 F(1) hybrids are the Nap, and 25 the Pol cytoplasm type, which is consistent with the information provided by the breeders. Out of 140 accessions tested, 100 (71.4%), 21 (15%) and 19 (13.6%) accessions possess Nap, Cam and Pol cytoplasm, respectively. All 19 accessions with Pol cytoplasm are from China. Pedigree analysis indicated that these accessions with Pol cytoplasm were either restorers for Pol CMS, including Shaan 2C, Huiyehui, 220, etc. or derived from hybrids with Pol CMS as female parent. Our molecular results are consistent with those of the classical testcross, suggesting the reliability of this method. The multiplex PCR assay method can be applied to CMS "three-line" breeding, selection and validation of hybrid rapeseed.

  3. A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.

    Directory of Open Access Journals (Sweden)

    Tanaka Yoshiyuki

    2012-07-01

    Full Text Available Abstract Background Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. Results Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura-type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3 kb, similarity >99.9%. It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size that are non-syntenic to normal-type genome, and the gene orf138

  4. Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications.

    Science.gov (United States)

    Mallik, Roop; Petrov, Dmitri; Lex, S A; King, S J; Gross, S P

    2005-12-06

    Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dynein's mechanochemical cycle. Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

  5. Intracellular organelles mediate cytoplasmic pulling force for centrosome centration in the Caenorhabditis elegans early embryo

    Science.gov (United States)

    Kimura, Akatsuki

    2010-01-01

    The centrosome is generally maintained at the center of the cell. In animal cells, centrosome centration is powered by the pulling force of microtubules, which is dependent on cytoplasmic dynein. However, it is unclear how dynein brings the centrosome to the cell center, i.e., which structure inside the cell functions as a substrate to anchor dynein. Here, we provide evidence that a population of dynein, which is located on intracellular organelles and is responsible for organelle transport toward the centrosome, generates the force required for centrosome centration in Caenorhabditis elegans embryos. By using the database of full-genome RNAi in C. elegans, we identified dyrb-1, a dynein light chain subunit, as a potential subunit involved in dynein anchoring for centrosome centration. DYRB-1 is required for organelle movement toward the minus end of the microtubules. The temporal correlation between centrosome centration and the net movement of organelle transport was found to be significant. Centrosome centration was impaired when Rab7 and RILP, which mediate the association between organelles and dynein in mammalian cells, were knocked down. These results indicate that minus end-directed transport of intracellular organelles along the microtubules is required for centrosome centration in C. elegans embryos. On the basis of this finding, we propose a model in which the reaction forces of organelle transport generated along microtubules act as a driving force that pulls the centrosomes toward the cell center. This is the first model, to our knowledge, providing a mechanical basis for cytoplasmic pulling force for centrosome centration. PMID:21173218

  6. Positioning lipid membrane domains in giant vesicles by micro-organization of aqueous cytoplasm mimic.

    Science.gov (United States)

    Cans, Ann-Sofie; Andes-Koback, Meghan; Keating, Christine D

    2008-06-11

    We report localization of lipid membrane microdomains to specific "poles" of asymmetric giant vesicles (GVs) in response to local internal composition. Interior aqueous microdomains were generated in a simple model cytoplasm composed of a poly(ethyleneglycol) (PEG)/dextran aqueous two-phase system (ATPS) encapsulated in the vesicles. The GV membrane composition used here was a modification of a DOPC/DPPC/cholesterol mixture known to form micrometer-scale liquid ordered and liquid disordered domains; we added lipids with PEG 2000 Da-modified headgroups. Osmotically induced budding of the ATPS-containing GVs led to structures where the PEG-rich and dextran-rich interior aqueous phases were in contact with different regions of the vesicle membrane. Liquid ordered (L o) membrane domains rich in PEG-terminated lipids preferentially coated the PEG-rich aqueous phase vesicle "body", while coexisting liquid disordered (L d) membrane domains coated the dextran-rich aqueous phase "bud". Membrane domain positioning resulted from interactions between lipid headgroups and the interior aqueous polymer solutions, e.g., PEGylated headgroups with PEG and dextran polymers. Heating resulted first in patchy membranes where L o and L d domains no longer showed any preference for coating the PEG-rich vs dextran-rich interior aqueous volumes, and eventually complete lipid mixing. Upon cooling lipid domains again coated their preferred interior aqueous microvolume. This work shows that nonspecific interactions between interior aqueous contents and the membrane that encapsulates them can drive local chemical heterogeneity, and offers a primitive experimental model for membrane and cytoplasmic polarity in biological cells.

  7. Exclusion of NFAT5 from mitotic chromatin resets its nucleo-cytoplasmic distribution in interphase.

    Directory of Open Access Journals (Sweden)

    Anaïs Estrada-Gelonch

    Full Text Available BACKGROUND: The transcription factor NFAT5 is a major inducer of osmoprotective genes and is required to maintain the proliferative capacity of cells exposed to hypertonic stress. In response to hypertonicity, NFAT5 translocates to the nucleus, binds to regulatory regions of osmoprotective genes and activates their transcription. Besides stimulus-specific regulatory mechanisms, the activity of transcription factors in cycling cells is also regulated by the passage through mitosis, when most transcriptional processes are downregulated. It was not known whether mitosis could be a point of control for NFAT5. METHODOLOGY/PRINCIPAL FINDINGS: Using confocal microscopy we observed that NFAT5 was excluded from chromatin during mitosis in both isotonic and hypertonic conditions. Analysis of NFAT5 deletions showed that exclusion was mediated by the carboxy-terminal domain (CTD. NFAT5 mutants lacking this domain showed constitutive binding to mitotic chromatin independent of tonicity, which caused them to localize in the nucleus and remain bound to chromatin in the subsequent interphase without hypertonic stimulation. We analyzed the contribution of the CTD, DNA binding, and nuclear import and export signals to the subcellular localization of this factor. Our results indicated that cytoplasmic localization of NFAT5 in isotonic conditions required both the exclusion from mitotic DNA and active nuclear export in interphase. Finally, we identified several regions within the CTD of NFAT5, some of them overlapping with transactivation domains, which were separately capable of causing its exclusion from mitotic chromatin. CONCLUSIONS/SIGNIFICANCE: Our results reveal a multipart mechanism regulating the subcellular localization of NFAT5. The transactivating module of NFAT5 switches its function from an stimulus-specific activator of transcription in interphase to an stimulus-independent repressor of binding to DNA in mitosis. This mechanism, together with export

  8. Transport of proteins to the plant vacuole is not by bulk flow through the secretory system, and requires positive sorting information.

    Science.gov (United States)

    Dorel, C; Voelker, T A; Herman, E M; Chrispeels, M J

    1989-02-01

    Plant cells, like other eukaryotic cells, use the secretory pathway to target proteins to the vacuolar/lysosomal compartment and to the extracellular space. We wished to determine whether the presence of a hydrophobic signal peptide would result in the transport of a reporter protein to vacuoles by bulk flow; to investigate this question, we expressed a chimeric gene in transgenic tobacco. The chimeric gene, Phalb, used for this study consists of the 1,188-bp 5' upstream sequence and the hydrophobic signal sequence of a vacuolar seed protein phytohemagglutinin, and the coding sequence of a cytosolic seed albumin (PA2). The chimeric protein PHALB cross-reacted with antibodies to PA2 and was found in the seeds of the transgenic plants (approximately 0.7% of total protein), but not in the leaves, roots, or flowers. Immunoblot analyses of seed extracts revealed four glycosylated polypeptides ranging in molecular weight from 29,000 to 32,000. The four polypeptides are glycoforms of a single polypeptide of Mr 27,000, and the heterogeneity is due to the presence of high mannose and endoglycosidase H-resistant glycans. The PHALB products reacted with an antiserum specific for complex plant glycans indicating that the glycans had been modified in the Golgi apparatus. Subcellular fractionation of glycerol extracts of mature seeds showed that only small amounts of PHALB accumulated in the protein storage vacuoles of the tobacco seeds. In homogenates made in an isotonic medium, very little PHALB was associated with the organelle fraction containing the endoplasmic reticulum and Golgi apparatus; most of it was in the soluble fraction. We conclude that PHALB passed through the Golgi apparatus, but did not arrive in the vacuoles. Transport to vacuoles is not by a bulk-flow mechanism, once proteins have entered the secretory system, and requires information beyond that provided by a hydrophobic signal peptide.

  9. Effects of alien and intraspecies cytoplasms on manifestation of nuclear genes for wheat resistance to brown rust: II. Specificity of cytoplasm influence on different Lr genes

    Energy Technology Data Exchange (ETDEWEB)

    Voluevich, E.A.; Buloichik, A.A.; Palilova, A.N. [Institute of Genetics and Cytology, Minsk (Belarus)

    1995-04-01

    Specificity of expression of the major nuclear genes Lr to two brown rust clones in hybrids with the same maternal cytoplasm was analyzed. It was evaluated by a resistant: susceptible ratio in the F{sub 2}. Reciprocal hybrids were obtained from the cross between the progeny of homozygous susceptible plants of the cultivar Penjamo 62 and its alloplasmatic lines carrying cytoplasms of Triticum dicoccoides var. fulvovillosum, Aegilops squarrosa var. typical, Agropyron trichophorum, and isogenic lines of the cultivar Thatcher (Th) with the Lr1, Lr9, Lr15, and Lr19 genes. It was shown that the effect of the Lr1 gene in the cytoplasm of cultivar Thatcher and in eu-, and alloplasmatic forms of Penjamo 62 was less expressed than that of other Lr genes. Cytoplasm of the alloplasmatic line (dicoccoides)-Penjamo 62 was the only exception: in the F{sub 2}, hybrids with Th (Lr1) had a higher yield of resistant forms than those with Th (Lr15). In the hybrid combinations studied, expression and/or transmission of the Lr19 gene was more significant than that of other genes. This gene had no advantages over Lr15 and Lr19 only in cytoplasm of the alloplasmatic line (squarrosa)-Penjamo 62. In certain hybrid cytoplasms, the display of the Lr1, Lr15, and Lr19 genes, in contrast to Lr9, varied with the virulence of the pathogen clones. 15 refs., 5 tabs.

  10. Cargo transport by cytoplasmic Dynein can center embryonic centrosomes.

    Directory of Open Access Journals (Sweden)

    Rafael A Longoria

    Full Text Available To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell's center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.

  11. Anti-neutrophil cytoplasmic antibodies stimulate release of neutrophil microparticles.

    LENUS (Irish Health Repository)

    Hong, Ying

    2012-01-01

    The mechanisms by which anti-neutrophil cytoplasmic antibodies (ANCAs) may contribute to the pathogenesis of ANCA-associated vasculitis are not well understood. In this study, both polyclonal ANCAs isolated from patients and chimeric proteinase 3-ANCA induced the release of neutrophil microparticles from primed neutrophils. These microparticles expressed a variety of markers, including the ANCA autoantigens proteinase 3 and myeloperoxidase. They bound endothelial cells via a CD18-mediated mechanism and induced an increase in endothelial intercellular adhesion molecule-1 expression, production of endothelial reactive oxygen species, and release of endothelial IL-6 and IL-8. Removal of the neutrophil microparticles by filtration or inhibition of reactive oxygen species production with antioxidants abolished microparticle-mediated endothelial activation. In addition, these microparticles promoted the generation of thrombin. In vivo, we detected more neutrophil microparticles in the plasma of children with ANCA-associated vasculitis compared with that in healthy controls or those with inactive vasculitis. Taken together, these results support a role for neutrophil microparticles in the pathogenesis of ANCA-associated vasculitis, potentially providing a target for future therapeutics.

  12. In silico classification of proteins from acidic and neutral cytoplasms.

    Directory of Open Access Journals (Sweden)

    Yaping Fang

    Full Text Available Protein acidostability is a common problem in biopharmaceutical and other industries. However, it remains a great challenge to engineer proteins for enhanced acidostability because our knowledge of protein acidostabilization is still very limited. In this paper, we present a comparative study of proteins from bacteria with acidic (AP and neutral cytoplasms (NP using an integrated statistical and machine learning approach. We construct a set of 393 non-redundant AP-NP ortholog pairs and calculate a total of 889 sequence based features for these proteins. The pairwise alignments of these ortholog pairs are used to build a residue substitution propensity matrix between APs and NPs. We use Gini importance provided by the Random Forest algorithm to rank the relative importance of these features. A scoring function using the 10 most significant features is developed and optimized using a hill climbing algorithm. The accuracy of the score function is 86.01% in predicting AP-NP ortholog pairs and is 76.65% in predicting non-ortholog AP-NP pairs, suggesting that there are significant differences between APs and NPs which can be used to predict relative acidostability of proteins. The overall trends uncovered in the study can be used as general guidelines for designing acidostable proteins. To best of our knowledge, this work represents the first systematic comparative study of the acidostable proteins and their non-acidostable orthologs.

  13. Does a parthenogenesis-inducing Wolbachia induce vestigial cytoplasmic incompatibility?

    Science.gov (United States)

    Kraaijeveld, Ken; Reumer, Barbara M.; Mouton, Laurence; Kremer, Natacha; Vavre, Fabrice; van Alphen, Jacques J. M.

    2011-03-01

    Wolbachia is a maternally inherited bacterium that manipulates the reproduction of its host. Recent studies have shown that male-killing strains can induce cytoplasmic incompatibility (CI) when introgressed into a resistant host. Phylogenetic studies suggest that transitions between CI and other Wolbachia phenotypes have also occurred frequently, raising the possibility that latent CI may be widespread among Wolbachia. Here, we investigate whether a parthenogenesis-inducing Wolbachia strain can also induce CI. Parthenogenetic females of the parasitoid wasp Asobara japonica regularly produce a small number of males that may be either infected or not. Uninfected males were further obtained through removal of the Wolbachia using antibiotics and from a naturally uninfected strain. Uninfected females that had mated with infected males produced a slightly, but significantly more male-biased sex ratio than uninfected females that had mated with uninfected males. This effect was strongest in females that mated with males that had a relatively high Wolbachia titer. Quantitative PCR indicated that infected males did not show higher ratios of nuclear versus mitochondrial DNA content. Wolbachia therefore does not cause diploidization of cells in infected males. While these results are consistent with CI, other alternatives such as production of abnormal sperm by infected males cannot be completely ruled out. Overall, the effect was very small (9%), suggesting that if CI is involved it may have degenerated through the accumulation of mutations.

  14. Human lung epithelial cells contain Mycobacterium tuberculosis in a late endosomal vacuole and are efficiently recognized by CD8⁺ T cells.

    Directory of Open Access Journals (Sweden)

    Melanie J Harriff

    Full Text Available Mycobacterium tuberculosis (Mtb is transmitted via inhalation of aerosolized particles. While alveolar macrophages are thought to play a central role in the acquisition and control of this infection, Mtb also has ample opportunity to interact with the airway epithelium. In this regard, we have recently shown that the upper airways are enriched with a population of non-classical, MR1-restricted, Mtb-reactive CD8⁺ T cells (MAIT cells. Additionally, we have demonstrated that Mtb-infected epithelial cells lining the upper airways are capable of stimulating IFNγ production by MAIT cells. In this study, we demonstrate that airway epithelial cells efficiently stimulate IFNγ release by MAIT cells as well as HLA-B45 and HLA-E restricted T cell clones. Characterization of the intracellular localization of Mtb in epithelial cells indicates that the vacuole occupied by Mtb in epithelial cells is distinct from DC in that it acquires Rab7 molecules and does not retain markers of early endosomes such as Rab5. The Mtb vacuole is also heterogeneous as there is a varying degree of association with Lamp1 and HLA-I. Although the Mtb vacuole shares markers associated with the late endosome, it does not acidify, and the bacteria are able to replicate within the cell. This work demonstrates that Mtb infected lung epithelial cells are surprisingly efficient at stimulating IFNγ release by CD8⁺ T cells.

  15. Patch clamp studies on root cell vacuoles of a salt-tolerant and a salt-sensitive plantago species : regulation of channel activity by salt stress.

    Science.gov (United States)

    Maathuis, F J; Prins, H B

    1990-01-01

    Plantago media L. and Plantago maritima L. differ in their strategy toward salt stress, a major difference being the uptake and distribution of ions. Patch clamp techniques were applied to root cell vacuoles to study the tonoplast channel characteristics. In both species the major channel found was a 60 to 70 picosiemens channel with a low ion selectivity. The conductance of this channel for Na(+) was the same as for K(+), P(K) (+)/P(Na) (+) = 1, whereas the cation/anion selectivity (P(K) (+)/P(c1) (-)) was about 5. Gating characteristics were voltage and calcium dependent. An additional smaller channel of 25 picosiemens was present in P. maritima. In the whole vacuole configuration, the summation of the single channel currents resulted in slowly activated inward currents (t((1/2)) = 1.2 second). Inwardly directed, ATP-dependent currents could be measured against a DeltapH gradient of 1.5 units over the tonoplast. This observation strongly indicated the physiological intactness of the used vacuoles. The open probability of the tonoplast channels dramatically decreased when plants were grown on NaCl, although single channel conductance and selectivity were not altered.

  16. Model-Assisted Analysis of Sugar Metabolism throughout Tomato Fruit Development Reveals Enzyme and Carrier Properties in Relation to Vacuole Expansion[W

    Science.gov (United States)

    Beauvoit, Bertrand P.; Colombié, Sophie; Monier, Antoine; Andrieu, Marie-Hélène; Biais, Benoit; Bénard, Camille; Chéniclet, Catherine; Dieuaide-Noubhani, Martine; Nazaret, Christine; Mazat, Jean-Pierre; Gibon, Yves

    2014-01-01

    A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division. PMID:25139005

  17. Selection of normal spermatozoa with a vacuole-free head (x6300) improves selection of spermatozoa with intact DNA in patients with high sperm DNA fragmentation rates.

    Science.gov (United States)

    Hammoud, I; Boitrelle, F; Ferfouri, F; Vialard, F; Bergere, M; Wainer, B; Bailly, M; Albert, M; Selva, J

    2013-06-01

    Intracytoplasmic morphologically selected sperm injection (IMSI, 6300× magnification with Nomarski contrast) of a normal spermatozoon with a vacuole-free head could improve the embryo's ability to grow to the blastocyst stage and then implant. However, the most relevant indications for IMSI remain to be determined. To evaluate the potential value of IMSI for patients with a high degree of sperm DNA fragmentation (n = 8), different types of spermatozoa were analysed in terms of DNA fragmentation. Motile normal spermatozoa with a vacuole-free head selected at 6300× magnification had a significantly lower mean DNA fragmentation rate (4.1 ± 1.1%, n = 191) than all other types of spermatozoa: non-selected spermatozoa (n = 8000; 26.1 ± 1.5% versus 4.1 ± 1.1%; P sperm DNA fragmentation rates, selection of normal spermatozoa with a vacuole-free head (6300×) yields the greatest likelihood of obtaining spermatozoa with non-fragmented DNA.

  18. Static Clathrin Assemblies at the Peripheral Vacuole-Plasma Membrane Interface of the Parasitic Protozoan Giardia lamblia.

    Science.gov (United States)

    Zumthor, Jon Paulin; Cernikova, Lenka; Rout, Samuel; Kaech, Andres; Faso, Carmen; Hehl, Adrian B

    2016-07-01

    Giardia lamblia is a parasitic protozoan that infects a wide range of vertebrate hosts including humans. Trophozoites are non-invasive but associate tightly with the enterocyte surface of the small intestine. This narrow ecological specialization entailed extensive morphological and functional adaptations during host-parasite co-evolution, including a distinctly polarized array of endocytic organelles termed peripheral vacuoles (PVs), which are confined to the dorsal cortical region exposed to the gut lumen and are in close proximity to the plasma membrane (PM). Here, we investigated the molecular consequences of these adaptations on the Giardia endocytic machinery and membrane coat complexes. Despite the absence of canonical clathrin coated vesicles in electron microscopy, Giardia possesses conserved PV-associated clathrin heavy chain (GlCHC), dynamin-related protein (GlDRP), and assembly polypeptide complex 2 (AP2) subunits, suggesting a novel function for GlCHC and its adaptors. We found that, in contrast to GFP-tagged AP2 subunits and DRP, CHC::GFP reporters have no detectable turnover in living cells, indicating fundamental differences in recruitment to the membrane and disassembly compared to previously characterized clathrin coats. Histochemical localization in electron tomography showed that these long-lived GlCHC assemblies localized at distinctive approximations between the plasma and PV membrane. A detailed protein interactome of GlCHC revealed all of the conserved factors in addition to novel or highly diverged proteins, including a putative clathrin light chain and lipid-binding proteins. Taken together, our data provide strong evidence for giardial CHC as a component of highly stable assemblies at PV-PM junctions that likely have a central role in organizing continuities between the PM and PV membranes for controlled sampling of the fluid environment. This suggests a novel function for CHC in Giardia and the extent of molecular remodeling of

  19. New insights into globoids of protein storage vacuoles in wheat aleurone using synchrotron soft X-ray microscopy.

    Science.gov (United States)

    Regvar, Marjana; Eichert, Diane; Kaulich, Burkhard; Gianoncelli, Alessandra; Pongrac, Paula; Vogel-Mikus, Katarina; Kreft, Ivan

    2011-07-01

    Mature developed seeds are physiologically and biochemically committed to store nutrients, principally as starch, protein, oils, and minerals. The composition and distribution of elements inside the aleurone cell layer reflect their biogenesis, structural characteristics, and physiological functions. It is therefore of primary importance to understand the mechanisms underlying metal ion accumulation, distribution, storage, and bioavailability in aleurone subcellular organelles for seed fortification purposes. Synchrotron radiation soft X-ray full-field imaging mode (FFIM) and low-energy X-ray fluorescence (LEXRF) spectromicroscopy were applied to characterize major structural features and the subcellular distribution of physiologically important elements (Zn, Fe, Na, Mg, Al, Si, and P). These direct imaging methods reveal the accumulation patterns between the apoplast and symplast, and highlight the importance of globoids with phytic acid mineral salts and walls as preferential storage structures. C, N, and O chemical topographies are directly linked to the structural backbone of plant substructures. Zn, Fe, Na, Mg, Al, and P were linked to globoid structures within protein storage vacuoles with variable levels of co-localization. Si distribution was atypical, being contained in the aleurone apoplast and symplast, supporting a physiological role for Si in addition to its structural function. These results reveal that the immobilization of metals within the observed endomembrane structures presents a structural and functional barrier and affects bioavailability. The combination of high spatial and chemical X-ray microscopy techniques highlights how in situ analysis can yield new insights into the complexity of the wheat aleurone layer, whose precise biochemical composition, morphology, and structural characteristics are still not unequivocally resolved.

  20. Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail.

    OpenAIRE

    1992-01-01

    Engagement of the cell surface receptor for interleukin 7 (IL-7R) provokes protein tyrosine phosphorylation, although the receptor lacks a kinase catalytic domain in its cytoplasmic tail. The molecular basis of this response is not known. Here we report that the IL-7R functions by recruiting p59fyn, an intracellular tyrosine kinase of the src family. Treatment of pre-B cells with IL-7 causes an enhancement of the catalytic activity of p59fyn, but not of the related kinase p62yes. IL-7-depende...

  1. Cotranscriptional assembly of mRNP complexes that determine the cytoplasmic fate of mRNA

    OpenAIRE

    Forget, Amélie; Chartrand, Pascal

    2011-01-01

    Unlike prokaryotes, in which transcription and translation are coupled, eukaryotes physically separate transcription in the nucleus from mRNA translation and degradation in the cytoplasm. However, recent evidence has revealed that the full picture is more complex and that the nuclear transcription machinery plays specific roles in regulating the cytoplasmic fate of mRNA.

  2. Sequential closure of the cytoplasm and then the periplasm during cell division in Escherichia coli.

    Science.gov (United States)

    Skoog, Karl; Söderström, Bill; Widengren, Jerker; von Heijne, Gunnar; Daley, Daniel O

    2012-02-01

    To visualize the latter stages of cell division in live Escherichia coli, we have carried out fluorescence recovery after photobleaching (FRAP) on 121 cells expressing cytoplasmic green fluorescent protein and periplasmic mCherry. Our data show conclusively that the cytoplasm is sealed prior to the periplasm during the division event.

  3. Nematode development after removal of egg cytoplasm: absence of localized unbound determinants.

    Science.gov (United States)

    Laufer, J S; von Ehrenstein, G

    1981-01-23

    Embryos of Caenorhabditis elegans develop into fertile adults after cell fragments, containing presumptive cytoplasm of somatic and germ line precursors, are extruded from uncleaved eggs or early blastomeres through laser-induced holes in the eggshells. This suggests that the determinate development of this worm is not dependent on the prelocalization of determinants in specific regions of the egg cytoplasm.

  4. Cytoplasmic pathway followed by chloride ions to enter the CFTR channel pore.

    Science.gov (United States)

    El Hiani, Yassine; Negoda, Alexander; Linsdell, Paul

    2016-05-01

    Most ATP-binding cassette (ABC) proteins function as ATP-dependent membrane pumps. One exception is the cystic fibrosis transmembrane conductance regulator (CFTR), an ABC protein that functions as a Cl(-) ion channel. As such, the CFTR protein must form a continuous pathway for the movement of Cl(-) ions from the cytoplasm to the extracellular solution when in its open channel state. Extensive functional investigations have characterized most parts of this Cl(-) permeation pathway. However, one region remains unexplored-the pathway connecting the cytoplasm to the membrane-spanning pore. We used patch clamp recording and extensive substituted cysteine accessibility mutagenesis to identify amino acid side-chains in cytoplasmic regions of CFTR that lie close to the pathway taken by Cl(-) ions as they pass from the cytoplasm through this pathway. Our results suggest that Cl(-) ions enter the permeation pathway via a single lateral tunnel formed by the cytoplasmic parts of the protein, and then follow a fairly direct central pathway towards the membrane-spanning parts of the protein. However, this pathway is not lined continuously by any particular part of the protein; instead, the contributions of different cytoplasmic regions of the protein appear to change as the permeation pathway approaches the membrane, which appears to reflect the ways in which different cytoplasmic regions of the protein are oriented towards its central axis. Our results allow us to define for the first time the complete Cl(-) permeation pathway in CFTR, from the cytoplasm to the extracellular solution.

  5. The distribution of special cytoplasmic differentiations of the egg during early cleavage in Limnaea stagnalis

    NARCIS (Netherlands)

    Raven, Chr.P.

    1967-01-01

    Uncleaved eggs of Limnaea stagnalis show a pattern of cytoplasmic differentiations in which there are 6 “subcortical accumulations” (SCA) in the equatorial region. SCA consist of a dense cytoplasmic matrix, containing a special kind of small granules. At certain stages also lens-shaped bodies, consi

  6. Molecular characterization of cytoplasmic male sterility (CMS) in perennial ryegrass ( Lolium perenne L.)

    DEFF Research Database (Denmark)

    Islam, Md. Shofiqul; Møller, Ian Max; Studer, Bruno;

    2011-01-01

    to increase biomass yield, improve nutritional value and tolerance towards abiotic and biotic stress. Cytoplasmic male sterility (CMS) is an efficient tool to control pollination for hybrid seed production. In order to identify the causative polymorphism of the CMS phenotype, a cytoplasmic male sterile plant...... genomes will enable to identify the causative polymorphism of CMS phenotype in perennial ryegrass....

  7. Effect of wild Helianthus cytoplasms on agronomic and oil characteristics of cultivated sunflower (H. annuus L.)

    Science.gov (United States)

    Sunflower (Helianthus annuus L.) productions reliance on a single source of cytoplasmic male-sterility, PET1, derived from H. petiolaris Nutt., makes the crop genetically vulnerable. Twenty diverse cytoplasmic substitution lines from annual and perennial wild species were compared with the inbred li...

  8. Cytoplasmic factors do not contribute to a maternal effect on ethanol teratogenesis.

    Science.gov (United States)

    Downing, C; Gilliam, D

    1999-01-01

    Both maternal and fetal genetic factors influence variations in response to prenatal ethanol exposure. To assess the effect of maternal genotype on the incidence of ethanol teratogenesis, a reciprocal cross study was conducted in an animal mode using the relatively susceptible C57BL/6J (B6) and the relatively resistant DBA/2J (D2) inbred mice. This mating pattern produced four embryonic genotypes: true-bred B6B6 and D2D2 litters and hybrid B6D2 and D2B6 litters. To examine the role of maternal egg cytoplasm as the source of variation that could account for a maternal effect, B6D2 and D2B6 F1 females were mated back to B6 males, which produced two additional embryonic genotypes: B6D2.B6 and D2B6.B6. Dams were intubated with either 5.8 g/kg of ethanol or an isocaloric amount of maltose-dextrin on day 9 of pregnancy. On day 18 of pregnancy, dams were sacrificed, fetuses were removed, weighed, sexed, and examined for gross morphological malformations. Every other fetus within a litter was prepared for either skeletal or soft tissue analysis. Results showed a higher rate of teratogenesis in the B6D2 group compared to the genetically similar D2B6 group, which indicates an influence of maternal genotype on susceptibility to ethanol teratogenesis. The percentage of affected male and female fetuses did not differ, which suggests that sex-linked factors are not responsible for the maternal effect. The backcross B6D2.B6 and D2B6.B6 litters did not differ significantly for any measure of teratogenesis, suggesting that differences in maternally transmitted cytoplasmic material are not the cause of the maternal effect. Factors that could account for the maternal effect are differences in the maternal uterine environment and genomic imprinting. Separating maternal from fetal-mediated mechanisms responsible for susceptibility to ethanol teratogenesis is needed for identifying mothers and infants at risk.

  9. Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs.

    Science.gov (United States)

    Coffe, G; Foucault, G; Raymond, M N; Pudles, J

    1983-12-01

    Sedimentation studies and [3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20-25% of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles.

  10. Cytoplasmic genetic variation and extensive cytonuclear interactions influence natural variation in the metabolome

    DEFF Research Database (Denmark)

    Joseph, Bindu; Corwin, Jason A.; Li, Baohua

    2013-01-01

    affects phenotypic variation. This showed that the cytoplasmic variation had effects similar to, if not larger than, the largest individual nuclear locus. Inclusion of cytoplasmic variation into the genetic model greatly increased the explained phenotypic variation. Cytoplasmic genetic variation...... was a central hub in the epistatic network controlling the plant metabolome. This epistatic influence manifested such that the cytoplasmic background could alter or hide pairwise epistasis between nuclear loci. Thus, cytoplasmic genetic variation plays a central role in controlling natural variation......Understanding genome to phenotype linkages has been greatly enabled by genomic sequencing. However, most genome analysis is typically confined to the nuclear genome. We conducted a metabolomic QTL analysis on a reciprocal RIL population structured to examine how variation in the organelle genomes...

  11. Cytoplasmic phospholipase A2 deletion enhances colon tumorigenesis.

    Science.gov (United States)

    Ilsley, Jillian N M; Nakanishi, Masako; Flynn, Christopher; Belinsky, Glenn S; De Guise, Sylvain; Adib, John N; Dobrowsky, Rick T; Bonventre, Joseph V; Rosenberg, Daniel W

    2005-04-01

    Cellular pools of free arachidonic acid are tightly controlled through enzymatic release of the fatty acid and subsequent utilization by downstream enzymes including the cyclooxygenases. Arachidonic acid cleavage from membrane phospholipids is accomplished by the actions of phospholipase A(2) (PLA(2)). Upon release, free arachidonic acid provides substrate for the synthesis of eicosanoids. However, under certain conditions, arachidonic acid may participate in ceramide-mediated apoptosis. Disruption of arachidonic acid homeostasis can shift the balance of cell turnover in favor of tumorigenesis, via overproduction of tumor-promoting eicosanoids or alternatively by limiting proapoptotic signals. In the following study, we evaluated the influence of genetic deletion of a key intracellular phospholipase, cytoplasmic PLA(2) (cPLA(2)), on azoxymethane-induced colon tumorigenesis. Heterozygous and null mice, upon treatment with the organotropic colon carcinogen, azoxymethane, developed a significant (P < 0.05) increase in colon tumor multiplicity (7.2-fold and 5.5-fold, respectively) relative to their wild-type littermates. This enhanced tumor sensitivity may be explained, in part, by the attenuated levels of apoptosis observed by terminal deoxynucleotidyl transferase-mediated nick end labeling staining within the colonic epithelium of heterozygous and null mice ( approximately 50% of wild type). The lower frequency of apoptotic cells corresponded with reduced ceramide levels (69% and 46% of wild-type littermates, respectively). Remarkably, increased tumorigenesis resulting from cPLA(2) deletion occurred despite a significant reduction in prostaglandin E(2) production, even in cyclooxygenase-2-overexpressing tumors. These data contribute new information that supports a fundamental role of cPLA(2) in the control of arachidonic acid homeostasis and cell turnover. Our findings indicate that the proapoptotic role of cPLA(2) in the colon may supercede its contribution to

  12. Urinary Biomarkers in Relapsing Antineutrophil Cytoplasmic Antibody-associated Vasculitis

    Science.gov (United States)

    Lieberthal, Jason G.; Cuthbertson, David; Carette, Simon; Hoffman, Gary S.; Khalidi, Nader A.; Koening, Curry L.; Langford, Carol A.; Maksimowicz-McKinnon, Kathleen; Seo, Philip; Specks, Ulrich; Ytterberg, Steven R.; Merkel, Peter A.; Monach, Paul A.

    2015-01-01

    Objective Glomerulonephritis (GN) is common in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), but tools for early detection of renal involvement are imperfect. We investigated 4 urinary proteins as markers of active renal AAV: alpha-1 acid glycoprotein (AGP), kidney injury molecule-1 (KIM-1), monocyte chemoattractant protein-1 (MCP-1), and neutrophil gelatinase-associated lipocalin (NGAL). Methods Patients with active renal AAV (n = 20), active nonrenal AAV (n = 16), and AAV in longterm remission (n = 14) were identified within a longitudinal cohort. Urinary biomarker concentrations (by ELISA) were normalized for urine creatinine. Marker levels during active AAV were compared to baseline remission levels (from 1–4 visits) for each patient. Areas under receiver-operating characteristic curves (AUC), sensitivities, specificities, and likelihood ratios (LR) comparing disease states were calculated. Results Baseline biomarker levels varied among patients. All 4 markers increased during renal flares (p < 0.05). MCP-1 discriminated best between active renal disease and remission: a 1.3-fold increase in MCP-1 had 94% sensitivity and 89% specificity for active renal disease (AUC = 0.93, positive LR 8.5, negative LR 0.07). Increased MCP-1 also characterized 50% of apparently nonrenal flares. Change in AGP, KIM-1, or NGAL showed more modest ability to distinguish active renal disease from remission (AUC 0.71–0.75). Hematuria was noted in 83% of active renal episodes, but also 43% of nonrenal flares and 25% of remission samples. Conclusion Either urinary MCP-1 is not specific for GN in AAV, or it identifies early GN not detected by standard assessment and thus has potential to improve care. A followup study with kidney biopsy as the gold standard is needed. PMID:23547217

  13. Nuclear reprogramming by interphase cytoplasm of two-cell mouse embryos.

    Science.gov (United States)

    Kang, Eunju; Wu, Guangming; Ma, Hong; Li, Ying; Tippner-Hedges, Rebecca; Tachibana, Masahito; Sparman, Michelle; Wolf, Don P; Schöler, Hans R; Mitalipov, Shoukhrat

    2014-05-01

    Successful mammalian cloning using somatic cell nuclear transfer (SCNT) into unfertilized, metaphase II (MII)-arrested oocytes attests to the cytoplasmic presence of reprogramming factors capable of inducing totipotency in somatic cell nuclei. However, these poorly defined maternal factors presumably decline sharply after fertilization, as the cytoplasm of pronuclear-stage zygotes is reportedly inactive. Recent evidence suggests that zygotic cytoplasm, if maintained at metaphase, can also support derivation of embryonic stem (ES) cells after SCNT, albeit at low efficiency. This led to the conclusion that critical oocyte reprogramming factors present in the metaphase but not in the interphase cytoplasm are 'trapped' inside the nucleus during interphase and effectively removed during enucleation. Here we investigated the presence of reprogramming activity in the cytoplasm of interphase two-cell mouse embryos (I2C). First, the presence of candidate reprogramming factors was documented in both intact and enucleated metaphase and interphase zygotes and two-cell embryos. Consequently, enucleation did not provide a likely explanation for the inability of interphase cytoplasm to induce reprogramming. Second, when we carefully synchronized the cell cycle stage between the transplanted nucleus (ES cell, fetal fibroblast or terminally differentiated cumulus cell) and the recipient I2C cytoplasm, the reconstructed SCNT embryos developed into blastocysts and ES cells capable of contributing to traditional germline and tetraploid chimaeras. Last, direct transfer of cloned embryos, reconstructed with ES cell nuclei, into recipients resulted in live offspring. Thus, the cytoplasm of I2C supports efficient reprogramming, with cell cycle synchronization between the donor nucleus and recipient cytoplasm as the most critical parameter determining success. The ability to use interphase cytoplasm in SCNT could aid efforts to generate autologous human ES cells for regenerative

  14. The fertilization—induced Ca2+ oscillation in mouse oocytes is cytoplasmic maturation dependent

    Institute of Scientific and Technical Information of China (English)

    DENGMANQI; FANGZHENSUN

    1996-01-01

    Mature eggs (at metaphase II stage) produce a series of Ca2+ oscillation at fertilization.To define whether the fertilization-induced Ca2+ oscillation is restrict to the metaphase II eggs and cell cycle dependent,mouse oocytes at prophase I (arrested at germinal vesicle stage),metaphase I,metaphase II,as well as the pronuclear embryos at interphase of the first mitotic division derived from fertilization of parthenogenetic activation were inseminated after removal of zona pellucida,The results show that the fertilization-induced Ca2+ oscillation is not specific to metaphase II eggs.This is supported by the fact that immature oocytes generated the Ca2+ oscillations at fertilization regardless of their nuclear progression from prophase I to metaphase I (in vitro matured) stage.More interestingly,it was first found that pronuclear embryos at interphase derived from parthenogenetic activation showed Ca2+ oscillations in response to fertilization while the zygotes at interphase did not after reinsemination or intracytoplasmic injection of sperm extracts which induce Ca2+ oscillations in MII eggs.This suggests that the ability of oocytes to generate Ca2+ oscillation in response to sperm penetration is not regulated in a cell cycle dependent manner but dependent on the cytoplasmic maturation.

  15. 青霉素处理检查和分离蓝藻细胞液泡%Determination and Isolation of Cell Vacuoles from Blue-green Algae by Penicillin Method

    Institute of Scientific and Technical Information of China (English)

    郭碧薇; 易平; 刘希玲; 郭厚良

    2003-01-01

    Growing in the liquid medium containing penicillin, the cells of the Cyanobacteria,Anabaena 7120, Nostoc flagelliforme, and Synechocystis 6803 were broken and vacuoles were released. Percentage of broken cells declined and percentage of broken cells increased with the growing days of the algae. The percentage of vacuoles to broken cells were respectively 0.7%, 0.8%, and 13.3% in the three types of algae Anabaena 7120, N.flagelliforme and Synechocystis 6803 which had grown for 3 days.

  16. Cytoplasmic localization of Hug1p, a negative regulator of the MEC1 pathway, coincides with the compartmentalization of Rnr2p–Rnr4p

    Energy Technology Data Exchange (ETDEWEB)

    Ainsworth, William B. [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 (United States); Hughes, Bridget Todd; Au, Wei Chun; Sakelaris, Sally [Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Kerscher, Oliver [Biology Department, The College of William and Mary, Williamsburg, VA 23185 (United States); Benton, Michael G., E-mail: benton@lsu.edu [Cain Department of Chemical Engineering, Louisiana State University, Baton Rouge, LA 70803 (United States); Basrai, Munira A., E-mail: basraim@mail.nih.gov [Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States)

    2013-10-04

    Highlights: •Hug1p overexpression sensitizes wild-type cells to DNA damage and hydroxyurea (HU). •Expression of Hug1p in response to HU treatment is delayed relative to Rnr3p. •MEC1 pathway genes are required for cytoplasmic localization of Hug1p. •Hug1p subcellular compartmentalization to the cytoplasm coincides with Rnr2p–Rnr4p. -- Abstract: The evolutionarily conserved MEC1 checkpoint pathway mediates cell cycle arrest and induction of genes including the RNR (Ribonucleotide reductase) genes and HUG1 (Hydroxyurea, ultraviolet, and gamma radiation) in response to DNA damage and replication arrest. Rnr complex activity is in part controlled by cytoplasmic localization of the Rnr2p–Rnr4p subunits and inactivation of negative regulators Sml1p and Dif1p upon DNA damage and hydroxyurea (HU) treatment. We previously showed that a deletion of HUG1 rescues lethality of mec1Δ and suppresses dun1Δ strains. In this study, multiple approaches demonstrate the regulatory response of Hug1p to DNA damage and HU treatment and support its role as a negative effector of the MEC1 pathway. Consistent with our hypothesis, wild-type cells are sensitive to DNA damage and HU when HUG1 is overexpressed. A Hug1 polyclonal antiserum reveals that HUG1 encodes a protein in budding yeast and its MEC1-dependent expression is delayed compared to the rapid induction of Rnr3p in response to HU treatment. Cell biology and subcellular fractionation experiments show localization of Hug1p-GFP to the cytoplasm upon HU treatment. The cytoplasmic localization of Hug1p-GFP is dependent on MEC1 pathway genes and coincides with the cytoplasmic localization of Rnr2p–Rnr4p. Taken together, the genetic interactions, gene expression, and localization studies support a novel role for Hug1p as a negative regulator of the MEC1 checkpoint response through its compartmentalization with Rnr2p–Rnr4p.

  17. High Temperature as a Mechanism for Plant Cytoplasm Preservation in Fossils

    Institute of Scientific and Technical Information of China (English)

    WANG Xin

    2007-01-01

    Because the cytoplasm of a plant normally degrades after the death of the plant, finding cytoplasm in a plant body after a prolonged period of time, especially in fossil plants, is unexpected.Recent work on several 100-Myr-old plant fossils from Kansas, USA indicates, however, that cells and their contents can be preserved. Most of the cells in these fossil plants appear to be in a state of plasmolysis, and these fossil cells bear a strong resemblance to laboratory-baked cells of extant plant tissues. Based on a comparison with extant material plus biophysical and biochemical analyses of the cytoplasm degrading process, a new hypothesis for cytoplasm preservation in nature is proposed: high temperature, a concomitant of commonly seen wildfires, may preserve cytopla