Sample records for acidic vesicular organelle

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

    PubMed

    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.

  2. Stochastic Model of Vesicular Sorting in Cellular Organelles

    NASA Astrophysics Data System (ADS)

    Vagne, Quentin; Sens, Pierre

    2018-02-01

    The proper sorting of membrane components by regulated exchange between cellular organelles is crucial to intracellular organization. This process relies on the budding and fusion of transport vesicles, and should be strongly influenced by stochastic fluctuations, considering the relatively small size of many organelles. We identify the perfect sorting of two membrane components initially mixed in a single compartment as a first passage process, and we show that the mean sorting time exhibits two distinct regimes as a function of the ratio of vesicle fusion to budding rates. Low ratio values lead to fast sorting but result in a broad size distribution of sorted compartments dominated by small entities. High ratio values result in two well-defined sorted compartments but sorting is exponentially slow. Our results suggest an optimal balance between vesicle budding and fusion for the rapid and efficient sorting of membrane components and highlight the importance of stochastic effects for the steady-state organization of intracellular compartments.

  3. Analysis of a vesicular glutamate transporter (VGLUT2) supports a cell-leakage mode in addition to vesicular packaging.

    PubMed

    Mackenzie, Bryan; Illing, Anthony C; Morris, Marie E K; Varoqui, Hélène; Erickson, Jeffrey D

    2008-02-01

    VGLUT2 is one of three vesicular glutamate transporters that play crucial roles in glutamatergic excitatory neurotransmission. We explored the functional properties of the rat VGLUT2 by heterologous expression of VGLUT2 in Xenopus oocytes. Immunocytochemical analysis indicated that most VGLUT2 protein was expressed in intracellular compartments but that some expression occurred also on the plasma membrane. Functional analysis revealed VGLUT2 to be active in two independent modes, namely, uptake into intracellular organelles and efflux at the plasma membrane. VGLUT-specific transport was identified based on the strong preference for glutamate over aspartate--in contrast to plasma-membrane or mitochondrial glutamate transporters--and sensitivity to known VGLUT blockers. VGLUT2 expression in oocytes (1) stimulated the influx of L-[(3)H]glutamate, but not D-[(3)H]aspartate, into digitonin-permeabilized oocytes and (2) stimulated efflux of L-glutamate, but not L-aspartate, from intact oocytes preinjected with (3)H-labeled amino acids. In the latter assay, cellular efflux of glutamate (which was blocked by rose bengal and trypan blue) may be analogous to vesicular packaging of glutamate. Our data are consistent with VGLUT2-mediated H(+)/L-glutamate antiport, but not antiport with chloride. Expression of mammalian VGLUT1 and VGLUT3 also stimulated L-[(3)H]glutamate efflux from Xenopus oocytes, suggesting that this phenomenon is a general feature of vesicular glutamate transporters. Our findings support the idea that vesicular glutamate transporters, when transiently expressed on the neuronal plasma membrane, may mediate Ca(2+)-independent glutamate leakage in addition to their traditional role of packaging glutamate into synaptic vesicles for Ca(2+)-dependent exocytosis.

  4. Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy.

    PubMed

    Thomé, Marcos P; Filippi-Chiela, Eduardo C; Villodre, Emilly S; Migliavaca, Celina B; Onzi, Giovana R; Felipe, Karina B; Lenz, Guido

    2016-12-15

    Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods. © 2016. Published by The Company of Biologists Ltd.

  5. Regulation of Organelle Acidity

    PubMed Central

    Grabe, Michael; Oster, George

    2001-01-01

    Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674–687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on the interplay of ion pumps and channels, the physical properties of the lumenal matrix, and the organelle geometry. The model successfully predicts experimentally measured steady-state and transient pH values and membrane potentials. We conclude that morphological differences among organelles are insufficient to explain the wide range of pHs present in the cell. Using sensitivity analysis, we quantified the influence of pH regulatory elements on the dynamics of acidification. We found that V-ATPase proton pump and proton leak densities are the two parameters that most strongly influence resting pH. Additionally, we modeled the pH response of the Golgi complex to varying external solutions, and our findings suggest that the membrane is permeable to more than one dominant counter ion. From this data, we determined a Golgi complex proton permeability of 8.1 × 10−6 cm/s. Furthermore, we analyzed the early-to-late transition in the endosomal pathway where Na,K-ATPases have been shown to limit acidification by an entire pH unit. Our model supports the role of the Na,K-ATPase in regulating endosomal pH by affecting the membrane potential. However, experimental data can only be reproduced by (1) positing the existence of a hypothetical voltage-gated chloride channel or (2) that newly formed vesicles have especially high potassium concentrations and small chloride conductance. PMID:11279253

  6. Membraneless organelles can melt nucleic acid duplexes and act as biomolecular filters

    NASA Astrophysics Data System (ADS)

    Nott, Timothy J.; Craggs, Timothy D.; Baldwin, Andrew J.

    2016-06-01

    Membraneless organelles are cellular compartments made from drops of liquid protein inside a cell. These compartments assemble via the phase separation of disordered regions of proteins in response to changes in the cellular environment and the cell cycle. Here we demonstrate that the solvent environment within the interior of these cellular bodies behaves more like an organic solvent than like water. One of the most-stable biological structures known, the DNA double helix, can be melted once inside the liquid droplet, and simultaneously structures formed from regulatory single-stranded nucleic acids are stabilized. Moreover, proteins are shown to have a wide range of absorption or exclusion from these bodies, and can act as importers for otherwise-excluded nucleic acids, which suggests the existence of a protein-mediated trafficking system. A common strategy in organic chemistry is to utilize different solvents to influence the behaviour of molecules and reactions. These results reveal that cells have also evolved this capability by exploiting the interiors of membraneless organelles.

  7. Organelle Communication at Membrane Contact Sites (MCS): From Curiosity to Center Stage in Cell Biology and Biomedical Research.

    PubMed

    Simmen, Thomas; Tagaya, Mitsuo

    2017-01-01

    Cell biology has long recognized that organelles can communicate with each other. Initially, such communication was thought to occur primarily via vesicular trafficking between biochemically distinct organelles. However, studies starting in the 1970s on lipid metabolism have unearthed another way how organelles can communicate and have spawned the field of membrane contact sites (MCS). While, initially, MCS had been recognized as fluid entities that mediate lipid and ion transport in an ad hoc manner, more recently MCS have been found to depend on protein-protein interactions that control themselves a variety of MCS functions. As a result, the cell biological definition of an intracellular organelle as an isolated membrane compartment is now being revised. Accordingly, the organelle definition now describes organelles as dynamic membrane compartments that function in a milieu of coordinated contacts with other organelles. Through these mercurial functions, MCS dictate the function of organelles to a large extent but also play important roles in a number of diseases, including type 2 diabetes, neurodegenerative diseases, infections, and cancer. This book assembles reviews that describe our quickly evolving knowledge about organellar communication on MCS and the significance of MCS for disease.

  8. Co-existence of Functionally Different Vesicular Neurotransmitter Transporters.

    PubMed

    Münster-Wandowski, Agnieszka; Zander, Johannes-Friedrich; Richter, Karin; Ahnert-Hilger, Gudrun

    2016-01-01

    The vesicular transmitter transporters VGLUT, VGAT, VMAT2 and VAChT, define phenotype and physiological properties of neuronal subtypes. VGLUTs concentrate the excitatory amino acid glutamate, VGAT the inhibitory amino acid GABA, VMAT2 monoamines, and VAChT acetylcholine (ACh) into synaptic vesicle (SV). Following membrane depolarization SV release their content into the synaptic cleft. A strict segregation of vesicular transporters is mandatory for the precise functioning of synaptic communication and of neuronal circuits. In the last years, evidence accumulates that subsets of neurons express more than one of these transporters leading to synaptic co-release of different and functionally opposing transmitters and modulation of synaptic plasticity. Synaptic co-existence of transporters may change during pathological scenarios in order to ameliorate misbalances in neuronal activity. In addition, evidence increases that transporters also co-exist on the same vesicle providing another layer of regulation. Generally, vesicular transmitter loading relies on an electrochemical gradient ΔμH(+) driven by the proton ATPase rendering the lumen of the vesicle with respect to the cytosol positive (Δψ) and acidic (ΔpH). While the activity of VGLUT mainly depends on the Δψ component, VMAT, VGAT and VAChT work best at a high ΔpH. Thus, a vesicular synergy of transporters depending on the combination may increase or decrease the filling of SV with the principal transmitter. We provide an overview on synaptic co-existence of vesicular transmitter transporters including changes in the excitatory/inhibitory balance under pathological conditions. Additionally, we discuss functional aspects of vesicular synergy of transmitter transporters.

  9. Co-existence of Functionally Different Vesicular Neurotransmitter Transporters

    PubMed Central

    Münster-Wandowski, Agnieszka; Zander, Johannes-Friedrich; Richter, Karin; Ahnert-Hilger, Gudrun

    2016-01-01

    The vesicular transmitter transporters VGLUT, VGAT, VMAT2 and VAChT, define phenotype and physiological properties of neuronal subtypes. VGLUTs concentrate the excitatory amino acid glutamate, VGAT the inhibitory amino acid GABA, VMAT2 monoamines, and VAChT acetylcholine (ACh) into synaptic vesicle (SV). Following membrane depolarization SV release their content into the synaptic cleft. A strict segregation of vesicular transporters is mandatory for the precise functioning of synaptic communication and of neuronal circuits. In the last years, evidence accumulates that subsets of neurons express more than one of these transporters leading to synaptic co-release of different and functionally opposing transmitters and modulation of synaptic plasticity. Synaptic co-existence of transporters may change during pathological scenarios in order to ameliorate misbalances in neuronal activity. In addition, evidence increases that transporters also co-exist on the same vesicle providing another layer of regulation. Generally, vesicular transmitter loading relies on an electrochemical gradient ΔμH+ driven by the proton ATPase rendering the lumen of the vesicle with respect to the cytosol positive (Δψ) and acidic (ΔpH). While the activity of VGLUT mainly depends on the Δψ component, VMAT, VGAT and VAChT work best at a high ΔpH. Thus, a vesicular synergy of transporters depending on the combination may increase or decrease the filling of SV with the principal transmitter. We provide an overview on synaptic co-existence of vesicular transmitter transporters including changes in the excitatory/inhibitory balance under pathological conditions. Additionally, we discuss functional aspects of vesicular synergy of transmitter transporters. PMID:26909036

  10. Protons Regulate Vesicular Glutamate Transporters through an Allosteric Mechanism.

    PubMed

    Eriksen, Jacob; Chang, Roger; McGregor, Matt; Silm, Katlin; Suzuki, Toshiharu; Edwards, Robert H

    2016-05-18

    The quantal nature of synaptic transmission requires a mechanism to transport neurotransmitter into synaptic vesicles without promoting non-vesicular efflux across the plasma membrane. Indeed, the vesicular transport of most classical transmitters involves a mechanism of H(+) exchange, which restricts flux to acidic membranes such as synaptic vesicles. However, vesicular transport of the principal excitatory transmitter glutamate depends primarily on membrane potential, which would drive non-vesicular efflux, and the role of protons is unclear. Adapting electrophysiology to record currents associated with the vesicular glutamate transporters (VGLUTs), we characterize a chloride conductance that is gated by lumenal protons and chloride and supports glutamate uptake. Rather than coupling stoichiometrically to glutamate flux, lumenal protons and chloride allosterically activate vesicular glutamate transport. Gating by protons serves to inhibit what would otherwise be substantial non-vesicular glutamate efflux at the plasma membrane, thereby restricting VGLUT activity to synaptic vesicles. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Investigating the effect of poly-l-lactic acid nanoparticles carrying hypericin on the flow-biased diffusive motion of HeLa cell organelles.

    PubMed

    Penjweini, Rozhin; Deville, Sarah; Haji Maghsoudi, Omid; Notelaers, Kristof; Ethirajan, Anitha; Ameloot, Marcel

    2017-07-19

    In this study, we investigate in human cervical epithelial HeLa cells the intracellular dynamics and the mutual interaction with the organelles of the poly-l-lactic acid nanoparticles (PLLA NPs) carrying the naturally occurring hydrophobic photosensitizer hypericin. Temporal and spatiotemporal image correlation spectroscopy was used for the assessment of the intracellular diffusion and directed motion of the nanocarriers by tracking the hypericin fluorescence. Using image cross-correlation spectroscopy and specific fluorescent labelling of endosomes, lysosomes and mitochondria, the NPs dynamics in association with the cell organelles was studied. Static colocalization experiments were interpreted according to the Manders' overlap coefficient. Nanoparticles associate with a small fraction of the whole-organelle population. The organelles moving with NPs exhibit higher directed motion compared to those moving without them. The rate of the directed motion drops substantially after the application of nocodazole. The random component of the organelle motions is not influenced by the NPs. Image correlation and cross-correlation spectroscopy are most appropriate to unravel the motion of the PLLA nanocarrier and to demonstrate that the rate of the directed motion of organelles is influenced by their interaction with the nanocarriers. Not all PLLA-hypericin NPs are associated with organelles. © 2017 Royal Pharmaceutical Society.

  12. The different facets of organelle interplay-an overview of organelle interactions.

    PubMed

    Schrader, Michael; Godinho, Luis F; Costello, Joseph L; Islinger, Markus

    2015-01-01

    Membrane-bound organelles such as mitochondria, peroxisomes, or the endoplasmic reticulum (ER) create distinct environments to promote specific cellular tasks such as ATP production, lipid breakdown, or protein export. During recent years, it has become evident that organelles are integrated into cellular networks regulating metabolism, intracellular signaling, cellular maintenance, cell fate decision, and pathogen defence. In order to facilitate such signaling events, specialized membrane regions between apposing organelles bear distinct sets of proteins to enable tethering and exchange of metabolites and signaling molecules. Such membrane associations between the mitochondria and a specialized site of the ER, the mitochondria associated-membrane (MAM), as well as between the ER and the plasma membrane (PAM) have been partially characterized at the molecular level. However, historical and recent observations imply that other organelles like peroxisomes, lysosomes, and lipid droplets might also be involved in the formation of such apposing membrane contact sites. Alternatively, reports on so-called mitochondria derived-vesicles (MDV) suggest alternative mechanisms of organelle interaction. Moreover, maintenance of cellular homeostasis requires the precise removal of aged organelles by autophagy-a process which involves the detection of ubiquitinated organelle proteins by the autophagosome membrane, representing another site of membrane associated-signaling. This review will summarize the available data on the existence and composition of organelle contact sites and the molecular specializations each site uses in order to provide a timely overview on the potential functions of organelle interaction.

  13. Visualizing multiple inter-organelle contact sites using the organelle-targeted split-GFP system.

    PubMed

    Kakimoto, Yuriko; Tashiro, Shinya; Kojima, Rieko; Morozumi, Yuki; Endo, Toshiya; Tamura, Yasushi

    2018-04-18

    Functional integrity of eukaryotic organelles relies on direct physical contacts between distinct organelles. However, the entity of organelle-tethering factors is not well understood due to lack of means to analyze inter-organelle interactions in living cells. Here we evaluate the split-GFP system for visualizing organelle contact sites in vivo and show its advantages and disadvantages. We observed punctate GFP signals from the split-GFP fragments targeted to any pairs of organelles among the ER, mitochondria, peroxisomes, vacuole and lipid droplets in yeast cells, which suggests that these organelles form contact sites with multiple organelles simultaneously although it is difficult to rule out the possibilities that these organelle contacts sites are artificially formed by the irreversible associations of the split-GFP probes. Importantly, split-GFP signals in the overlapped regions of the ER and mitochondria were mainly co-localized with ERMES, an authentic ER-mitochondria tethering structure, suggesting that split-GFP assembly depends on the preexisting inter-organelle contact sites. We also confirmed that the split-GFP system can be applied to detection of the ER-mitochondria contact sites in HeLa cells. We thus propose that the split-GFP system is a potential tool to observe and analyze inter-organelle contact sites in living yeast and mammalian cells.

  14. Increased Long Chain acyl-Coa Synthetase Activity and Fatty Acid Import Is Linked to Membrane Synthesis for Development of Picornavirus Replication Organelles

    PubMed Central

    Scott, Alison J.; Ford, Lauren A.; Pei, Zhengtong; Watkins, Paul A.; Ernst, Robert K.; Belov, George A.

    2013-01-01

    All positive strand (+RNA) viruses of eukaryotes replicate their genomes in association with membranes. The mechanisms of membrane remodeling in infected cells represent attractive targets for designing future therapeutics, but our understanding of this process is very limited. Elements of autophagy and/or the secretory pathway were proposed to be hijacked for building of picornavirus replication organelles. However, even closely related viruses differ significantly in their requirements for components of these pathways. We demonstrate here that infection with diverse picornaviruses rapidly activates import of long chain fatty acids. While in non-infected cells the imported fatty acids are channeled to lipid droplets, in infected cells the synthesis of neutral lipids is shut down and the fatty acids are utilized in highly up-regulated phosphatidylcholine synthesis. Thus the replication organelles are likely built from de novo synthesized membrane material, rather than from the remodeled pre-existing membranes. We show that activation of fatty acid import is linked to the up-regulation of cellular long chain acyl-CoA synthetase activity and identify the long chain acyl-CoA syntheatse3 (Acsl3) as a novel host factor required for polio replication. Poliovirus protein 2A is required to trigger the activation of import of fatty acids independent of its protease activity. Shift in fatty acid import preferences by infected cells results in synthesis of phosphatidylcholines different from those in uninfected cells, arguing that the viral replication organelles possess unique properties compared to the pre-existing membranes. Our data show how poliovirus can change the overall cellular membrane homeostasis by targeting one critical process. They explain earlier observations of increased phospholipid synthesis in infected cells and suggest a simple model of the structural development of the membranous scaffold of replication complexes of picorna-like viruses, that may be

  15. Biologically active peptides of the vesicular stomatitis virus glycoprotein.

    PubMed Central

    Schlegel, R; Wade, M

    1985-01-01

    A peptide corresponding to the amino-terminal 25 amino acids of the mature vesicular stomatitis virus glycoprotein has recently been shown to be a pH-dependent hemolysin. In the present study, we analyzed smaller constituent peptides and found that the hemolytic domain resides within the six amino-terminal amino acids. Synthesis of variant peptides indicates that the amino-terminal lysine can be replaced by another positively charged amino acid (arginine) but that substitution with glutamic acid results in the total loss of the hemolytic function. Peptide-induced hemolysis was dependent upon buffer conditions and was inhibited when isotonicity was maintained with mannitol, sucrose, or raffinose. In sucrose, all hemolytic peptides were also observed to mediate hemagglutination. The large 25-amino acid peptide is also a pH-dependent cytotoxin for mammalian cells and appears to effect gross changes in cell permeability. Conservation of the amino terminus of vesicular stomatitis virus and rabies virus suggests that the membrane-destabilizing properties of this domain may be important for glycoprotein function. Images PMID:2981356

  16. Regulation of Monocarboxylic Acid Transporter-1 by cAMP Dependent Vesicular Trafficking in Brain Microvascular Endothelial Cells

    PubMed Central

    Uhernik, Amy L.; Li, Lun; LaVoy, Nathan; Velasquez, Micah J.; Smith, Jeffrey P.

    2014-01-01

    In this study, a detailed characterization of Monocarboxylic Acid Transporter-1 (Mct1) in cytoplasmic vesicles of cultured rat brain microvascular endothelial cells shows them to be a diverse population of endosomes intrinsic to the regulation of the transporter by a brief 25 to 30 minute exposure to the membrane permeant cAMP analog, 8Br-cAMP. The vesicles are heterogeneous in size, mobility, internal pH, and co-localize with discreet markers of particular types of endosomes including early endosomes, clathrin coated vesicles, caveolar vesicles, trans-golgi, and lysosomes. The vesicular localization of Mct1 was not dependent on its N or C termini, however, the size and pH of Mct1 vesicles was increased by deletion of either terminus demonstrating a role for the termini in vesicular trafficking of Mct1. Using a novel BCECF-AM based assay developed in this study, 8Br-cAMP was shown to decrease the pH of Mct1 vesicles after 25 minutes. This result and method were confirmed in experiments with a ratiometric pH-sensitive EGFP-mCherry dual tagged Mct1 construct. Overall, the results indicate that cAMP signaling reduces the functionality of Mct1 in cerebrovascular endothelial cells by facilitating its entry into a highly dynamic vesicular trafficking pathway that appears to lead to the transporter's trafficking to autophagosomes and lysosomes. PMID:24454947

  17. Polymeric Nucleic Acid Vehicles Exploit Active Inter-Organelle Trafficking Mechanisms

    PubMed Central

    Fichter, Katye M.; Ingle, Nilesh. P.; McLendon, Patrick M.; Reineke, Theresa M.

    2013-01-01

    Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery. PMID:23234474

  18. Designer amphiphilic proteins as building blocks for the intracellular formation of organelle-like compartments

    NASA Astrophysics Data System (ADS)

    Huber, Matthias C.; Schreiber, Andreas; von Olshausen, Philipp; Varga, Balázs R.; Kretz, Oliver; Joch, Barbara; Barnert, Sabine; Schubert, Rolf; Eimer, Stefan; Kele, Péter; Schiller, Stefan M.

    2015-01-01

    Nanoscale biological materials formed by the assembly of defined block-domain proteins control the formation of cellular compartments such as organelles. Here, we introduce an approach to intentionally ‘program’ the de novo synthesis and self-assembly of genetically encoded amphiphilic proteins to form cellular compartments, or organelles, in Escherichia coli. These proteins serve as building blocks for the formation of artificial compartments in vivo in a similar way to lipid-based organelles. We investigated the formation of these organelles using epifluorescence microscopy, total internal reflection fluorescence microscopy and transmission electron microscopy. The in vivo modification of these protein-based de novo organelles, by means of site-specific incorporation of unnatural amino acids, allows the introduction of artificial chemical functionalities. Co-localization of membrane proteins results in the formation of functionalized artificial organelles combining artificial and natural cellular function. Adding these protein structures to the cellular machinery may have consequences in nanobiotechnology, synthetic biology and materials science, including the constitution of artificial cells and bio-based metamaterials.

  19. Eicosapentaenoic acid prevents TCDD-induced oxidative stress and inflammatory response by modulating MAP kinases and redox-sensitive transcription factors

    PubMed Central

    Palanisamy, Kalaiselvi; Krishnaswamy, Rajashree; Paramasivan, Poornima; Chih-Yang, Huang; Vishwanadha, Vijaya Padma

    2015-01-01

    Background and Purpose Oxidative stress and subsequent activation of inflammatory responses is a widely accepted consequence of exposure to environmental toxins. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin), a well-known environmental toxin, exerts its toxicity through many signalling mechanisms, with liver being the principal organ affected. However, an effective antidote to TCDD-induced toxicity is unknown. The present study evaluated the effect of eicosapentaenoic acid (EPA), an n3 fatty acid, on TCDD-induced toxicity. Experimental Approach In cultures of HepG2 cells, the EPA/AA ratio was determined using gas chromatography, oxidative stress and inflammatory responses through reactive oxygen species (ROS) levels, antioxidant status, [Ca2+]i, nuclear migration of two redox-sensitive transcription factors, NF-κB p65 and Nrf-2, expression of MAP kinase (p-Erk, p-p38), NF-κB p65, COX-2 and Nrf-2. Cellular changes in ΔΨm, acidic vesicular organelle formation, cell cycle analysis and scanning electron microscopy analysis were performed. Key Results EPA offered significant cytoprotection by increasing EPA/AA ratios in cell membranes, inhibiting ROS generation, enhancing antioxidant status and modulating nuclear translocation of redox-sensitive transcription factors (NF-κB p65 and Nrf-2) and expression of NF-κB p65, COX-2 and Nrf-2. Furthermore, TCDD-induced upstream events of MAPK phosphorylation, the increase in [Ca2+]i levels and cell surface changes in microvilli were significantly inhibited by EPA. EPA treatment maintained ΔΨm and prevented formation of acidic vesicular organelles. Conclusion and Implications The present study demonstrates for the first time some underlying molecular mechanisms of cytoprotection exerted by EPA against TCDD-induced oxidative stress and inflammatory responses. PMID:26177858

  20. Fluorescent Probes for Sensing and Imaging within Specific Cellular Organelles.

    PubMed

    Zhu, Hao; Fan, Jiangli; Du, Jianjun; Peng, Xiaojun

    2016-10-18

    long fluorescence lifetimes, which are crucial for long-time tracking of biological processes, tissue and body imaging with deep penetration and low autofluorescence, and time-resolved fluorescence imaging. Research on fluorescent probes with both analyte responsiveness and organelle targetability is a new and emerging area that has attracted increasing attention over the past few years. We have extended the diversity by developing organelle-specific responsive probes capable of detecting changes in biomolecular levels (reactive oxygen species, fluoride ion, hydrogen sulfide, zinc cation, thiol-containing amino acids, and cyclooxygenase-2) and the microenvironment (viscosity, polarity, and pH). Future research should give more considerations of the "low-concern" organelles, such as the Golgi apparatus, the endoplasmic reticulum, and ribosomes. In addition, given the tiny sizes of subcellular organelles (20-1000 nm), we anticipate that clearer visulization of the cellular events within specific organelles will rely on super-resolution optical microscopy with nanoscopic-scale resolution.

  1. Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots.

    PubMed

    Guttenberger, M

    2000-08-01

    The most widespread type of mycorrhiza is the so-called vesicular-arbuscular mycorrhiza. In this endomycorrhiza, fungal hyphae penetrate plant cell walls in the root cortex. There they form densely branched arbuscules. Fungus and plant plasma membrane are separated by a common interfacial apoplast. The pH of the compartment between the symbionts is of pivotal importance for nutrient transfer. Histochemical experiments were conducted to check for an acidic nature of the interface in the model system Glomus versiforme (Karst.) Berch-Allium porrum L. Two chemically different acidotropic dyes (neutral red and LysoSensor Green DND-189) stained the arbuscules intensely. The staining of arbuscules could be eliminated by addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) or treatments leading to membrane rupture. Therefore, the staining of the arbuscules was based on the ion-trap mechanism, which indicates acidic, membrane-bound compartments. Microscopic examination of stained arbuscules at high optical resolution revealed a peripheral accumulation of the dye. Since plasmolysis rapidly destained the arbuscules, it is concluded that the dyes accumulate in the arbuscular interface, indicating the highly acidic nature of this compartment. The findings are discussed with respect to their relevance for the nutrient transfer in mycorrhizas. In addition, evidence for a discontinuity in the arbuscular interface between the stem and the branches of the arbuscule is given.

  2. Mitochondria are the main target organelle for trivalent monomethylarsonous acid (MMA(III))-induced cytotoxicity.

    PubMed

    Naranmandura, Hua; Xu, Shi; Sawata, Takashi; Hao, Wen Hui; Liu, Huan; Bu, Na; Ogra, Yasumitsu; Lou, Yi Jia; Suzuki, Noriyuki

    2011-07-18

    Excessive generation of reactive oxygen species (ROS) is considered to play an important role in arsenic-induced carcinogenicity in the liver, lungs, and urinary bladder. However, little is known about the mechanism of ROS-based carcinogenicity, including where the ROS are generated, and which arsenic species are the most effective ROS inducers. In order to better understand the mechanism of arsenic toxicity, rat liver RLC-16 cells were exposed to arsenite (iAs(III)) and its intermediate metabolites [i.e., monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III))]. MMA(III) (IC(50) = 1 μM) was found to be the most toxic form, followed by DMA(III) (IC(50) = 2 μM) and iAs(III) (IC(50) = 18 μM). Following exposure to MMA(III), ROS were found to be generated primarily in the mitochondria. DMA(III) exposure resulted in ROS generation in other organelles, while no ROS generation was seen following exposures to low levels of iAs(III). This suggests the mechanisms of induction of ROS are different among the three arsenicals. The effects of iAs(III), MMA(III), and DMA(III) on activities of complexes I-IV in the electron transport chain (ETC) of rat liver submitochondrial particles and on the stimulation of ROS production in intact mitochondria were also studied. Activities of complexes II and IV were significantly inhibited by MMA(III), but only the activity of complexes II was inhibited by DMA(III). Incubation with iAs(III) had no inhibitory effects on any of the four complexes. Generation of ROS in intact mitochondria was significantly increased following incubation with MMA(III), while low levels of ROS generation were observed following incubation with DMA(III). ROS was not produced in mitochondria following exposure to iAs(III). The mechanism underlying cell death is different among As(III), MMA(III), and DMA(III), with mitochondria being one of the primary target organelles for MMA(III)-induced cytotoxicity. © 2011 American Chemical Society

  3. Expression of Glutamate and Inhibitory Amino Acid Vesicular Transporters in the Rodent Auditory Brainstem

    PubMed Central

    Ito, Tetsufumi; Bishop, Deborah C.; Oliver, Douglas L.

    2011-01-01

    Glutamate is the main excitatory neurotransmitter in the auditory system, but associations between glutamatergic neuronal populations and the distribution of their synaptic terminations have been difficult. Different subsets of glutamatergic terminals employ one of three vesicular glutamate transporters (VGLUT) to load synaptic vesicles. Recently, VGLUT1 and VGLUT2 terminals were found to have different patterns of organization in the inferior colliculus suggesting that there are different types of glutamatergic neurons in the brainstem auditory system with projections to the colliculus. To positively identify VGLUT-expressing neurons as well as inhibitory neurons in the auditory brainstem, we used in situ hybridization to identify the mRNA for VGLUT1, VGLUT2, and VIAAT (the vesicular inhibitory amino acid transporter used by GABAergic and glycinergic terminals). Similar expression patterns were found in subsets of glutamatergic and inhibitory neurons in the auditory brainstem and thalamus of adult rats and mice. Four patterns of gene expression were seen in individual neurons. 1) VGLUT2 expressed alone was the prevalent pattern. 2) VGLUT1 co-expressed with VGLUT2 was seen in scattered neurons in most nuclei but was common in the medial geniculate body and ventral cochlear nucleus. 3) VGLUT1 expressed alone was found only in granule cells. 4) VIAAT expression was common in most nuclei but dominated in some. These data show that the expression of the VGLUT1/2 and VIAAT genes can identify different subsets of auditory neurons. This may facilitate the identification of different components in auditory circuits. PMID:21165977

  4. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine by...

  5. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if the...

  6. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if the...

  7. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine by...

  8. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine by...

  9. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine by...

  10. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if the...

  11. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if the...

  12. 9 CFR 311.32 - Vesicular diseases.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Vesicular diseases. 311.32 Section 311... CERTIFICATION DISPOSAL OF DISEASED OR OTHERWISE ADULTERATED CARCASSES AND PARTS § 311.32 Vesicular diseases. (a) Any carcass affected with vesicular disease shall be condemned if the condition is acute and if the...

  13. 9 CFR 309.15 - Vesicular diseases.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Vesicular diseases. 309.15 Section 309... CERTIFICATION ANTE-MORTEM INSPECTION § 309.15 Vesicular diseases. (a) Immediate notification shall be given by... any livestock is found to be affected with a vesicular disease. (b) No livestock under quarantine by...

  14. Integrated femtosecond stimulated Raman scattering and two-photon fluorescence imaging of subcellular lipid and vesicular structures

    NASA Astrophysics Data System (ADS)

    Li, Xuesong; Lam, Wen Jiun; Cao, Zhe; Hao, Yan; Sun, Qiqi; He, Sicong; Mak, Ho Yi; Qu, Jianan Y.

    2015-11-01

    The primary goal of this study is to demonstrate that stimulated Raman scattering (SRS) as a new imaging modality can be integrated into a femtosecond (fs) nonlinear optical (NLO) microscope system. The fs sources of high pulse peak power are routinely used in multimodal nonlinear microscopy to enable efficient excitation of multiple NLO signals. However, with fs excitations, the SRS imaging of subcellular lipid and vesicular structures encounters significant interference from proteins due to poor spectral resolution and a lack of chemical specificity, respectively. We developed a unique NLO microscope of fs excitation that enables rapid acquisition of SRS and multiple two-photon excited fluorescence (TPEF) signals. In the in vivo imaging of transgenic C. elegans animals, we discovered that by cross-filtering false positive lipid signals based on the TPEF signals from tryptophan-bearing endogenous proteins and lysosome-related organelles, the imaging system produced highly accurate assignment of SRS signals to lipid. Furthermore, we demonstrated that the multimodal NLO microscope system could sequentially image lipid structure/content and organelles, such as mitochondria, lysosomes, and the endoplasmic reticulum, which are intricately linked to lipid metabolism.

  15. Sharing the cell's bounty - organelle inheritance in yeast.

    PubMed

    Knoblach, Barbara; Rachubinski, Richard A

    2015-02-15

    Eukaryotic cells replicate and partition their organelles between the mother cell and the daughter cell at cytokinesis. Polarized cells, notably the budding yeast Saccharomyces cerevisiae, are well suited for the study of organelle inheritance, as they facilitate an experimental dissection of organelle transport and retention processes. Much progress has been made in defining the molecular players involved in organelle partitioning in yeast. Each organelle uses a distinct set of factors - motor, anchor and adaptor proteins - that ensures its inheritance by future generations of cells. We propose that all organelles, regardless of origin or copy number, are partitioned by the same fundamental mechanism involving division and segregation. Thus, the mother cell keeps, and the daughter cell receives, their fair and equitable share of organelles. This mechanism of partitioning moreover facilitates the segregation of organelle fragments that are not functionally equivalent. In this Commentary, we describe how this principle of organelle population control affects peroxisomes and other organelles, and outline its implications for yeast life span and rejuvenation. © 2015. Published by The Company of Biologists Ltd.

  16. Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

    PubMed

    Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

    2014-01-01

    How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

  17. Mechanisms of Polarized Organelle Distribution in Neurons

    PubMed Central

    Britt, Dylan J.; Farías, Ginny G.; Guardia, Carlos M.; Bonifacino, Juan S.

    2016-01-01

    Neurons are highly polarized cells exhibiting axonal and somatodendritic domains with distinct complements of cytoplasmic organelles. Although some organelles are widely distributed throughout the neuronal cytoplasm, others are segregated to either the axonal or somatodendritic domains. Recent findings show that organelle segregation is largely established at a pre-axonal exclusion zone (PAEZ) within the axon hillock. Polarized sorting of cytoplasmic organelles at the PAEZ is proposed to depend mainly on their selective association with different microtubule motors and, in turn, with distinct microtubule arrays. Somatodendritic organelles that escape sorting at the PAEZ can be subsequently retrieved at the axon initial segment (AIS) by a microtubule- and/or actin-based mechanism. Dynamic sorting along the PAEZ-AIS continuum can thus explain the polarized distribution of cytoplasmic organelles between the axonal and somatodendritic domains. PMID:27065809

  18. The Plant Organelles Database 3 (PODB3) update 2014: integrating electron micrographs and new options for plant organelle research.

    PubMed

    Mano, Shoji; Nakamura, Takanori; Kondo, Maki; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nagatani, Akira; Nishimura, Mikio

    2014-01-01

    The Plant Organelles Database 2 (PODB2), which was first launched in 2006 as PODB, provides static image and movie data of plant organelles, protocols for plant organelle research and external links to relevant websites. PODB2 has facilitated plant organellar research and the understanding of plant organelle dynamics. To provide comprehensive information on plant organelles in more detail, PODB2 was updated to PODB3 (http://podb.nibb.ac.jp/Organellome/). PODB3 contains two additional components: the electron micrograph database and the perceptive organelles database. Through the electron micrograph database, users can examine the subcellular and/or suborganellar structures in various organs of wild-type and mutant plants. The perceptive organelles database provides information on organelle dynamics in response to external stimuli. In addition to the extra components, the user interface for access has been enhanced in PODB3. The data in PODB3 are directly submitted by plant researchers and can be freely downloaded for use in further analysis. PODB3 contains all the information included in PODB2, and the volume of data and protocols deposited in PODB3 continue to grow steadily. We welcome contributions of data from all plant researchers to enhance the utility and comprehensiveness of PODB3.

  19. The endoplasmic reticulum is a target organelle for trivalent dimethylarsinic acid (DMA{sup III})-induced cytotoxicity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Naranmandura, Hua, E-mail: narenman@zju.edu.cn; Xu, Shi; Koike, Shota

    The purpose of present study was to characterize the endoplasmic reticulum stress and generation of ROS in rat liver RLC-16 cells by exposing to trivalent dimethylarsinous acid (DMA{sup III}) and compared with that of trivalent arsenite (iAs{sup III}) and monomethylarsonous acid (MMA{sup III}). Protein kinase-like endoplasmic reticulum kinase (PERK) phosphorylation was significantly induced in cells exposed to DMA{sup III}, while there was no change in phosphorylated PERK (P-PERK) detected in cells after exposure to iAs{sup III} or MMA{sup III}. The generation of reactive oxygen species (ROS) after DMA{sup III} exposure was found to take place specifically in the endoplasmic reticulummore » (ER), while previous reports showed that ROS was generated in mitochondria following exposure to MMA{sup III}. Meanwhile, cycloheximide (CHX) which is an inhibitor of protein biosynthesis strongly inhibited the DMA{sup III}-induced intracellular ROS generation in the ER and the phosphorylation of PERK, suggesting the induction of ER stress probably occurs through the inhibition of the protein folding process. Activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) mRNA were induced by all three arsenic species, however, evidence suggested that they might be induced by different pathways in the case of iAs{sup III} and MMA{sup III}. In addition, ER resident molecular chaperone glucose-regulated protein78 (GRP78) was not affected by trivalent arsenicals, while it was induced in positive control only at high concentration (Thapsigargin;Tg), suggesting the GRP78 is less sensitive to low levels of ER stress. In summary, our findings demonstrate that the endoplasmic reticulum is a target organelle for DMA{sup III}-induced cytotoxicity. Highlights: ►ER is a target organelle for trivalent DMA{sup III}-induced cytotoxicity. ►Generation of ROS in ER can be induced specially by trivalent DMA{sup III}. ►ER-stress and generation of ROS are caused by the increase

  20. The border-to-border distribution method for analysis of cytoplasmic particles and organelles.

    PubMed

    Yacovone, Shalane K; Ornelles, David A; Lyles, Douglas S

    2016-02-01

    Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists of four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest, and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.

  1. Organelle communication: signaling crossroads between homeostasis and disease.

    PubMed

    Bravo-Sagua, Roberto; Torrealba, Natalia; Paredes, Felipe; Morales, Pablo E; Pennanen, Christian; López-Crisosto, Camila; Troncoso, Rodrigo; Criollo, Alfredo; Chiong, Mario; Hill, Joseph A; Simmen, Thomas; Quest, Andrew F; Lavandero, Sergio

    2014-05-01

    Cellular organelles do not function as isolated or static units, but rather form dynamic contacts between one another that can be modulated according to cellular needs. The physical interfaces between organelles are important for Ca2+ and lipid homeostasis, and serve as platforms for the control of many essential functions including metabolism, signaling, organelle integrity and execution of the apoptotic program. Emerging evidence also highlights the importance of organelle communication in disorders such as Alzheimer's disease, pulmonary arterial hypertension, cancer, skeletal and cardiac muscle dysfunction. Here, we provide an overview of the current literature on organelle communication and the link to human pathologies. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. A putative vesicular transporter expressed in Drosophila mushroom bodies that mediates sexual behavior may define a novel neurotransmitter system

    PubMed Central

    Brooks, Elizabeth S.; Greer, Christina L.; Romero-Calderón, Rafael; Serway, Christine N.; Grygoruk, Anna; Haimovitz, Jasmine M.; Nguyen, Bac T.; Najibi, Rod; Tabone, Christopher J.; de Belle, J. Steven; Krantz, David E.

    2011-01-01

    Summary Storage and release of classical and amino acid neurotransmitters requires vesicular transporters. Some neurons lack known vesicular transporters, suggesting additional neurotransmitter systems remain unidentified. Insect mushroom bodies (MBs) are critical for several behaviors, including learning, but the neurotransmitters released by the intrinsic Kenyon cells (KCs) remain unknown. Likewise, KCs do not express a known vesicular transporter. We report the identification of a novel Drosophila gene portabella (prt) that is structurally similar to known vesicular transporters. Both larval and adult brains express PRT in the KCs of the MBs. Additional PRT cells project to the central complex and optic ganglia. prt mutation causes an olfactory learning deficit and an unusual defect in the male’s position during copulation that is rescued by expression in KCs. Since prt is expressed in neurons that lack other known vesicular transporters or neurotransmitters, it may define a previously unknown neurotransmitter system responsible for sexual behavior and a component of olfactory learning. PMID:22017990

  3. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles

    PubMed Central

    Wang, Chensu; Wang, Yiguang; Li, Yang; Bodemann, Brian; Zhao, Tian; Ma, Xinpeng; Huang, Gang; Hu, Zeping; DeBerardinis, Ralph J.; White, Michael A.; Gao, Jinming

    2015-01-01

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells. Deployment in cells allows quantification of the proton accumulation rate in endosomes; illumination of previously unrecognized regulatory mechanisms coupling pH transitions to endosomal coat protein exchange; discovery of distinct pH thresholds required for mTORC1 activation by free amino acids versus proteins; broad-scale characterization of the consequence of endosomal pH transitions on cellular metabolomic profiles; and functionalization of a context-specific metabolic vulnerability in lung cancer cells. Together, these biological applications indicate the robustness and adaptability of this nanotechnology-enabled ‘detection and perturbation' strategy. PMID:26437053

  4. Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules.

    PubMed

    Valach, Matus; Burger, Gertraud; Gray, Michael W; Lang, B Franz

    2014-12-16

    5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  5. Comparative bioinformatics analyses and profiling of lysosome-related organelle proteomes

    NASA Astrophysics Data System (ADS)

    Hu, Zhang-Zhi; Valencia, Julio C.; Huang, Hongzhan; Chi, An; Shabanowitz, Jeffrey; Hearing, Vincent J.; Appella, Ettore; Wu, Cathy

    2007-01-01

    Complete and accurate profiling of cellular organelle proteomes, while challenging, is important for the understanding of detailed cellular processes at the organelle level. Mass spectrometry technologies coupled with bioinformatics analysis provide an effective approach for protein identification and functional interpretation of organelle proteomes. In this study, we have compiled human organelle reference datasets from large-scale proteomic studies and protein databases for seven lysosome-related organelles (LROs), as well as the endoplasmic reticulum and mitochondria, for comparative organelle proteome analysis. Heterogeneous sources of human organelle proteins and rodent homologs are mapped to human UniProtKB protein entries based on ID and/or peptide mappings, followed by functional annotation and categorization using the iProXpress proteomic expression analysis system. Cataloging organelle proteomes allows close examination of both shared and unique proteins among various LROs and reveals their functional relevance. The proteomic comparisons show that LROs are a closely related family of organelles. The shared proteins indicate the dynamic and hybrid nature of LROs, while the unique transmembrane proteins may represent additional candidate marker proteins for LROs. This comparative analysis, therefore, provides a basis for hypothesis formulation and experimental validation of organelle proteins and their functional roles.

  6. Organelle redox autonomy during environmental stress.

    PubMed

    Bratt, Avishay; Rosenwasser, Shilo; Meyer, Andreas; Fluhr, Robert

    2016-09-01

    Oxidative stress is generated in plants because of inequalities in the rate of reactive oxygen species (ROS) generation and scavenging. The subcellular redox state under various stress conditions was assessed using the redox reporter roGFP2 targeted to chloroplastic, mitochondrial, peroxisomal and cytosolic compartments. In parallel, the vitality of the plant was measured by ion leakage. Our results revealed that during certain physiological stress conditions the changes in roGFP2 oxidation are comparable to application of high concentrations of exogenous H2 O2 . Under each stress, particular organelles were affected. Conditions of extended dark stress, or application of elicitor, impacted chiefly on the status of peroxisomal redox state. In contrast, conditions of drought or high light altered the status of mitochondrial or chloroplast redox state, respectively. Amalgamation of the results from diverse environmental stresses shows cases of organelle autonomy as well as multi-organelle oxidative change. Importantly, organelle-specific oxidation under several stresses proceeded cell death as measured by ion leakage, suggesting early roGFP oxidation as predictive of cell death. The measurement of redox state in multiple compartments enables one to look at redox state connectivity between organelles in relation to oxidative stress as well as assign a redox fingerprint to various types of stress conditions. © 2016 John Wiley & Sons Ltd.

  7. Intracellular processing of epidermal growth factor. I. Acidification of 125I-epidermal growth factor in intracellular organelles.

    PubMed

    Matrisian, L M; Planck, S R; Magun, B E

    1984-03-10

    We previously reported that 125I-labeled epidermal growth factor is processed intracellularly to acidic macromolecules in Rat-1 fibroblasts. The present study defines the precursor-product relationship and localization of the processing steps to subcellular organelles by the use of a single isoelectric species of 125I-epidermal growth factor and Percoll gradient fractionation. The native pI 4.55 125I-epidermal growth factor was rapidly processed to a pI 4.2 species on or near the cell surface and in organelles corresponding to clathrin-coated vesicles, Golgi, and endoplasmic reticulum. This species was then processed to a pI 4.35 species in similar organelles. The pI 4.2 and 4.35 species were converted to a pI 4.0 species in dense, lysosome-like organelles. This species was ultimately degraded and exocytosed from the cell as low molecular weight products.

  8. Transmission and pathogenesis of vesicular stomatitis viruses

    USDA-ARS?s Scientific Manuscript database

    Vesicular Stomatitis (VS) is caused by the Vesicular Stomatitis Virus (VSV), a negative single stranded RNA arthropod-borne virus member of the Family Rhabdoviridae. The virion is composed of the host derived plasma membrane, the envelope, and an internal ribonucleoprotein core. The envelope contain...

  9. Boutons containing vesicular zinc define a subpopulation of synapses with low AMPAR content in rat hippocampus.

    PubMed

    Sindreu, Carlos Balet; Varoqui, Hélène; Erickson, Jeffrey D; Pérez-Clausell, Jeús

    2003-08-01

    Cortical regions of the brain stand out for their high content in synaptic zinc, which may thus be involved in synaptic function. The relative number, chemical nature and transmitter receptor profile of synapses that sequester vesicular zinc are largely unknown. To address this, we combined pre-embedding zinc histochemistry and post-embedding immunogold electron microscopy in rat hippocampus. All giant mossy fibre (MF) terminals in the CA3 region and approximately 45% of boutons making axospinous synapses in stratum radiatum in CA1 contained synaptic vesicles that stained for zinc. Both types of zinc-positive boutons selectively expressed the vesicular zinc transporter ZnT-3. Zinc-positive boutons further immunoreacted to the vesicular glutamate transporter VGLUT-1, but not to the transmitter gamma-aminobutyric acid. Most dendritic spines in CA1 immunoreacted to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunits GluR1-3 (approximately 80%) and to N-methyl-D-aspartate receptor (NMDAR) subunits NR1 + NR2A/B (approximately 90%). Synapses made by zinc-positive boutons contained 40% less AMPAR particles than those made by zinc-negative boutons, whereas NMDAR counts were similar. Further analysis indicated that this was due to the reduced synaptic expression of both GluR1 and GluR2 subunits. Hence, the levels of postsynaptic AMPARs may vary according to the presence of vesicular zinc in excitatory afferents to CA1. Zinc-positive and zinc-negative synapses may represent two glutamatergic subpopulations with distinct synaptic signalling.

  10. Laser-induced thermal ablation of cancerous cell organelles.

    PubMed

    Letfullin, Renat R; Szatkowski, Scott A

    2017-07-01

    By exploiting the physical changes experienced by cancerous organelles, we investigate the feasibility of destroying cancerous cells by single and multipulse modes of laser heating. Our procedure consists of two primary steps: determining the normal and cancerous organelles optical properties and simulating the heating of all of the major organelles in the cell to find the treatment modes for the laser ablation of cancerous organelles without harming healthy cells. Our simulations show that the cancerous nucleus can be selectively heated to damaging temperatures, making this nucleus a feasible therapeutic particle and removing the need for nanoparticle injection. Because of the removal of this extra step, the procedure we propose is simpler and safer for the patient.

  11. Mind the Organelle Gap - Peroxisome Contact Sites in Disease.

    PubMed

    Castro, Inês Gomes; Schuldiner, Maya; Zalckvar, Einat

    2018-03-01

    The eukaryotic cell is organized as a complex grid system where membrane-bound cellular compartments, organelles, must be localized to the right place at the right time. One way to facilitate correct organelle localization and organelle cooperation is through membrane contact sites, areas of close proximity between two organelles that are bridged by protein/lipid complexes. It is now clear that all organelles physically contact each other. The main focus of this review is contact sites of peroxisomes, central metabolic hubs whose defects lead to a variety of diseases. New peroxisome contacts, their tethering complexes and functions have been recently discovered. However, if and how peroxisome contacts contribute to the development of peroxisome-related diseases is still a mystery. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Evolving a photosynthetic organelle.

    PubMed

    Nakayama, Takuro; Archibald, John M

    2012-04-24

    The evolution of plastids from cyanobacteria is believed to represent a singularity in the history of life. The enigmatic amoeba Paulinella and its 'recently' acquired photosynthetic inclusions provide a fascinating system through which to gain fresh insight into how endosymbionts become organelles.The plastids, or chloroplasts, of algae and plants evolved from cyanobacteria by endosymbiosis. This landmark event conferred on eukaryotes the benefits of photosynthesis--the conversion of solar energy into chemical energy--and in so doing had a huge impact on the course of evolution and the climate of Earth 1. From the present state of plastids, however, it is difficult to trace the evolutionary steps involved in this momentous development, because all modern-day plastids have fully integrated into their hosts. Paulinella chromatophora is a unicellular eukaryote that bears photosynthetic entities called chromatophores that are derived from cyanobacteria and has thus received much attention as a possible example of an organism in the early stages of organellogenesis. Recent studies have unlocked the genomic secrets of its chromatophore 23 and provided concrete evidence that the Paulinella chromatophore is a bona fide photosynthetic organelle 4. The question is how Paulinella can help us to understand the process by which an endosymbiont is converted into an organelle.

  13. Effects of 4-aminopyridine on organelle movement in cultured mouse dorsal root ganglion neurites.

    PubMed

    Hiruma, Hiromi; Kawakami, Tadashi

    2010-03-01

    Aminopyridines, widely used as a K(+) channel blocker, are membrane-permeable weak bases and have the ability to form vacuoles in the cytoplasm. The vacuoles originate from acidic organelles such as lysosomes. Here, we investigated the effects of 4-aminopyridine (4-AP) on organelle movement in neurites of cultured mouse dorsal root ganglion (DRG) neurons by using video-enhanced microscopy. Some experiments were carried out using fluorescent dyes for lysosomes and mitochondria and confocal microscopy. Treatment of DRG neurons with 4 mM 4-AP caused Brownian movement of some lysosomes within 5 min. The Brownian movement gradually became rapid and vacuoles were formed around individual lysosomes 10-20 min after the start of treatment. Axonal transport of organelles was inhibited by 4-AP. Lysosomes showing Brownian movement were not transported in longitudinal direction of the neurite and the transport of mitochondria was interrupted by vacuoles. The 4-AP-induced Brownian movement of lysosomes with vacuole formation and inhibition of axonal transport were prevented by the simultaneous treatment with vacuolar H(+) ATPase inhibitor bafilomycin A1 or in Cl(-)-free SO(4)(2-) medium. These results indicate that changes in organelle movement by 4-AP are related to vacuole formation and the vacuolar H(+) ATPase and Cl(-) are required for the effects of 4-AP.

  14. Single-organelle tracking by two-photon conversion

    NASA Astrophysics Data System (ADS)

    Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Fukui, Kiichi; Shin-Ichi Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Isobe, Keisuke; Itoh, Kazuyoshi

    2007-03-01

    Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

  15. Organelle-localized potassium transport systems in plants.

    PubMed

    Hamamoto, Shin; Uozumi, Nobuyuki

    2014-05-15

    Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K(+) transport systems allowing K(+) to move across the membrane. K(+) transport systems in plant organelles act coordinately with the plasma membrane intrinsic K(+) transport systems to maintain cytosolic K(+) concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K(+) channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K(+) homeostasis of the cytoplasm. The initial electrophysiological measurements of K(+) transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K(+) transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K(+) transport system has been isolated from cyanobacteria, which may add to our understanding of K(+) flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K(+) transport proteins. Copyright © 2014 Elsevier GmbH. All rights reserved.

  16. Homeostatic scaling of vesicular glutamate and GABA transporter expression in rat neocortical circuits.

    PubMed

    De Gois, Stéphanie; Schäfer, Martin K-H; Defamie, Norah; Chen, Chu; Ricci, Anthony; Weihe, Eberhard; Varoqui, Hélène; Erickson, Jeffrey D

    2005-08-03

    Homeostatic control of pyramidal neuron firing rate involves a functional balance of feedforward excitation and feedback inhibition in neocortical circuits. Here, we reveal a dynamic scaling in vesicular excitatory (vesicular glutamate transporters VGLUT1 and VGLUT2) and inhibitory (vesicular inhibitory amino acid transporter VIAAT) transporter mRNA and synaptic protein expression in rat neocortical neuronal cultures, using a well established in vitro protocol to induce homeostatic plasticity. During the second and third week of synaptic differentiation, the predominant vesicular transporters expressed in neocortical neurons, VGLUT1 and VIAAT, are both dramatically upregulated. In mature cultures, VGLUT1 and VIAAT exhibit bidirectional and opposite regulation by prolonged activity changes. Endogenous coregulation during development and homeostatic scaling of the expression of the transporters in functionally differentiated cultures may serve to control vesicular glutamate and GABA filling and adjust functional presynaptic excitatory/inhibitory balance. Unexpectedly, hyperexcitation in differentiated cultures triggers a striking increase in VGLUT2 mRNA and synaptic protein, whereas decreased excitation reduces levels. VGLUT2 mRNA and protein are expressed in subsets of VGLUT1-encoded neocortical neurons that we identify in primary cultures and in neocortex in situ and in vivo. After prolonged hyperexcitation, downregulation of VGLUT1/synaptophysin intensity ratios at most synapses is observed, whereas a subset of VGLUT1-containing boutons selectively increase the expression of VGLUT2. Bidirectional and opposite regulation of VGLUT1 and VGLUT2 by activity may serve as positive or negative feedback regulators for cortical synaptic transmission. Intracortical VGLUT1/VGLUT2 coexpressing neurons have the capacity to independently modulate the level of expression of either transporter at discrete synapses and therefore may serve as a plastic interface between subcortical

  17. Cell biology of prokaryotic organelles.

    PubMed

    Murat, Dorothee; Byrne, Meghan; Komeili, Arash

    2010-10-01

    Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance.

  18. The development of benzo- and naphtho-fused quinoline-2,4-dicarboxylic acids as vesicular glutamate transporter (VGLUT) inhibitors reveals a possible role for neuroactive steroids

    PubMed Central

    Carrigan, Christina N.; Patel, Sarjubhai A.; Cox, Holly D.; Bolstad, Erin S.; Gerdes, John M.; Smith, Wesley E.; Bridges, Richard J.

    2014-01-01

    Substituted quinoline-2,4-dicarboxylates (QDCs) are conformationally-restricted mimics of glutamate that were previously reported to selectively block the glutamate vesicular transporters (VGLUTs). We find that expanding the QDC scaffold to benzoquinoline dicarboxylic acids (BQDC) and naphthoquinoline dicarboxylic acids (NQDCs) improves inhibitory activity with the NQDCs showing IC50 ~ 70 µM. Modeling overlay studies showed that the polycyclic QDCs resembled steroid structures and led to the identification and testing of estrone sulfate, pregnenolone sulfate and pregnanolone sulfate that blocked the uptake of l-Glu by 50%, 70% and 85% of control, respectively. Pregnanolone sulfate was further characterized by kinetic pharmacological determinations that demonstrated competitive inhibition and a Ki of ≈ 20 µM. PMID:24424130

  19. Metabolic Reprogramming and Oncogenesis: One Hallmark, Many Organelles.

    PubMed

    Costa, A S H; Frezza, C

    2017-01-01

    The process of tumorigenesis can be described by a series of molecular features, among which alteration of cellular metabolism has recently emerged. This metabolic rewiring fulfills the energy and biosynthetic demands of fast proliferating cancer cells and amplifies their metabolic repertoire to survive and proliferate in the poorly oxygenated and nutrient-deprived tumor microenvironment. During the last decade, the complex reprogramming of cancer cell metabolism has been widely investigated, revealing cancer-specific metabolic alterations. These include dysregulation of glucose and glutamine metabolism, alterations of lipid synthesis and oxidation, and a complex rewiring of mitochondrial function. However, mitochondria are not the only metabolically active organelles within the cell, and other organelles, including lysosomes, peroxisomes, and endoplasmic reticulum, harbor components of the metabolic network. Of note, dysregulation of the function of these organelles is increasingly recognized in cancer cells. However, to what extent these organelles contribute to the metabolic reprogramming of cancer is not fully understood. In this review, we describe the main metabolic functions of these organelles and provide insights into how they communicate to orchestrate a coordinated metabolic reprogramming during transformation. © 2017 Elsevier Inc. All rights reserved.

  20. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    PubMed Central

    Luzio, J. Paul; Hackmann, Yvonne; Dieckmann, Nele M.G.; Griffiths, Gillian M.

    2014-01-01

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. PMID:25183830

  1. Nanomanipulation-Coupled Matrix-Assisted Laser Desorption/ Ionization-Direct Organelle Mass Spectrometry: A Technique for the Detailed Analysis of Single Organelles

    NASA Astrophysics Data System (ADS)

    Phelps, Mandy S.; Sturtevant, Drew; Chapman, Kent D.; Verbeck, Guido F.

    2016-02-01

    We describe a novel technique combining precise organelle microextraction with deposition and matrix-assisted laser desorption/ionization (MALDI) for a rapid, minimally invasive mass spectrometry (MS) analysis of single organelles from living cells. A dual-positioner nanomanipulator workstation was utilized for both extraction of organelle content and precise co-deposition of analyte and matrix solution for MALDI-direct organelle mass spectrometry (DOMS) analysis. Here, the triacylglycerol (TAG) profiles of single lipid droplets from 3T3-L1 adipocytes were acquired and results validated with nanoelectrospray ionization (NSI) MS. The results demonstrate the utility of the MALDI-DOMS technique as it enabled longer mass analysis time, higher ionization efficiency, MS imaging of the co-deposited spot, and subsequent MS/MS capabilities of localized lipid content in comparison to NSI-DOMS. This method provides selective organellar resolution, which complements current biochemical analyses and prompts for subsequent subcellular studies to be performed where limited samples and analyte volume are of concern.

  2. Vesicular secretion of auxin

    PubMed Central

    Schlicht, Markus; Volkmann, Dieter; Mancuso, Stefano

    2008-01-01

    The plant hormone auxin is secreted in root apices via phospholipase Dζ2 (PLDζ2) activity which produces specific population of phosphatidic acid that stimulates secretion of vesicles enriched with auxin. These vesicles were reported to be localized at plant synapses which are active in auxin secretion, especially at the transition zone of the root apex. There are several implications of this vesicular secretion of auxin. In root apices, auxin emerges as plant neurotransmitter-like signal molecule which coordinates activities of adjacent cells via electric and chemical signaling. Putative quantal release of auxin after electrical stimulation, if confirmed, would be part of neuronal communication between plant cells. As auxin transport across plant synapses is tightly linked with integrated sensory perception of environment, especially of omnipresent gravity and light, this process is proposed to mediate the plant perception of environment. These neuronal features allow sessile plants to integrate multitude of sensory signals into the adaptive behavior of whole plants and the animal-like exploratory behavior of growing roots. PMID:19704646

  3. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells

    NASA Astrophysics Data System (ADS)

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  4. A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells.

    PubMed

    Saha, Sonali; Prakash, Ved; Halder, Saheli; Chakraborty, Kasturi; Krishnan, Yamuna

    2015-07-01

    The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.

  5. Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

    PubMed Central

    Beznoussenko, Galina V.; Pilyugin, Sergei S.; Geerts, Willie J. C.; Kozlov, Michael M.; Burger, Koert N. J.; Luini, Alberto; Derganc, Jure; Mironov, Alexander A.

    2015-01-01

    Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant. PMID:25761238

  6. Prion protein conversion induced by trivalent iron in vesicular trafficking.

    PubMed

    Choi, Bo-Ran; Lee, Jeongmin; Kim, Su Yeon; Yim, Inbeen; Kim, Eun-Hee; Woo, Hee-Jong

    2013-03-15

    Iron dyshomeostasis has been observed in prion diseases; however, little is known regarding the contribution of the oxidation state of iron to prion protein (PrP) conversion. In this study, PrP(C)-deficient HpL3-4 cells were exposed to divalent [Fe(II)] or trivalent [Fe(III)] iron, followed by exogenous recombinant PrP (rPrP) treatment. We then analyzed the accumulation of internalized rPrP and its biochemical properties, including its resistance to both proteinase K (PK) digestion and detergent solubility. Fe(III), but not Fe(II), induced the accumulation of internalized rPrP, which was partially converted to detergent-insoluble and PK-resistant PrP (PrP(res)). The Fe(III)-induced PrP(res) generation required an intact cell structure, and it was hindered by U18666A, an inhibitor of vesicular trafficking, but not by NH4Cl, an inhibitor of endolysosomal acidification. These observations implicated that the Fe(III)-mediated PrP(res) conversion likely occurs during endosomal vesicular trafficking rather than in the acidic environment of lysosomes. Copyright © 2013 Elsevier Inc. All rights reserved.

  7. Tetrapeptide Inhibitors of the Glutamate Vesicular Transporter (VGLUT)

    PubMed Central

    Patel, Sarjubhai A.; Nagy, Jon O.; Bolstad, Erin D.; Gerdes, John M.; Thompson, Charles M.

    2007-01-01

    Quinoline-2,4-dicaboxylic acids (QDCs) bearing lipophilic substituents in the 6- or 7-position were shown to be inhibitors of the glutamate vesicular transporter (VGLUT). Using the arrangement of the QDC lipophilic substituents as a template, libraries of X1X2EF and X1X2EW tetrapeptides were synthesized and tested as VGLUT inhibitors. The peptides QIEW and WNEF were found to be the most potent. Further stereochemical deconvolution of these two peptides showed dQlIdElW to be the best inhibitor (Ki = 828 ± 252 μM). Modeling and overlay of the tetrapeptide inhibitors with the existing pharmacophore showed that H-bonding and lipophilic residues are important for VGLUT binding. PMID:17662605

  8. Metabolic Interplay between Peroxisomes and Other Subcellular Organelles Including Mitochondria and the Endoplasmic Reticulum

    PubMed Central

    Wanders, Ronald J. A.; Waterham, Hans R.; Ferdinandusse, Sacha

    2016-01-01

    Peroxisomes are unique subcellular organelles which play an indispensable role in several key metabolic pathways which include: (1.) etherphospholipid biosynthesis; (2.) fatty acid beta-oxidation; (3.) bile acid synthesis; (4.) docosahexaenoic acid (DHA) synthesis; (5.) fatty acid alpha-oxidation; (6.) glyoxylate metabolism; (7.) amino acid degradation, and (8.) ROS/RNS metabolism. The importance of peroxisomes for human health and development is exemplified by the existence of a large number of inborn errors of peroxisome metabolism in which there is an impairment in one or more of the metabolic functions of peroxisomes. Although the clinical signs and symptoms of affected patients differ depending upon the enzyme which is deficient and the extent of the deficiency, the disorders involved are usually (very) severe diseases with neurological dysfunction and early death in many of them. With respect to the role of peroxisomes in metabolism it is clear that peroxisomes are dependent on the functional interplay with other subcellular organelles to sustain their role in metabolism. Indeed, whereas mitochondria can oxidize fatty acids all the way to CO2 and H2O, peroxisomes are only able to chain-shorten fatty acids and the end products of peroxisomal beta-oxidation need to be shuttled to mitochondria for full oxidation to CO2 and H2O. Furthermore, NADH is generated during beta-oxidation in peroxisomes and beta-oxidation can only continue if peroxisomes are equipped with a mechanism to reoxidize NADH back to NAD+, which is now known to be mediated by specific NAD(H)-redox shuttles. In this paper we describe the current state of knowledge about the functional interplay between peroxisomes and other subcellular compartments notably the mitochondria and endoplasmic reticulum for each of the metabolic pathways in which peroxisomes are involved. PMID:26858947

  9. Fatty Acids Suppress Autophagic Turnover in β-Cells*

    PubMed Central

    Las, Guy; Serada, Sam B.; Wikstrom, Jakob D.; Twig, Gilad; Shirihai, Orian S.

    2011-01-01

    Recent studies have shown that autophagy is essential for proper β-cell function and survival. However, it is yet unclear under what pathogenic conditions autophagy is inhibited in β-cells. Here, we report that long term exposure to fatty acids and glucose block autophagic flux in β-cells, contributing to their toxic effect. INS1 cells expressing GFP-LC3 (an autophagosome marker) were treated with 0.4 mm palmitate, 0.4 mm oleate, and various concentrations of glucose for 22 h. Kinetics of the effect of fatty acids on autophagy showed a biphasic response. During the second phase of autophagy, the size of autophagosomes and the content of autophagosome substrates (GFP-LC3, p62) and endogenous LC3 was increased. During the same phase, fatty acids suppressed autophagic degradation of long lived protein in both INS1 cells and islets. In INS1 cells, palmitate induced a 3-fold decrease in the number and the acidity of Acidic Vesicular Organelles. This decrease was associated with a suppression of hydrolase activity, suppression of endocytosis, and suppression of oxidative phosphorylation. The combination of fatty acids with glucose synergistically suppressed autophagic turnover, concomitantly suppressing insulin secretion. Rapamycin treatment resulted in partial reversal of the inhibition of autophagic flux, the inhibition of insulin secretion, and the increase in cell death. Our results indicate that excess nutrient could impair autophagy in the long term, hence contributing to nutrient-induced β-cell dysfunction. This may provide a novel mechanism that connects diet-induced obesity and diabetes. PMID:21859708

  10. Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

    PubMed Central

    Pramanik, Smritimoy; Tateishi-Karimata, Hisae; Sugimoto, Naoki

    2014-01-01

    Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces. PMID:25336617

  11. Structural associations between organelle membranes in nectary parenchyma cells.

    PubMed

    Machado, Silvia Rodrigues; Gregório, Elisa A; Rodrigues, Tatiane M

    2018-05-01

    The close association between membranes and organelles, and the intense chloroplast remodeling in parenchyma cells of extrafloral nectaries occurred only at the secretion time and suggest a relationship with the nectar secretion. Associations between membranes and organelles have been well documented in different tissues and cells of plants, but poorly explored in secretory cells. Here, we described the close physical juxtaposition between membranes and organelles, mainly with chloroplasts, in parenchyma cells of Citharexylum myrianthum (Verbenaeceae) extrafloral nectaries under transmission electron microscopy, using conventional and microwave fixation. At the time of nectar secretion, nectary parenchyma cells exhibit a multitude of different organelle and membrane associations as mitochondria-mitochondria, mitochondria-endoplasmic reticulum, mitochondria-chloroplast, chloroplast-nuclear envelope, mitochondria-nuclear envelope, chloroplast-plasmalemma, chloroplast-chloroplast, chloroplast-tonoplast, chloroplast-peroxisome, and mitochondria-peroxisome. These associations were visualized as amorphous electron-dense material, a network of dense fibrillar material and/or dense bridges. Chloroplasts exhibited protrusions variable in shape and extension, which bring them closer to each other and to plasmalemma, tonoplast, and nuclear envelope. Parenchyma cells in the pre- and post-secretory stages did not exhibit any association or juxtaposition of membranes and organelles, and chloroplast protrusions were absent. Chloroplasts had peripheral reticulum that was more developed in the secretory stage. We propose that such subcellular phenomena during the time of nectar secretion optimize the movement of signaling molecules and the exchange of metabolites. Our results open new avenues on the potential mechanisms of organelle contact in parenchyma nectary cells, and reveal new attributes of the secretory cells on the subcellular level.

  12. Cytoplasmic Flow Enhances Organelle Dispersion in Eukaryotic Cells

    NASA Astrophysics Data System (ADS)

    Koslover, Elena; Mogre, Saurabh; Chan, Caleb; Theriot, Julie

    The cytoplasm of a living cell is an active environment through which intracellular components move and mix. We explore, using theoretical modeling coupled with microrheological measurements, the efficiency of particle dispersion via different modes of transport within this active environment. In particular, we focus on the role of cytoplasmic flow over different scales in contributing to organelle transport within two different cell types. In motile neutrophil cells, we show that bulk fluid flow associated with rapid cell deformation enhances particle transport to and from the cell periphery. In narrow fungal hyphae, localized flows due to hydrodynamic entrainment are shown to contribute to optimally efficient organelle dispersion. Our results highlight the importance of non-traditional modes of transport associated with flow of the cytoplasmic fluid in the distribution of organelles throughout eukaryotic cells.

  13. Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists.

    PubMed

    Sanitá Lima, Matheus; Smith, David Roy

    2017-11-06

    Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq) data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb), indicating that most of the organelle DNA-coding and noncoding-is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb) and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells. Copyright © 2017 Sanitá Lima and Smith.

  14. Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation.

    PubMed

    Schwab, S M; Menge, J A; Leonard, R T

    1983-11-01

    A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular-arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study.

  15. Integrated regulation of motor-driven organelle transport by scaffolding proteins.

    PubMed

    Fu, Meng-meng; Holzbaur, Erika L F

    2014-10-01

    Intracellular trafficking pathways, including endocytosis, autophagy, and secretion, rely on directed organelle transport driven by the opposing microtubule motor proteins kinesin and dynein. Precise spatial and temporal targeting of vesicles and organelles requires the integrated regulation of these opposing motors, which are often bound simultaneously to the same cargo. Recent progress demonstrates that organelle-associated scaffolding proteins, including Milton/TRAKs (trafficking kinesin-binding protein), JIP1, JIP3 (JNK-interacting proteins), huntingtin, and Hook1, interact with molecular motors to coordinate activity and sustain unidirectional transport. Scaffolding proteins also bind to upstream regulatory proteins, including kinases and GTPases, to modulate transport in the cell. This integration of regulatory control with motor activity allows for cargo-specific changes in the transport or targeting of organelles in response to cues from the complex cellular environment. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. The Journey of the Autophagosome through Mammalian Cell Organelles and Membranes.

    PubMed

    Molino, Diana; Zemirli, Naïma; Codogno, Patrice; Morel, Etienne

    2017-02-17

    Autophagy is an intracellular degradation process carried out by a double-membrane organelle, termed the autophagosome, which sequesters cytoplasmic material destined for lysosomal degradation and recycling. Autophagy and autophagosome biogenesis are highly conserved processes in eukaryotes and are essential for cell survival, stress responses, and homeostasis. Autophagosomes are dynamic and complex organelles that can originate from several different membrane compartments. Autophagosomes traffic through the cell to fuse with lysosomes or other compartments. Despite identification of key proteins necessary for autophagosome assembly and transport, such as those encoded by the autophagy-related genes, the relationship and interdependence of the autophagosome with other intracellular endo-membranes, including those of organelles involved in exocytosis and endocytic trafficking pathways, are still poorly understood. Here we discuss formation of autophagosomes, the journey of these organelles through the cell, and their close interplay with other mammalian organelles from points of view of signalization platforms and membrane dynamics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Experimental infection of Didelphis marsupialis with Vesicular Stomatitis New Jersey Virus

    USDA-ARS?s Scientific Manuscript database

    Although vesicular stomatitis has been present for many years in the Americas, many aspects of its natural history remain undefined. In this study we challenged five adult Virginia opossums (Didelphis marsupialis) with vesicular stomatitis New Jersey serotype virus (VSNJV). Opossums had no detecta...

  18. Vesicular syphilid in a seropositive patient.

    PubMed

    Arora, Shikha; Dhali, Tapan K; Haroon, Mohammad A

    2013-11-01

    Syphilis is a sexually transmitted infection with various stages of evolution and a myriad of presentations. To avoid a delay in diagnosis, it is important to recognize secondary syphilis presenting with vesicular lesions. A patient presented with maculopapular rash of recent onset with several vesicles and related the eruption to paracetamol taken one day before. The differential diagnoses considered were drug eruption, pityriasis lichenoides et varioliformis acuta, pityriasis rosea and secondary syphilis. HIV, VDRL (1:256) and TPHA tests were positive and histopathology revealed lymphohistiocytic infiltrate and plasma cells. Thus, a diagnosis of secondary syphilis coexisting with HIV was confirmed. The patient was administered benzathine penicillin and anti-retroviral therapy was started. He responded very well to treatment. We report this case because of the rarity of vesicular eruption in secondary syphilis.

  19. Histone H3 is absent from organelle nucleoids in BY-2 cultured tobacco cells.

    PubMed

    Takusagawa, Mari; Tamotsu, Satoshi; Sakai, Atsushi

    2013-07-01

    The core histone proteins (H2A, H2B, H3 and H4) are nuclear-localised proteins that play a central role in the formation of nucleosome structure. They have long been considered to be absent from extra-nuclear, DNA-containing organelles; that is plastids and mitochondria. Recently, however, the targeting of core histone H3 to mitochondria, and the presence of nucleosome-like structures in mitochondrial nucleoids, were proposed in cauliflower and tobacco respectively. Thus, we examined whether histone H3 was present in plant organelles and participated in the organisation of nucleoid structure, using highly purified organelles and organelle nucleoids isolated from BY-2 cultured tobacco cells. Immunofluorescence microscopic observations and Western blotting analyses demonstrated that histone H3 was absent from organelles and organelle nucleoids, consistent with the historical hypothesis. Thus, the organisation of organelle nucleoids, including putative nucleosome-like repetitive structures, should be constructed and maintained without participation of histone H3. © 2013 International Federation for Cell Biology.

  20. Cellular Distribution and Subcellular Localization of Molecular Components of Vesicular Transmitter Release in Horizontal Cells of Rabbit Retina

    PubMed Central

    HIRANO, ARLENE A.; BRANDSTÄTTER, JOHANN H.; BRECHA, NICHOLAS C.

    2010-01-01

    The mechanism underlying transmitter release from retinal horizontal cells is poorly understood. We investigated the possibility of vesicular transmitter release from mammalian horizontal cells by examining the expression of synaptic proteins that participate in vesicular transmitter release at chemical synapses. Using immunocytochemistry, we evaluated the cellular and subcellular distribution of complexin I/II, syntaxin-1, and synapsin I in rabbit retina. Strong labeling for complexin I/II, proteins that regulate a late step in vesicular transmitter release, was found in both synaptic layers of the retina, and in somata of A- and B-type horizontal cells, of γ-aminobutyric acid (GABA)- and glycinergic amacrine cells, and of ganglion cells. Immunoelectron microscopy demonstrated the presence of complexin I/II in horizontal cell processes postsynaptic to rod and cone ribbon synapses. Syntaxin-1, a core protein of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex known to bind to complexin, and synapsin I, a synaptic vesicle-associated protein involved in the Ca2+-dependent recruitment of synaptic vesicles for transmitter release, were also present in the horizontal cells and their processes at photoreceptor synapses. Photoreceptors and bipolar cells did not express any of these proteins at their axon terminals. The presence of complexin I/II, syntaxin-1, and synapsin I in rabbit horizontal cell processes and tips suggests that a vesicular mechanism may underlie transmitter release from mammalian horizontal cells. PMID:15912504

  1. High Speed Size Sorting of Subcellular Organelles by Flow Field-Flow Fractionation.

    PubMed

    Yang, Joon Seon; Lee, Ju Yong; Moon, Myeong Hee

    2015-06-16

    Separation/isolation of subcellular species, such as mitochondria, lysosomes, peroxisomes, Golgi apparatus, and others, from cells is important for gaining an understanding of the cellular functions performed by specific organelles. This study introduces a high speed, semipreparative scale, biocompatible size sorting method for the isolation of subcellular organelle species from homogenate mixtures of HEK 293T cells using flow field-flow fractionation (FlFFF). Separation of organelles was achieved using asymmetrical FlFFF (AF4) channel system at the steric/hyperlayer mode in which nuclei, lysosomes, mitochondria, and peroxisomes were separated in a decreasing order of hydrodynamic diameter without complicated preprocessing steps. Fractions in which organelles were not clearly separated were reinjected to AF4 for a finer separation using the normal mode, in which smaller sized species can be well fractionated by an increasing order of diameter. The subcellular species contained in collected AF4 fractions were examined with scanning electron microscopy to evaluate their size and morphology, Western blot analysis using organelle specific markers was used for organelle confirmation, and proteomic analysis was performed with nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS/MS). Since FlFFF operates with biocompatible buffer solutions, it offers great flexibility in handling subcellular components without relying on a high concentration sucrose solution for centrifugation or affinity- or fluorescence tag-based sorting methods. Consequently, the current study provides an alternative, competitive method for the isolation/purification of subcellular organelle species in their intact states.

  2. Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis.

    PubMed

    Yaffe, Dana; Vergara-Jaque, Ariela; Forrest, Lucy R; Schuldiner, Shimon

    2016-11-22

    Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H + per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen- to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H + -coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation.

  3. Multimodal transport and dispersion of organelles in narrow tubular cells

    NASA Astrophysics Data System (ADS)

    Mogre, Saurabh S.; Koslover, Elena F.

    2018-04-01

    Intracellular components explore the cytoplasm via active motor-driven transport in conjunction with passive diffusion. We model the motion of organelles in narrow tubular cells using analytical techniques and numerical simulations to study the efficiency of different transport modes in achieving various cellular objectives. Our model describes length and time scales over which each transport mode dominates organelle motion, along with various metrics to quantify exploration of intracellular space. For organelles that search for a specific target, we obtain the average capture time for given transport parameters and show that diffusion and active motion contribute to target capture in the biologically relevant regime. Because many organelles have been found to tether to microtubules when not engaged in active motion, we study the interplay between immobilization due to tethering and increased probability of active transport. We derive parameter-dependent conditions under which tethering enhances long-range transport and improves the target capture time. These results shed light on the optimization of intracellular transport machinery and provide experimentally testable predictions for the effects of transport regulation mechanisms such as tethering.

  4. Seeing is believing: on the use of image databases for visually exploring plant organelle dynamics.

    PubMed

    Mano, Shoji; Miwa, Tomoki; Nishikawa, Shuh-ichi; Mimura, Tetsuro; Nishimura, Mikio

    2009-12-01

    Organelle dynamics vary dramatically depending on cell type, developmental stage and environmental stimuli, so that various parameters, such as size, number and behavior, are required for the description of the dynamics of each organelle. Imaging techniques are superior to other techniques for describing organelle dynamics because these parameters are visually exhibited. Therefore, as the results can be seen immediately, investigators can more easily grasp organelle dynamics. At present, imaging techniques are emerging as fundamental tools in plant organelle research, and the development of new methodologies to visualize organelles and the improvement of analytical tools and equipment have allowed the large-scale generation of image and movie data. Accordingly, image databases that accumulate information on organelle dynamics are an increasingly indispensable part of modern plant organelle research. In addition, image databases are potentially rich data sources for computational analyses, as image and movie data reposited in the databases contain valuable and significant information, such as size, number, length and velocity. Computational analytical tools support image-based data mining, such as segmentation, quantification and statistical analyses, to extract biologically meaningful information from each database and combine them to construct models. In this review, we outline the image databases that are dedicated to plant organelle research and present their potential as resources for image-based computational analyses.

  5. Development of a novel real-time RT-PCR assay to detect Seneca Valley virus-1 associated with emerging cases of vesicular disease in pigs.

    PubMed

    Fowler, Veronica L; Ransburgh, Russell H; Poulsen, Elizabeth G; Wadsworth, Jemma; King, Donald P; Mioulet, Valerie; Knowles, Nick J; Williamson, Susanna; Liu, Xuming; Anderson, Gary A; Fang, Ying; Bai, Jianfa

    2017-01-01

    Seneca Valley virus 1 (SVV-1) can cause vesicular disease that is clinically indistinguishable from foot-and-mouth disease, vesicular stomatitis and swine vesicular disease. SVV-1-associated disease has been identified in pigs in several countries, namely USA, Canada, Brazil and China. Diagnostic tests are required to reliably detect this emerging virus, and this report describes the development and evaluation of a novel real-time (r) reverse-transcription (RT) PCR assay (rRT-PCR), targeting the viral polymerase gene (3D) of SVV-1. This new assay detected all historical and contemporary SVV-1 isolates examined (n=8), while no cross-reactivity was observed with nucleic acid templates prepared from other vesicular disease viruses or common swine pathogens. The analytical sensitivity of the rRT-PCR was 0.79 TCID 50 /ml and the limit of detection was equivalent using two different rRT-PCR master-mixes. The performance of the test was further evaluated using pig nasal (n=25) and rectal swab samples (n=25), where concordant results compared to virus sequencing were generated for 43/50 samples. The availability of this assay, will enable laboratories to rapidly detect SVV-1 in cases of vesicular disease in pigs, negated for notifiable diseases, and could enable existing knowledge gaps to be investigated surrounding the natural epidemiology of SVV-1. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Melanosomes – dark organelles enlighten endosomal membrane transport

    PubMed Central

    Raposo, Graça; Marks, Michael S.

    2009-01-01

    Melanosomes are tissue-specific “lysosome-related” organelles of pigment cells in which melanins are synthesized and stored. Analyses of the trafficking and fate of melanosomal components are beginning to reveal how melanosomes are formed through novel pathways from early endosomal intermediates. These studies unveil generalized structural and functional modifications of the endosomal system in specialized cells, and provide unexpected insights into the biogenesis of multivesicular bodies and how compartmentalization regulates protein refolding. Moreover, genetic disorders that affect the biogenesis of melanosomes and other lysosome-related organelles have shed light into the molecular machinery that controls specialized endosomal sorting events. PMID:17878918

  7. An organelle-specific protein landscape identifies novel diseases and molecular mechanisms

    PubMed Central

    Boldt, Karsten; van Reeuwijk, Jeroen; Lu, Qianhao; Koutroumpas, Konstantinos; Nguyen, Thanh-Minh T.; Texier, Yves; van Beersum, Sylvia E. C.; Horn, Nicola; Willer, Jason R.; Mans, Dorus A.; Dougherty, Gerard; Lamers, Ideke J. C.; Coene, Karlien L. M.; Arts, Heleen H.; Betts, Matthew J.; Beyer, Tina; Bolat, Emine; Gloeckner, Christian Johannes; Haidari, Khatera; Hetterschijt, Lisette; Iaconis, Daniela; Jenkins, Dagan; Klose, Franziska; Knapp, Barbara; Latour, Brooke; Letteboer, Stef J. F.; Marcelis, Carlo L.; Mitic, Dragana; Morleo, Manuela; Oud, Machteld M.; Riemersma, Moniek; Rix, Susan; Terhal, Paulien A.; Toedt, Grischa; van Dam, Teunis J. P.; de Vrieze, Erik; Wissinger, Yasmin; Wu, Ka Man; Apic, Gordana; Beales, Philip L.; Blacque, Oliver E.; Gibson, Toby J.; Huynen, Martijn A.; Katsanis, Nicholas; Kremer, Hannie; Omran, Heymut; van Wijk, Erwin; Wolfrum, Uwe; Kepes, François; Davis, Erica E.; Franco, Brunella; Giles, Rachel H.; Ueffing, Marius; Russell, Robert B.; Roepman, Ronald; Al-Turki, Saeed; Anderson, Carl; Antony, Dinu; Barroso, Inês; Bentham, Jamie; Bhattacharya, Shoumo; Carss, Keren; Chatterjee, Krishna; Cirak, Sebahattin; Cosgrove, Catherine; Danecek, Petr; Durbin, Richard; Fitzpatrick, David; Floyd, Jamie; Reghan Foley, A.; Franklin, Chris; Futema, Marta; Humphries, Steve E.; Hurles, Matt; Joyce, Chris; McCarthy, Shane; Mitchison, Hannah M.; Muddyman, Dawn; Muntoni, Francesco; O'Rahilly, Stephen; Onoufriadis, Alexandros; Payne, Felicity; Plagnol, Vincent; Raymond, Lucy; Savage, David B.; Scambler, Peter; Schmidts, Miriam; Schoenmakers, Nadia; Semple, Robert; Serra, Eva; Stalker, Jim; van Kogelenberg, Margriet; Vijayarangakannan, Parthiban; Walter, Klaudia; Whittall, Ros; Williamson, Kathy

    2016-01-01

    Cellular organelles provide opportunities to relate biological mechanisms to disease. Here we use affinity proteomics, genetics and cell biology to interrogate cilia: poorly understood organelles, where defects cause genetic diseases. Two hundred and seventeen tagged human ciliary proteins create a final landscape of 1,319 proteins, 4,905 interactions and 52 complexes. Reverse tagging, repetition of purifications and statistical analyses, produce a high-resolution network that reveals organelle-specific interactions and complexes not apparent in larger studies, and links vesicle transport, the cytoskeleton, signalling and ubiquitination to ciliary signalling and proteostasis. We observe sub-complexes in exocyst and intraflagellar transport complexes, which we validate biochemically, and by probing structurally predicted, disruptive, genetic variants from ciliary disease patients. The landscape suggests other genetic diseases could be ciliary including 3M syndrome. We show that 3M genes are involved in ciliogenesis, and that patient fibroblasts lack cilia. Overall, this organelle-specific targeting strategy shows considerable promise for Systems Medicine. PMID:27173435

  8. Effects of cell geometry on reversible vesicular transport

    NASA Astrophysics Data System (ADS)

    Karamched, Bhargav R.; Bressloff, Paul C.

    2017-02-01

    A major question in cell biology concerns the biophysical mechanism underlying delivery of newly synthesized macromolecules to specific targets within a cell. A recent modeling paper investigated this phenomenon in the context of vesicular delivery to en passant synapses in neurons (Bressloff and Levien 2015 Phys. Rev. Lett.). It was shown how reversibility in vesicular delivery to synapses could play a crucial role in achieving uniformity in the distribution of resources throughout an axon, which is consistent with experimental observations in C. elegans and Drosophila. In this work we generalize the previous model by investigating steady-state vesicular distributions on a Cayley tree, a disk, and a sphere. We show that for irreversible transport on a tree, branching increases the rate of decay of the steady-state distribution of vesicles. On the other hand, the steady-state profiles for reversible transport are similar to the 1D case. In the case of higher-dimensional geometries, we consider two distinct types of radially-symmetric microtubular network: (i) a continuum and (ii) a discrete set. In the continuum case, we model the motor-cargo dynamics using a phenomenologically-based advection-diffusion equation in polar (2D) and spherical (3D) coordinates. On the other-hand, in the discrete case, we derive the population model from a stochastic model of a single motor switching between ballistic motion and diffusion. For all of the geometries we find that reversibility in vesicular delivery to target sites allows for a more uniform distribution of vesicles, provided that cargo-carrying motors are not significantly slowed by their cargo. In each case we characterize the loss of uniformity as a function of the dispersion in velocities.

  9. Limited distal organelles and synaptic function in extensive monoaminergic innervation.

    PubMed

    Tao, Juan; Bulgari, Dinara; Deitcher, David L; Levitan, Edwin S

    2017-08-01

    Organelles such as neuropeptide-containing dense-core vesicles (DCVs) and mitochondria travel down axons to supply synaptic boutons. DCV distribution among en passant boutons in small axonal arbors is mediated by circulation with bidirectional capture. However, it is not known how organelles are distributed in extensive arbors associated with mammalian dopamine neuron vulnerability, and with volume transmission and neuromodulation by monoamines and neuropeptides. Therefore, we studied presynaptic organelle distribution in Drosophila octopamine neurons that innervate ∼20 muscles with ∼1500 boutons. Unlike in smaller arbors, distal boutons in these arbors contain fewer DCVs and mitochondria, although active zones are present. Absence of vesicle circulation is evident by proximal nascent DCV delivery, limited impact of retrograde transport and older distal DCVs. Traffic studies show that DCV axonal transport and synaptic capture are not scaled for extensive innervation, thus limiting distal delivery. Activity-induced synaptic endocytosis and synaptic neuropeptide release are also reduced distally. We propose that limits in organelle transport and synaptic capture compromise distal synapse maintenance and function in extensive axonal arbors, thereby affecting development, plasticity and vulnerability to neurodegenerative disease. © 2017. Published by The Company of Biologists Ltd.

  10. Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

    PubMed Central

    1987-01-01

    We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they

  11. Primary structure of prostaglandin G/H synthase from sheep vesicular gland determined from the complementary DNA sequence.

    PubMed Central

    DeWitt, D L; Smith, W L

    1988-01-01

    Prostaglandin G/H synthase (8,11,14-icosatrienoate, hydrogen-donor:oxygen oxidoreductase, EC 1.14.99.1) catalyzes the first step in the formation of prostaglandins and thromboxanes, the conversion of arachidonic acid to prostaglandin endoperoxides G and H. This enzyme is the site of action of nonsteroidal anti-inflammatory drugs. We have isolated a 2.7-kilobase complementary DNA (cDNA) encompassing the entire coding region of prostaglandin G/H synthase from sheep vesicular glands. This cDNA, cloned from a lambda gt 10 library prepared from poly(A)+ RNA of vesicular glands, hybridizes with a single 2.75-kilobase mRNA species. The cDNA clone was selected using oligonucleotide probes modeled from amino acid sequences of tryptic peptides prepared from the purified enzyme. The full-length cDNA encodes a protein of 600 amino acids, including a signal sequence of 24 amino acids. Identification of the cDNA as coding for prostaglandin G/H synthase is based on comparison of amino acid sequences of seven peptides comprising 103 amino acids with the amino acid sequence deduced from the nucleotide sequence of the cDNA. The molecular weight of the unglycosylated enzyme lacking the signal peptide is 65,621. The synthase is a glycoprotein, and there are three potential sites for N-glycosylation, two of them in the amino-terminal half of the molecule. The serine reported to be acetylated by aspirin is at position 530, near the carboxyl terminus. There is no significant similarity between the sequence of the synthase and that of any other protein in amino acid or nucleotide sequence libraries, and a heme binding site(s) is not apparent from the amino acid sequence. The availability of a full-length cDNA clone coding for prostaglandin G/H synthase should facilitate studies of the regulation of expression of this enzyme and the structural features important for catalysis and for interaction with anti-inflammatory drugs. Images PMID:3125548

  12. Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Melo, Rossana C.N., E-mail: rossana.melo@ufjf.edu.br; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Avenue, CLS 943, Boston, MA 02215; Weller, Peter F.

    Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombreromore » Vesicles – EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. - Highlights: • Application of EM to understand the complex secretory pathway in human eosinophils. • EM techniques reveal an active vesicular system associated with secretory granules. • Tubular vesicles are involved in the transport of granule-derived immune mediators.« less

  13. Vesicular stomatitis forecasting based on Google Trends

    PubMed Central

    Lu, Yi; Zhou, GuangYa; Chen, Qin

    2018-01-01

    Background Vesicular stomatitis (VS) is an important viral disease of livestock. The main feature of VS is irregular blisters that occur on the lips, tongue, oral mucosa, hoof crown and nipple. Humans can also be infected with vesicular stomatitis and develop meningitis. This study analyses 2014 American VS outbreaks in order to accurately predict vesicular stomatitis outbreak trends. Methods American VS outbreaks data were collected from OIE. The data for VS keywords were obtained by inputting 24 disease-related keywords into Google Trends. After calculating the Pearson and Spearman correlation coefficients, it was found that there was a relationship between outbreaks and keywords derived from Google Trends. Finally, the predicted model was constructed based on qualitative classification and quantitative regression. Results For the regression model, the Pearson correlation coefficients between the predicted outbreaks and actual outbreaks are 0.953 and 0.948, respectively. For the qualitative classification model, we constructed five classification predictive models and chose the best classification predictive model as the result. The results showed, SN (sensitivity), SP (specificity) and ACC (prediction accuracy) values of the best classification predictive model are 78.52%,72.5% and 77.14%, respectively. Conclusion This study applied Google search data to construct a qualitative classification model and a quantitative regression model. The results show that the method is effective and that these two models obtain more accurate forecast. PMID:29385198

  14. Renal epithelial cells can release ATP by vesicular fusion

    PubMed Central

    Bjaelde, Randi G.; Arnadottir, Sigrid S.; Overgaard, Morten T.; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    Renal epithelial cells have the ability to release nucleotides as paracrine factors. In the intercalated cells of the collecting duct, ATP is released by connexin30 (cx30), which is selectively expressed in this cell type. However, ATP is released by virtually all renal epithelia and the aim of the present study was to identify possible alternative nucleotide release pathways in a renal epithelial cell model. We used MDCK (type1) cells to screen for various potential ATP release pathways. In these cells, inhibition of the vesicular H+-ATPases (bafilomycin) reduced both the spontaneous and hypotonically (80%)-induced nucleotide release. Interference with vesicular fusion using N-ethylamide markedly reduced the spontaneous nucleotide release, as did interference with trafficking from the endoplasmic reticulum to the Golgi apparatus (brefeldin A1) and vesicular transport (nocodazole). These findings were substantiated using a siRNA directed against SNAP-23, which significantly reduced spontaneous ATP release. Inhibition of pannexin and connexins did not affect the spontaneous ATP release in this cell type, which consists of ~90% principal cells. TIRF-microscopy of either fluorescently-labeled ATP (MANT-ATP) or quinacrine-loaded vesicles, revealed that spontaneous release of single vesicles could be promoted by either hypoosmolality (50%) or ionomycin. This vesicular release decreased the overall cellular fluorescence by 5.8 and 7.6% respectively. In summary, this study supports the notion that spontaneous and induced ATP release can occur via exocytosis in renal epithelial cells. PMID:24065923

  15. Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A

    DOE PAGES

    Montiel, Nestor; Buckley, Alexandra; Guo, Baoqing; ...

    2016-07-01

    Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. Here we report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions.

  16. Vesicular Disease in 9-Week-Old Pigs Experimentally Infected with Senecavirus A

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Montiel, Nestor; Buckley, Alexandra; Guo, Baoqing

    Senecavirus A has been infrequently associated with vesicular disease in swine since 1988. However, clinical disease has not been reproduced after experimental infection with this virus. Here we report vesicular disease in 9-week-old pigs after Sencavirus A infection by the intranasal route under experimental conditions.

  17. [Hemapheresis using vesicular plant separation materials].

    PubMed

    Mavrina, L; Ehwald, R; Matthes, G; Stamminger, G

    1990-01-01

    The present paper deals with the separation of cells from soluble compounds of blood by means of exclusion chromatography using a recently described vesicular packing material made from the cell wall framework of the small duckweed Wolffia arrhiza. The cells of the periphere blood are hardly retarded in passing through a packing of the vesicular material and eluted as sharp peak at an elution volume which is near to 30% of the column volume. The behavior of cells is similar to that of the excluded high molecular weight plasma proteins (e.g. serumalbumin). Low molecular weight solutes (e.g. salts, glucose, urea, kreatinin), but also substances of considerable molecular weight (e.g. myoglobin and Vitamin B12) which are usually difficult to separate by dialysis from serum, are eluted at nearly 100% of the packing volume and may be separated completely from cells and high molecular weight proteins. In vitro-Tests did not show a reduced vitality of eluted blood cells.

  18. Vesicular disease in 9-week-old pigs experimentally infected with Senecavirus A

    USDA-ARS?s Scientific Manuscript database

    Introduction: Senecavirus A (SVA), a picornavirus, has been infrequently associated with cases of idiopathic vesicular disease (IVD) in pigs in the US and Canada since 1988. In 2014 and 2015 there was surge of IVD cases in Brazil and US, respectively. SVA was identified in serum, vesicular fluid, an...

  19. C. elegans Major Fats Are Stored in Vesicles Distinct from Lysosome-Related Organelles

    PubMed Central

    O’Rourke, Eyleen J.; Soukas, Alexander A.; Carr, Christopher E.; Ruvkun, Gary

    2010-01-01

    SUMMARY Genetic conservation allows ancient features of fat storage endocrine pathways to be explored in C. elegans. Multiple studies have used Nile red or BODIPY-labeled fatty acids to identify regulators of fat mass. When mixed with their food, E. coli bacteria, Nile red, and BODIPY-labeled fatty acids stain multiple spherical cellular structures in the C. elegans major fat storage organ, the intestine. However, here we demonstrate that, in the conditions previously reported, the lysosome-related organelles stained by Nile red and BODIPY-labeled fatty acids are not the C. elegans major fat storage compartment. We show that the major fat stores are contained in a distinct cellular compartment that is not stained by Nile red. Using biochemical assays, we validate oil red O staining as a method to assess major fat stores in C. elegans, allowing for efficient and accurate genetic and functional genomic screens for genes that control fat accumulation at the organismal level. PMID:19883620

  20. Monte carlo simulation of vesicular release, spatiotemporal distribution of glutamate in synaptic cleft and generation of postsynaptic currents.

    PubMed

    Glavinovíc, M I

    1999-02-01

    The release of vesicular glutamate, spatiotemporal changes in glutamate concentration in the synaptic cleft and the subsequent generation of fast excitatory postsynaptic currents at a hippocampal synapse were modeled using the Monte Carlo method. It is assumed that glutamate is released from a spherical vesicle through a cylindrical fusion pore into the synaptic cleft and that S-alpha-amino-3-hydroxy -5-methyl-4-isoxazolepropionic acid (AMPA) receptors are uniformly distributed postsynaptically. The time course of change in vesicular concentration can be described by a single exponential, but a slow tail is also observed though only following the release of most of the glutamate. The time constant of decay increases with vesicular size and a lower diffusion constant, and is independent of the initial concentration, becoming markedly shorter for wider fusion pores. The cleft concentration at the fusion pore mouth is not negligible compared to vesicular concentration, especially for wider fusion pores. Lateral equilibration of glutamate is rapid, and within approximately 50 micros all AMPA receptors on average see the same concentration of glutamate. Nevertheless the single-channel current and the number of channels estimated from mean-variance plots are unreliable and different when estimated from rise- and decay-current segments. Greater saturation of AMPA receptor channels provides higher but not more accurate estimates. Two factors contribute to the variability of postsynaptic currents and render the mean-variance nonstationary analysis unreliable, even when all receptors see on average the same glutamate concentration. Firstly, the variability of the instantaneous cleft concentration of glutamate, unlike the mean concentration, first rapidly decreases before slowly increasing; the variability is greater for fewer molecules in the cleft and is spatially nonuniform. Secondly, the efficacy with which glutamate produces a response changes with time. Understanding

  1. Transmission of vesicular stomatitis New Jersey virus to cattle by the biting midge Culicoides sonorensis (Diptera: Ceratopogonidae).

    PubMed

    Perez de Leon, Adalberto A; Tabachnick, Walter J

    2006-03-01

    Laboratory-reared Culicoides sonorensis Wirth & Jones were infected with vesicular stomatitis virus serotype New Jersey (family Rhabdoviridae, genus Vesiculovirus, VSNJV) through intrathoracic inoculation. After 10-d incubation at 25 degrees C, these insects were allowed to blood feed on four steers. Two other steers were exposed to VSNJV through intralingual inoculation with 10(8) tissue culture infective dose50 VSNJV. All six steers became seropositive for VSNJV. The results demonstrate the ability of C. sonorensis to transmit VSNJV to livestock. Only the animals intralingually inoculated with VSNJV showed clinical signs in the form of vesicles at the site of inoculation. Uninfected C. sonorensis allowed to feed on the exposed animals did not become infected with VSNJV. Animals infected by C. sonorensis showed a slower antibody response compared with intralingually inoculated animals. This is probably because of different amounts of virus received via insect transmission and syringe inoculation. A significant difference was found in the serum acute-phase protein alpha-1-acid glycoprotein in animals that received VSNJV through C. sonorensis transmission. These animals had previously been exposed to insect attack in the field compared with intralingually inoculated animals and C. sonorensis-infected animals that had been protected from insect attack. The failure to observe clinical signs of vesicular stomatitis through transmission of VSNJV by C. sonorensis may explain widespread subclinical infections during vesicular stomatitis epidemics.

  2. Vesicular thick-walled swollen hyphae in pulmonary zygomycosis.

    PubMed

    Kimura, Masatomo; Ito, Hiroyuki

    2009-03-01

    An autopsy case of pulmonary zygomycosis in a patient with rheumatoid arthritis on immunosuppressive therapy is presented herein. There was a pulmonary cavitated infarct caused by mycotic thrombosis. Thin-walled narrow hyphae and vesicular thick-walled swollen hyphae were found on the pleural surface and in the necrotic tissue at the periphery of the cavity. Findings of such shaped fungal elements may cause erroneous histopathological diagnosis because pauciseptate broad thin-walled hyphae are usually the only detectable fungal elements in zygomycosis tissue. Although immunohistochemistry confirmed these unusual elements to be zygomycetous in the present case, it is important for the differential diagnosis to be aware that zygomycetes can form thin narrow hyphae and vesicular thick-walled swollen hyphae.

  3. Model of reversible vesicular transport with exclusion

    NASA Astrophysics Data System (ADS)

    Bressloff, Paul C.; Karamched, Bhargav R.

    2016-08-01

    A major question in neurobiology concerns the mechanics behind the motor-driven transport and delivery of vesicles to synaptic targets along the axon of a neuron. Experimental evidence suggests that the distribution of vesicles along the axon is relatively uniform and that vesicular delivery to synapses is reversible. A recent modeling study has made explicit the crucial role that reversibility in vesicular delivery to synapses plays in achieving uniformity in vesicle distribution, so called synaptic democracy (Bressloff et al 2015 Phys. Rev. Lett. 114 168101). In this paper we generalize the previous model by accounting for exclusion effects (hard-core repulsion) that may occur between molecular motor-cargo complexes (particles) moving along the same microtubule track. The resulting model takes the form of an exclusion process with four internal states, which distinguish between motile and stationary particles, and whether or not a particle is carrying vesicles. By applying a mean field approximation and an adiabatic approximation we reduce the system of ODEs describing the evolution of occupation numbers of the sites on a 1D lattice to a system of hydrodynamic equations in the continuum limit. We find that reversibility in vesicular delivery allows for synaptic democracy even in the presence of exclusion effects, although exclusion does exacerbate nonuniform distributions of vesicles in an axon when compared with a model without exclusion. We also uncover the relationship between our model and other models of exclusion processes with internal states.

  4. Organelles – understanding noise and heterogeneity in cell biology at an intermediate scale

    PubMed Central

    Chang, Amy Y.

    2017-01-01

    ABSTRACT Many studies over the years have shown that non-genetic mechanisms for producing cell-to-cell variation can lead to highly variable behaviors across genetically identical populations of cells. Most work to date has focused on gene expression noise as the primary source of phenotypic heterogeneity, yet other sources may also contribute. In this Commentary, we explore organelle-level heterogeneity as a potential secondary source of cellular ‘noise’ that contributes to phenotypic heterogeneity. We explore mechanisms for generating organelle heterogeneity and present evidence of functional links between organelle morphology and cellular behavior. Given the many instances in which molecular-level heterogeneity has been linked to phenotypic heterogeneity, we posit that organelle heterogeneity may similarly contribute to overall phenotypic heterogeneity and underline the importance of studying organelle heterogeneity to develop a more comprehensive understanding of phenotypic heterogeneity. Finally, we conclude with a discussion of the medical challenges associated with phenotypic heterogeneity and outline how improved methods for characterizing and controlling this heterogeneity may lead to improved therapeutic strategies and outcomes for patients. PMID:28183729

  5. Membrane contact sites, ancient and central hubs of cellular lipid logistics.

    PubMed

    Jain, Amrita; Holthuis, Joost C M

    2017-09-01

    Membrane contact sites (MCSs) are regions where two organelles are closely apposed to facilitate molecular communication and promote a functional integration of compartmentalized cellular processes. There is growing evidence that MCSs play key roles in controlling intracellular lipid flows and distributions. Strikingly, even organelles connected by vesicular trafficking exchange lipids en bulk via lipid transfer proteins that operate at MCSs. Herein, we describe how MCSs developed into central hubs of lipid logistics during the evolution of eukaryotic cells. We then focus on how modern eukaryotes exploit MCSs to help solve a major logistical problem, namely to preserve the unique lipid mixtures of their early and late secretory organelles in the face of extensive vesicular trafficking. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann. Copyright © 2017. Published by Elsevier B.V.

  6. Complete Genome Sequences of Two Vesicular Stomatitis Virus Isolates Collected in Mexico.

    PubMed

    Velazquez-Salinas, Lauro; Isa, Pavel; Pauszek, Steven J; Rodriguez, Luis L

    2017-09-14

    We report two full-genome sequences of vesicular stomatitis New Jersey virus (VSNJV) obtained by Illumina next-generation sequencing of RNA isolated from epithelial suspensions of cattle naturally infected in Mexico. These genomes represent the first full-genome sequences of vesicular stomatitis New Jersey viruses circulating in Mexico deposited in the GenBank database.

  7. Vesicular trafficking of immune mediators in human eosinophils revealed by immunoelectron microscopy.

    PubMed

    Melo, Rossana C N; Weller, Peter F

    2016-10-01

    Electron microscopy (EM)-based techniques are mostly responsible for our current view of cell morphology at the subcellular level and continue to play an essential role in biological research. In cells from the immune system, such as eosinophils, EM has helped to understand how cells package and release mediators involved in immune responses. Ultrastructural investigations of human eosinophils enabled visualization of secretory processes in detail and identification of a robust, vesicular trafficking essential for the secretion of immune mediators via a non-classical secretory pathway associated with secretory (specific) granules. This vesicular system is mainly organized as large tubular-vesicular carriers (Eosinophil Sombrero Vesicles - EoSVs) actively formed in response to cell activation and provides a sophisticated structural mechanism for delivery of granule-stored mediators. In this review, we highlight the application of EM techniques to recognize pools of immune mediators at vesicular compartments and to understand the complex secretory pathway within human eosinophils involved in inflammatory and allergic responses. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Contributions of hydrology to Vesicular Stomatitis Virus emergence in the Western United States

    USDA-ARS?s Scientific Manuscript database

    Relationships between environmental variables associated with the spread of vector-borne pathogens, such as RNA viruses transmitted to humans and animals, remain poorly understood. Vesicular stomatitis (VS) is caused by a vector-borne, zoonotic RNA virus (VSV), and is the most common vesicular dise...

  9. Complete Genome Sequences of Two Vesicular Stomatitis Virus Isolates Collected in Mexico

    PubMed Central

    Isa, Pavel; Pauszek, Steven J.; Rodriguez, Luis L.

    2017-01-01

    ABSTRACT We report two full-genome sequences of vesicular stomatitis New Jersey virus (VSNJV) obtained by Illumina next-generation sequencing of RNA isolated from epithelial suspensions of cattle naturally infected in Mexico. These genomes represent the first full-genome sequences of vesicular stomatitis New Jersey viruses circulating in Mexico deposited in the GenBank database. PMID:28912331

  10. ChloroMitoCU: Codon patterns across organelle genomes for functional genomics and evolutionary applications.

    PubMed

    Sablok, Gaurav; Chen, Ting-Wen; Lee, Chi-Ching; Yang, Chi; Gan, Ruei-Chi; Wegrzyn, Jill L; Porta, Nicola L; Nayak, Kinshuk C; Huang, Po-Jung; Varotto, Claudio; Tang, Petrus

    2017-06-01

    Organelle genomes are widely thought to have arisen from reduction events involving cyanobacterial and archaeal genomes, in the case of chloroplasts, or α-proteobacterial genomes, in the case of mitochondria. Heterogeneity in base composition and codon preference has long been the subject of investigation of topics ranging from phylogenetic distortion to the design of overexpression cassettes for transgenic expression. From the overexpression point of view, it is critical to systematically analyze the codon usage patterns of the organelle genomes. In light of the importance of codon usage patterns in the development of hyper-expression organelle transgenics, we present ChloroMitoCU, the first-ever curated, web-based reference catalog of the codon usage patterns in organelle genomes. ChloroMitoCU contains the pre-compiled codon usage patterns of 328 chloroplast genomes (29,960 CDS) and 3,502 mitochondrial genomes (49,066 CDS), enabling genome-wide exploration and comparative analysis of codon usage patterns across species. ChloroMitoCU allows the phylogenetic comparison of codon usage patterns across organelle genomes, the prediction of codon usage patterns based on user-submitted transcripts or assembled organelle genes, and comparative analysis with the pre-compiled patterns across species of interest. ChloroMitoCU can increase our understanding of the biased patterns of codon usage in organelle genomes across multiple clades. ChloroMitoCU can be accessed at: http://chloromitocu.cgu.edu.tw/. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  11. Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells

    PubMed Central

    Kobayashi, Yuki; Kanesaki, Yu; Tanaka, Ayumi; Kuroiwa, Haruko; Kuroiwa, Tsuneyoshi; Tanaka, Kan

    2009-01-01

    Eukaryotic cells arose from an ancient endosymbiotic association of prokaryotes, with plant cells harboring 3 genomes as the remnants of such evolution. In plant cells, plastid and mitochondrial DNA replication [organelle DNA replication (ODR)] occurs in advance of the subsequent cell cycles composed of nuclear DNA replication (NDR) and cell division. However, the mechanism by which replication of these genomes with different origins is coordinated is largely unknown. Here, we show that NDR is regulated by a tetrapyrrole signal in plant cells, which has been suggested as an organelle-to-nucleus retrograde signal. In synchronized cultures of the primitive red alga Cyanidioschyzon merolae, specific inhibition of A-type cyclin-dependent kinase (CDKA) prevented NDR but not ODR after onset of the cell cycle. In contrast, inhibition of ODR by nalidixic acid also resulted in inhibition of NDR, indicating a strict dependence of NDR on ODR. The requirement of ODR for NDR was bypassed by addition of the tetrapyrrole intermediates protoporphyrin IX (ProtoIX) or Mg-ProtoIX, both of which activated CDKA without inducing ODR. This scheme was also observed in cultured tobacco cells (BY-2), where inhibition of ODR by nalidixic acid prevented CDKA activation and NDR, and these inhibitions were circumvented by Mg-ProtoIX without inducing ODR. We thus show that tetrapyrrole-mediated organelle–nucleus replicational coupling is an evolutionary conserved process among plant cells. PMID:19141634

  12. Imaging trace element distributions in single organelles and subcellular features

    NASA Astrophysics Data System (ADS)

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; Rose, Volker; Vogt, Stefan; El-Muayed, Malek

    2016-02-01

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro- and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (which some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. It could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.

  13. Imaging trace element distributions in single organelles and subcellular features

    DOE PAGES

    Kashiv, Yoav; Austin, Jotham R.; Lai, Barry; ...

    2016-02-25

    The distributions of chemical elements within cells are of prime importance in a wide range of basic and applied biochemical research. An example is the role of the subcellular Zn distribution in Zn homeostasis in insulin producing pancreatic beta cells and the development of type 2 diabetes mellitus. We combined transmission electron microscopy with micro-and nano-synchrotron X-ray fluorescence to image unequivocally for the first time, to the best of our knowledge, the natural elemental distributions, including those of trace elements, in single organelles and other subcellular features. Detected elements include Cl, K, Ca, Co, Ni, Cu, Zn and Cd (whichmore » some cells were supplemented with). Cell samples were prepared by a technique that minimally affects the natural elemental concentrations and distributions, and without using fluorescent indicators. In conclusion, it could likely be applied to all cell types and provide new biochemical insights at the single organelle level not available from organelle population level studies.« less

  14. Peroxisomes are oxidative organelles.

    PubMed

    Antonenkov, Vasily D; Grunau, Silke; Ohlmeier, Steffen; Hiltunen, J Kalervo

    2010-08-15

    Peroxisomes are multifunctional organelles with an important role in the generation and decomposition of reactive oxygen species (ROS). In this review, the ROS-producing enzymes, as well as the antioxidative defense system in mammalian peroxisomes, are described. In addition, various conditions leading to disturbances in peroxisomal ROS metabolism, such as abnormal peroxisomal biogenesis, hypocatalasemia, and proliferation of peroxisomes are discussed. We also review the role of mammalian peroxisomes in some physiological and pathological processes involving ROS that lead to mitochondrial abnormalities, defects in cell proliferation, and alterations in the central nervous system, alcoholic cardiomyopathy, and aging. Antioxid.

  15. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... vesicular disease exists; importations prohibited. 94.14 Section 94.14 Animals and Animal Products ANIMAL... (INCLUDING POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY...

  16. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... vesicular disease exists; importations prohibited. 94.14 Section 94.14 Animals and Animal Products ANIMAL... (INCLUDING POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY...

  17. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... vesicular disease exists; importations prohibited. 94.14 Section 94.14 Animals and Animal Products ANIMAL... (INCLUDING POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY...

  18. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... vesicular disease exists; importations prohibited. 94.14 Section 94.14 Animals and Animal Products ANIMAL... (INCLUDING POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY...

  19. Checkpoints for vesicular traffic?

    PubMed

    Fiset, A; Faure, R

    2001-01-01

    During interphase the transport of material between different intracellular organelles requires accurate regulation of fusiogenic domains. Recent studies on hepatic endosomes indicated that compartmentalized Cdk2-cyclin E complexes act by braking fusion events. These Cdk2 complexes integrate tyrosine phosphorylation and dephosphorylation inputs, resulting in the control of the number of rounds of fusion at discrete domains. This leads to changes in the intracellular location of internalized receptors and ultimately their biological response.

  20. Reduction of Cr (VI) into Cr (III) by organelles of Chlorella vulgaris in aqueous solution: An organelle-level attempt.

    PubMed

    Chen, Zunwei; Song, Shufang; Wen, Yuezhong

    2016-12-01

    The priority pollutant chromium (Cr) was ubiquitous and great efforts have been made to reduce Cr (VI) into less-toxic Cr (III) by alga for the convenient availability and low expense. However, the functional role of organelle inside the algal cell in Cr (VI) reduction was poorly understood. In this study, organelles in green algae Chlorella vulgaris were extracted and further decorated for Cr (VI) reduction tests. Results showed that the chloroplast exhibited not only adsorption ability of total Cr (21.18% comparing to control) but also reduction potential of Cr (VI) (almost 70% comparing to control), whose most suitable working concentration was at 17μg/mL. Furtherly, the isolated thylakoid membrane (ITM) showed better Cr (VI) reduction potential with the presence of sodium alginate (SA), even though the Hill reaction activity (HRA) was inhibited. As for photosystem II (PSII), the addition of mesoporous silica SBA-15 enhanced the reduction ability through improving the light-harvesting complex (LHC) II efficiency and electron transport rate. On the whole, the reduction ability order of the three kinds of materials based on chloroplast in C. vulgaris was PSII@SBA-15>Chloroplast>ITM@SA. The attempt made in this study to reduce the Cr (VI) with C. vulgaris organelles might not only offer basement to detect the potential action mechanism of Cr (VI) reduction by C. vulgaris but also provide a new sight for the scavenge of heavy metal with biological materials. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Mapping the distribution of vesicular textures on silicic lavas using the Thermal Infrared Multispectral Scanner

    NASA Technical Reports Server (NTRS)

    Ondrusek, Jaime; Christensen, Philip R.; Fink, Jonathan H.

    1993-01-01

    To investigate the effect of vesicularity on TIMS (Thermal Infrared Multispectral Scanner) imagery independent of chemical variations, we studied a large rhyolitic flow of uniform composition but textural heterogeneity. The imagery was recalibrated so that the digital number values for a lake in the scene matched a calculated ideal spectrum for water. TIMS spectra for the lava show useful differences in coarsely and finely vesicular pumice data, particularly in TIMS bands 3 and 4. Images generated by ratioing these bands accurately map out those areas known from field studies to be coarsely vesicular pumice. These texture-related emissivity variations are probably due to the larger vesicles being relatively deeper and separated by smaller septa leaving less smooth glass available to give the characteristic emission of the lava. In studies of inaccessible lava flows (as on Mars) areas of coarsely vesicular pumice must be identified and avoided before chemical variations can be interpreted. Remotely determined distributions of vesicular and glassy textures can also be related to the volatile contents and potential hazards associated with the emplacement of silicic lava flows on Earth.

  2. A workflow for the automatic segmentation of organelles in electron microscopy image stacks

    PubMed Central

    Perez, Alex J.; Seyedhosseini, Mojtaba; Deerinck, Thomas J.; Bushong, Eric A.; Panda, Satchidananda; Tasdizen, Tolga; Ellisman, Mark H.

    2014-01-01

    Electron microscopy (EM) facilitates analysis of the form, distribution, and functional status of key organelle systems in various pathological processes, including those associated with neurodegenerative disease. Such EM data often provide important new insights into the underlying disease mechanisms. The development of more accurate and efficient methods to quantify changes in subcellular microanatomy has already proven key to understanding the pathogenesis of Parkinson's and Alzheimer's diseases, as well as glaucoma. While our ability to acquire large volumes of 3D EM data is progressing rapidly, more advanced analysis tools are needed to assist in measuring precise three-dimensional morphologies of organelles within data sets that can include hundreds to thousands of whole cells. Although new imaging instrument throughputs can exceed teravoxels of data per day, image segmentation and analysis remain significant bottlenecks to achieving quantitative descriptions of whole cell structural organellomes. Here, we present a novel method for the automatic segmentation of organelles in 3D EM image stacks. Segmentations are generated using only 2D image information, making the method suitable for anisotropic imaging techniques such as serial block-face scanning electron microscopy (SBEM). Additionally, no assumptions about 3D organelle morphology are made, ensuring the method can be easily expanded to any number of structurally and functionally diverse organelles. Following the presentation of our algorithm, we validate its performance by assessing the segmentation accuracy of different organelle targets in an example SBEM dataset and demonstrate that it can be efficiently parallelized on supercomputing resources, resulting in a dramatic reduction in runtime. PMID:25426032

  3. 9 CFR 94.14 - Swine from regions where swine vesicular disease exists; importations prohibited.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... vesicular disease exists; importations prohibited. 94.14 Section 94.14 Animals and Animal Products ANIMAL... (INCLUDING POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, NEWCASTLE DISEASE, HIGHLY PATHOGENIC AVIAN INFLUENZA, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE...

  4. Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells.

    PubMed

    Roudeau, Stéphane; Carmona, Asuncion; Perrin, Laura; Ortega, Richard

    2014-11-01

    X-ray chemical element imaging has the potential to enable fundamental breakthroughs in the understanding of biological systems because chemical element interactions with organelles can be studied at the sub-cellular level. What is the distribution of trace metals in cells? Do some elements accumulate within sub-cellular organelles? What are the chemical species of the elements in these organelles? These are some of the fundamental questions that can be addressed by use of X-ray chemical element imaging with synchrotron radiation beams. For precise location of the distribution of the elements, identification of cellular organelles is required; this can be achieved, after appropriate labelling, by use of fluorescence microscopy. As will be discussed, this approach imposes some limitations on sample preparation. For example, standard immunolabelling procedures strongly modify the distribution of the elements in cells as a result of the chemical fixation and permeabilization steps. Organelle location can, however, be performed, by use of a variety of specific fluorescent dyes or fluorescent proteins, on living cells before cryogenic fixation, enabling preservation of element distribution. This article reviews the methods used for fluorescent organelle labelling and X-ray chemical element imaging and speciation of single cells. Selected cases from our work and from other research groups are presented to illustrate the potential of the combination of the two techniques.

  5. Updating Our View of Organelle Genome Nucleotide Landscape

    PubMed Central

    Smith, David Roy

    2012-01-01

    Organelle genomes show remarkable variation in architecture and coding content, yet their nucleotide composition is relatively unvarying across the eukaryotic domain, with most having a high adenine and thymine (AT) content. Recent studies, however, have uncovered guanine and cytosine (GC)-rich mitochondrial and plastid genomes. These sequences come from a small but eclectic list of species, including certain green plants and animals. Here, I review GC-rich organelle DNAs and the insights they have provided into the evolution of nucleotide landscape. I emphasize that GC-biased mitochondrial and plastid DNAs are more widespread than once thought, sometimes occurring together in the same species, and suggest that the forces biasing their nucleotide content can differ both among and within lineages, and may be associated with specific genome architectural features and life history traits. PMID:22973299

  6. Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly "Targeted" Drug Delivery Systems.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2016-01-04

    Many drugs have been designed to act on intracellular targets and to affect intracellular processes inside target cells. For the desired effects to be exerted, these drugs should permeate target cells and reach specific intracellular organelles. This subcellular drug targeting approach has been proposed for enhancement of accumulation of these drugs in target organelles and improved efficiency. This approach is based on drug encapsulation in drug delivery systems (DDSs) and/or their decoration with specific targeting moieties that are intended to enhance the drug/DDS accumulation in the intracellular organelle of interest. During recent years, there has been a constant increase in interest in DDSs targeted to specific intracellular organelles, and many different approaches have been proposed for attaining efficient drug delivery to specific organelles of interest. However, it appears that in many studies insufficient efforts have been devoted to quantitative analysis of the major formulation parameters of the DDSs disposition (efficiency of DDS endocytosis and endosomal escape, intracellular trafficking, and efficiency of DDS delivery to the target organelle) and of the resulting pharmacological effects. Thus, in many cases, claims regarding efficient delivery of drug/DDS to a specific organelle and efficient subcellular targeting appear to be exaggerated. On the basis of the available experimental data, it appears that drugs/DDS decoration with specific targeting residues can affect their intracellular fate and result in preferential drug accumulation within an organelle of interest. However, it is not clear whether these approaches will be efficient in in vivo settings and be translated into preclinical and clinical applications. Studies that quantitatively assess the mechanisms, barriers, and efficiencies of subcellular drug delivery and of the associated toxic effects are required to determine the therapeutic potential of subcellular DDS targeting.

  7. Discerning the Chemistry in Individual Organelles with Small-Molecule Fluorescent Probes.

    PubMed

    Xu, Wang; Zeng, Zebing; Jiang, Jian-Hui; Chang, Young-Tae; Yuan, Lin

    2016-10-24

    Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

    PubMed Central

    Puchner, Elias M.; Walter, Jessica M.; Kasper, Robert; Huang, Bo; Lim, Wendell A.

    2013-01-01

    Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes. PMID:24043832

  9. Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

    2016-04-01

    The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

  10. Widespread occurrence of organelle genome-encoded 5S rRNAs including permuted molecules

    PubMed Central

    Valach, Matus; Burger, Gertraud; Gray, Michael W.; Lang, B. Franz

    2014-01-01

    5S Ribosomal RNA (5S rRNA) is a universal component of ribosomes, and the corresponding gene is easily identified in archaeal, bacterial and nuclear genome sequences. However, organelle gene homologs (rrn5) appear to be absent from most mitochondrial and several chloroplast genomes. Here, we re-examine the distribution of organelle rrn5 by building mitochondrion- and plastid-specific covariance models (CMs) with which we screened organelle genome sequences. We not only recover all organelle rrn5 genes annotated in GenBank records, but also identify more than 50 previously unrecognized homologs in mitochondrial genomes of various stramenopiles, red algae, cryptomonads, malawimonads and apusozoans, and surprisingly, in the apicoplast (highly derived plastid) genomes of the coccidian pathogens Toxoplasma gondii and Eimeria tenella. Comparative modeling of RNA secondary structure reveals that mitochondrial 5S rRNAs from brown algae adopt a permuted triskelion shape that has not been seen elsewhere. Expression of the newly predicted rrn5 genes is confirmed experimentally in 10 instances, based on our own and published RNA-Seq data. This study establishes that particularly mitochondrial 5S rRNA has a much broader taxonomic distribution and a much larger structural variability than previously thought. The newly developed CMs will be made available via the Rfam database and the MFannot organelle genome annotator. PMID:25429974

  11. The big and intricate dreams of little organelles: Embracing complexity in the study of membrane traffic.

    PubMed

    Liu, Allen P; Botelho, Roberto J; Antonescu, Costin N

    2017-09-01

    Compartmentalization of eukaryotic cells into dynamic organelles that exchange material through regulated membrane traffic governs virtually every aspect of cellular physiology including signal transduction, metabolism and transcription. Much has been revealed about the molecular mechanisms that control organelle dynamics and membrane traffic and how these processes are regulated by metabolic, physical and chemical cues. From this emerges the understanding of the integration of specific organellar phenomena within complex, multiscale and nonlinear regulatory networks. In this review, we discuss systematic approaches that revealed remarkable insight into the complexity of these phenomena, including the use of proximity-based proteomics, high-throughput imaging, transcriptomics and computational modeling. We discuss how these methods offer insights to further understand molecular versatility and organelle heterogeneity, phenomena that allow a single organelle population to serve a range of physiological functions. We also detail on how transcriptional circuits drive organelle adaptation, such that organelles may shift their function to better serve distinct differentiation and stress conditions. Thus, organelle dynamics and membrane traffic are functionally heterogeneous and adaptable processes that coordinate with higher-order system behavior to optimize cell function under a range of contexts. Obtaining a comprehensive understanding of organellar phenomena will increasingly require combined use of reductionist and system-based approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Nanohole Array-directed Trapping of Mammalian Mitochondria Enabling Single Organelle Analysis

    PubMed Central

    Kumar, Shailabh; Wolken, Gregory G.; Wittenberg, Nathan J.; Arriaga, Edgar A.; Oh, Sang-Hyun

    2016-01-01

    We present periodic nanohole arrays fabricated in free-standing metal-coated nitride films as a platform for trapping and analyzing single organelles. When a microliter-scale droplet containing mitochondria is dispensed above the nanohole array, the combination of evaporation and capillary flow directs individual mitochondria to the nanoholes. Mammalian mitochondria arrays were rapidly formed on chip using this technique without any surface modification steps, microfluidic interconnects or external power sources. The trapped mitochondria were depolarized on chip using an ionophore with results showing that the organelle viability and behavior were preserved during the on-chip assembly process. Fluorescence signal related to mitochondrial membrane potential was obtained from single mitochondria trapped in individual nanoholes revealing statistical differences between the behavior of polarized vs. depolarized mammalian mitochondria. This technique provides a fast and stable route for droplet-based directed localization of organelles-on-a-chip with minimal limitations and complexity, as well as promotes integration with other optical or electrochemical detection techniques. PMID:26593329

  13. Organelle DNA variation and systematic relationships in the genus Zea: Teosinte

    PubMed Central

    Timothy, D. H.; Levings, C. S.; Pring, D. R.; Conde, M. F.; Kermicle, J. L.

    1979-01-01

    Chloroplast and mitochondrial DNAs from six races of annual teosinte (Guatemala, Huehuetenango, Balsas, Central Plateau, Chalco, and Nobogame), perennial teosinte, and maize were compared and grouped by restriction endonuclease fragment analyses. Three groups of chloroplast DNAs were detected: (i) perennial teosinte and Guatemala; (ii) Balsas and Huehuetenango; and (iii) all other teosintes. Four groups of mitochondrial DNAs were separated: (i) perennial teosinte; (ii) Guatemala; (iii) Nobogame; and (iv) all other teosintes. Separation of the teosinte and maize organelle DNAs into five groups (Guatemala; perennial teosinte; Balsas and Huehuetenango; Central Plateau and Chalco; Nobogame and maize) approximated the biosystematic relationships of the taxa. It was suggested that the evolutions of the chloroplast and mitochondrial DNAs may be independent of each other, that variation of organelle DNA within a species complex of an organism may be the common condition, and that the DNAs of the organelle and nuclear systems evolve in reasonable harmony. Images PMID:16592708

  14. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED...

  15. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... where swine vesicular disease exists. 94.12 Section 94.12 Animals and Animal Products ANIMAL AND PLANT... POULTRY) AND ANIMAL PRODUCTS RINDERPEST, FOOT-AND-MOUTH DISEASE, EXOTIC NEWCASTLE DISEASE, AFRICAN SWINE FEVER, CLASSICAL SWINE FEVER, SWINE VESICULAR DISEASE, AND BOVINE SPONGIFORM ENCEPHALOPATHY: PROHIBITED...

  16. Experimental infection of Didelphis marsupialis with vesicular stomatitis New Jersey virus.

    PubMed

    Trujillo, Carlos M; Rodriguez, Luis; Rodas, Juan D; Arboleda, John Jairo

    2010-01-01

    Although vesicular stomatitis has been present for many years in the Americas, many aspects of its natural history remain undefined. In this study, we challenged five adult Virginia opossums (Didelphis marsupialis) with vesicular stomatitis New Jersey serotype virus (VSNJV). Opossums had no detectable antibodies against VSNJV prior to being inoculated with 10(6.5) median tissue culture infective doses (TCID(50)) of VSNJV by two routes; intraepithelial/subepithelial (IE/SE) inoculation and scarification in the muzzle (SM). Clinical response was monitored daily and animals were tested for viral shedding. All infected animals developed vesicles and ulcers on the tongue and inflammation of the nasal alar folds. Virus was isolated from esophagus-pharynx, nasal, and from ocular swabs and lesions samples. The failure to detect viremia in these animals indicates that a source other than blood may be required for transmission to insect vectors. Our results suggest that D. marsupialis could play a role in the maintenance of VSNJV outside of domestic animal populations and could provide a model to study vesicular stomatitis virus pathogenesis.

  17. Action potential-independent and pharmacologically unique vesicular serotonin release from dendrites

    PubMed Central

    Colgan, Lesley A.; Cavolo, Samantha L.; Commons, Kathryn G.; Levitan, Edwin S.

    2012-01-01

    Serotonin released within the dorsal raphe nucleus (DR) induces feedback inhibition of serotonin neuron activity and consequently regulates mood-controlling serotonin release throughout the forebrain. Serotonin packaged in vesicles is released in response to action potentials by the serotonin neuron soma and terminals, but the potential for release by dendrites is unknown. Here three-photon (3P) microscopy imaging of endogenous serotonin in living rat brain slice, immunofluorescence and immuno-gold electron microscopy detection of VMAT2 (vesicular monoamine transporter 2) establish the presence of vesicular serotonin within DR dendrites. Furthermore, activation of glutamate receptors is shown to induce vesicular serotonin release from dendrites. However, unlike release from the soma and terminals, dendritic serotonin release is independent of action potentials, relies on L-type Ca2+ channels, is induced preferentially by NMDA, and displays distinct sensitivity to the selective serotonin reuptake inhibitor (SSRI) antidepressant fluoxetine. The unique control of dendritic serotonin release has important implications for DR physiology and the antidepressant action of SSRIs, dihydropyridines and NMDA receptor antagonists. PMID:23136413

  18. The Symbiosome: Legume and Rhizobia Co-evolution toward a Nitrogen-Fixing Organelle?

    PubMed Central

    Coba de la Peña, Teodoro; Fedorova, Elena; Pueyo, José J.; Lucas, M. Mercedes

    2018-01-01

    In legume nodules, symbiosomes containing endosymbiotic rhizobial bacteria act as temporary plant organelles that are responsible for nitrogen fixation, these bacteria develop mutual metabolic dependence with the host legume. In most legumes, the rhizobia infect post-mitotic cells that have lost their ability to divide, although in some nodules cells do maintain their mitotic capacity after infection. Here, we review what is currently known about legume symbiosomes from an evolutionary and developmental perspective, and in the context of the different interactions between diazotroph bacteria and eukaryotes. As a result, it can be concluded that the symbiosome possesses organelle-like characteristics due to its metabolic behavior, the composite origin and differentiation of its membrane, the retargeting of host cell proteins, the control of microsymbiont proliferation and differentiation by the host legume, and the cytoskeletal dynamics and symbiosome segregation during the division of rhizobia-infected cells. Different degrees of symbiosome evolution can be defined, specifically in relation to rhizobial infection and to the different types of nodule. Thus, our current understanding of the symbiosome suggests that it might be considered a nitrogen-fixing link in organelle evolution and that the distinct types of legume symbiosomes could represent different evolutionary stages toward the generation of a nitrogen-fixing organelle. PMID:29403508

  19. Lens fibre cell differentiation and organelle loss: many paths lead to clarity

    PubMed Central

    Wride, Michael A.

    2011-01-01

    The programmed removal of organelles from differentiating lens fibre cells contributes towards lens transparency through formation of an organelle-free zone (OFZ). Disruptions in OFZ formation are accompanied by the persistence of organelles in lens fibre cells and can contribute towards cataract. A great deal of work has gone into elucidating the nature of the mechanisms and signalling pathways involved. It is apparent that multiple, parallel and redundant pathways are involved in this process and that these pathways form interacting networks. Furthermore, it is possible that the pathways can functionally compensate for each other, for example in mouse knockout studies. This makes sense given the importance of lens clarity in an evolutionary context. Apoptosis signalling and proteolytic pathways have been implicated in both lens fibre cell differentiation and organelle loss, including the Bcl-2 and inhibitor of apoptosis families, tumour necrosis factors, p53 and its regulators (such as Mdm2) and proteolytic enzymes, including caspases, cathepsins, calpains and the ubiquitin–proteasome pathway. Ongoing approaches being used to dissect the molecular pathways involved, such as transgenics, lens-specific gene deletion and zebrafish mutants, are discussed here. Finally, some of the remaining unresolved issues and potential areas for future studies are highlighted. PMID:21402582

  20. Vesicular and non-vesicular glutamate release in the nucleus accumbens in conditions of a forced change of behavioral strategy.

    PubMed

    Saul'skaya, N B; Mikhailova, M O

    2005-09-01

    Studies on Sprague-Dawley rats used intracerebral dialysis and high-performance liquid chromatography to identify sources of glutamate release into the intercellular space of the nucleus accumbens during forced correction of food-related behavior, i.e., on presentation to the feeding rat of a conditioned signal previously combined with a pain stimulus or on replacement of a food reinforcement with an inedible food substitute. The results showed that glutamate release observed in the nucleus accumbens during these tests can be prevented by tetrodotoxin (1 microM), which blocks exocytosis, but not by (S)-4-carboxyphenylglycine (5 microM), which blocks non-vesicular glutamate release. Conversely, administration of (S)-4-carboxyphenylglycine halved baseline glutamate release, while administration of tetrodotoxin had no effect on this process. These data provide evidence that different mechanisms control glutamate release into the intercellular space of this nucleus in baseline conditions and in conditions of evoked correction of feeding behavior: the source of baseline glutamate release is non-vesicular glutamate release, while glutamate release seen during forced correction of feeding behavior results from increases in synaptic release.

  1. Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

    PubMed Central

    Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

    2014-01-01

    Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

  2. Rheumatoid arthritis and pseudo-vesicular skin plaques: rheumatoid neutrophilic dermatosis.

    PubMed

    Manriquez, Juan; Giesen, Laura; Puerto, Constanza Del; Gonzalez, Sergio

    2016-01-01

    A 54 year-old woman with a 3-year history of rheumatoid arthritis (RA) consulted us because of weight loss, fever and skin eruption. On physical examination, erythematous plaques with a pseudo-vesicular appearance were seen on the back of both shoulders. Histological examination was consistent with rheumatoid neutrophilic dermatosis (RND). After 3 days of prednisone treatment, the skin eruption resolved. RND is a rare cutaneous manifestation of seropositive RA, characterized by asymptomatic, symmetrical erythematous plaques with a pseudo-vesicular appearance. Histology characteristically reveals a dense, neutrophilic infiltrate with leucocitoclasis but without other signs of vasculitis. Lesions may resolve spontaneously or with RA treatment. This case illustrates an uncommon skin manifestation of active rheumatoid arthritis.

  3. Increased vesicular glutamate transporter expression causes excitotoxic neurodegeneration.

    PubMed

    Daniels, Richard W; Miller, Bradley R; DiAntonio, Aaron

    2011-02-01

    Increases in vesicular glutamate transporter (VGLUT) levels are observed after a variety of insults including hypoxic injury, stress, methamphetamine treatment, and in genetic seizure models. Such overexpression can cause an increase in the amount of glutamate released from each vesicle, but it is unknown whether this is sufficient to induce excitotoxic neurodegeneration. Here we show that overexpression of the Drosophila vesicular glutamate transporter (DVGLUT) leads to excess glutamate release, with some vesicles releasing several times the normal amount of glutamate. Increased DVGLUT expression also leads to an age-dependent loss of motor function and shortened lifespan, accompanied by a progressive neurodegeneration in the postsynaptic targets of the DVGLUT-overexpressing neurons. The early onset lethality, behavioral deficits, and neuronal pathology require overexpression of a functional DVGLUT transgene. Thus overexpression of DVGLUT is sufficient to generate excitotoxic neuropathological phenotypes and therefore reducing VGLUT levels after nervous system injury or stress may mitigate further damage. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Novel Quantitative Autophagy Analysis by Organelle Flow Cytometry after Cell Sonication

    PubMed Central

    Degtyarev, Michael; Reichelt, Mike; Lin, Kui

    2014-01-01

    Autophagy is a dynamic process of bulk degradation of cellular proteins and organelles in lysosomes. Current methods of autophagy measurement include microscopy-based counting of autophagic vacuoles (AVs) in cells. We have developed a novel method to quantitatively analyze individual AVs using flow cytometry. This method, OFACS (organelle flow after cell sonication), takes advantage of efficient cell disruption with a brief sonication, generating cell homogenates with fluorescently labeled AVs that retain their integrity as confirmed with light and electron microscopy analysis. These AVs could be detected directly in the sonicated cell homogenates on a flow cytometer as a distinct population of expected organelle size on a cytometry plot. Treatment of cells with inhibitors of autophagic flux, such as chloroquine or lysosomal protease inhibitors, increased the number of particles in this population under autophagy inducing conditions, while inhibition of autophagy induction with 3-methyladenine or knockdown of ATG proteins prevented this accumulation. This assay can be easily performed in a high-throughput format and opens up previously unexplored avenues for autophagy analysis. PMID:24489953

  5. Practical Microform Materials for Libraries: Silver, Diazo, Vesicular.

    ERIC Educational Resources Information Center

    Veaner, Allen B.

    1982-01-01

    Remarks on the relative permanence and durability of three types of film in use in library microform reproduction (silver, diazo, and vesicular) and points out some technical and economic facts that govern the choice of microform materials for libraries. A 6-item reference list is included. (Author/JL)

  6. Amyloplast sedimentation and organelle saltation in living corn columella cells

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Suyemoto, M. M.; Leopold, A. C.

    1986-01-01

    Amyloplast sedimentation during gravistimulation and organelle movements was studied in living central rootcap cells of Zea mays L. cv. Merit. Cells from sectioned roots were viewed with a horizontally-mounted videomicroscope. The kinetics of gravity-induced amyloplast sedimentation were comparable to those calculated from experiments using fixed material. Individual amyloplasts fell at an average velocity of 5.5 micrometers min-1; the maximal velocity of fall measured was 18.0 micrometers min-1. Amyloplasts often rotated, sometimes rose in the cytoplasm, and occasionally underwent sudden rapid movements as fast as 58 micrometers min-1. Saltations of other organelles were frequently observed. This appears to be the first report of cytoplasmic streaming in the presumptive statocytes of roots.

  7. Infection of guinea pigs with vesicular stomatitis New Jersey virus Transmitted by Culicoides sonorensis (Diptera: Ceratopogonidae).

    PubMed

    Pérez De León, Adalberto A; O'Toole, Donal; Tabachnick, Walter J

    2006-05-01

    Intrathoracically inoculated Culicoides sonorensis Wirth & Jones were capable of transmitting vesicular stomatitis New Jersey virus (family Rhabdoviridae, genus Vesiculovirus, VSNJV) during blood feeding on the abdomen of six guinea pigs. None of the guinea pigs infected in this manner developed clinical signs of vesicular stomatitis despite seroconversion for VSNJV. Guinea pigs infected by intradermal inoculations of VSNJV in the abdomen also failed to develop clinical signs of vesicular stomatitis. Three guinea pigs given intradermal inoculations of VSNJV in the foot pad developed lesions typical of vesicular stomatitis. Transmission by the bite of C. sonorensis may have facilitated guinea pig infection with VSNJV because a single infected C. sonorensis caused seroconversion and all guinea pigs infected by insect bite seroconverted compared with 50% of the guinea pigs infected by intradermal inoculation with a higher titer VSNJV inoculum. The role of C. sonorensis in the transmission of VSNJV is discussed.

  8. Artificially-induced organelles are optimal targets for optical trapping experiments in living cells

    PubMed Central

    López-Quesada, C.; Fontaine, A.-S.; Farré, A.; Joseph, M.; Selva, J.; Egea, G.; Ludevid, M. D.; Martín-Badosa, E.; Montes-Usategui, M.

    2014-01-01

    Optical trapping supplies information on the structural, kinetic or rheological properties of inner constituents of the cell. However, the application of significant forces to intracellular objects is notoriously difficult due to a combination of factors, such as the small difference between the refractive indices of the target structures and the cytoplasm. Here we discuss the possibility of artificially inducing the formation of spherical organelles in the endoplasmic reticulum, which would contain densely packed engineered proteins, to be used as optimized targets for optical trapping experiments. The high index of refraction and large size of our organelles provide a firm grip for optical trapping and thereby allow us to exert large forces easily within safe irradiation limits. This has clear advantages over alternative probes, such as subcellular organelles or internalized synthetic beads. PMID:25071944

  9. Gas transport and vesicularity in low-viscosity liquids

    NASA Astrophysics Data System (ADS)

    Pioli, Laura; Bonadonna, Costanza; Abdulkareem, Lokman; Azzopardi, Barry; Phillips, Jeremy

    2010-05-01

    Vesicle textures of basaltic scoria preserve information on magma bubble content at fragmentation and are commonly used to constrain degassing, vesiculation and magma permeability. These studies are based on the assumption that microscale textures are representative of the conduit-scale structures and processes. However, the conditions for which this assumption is valid have not been investigated in detail. We have investigated conduit-scale structures by performing a series of experiments of separate two-phase flows in a 6.5-m high cylindrical bubble column using a combination of air with pure glucose syrup, water-syrup mixtures and pure water to reproduce open-system degassing and strombolian activity conditions in the upper volcanic conduit (i.e. at very low or zero liquid fluxes). We have varied gas fluxes, initial liquid height, gas inlet configuration and liquid viscosity and analyzed flow regimes and properties. Temperature and pressure were measured at several heights along the pipe and vesicularity was calculated using pressure data, liquid level measurements and an Electrical Capacitance tomography (ECT) system, which measures instantaneous vesicularity and phase distribution from capacitance measurements between pairs of electrodes placed uniformly around the pipe circumference. The aim of the experiments was to identify the effect of gas-flow rates on the flow regimes (i.e. bubbly, slug, churn and annular), the main degassing structures and the total gas content of the column. The effect of increasing and decreasing gas flow rates was also studied to check hysteresis effects. Results indicate that the vesicularity of the liquid column depends primarily on gas flux, whereas flow regimes exert a minor control. In fact, vesicularity increases with gas flux following a power-law trend whose exponent depends on the viscosity of the liquid. In addition, distributions of instantaneous gas fraction in the column cross section during syrup experiments have shown

  10. Ursodeoxycholic and deoxycholic acids: Differential effects on intestinal Ca(2+) uptake, apoptosis and autophagy of rat intestine.

    PubMed

    Rodríguez, Valeria A; Rivoira, María A; Pérez, Adriana del V; Marchionatti, Ana M; Tolosa de Talamoni, Nori G

    2016-02-01

    The aim of this work was to study the effect of sodium deoxycholate (NaDOC) and ursodeoxycholic acid (UDCA) on Ca(2+) uptake by enterocytes and the underlying mechanisms. Rats were divided into four groups: a) controls, b) treated with NaDOC, c) treated with UDCA d) treated with NaDOC and UDCA. Ca(2+) uptake was studied in enterocytes with different degrees of maturation. Apoptosis, autophagy and NO content and iNOS protein expression were evaluated. NaDOC decreased and UDCA increased Ca(2+) uptake only in mature enterocytes. The enhancement of protein expression of Fas, FasL, caspase-8 and caspase-3 activity by NaDOC indicates triggering of the apoptotic extrinsic pathway, which was blocked by UDCA. NO content and iNOS protein expression were enhanced by NaDOC, and avoided by UDCA. The increment of acidic vesicular organelles and LC3 II produced by NaDOC was also prevented by UDCA. In conclusion, the inhibitory effects of NaDOC on intestinal Ca(2+) absorption occur by decreasing the Ca(2+) uptake by mature enterocytes. NaDOC triggers apoptosis and autophagy, in part as a result of nitrosative stress. In contrast, UDCA increases the Ca(2+) uptake by mature enterocytes, and in combination with NaDOC acts as an antiapoptotic and antiautophagic agent normalizing the transcellular Ca(2+) pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Nanoscale distribution of presynaptic Ca(2+) channels and its impact on vesicular release during development.

    PubMed

    Nakamura, Yukihiro; Harada, Harumi; Kamasawa, Naomi; Matsui, Ko; Rothman, Jason S; Shigemoto, Ryuichi; Silver, R Angus; DiGregorio, David A; Takahashi, Tomoyuki

    2015-01-07

    Synaptic efficacy and precision are influenced by the coupling of voltage-gated Ca(2+) channels (VGCCs) to vesicles. But because the topography of VGCCs and their proximity to vesicles is unknown, a quantitative understanding of the determinants of vesicular release at nanometer scale is lacking. To investigate this, we combined freeze-fracture replica immunogold labeling of Cav2.1 channels, local [Ca(2+)] imaging, and patch pipette perfusion of EGTA at the calyx of Held. Between postnatal day 7 and 21, VGCCs formed variable sized clusters and vesicular release became less sensitive to EGTA, whereas fixed Ca(2+) buffer properties remained constant. Experimentally constrained reaction-diffusion simulations suggest that Ca(2+) sensors for vesicular release are located at the perimeter of VGCC clusters (<30 nm) and predict that VGCC number per cluster determines vesicular release probability without altering release time course. This "perimeter release model" provides a unifying framework accounting for developmental changes in both synaptic efficacy and time course. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. A Conserved, Mg2+-Dependent Exonuclease Degrades Organelle DNA during Arabidopsis Pollen Development[C][W

    PubMed Central

    Matsushima, Ryo; Tang, Lay Yin; Zhang, Lingang; Yamada, Hiroshi; Twell, David; Sakamoto, Wataru

    2011-01-01

    In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg2+-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear. PMID:21521697

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

    PubMed

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Evolutionary genomics: is Buchnera a bacterium or an organelle?

    PubMed

    Andersson, J O

    2000-11-30

    The first genome sequence of an intracellular bacterial symbiont of a eukaryotic cell has been determined. The Buchnera genome shares features with the genomes of both intracellular pathogenic bacteria and eukaryotic organelles, and it may represent an intermediate between the two.

  15. Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy

    PubMed Central

    2016-01-01

    The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip–sample interaction in both lateral and vertical directions. Here, long-range tip–sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic “organelles”, chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles. PMID:28114766

  16. The sigma-1 receptor chaperone as an inter-organelle signaling modulator

    PubMed Central

    Su, Tsung-Ping; Hayashi, Teruo; Maurice, Tangui; Buch, Shilpa; Ruoho, Arnold E.

    2010-01-01

    Inter-organelle signaling plays important roles in many physiological functions. Endoplasmic reticulum (ER)-mitochondrion signaling affects intra-mitochondrial calcium (Ca2+) homeostasis and cellular bioenergetics. ER-nucleus signaling attenuates ER stress. ER-plasma membrane signaling regulates cytosolic Ca2+ homeostasis, and ER-mitochondrion-plasma membrane signaling regulates hippocampal dendritic spine formation. Here we propose that the sigma-1 receptor (Sig-1R), an ER chaperone protein, acts as an inter-organelle signaling modulator. Sig-1Rs normally reside at the ER-mitochondrion contact called the MAM (mitochondrion-associated ER membrane), where Sig-1Rs regulate ER-mitochondrion signaling and the ER-nucleus cross-talk. When cells are stimulated by ligands or undergo prolonged stress, Sig-1Rs translocate from the MAM to the ER reticular network and plasmalemma/plasma membrane to regulate a variety of functional proteins, including ion channels, receptors, and kinases. Thus, the Sig-1R serves as an inter-organelle signaling modulator locally at the MAM and remotely at the plasmalemma/plasma membrane. Many pharmacological/physiological effects of Sig-1Rs may relate to this unique action of Sig-1Rs. PMID:20869780

  17. A nanobuffer reporter library for fine-scale imaging and perturbation of endocytic organelles | Office of Cancer Genomics

    Cancer.gov

    Endosomes, lysosomes and related catabolic organelles are a dynamic continuum of vacuolar structures that impact a number of cell physiological processes such as protein/lipid metabolism, nutrient sensing and cell survival. Here we develop a library of ultra-pH-sensitive fluorescent nanoparticles with chemical properties that allow fine-scale, multiplexed, spatio-temporal perturbation and quantification of catabolic organelle maturation at single organelle resolution to support quantitative investigation of these processes in living cells.

  18. Differential sorting of the vesicular glutamate transporter 1 into a defined vesicular pool is regulated by light signaling involving the clock gene Period2.

    PubMed

    Yelamanchili, Sowmya V; Pendyala, Gurudutt; Brunk, Irene; Darna, Mahesh; Albrecht, Urs; Ahnert-Hilger, Gudrun

    2006-06-09

    Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes.

  19. Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

    NASA Astrophysics Data System (ADS)

    Aumiller, William M.; Keating, Christine D.

    2016-02-01

    Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

  20. DNA-Mediated Self-Organization of Polymeric Nanocompartments Leads to Interconnected Artificial Organelles.

    PubMed

    Liu, Juan; Postupalenko, Viktoriia; Lörcher, Samuel; Wu, Dalin; Chami, Mohamed; Meier, Wolfgang; Palivan, Cornelia G

    2016-11-09

    Self-organization of nanocomponents was mainly focused on solid nanoparticles, quantum dots, or liposomes to generate complex architectures with specific properties, but intrinsically limited or not developed enough, to mimic sophisticated structures with biological functions in cells. Here, we present a biomimetic strategy to self-organize synthetic nanocompartments (polymersomes) into clusters with controlled properties and topology by exploiting DNA hybridization to interconnect polymersomes. Molecular and external factors affecting the self-organization served to design clusters mimicking the connection of natural organelles: fine-tune of the distance between tethered polymersomes, different topologies, no fusion of clustered polymersomes, and no aggregation. Unexpected, extended DNA bridges that result from migration of the DNA strands inside the thick polymer membrane (about 12 nm) represent a key stability and control factor, not yet exploited for other synthetic nano-object networks. The replacement of the empty polymersomes with artificial organelles, already reported for single polymersome architecture, will provide an excellent platform for the development of artificial systems mimicking natural organelles or cells and represents a fundamental step in the engineering of molecular factories.

  1. Formation and Stability of Prebiotically Relevant Vesicular Systems in Terrestrial Geothermal Environments

    PubMed Central

    Joshi, Manesh Prakash; Samanta, Anupam; Tripathy, Gyana Ranjan; Rajamani, Sudha

    2017-01-01

    Terrestrial geothermal fields and oceanic hydrothermal vents are considered as candidate environments for the emergence of life on Earth. Nevertheless, the ionic strength and salinity of oceans present serious limitations for the self-assembly of amphiphiles, a process that is fundamental for the formation of first protocells. Consequently, we systematically characterized the efficiency of amphiphile assembly, and vesicular stability, in terrestrial geothermal environments, both, under simulated laboratory conditions and in hot spring water samples (collected from Ladakh, India, an Astrobiologically relevant site). Combinations of prebiotically pertinent fatty acids and their derivatives were evaluated for the formation of vesicles in aforesaid scenarios. Additionally, the stability of these vesicles was characterized over multiple dehydration-rehydration cycles, at elevated temperatures. Among the combinations that were tested, mixtures of fatty acid and its glycerol derivatives were found to be the most robust, also resulting in vesicles in all of the hot spring waters that were tested. Importantly, these vesicles were stable at high temperatures, and this fatty acid system retained its vesicle forming propensity, even after multiple cycles of dehydration-rehydration. The remaining systems, however, formed vesicles only in bicine buffer. Our results suggest that certain prebiotic compartments would have had a selective advantage in terrestrial geothermal niches. Significantly, our study highlights the importance of validating results that are obtained under ‘buffered’ laboratory conditions, by verifying their plausibility in prebiotically analogous environments. PMID:29189763

  2. Formation and Stability of Prebiotically Relevant Vesicular Systems in Terrestrial Geothermal Environments.

    PubMed

    Joshi, Manesh Prakash; Samanta, Anupam; Tripathy, Gyana Ranjan; Rajamani, Sudha

    2017-11-30

    Terrestrial geothermal fields and oceanic hydrothermal vents are considered as candidate environments for the emergence of life on Earth. Nevertheless, the ionic strength and salinity of oceans present serious limitations for the self-assembly of amphiphiles, a process that is fundamental for the formation of first protocells. Consequently, we systematically characterized the efficiency of amphiphile assembly, and vesicular stability, in terrestrial geothermal environments, both, under simulated laboratory conditions and in hot spring water samples (collected from Ladakh, India, an Astrobiologically relevant site). Combinations of prebiotically pertinent fatty acids and their derivatives were evaluated for the formation of vesicles in aforesaid scenarios. Additionally, the stability of these vesicles was characterized over multiple dehydration-rehydration cycles, at elevated temperatures. Among the combinations that were tested, mixtures of fatty acid and its glycerol derivatives were found to be the most robust, also resulting in vesicles in all of the hot spring waters that were tested. Importantly, these vesicles were stable at high temperatures, and this fatty acid system retained its vesicle forming propensity, even after multiple cycles of dehydration-rehydration. The remaining systems, however, formed vesicles only in bicine buffer. Our results suggest that certain prebiotic compartments would have had a selective advantage in terrestrial geothermal niches. Significantly, our study highlights the importance of validating results that are obtained under 'buffered' laboratory conditions, by verifying their plausibility in prebiotically analogous environments.

  3. Organelle-targeting surface-enhanced Raman scattering (SERS) nanosensors for subcellular pH sensing.

    PubMed

    Shen, Yanting; Liang, Lijia; Zhang, Shuqin; Huang, Dianshuai; Zhang, Jing; Xu, Shuping; Liang, Chongyang; Xu, Weiqing

    2018-01-25

    The pH value of subcellular organelles in living cells is a significant parameter in the physiological activities of cells. Its abnormal fluctuations are commonly believed to be associated with cancers and other diseases. Herein, a series of surface-enhanced Raman scattering (SERS) nanosensors with high sensitivity and targeting function was prepared for the quantification and monitoring of pH values in mitochondria, nucleus, and lysosome. The nanosensors were composed of gold nanorods (AuNRs) functionalized with a pH-responsive molecule (4-mercaptopyridine, MPy) and peptides that could specifically deliver the AuNRs to the targeting subcellular organelles. The localization of our prepared nanoprobes in specific organelles was confirmed by super-high resolution fluorescence imaging and bio-transmission electron microscopy (TEM) methods. By the targeting ability, the pH values of the specific organelles can be determined by monitoring the vibrational spectral changes of MPy with different pH values. Compared to the cases of reported lysosome and cytoplasm SERS pH sensors, more accurate pH values of mitochondria and nucleus, which could be two additional intracellular tracers for subcellular microenvironments, were disclosed by this SERS approach, further improving the accuracy of discrimination of related diseases. Our sensitive SERS strategy can also be employed to explore crucial physiological and biological processes that are related to subcellular pH fluctuations.

  4. Plasma biomarkers of decreased vesicular storage distinguish Parkinson disease with orthostatic hypotension from the parkinsonian form of multiple system atrophy.

    PubMed

    Goldstein, David S; Kopin, Irwin J; Sharabi, Yehonatan; Holmes, Courtney

    2015-02-01

    Parkinson disease with orthostatic hypotension (PD + OH) and the parkinsonian form of multiple system atrophy (MSA-P) can be difficult to distinguish clinically. Recent studies indicate that PD entails a vesicular storage defect in catecholaminergic neurons. Although cardiac sympathetic neuroimaging by (18)F-dopamine positron emission tomography can identify decreased vesicular storage, this testing is not generally available. We assessed whether plasma biomarkers of a vesicular storage defect can separate PD + OH from MSA-P. We conceptualized that after F-dopamine injection, augmented production of F-dihydroxyphenylacetic acid (F-DOPAC) indicates decreased vesicular storage, and we therefore predicted that arterial plasma F-DOPAC would be elevated in PD + OH but not in MSA-P. We measured arterial plasma F-DOPAC after (18)F-dopamine administration (infused i.v. over 3 min) in patients with PD + OH (N = 12) or MSA-P (N = 21) and in healthy control subjects (N = 26). Peak F-DOPAC:dihydroxyphenylglycol (DHPG) was also calculated to adjust for effects of denervation on F-DOPAC production. Plasma F-DOPAC accumulated rapidly after initiation of (18)F-dopamine infusion. Peak F-DOPAC (5-10 min) in PD + OH averaged three times that in MSA-P (P < 0.0001). Among MSA-P patients, none had peak F-DOPAC > 300 nCi-kg/cc-mCi, in contrast with 7 of 12 PD + OH patients (χ(2) = 16.6, P < 0.0001). DHPG was lower in PD + OH (3.83 ± 0.36 nmol/L) than in MSA-P (5.20 ± 0.29 nmol/L, P = 0.007). All MSA-P patients had peak F-DOPAC:DHPG < 60, in contrast with 9 of 12 PD + OH patients (χ(2) = 17.5, P < 0.0001). Adjustment of peak F-DOPAC for DHPG increased test sensitivity from 58 to 81% at similar high specificity. After F-dopamine injection, plasma F-DOPAC and F-DOPAC:DHPG distinguish PD + OH from MSA-P.

  5. Vesicular exanthema of swine.

    PubMed

    Smith, A W; Akers, T G

    1976-10-01

    Vesicular exanthema of swine (VES) was first recognized in 1932. At the time, eradication measures and, later, quarantine procedures were instituted and extension of the disease to surrounding farms appeared to have been prevented. Between 1932 and 1936, however, seemingly unrelated epizootics continued among swine herds being fed raw garbage. In 1936, VES disappeared only to reappear in 1939. The disease was contained within California until 1952, at which time it spread to all the major swine producing areas of the United States. The disease was eradicated in 1959, through the enforcement of laws prohibiting the feeding of raw garbage to swine. Other than the association with raw garbage, a reservoir for VES virus (VESV) was never found. In 1972, a virus isolated from California sea lions--and thus named the San Miguel sea lion virus (SMSV)--proved to be distinguishable from VESV. When SMSV was injected into swine, clinical signs of vesicular exanthema developed, leading to the conclusion that, for all practical purposes, SMSV and VESV were the same. To date, 5 species of marine mammals and 2 species of terrestrial mammals, including feral swine, have been shown to possess antibodies to 1 or more of the 4 distinct SMSV serotypes. Current evidence suggests that SMSV infections occur among both terrestrial and marine mammals inhabiting the California coastal zones. This and the practice of shipping frozen meats known to contain SMSV to mink ranches in Utah point to the possibility that domestic swine in the United States are occasionally being exposed to SMSV. Although marine mammals are a source of SMSV, the primary virus reservoir is thought to be 1 or more submammalian marine species common to the southern California coastline. Such a primary reservoir presumably is the source of a new SMSV serotypes infecting marine mammals and may have been the original source of the VESV serotypes that infected swine through the intermediary of raw garbage.

  6. Distinct Protein Sorting and Localization to Premelanosomes, Melanosomes, and Lysosomes in Pigmented Melanocytic Cells✪

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Murphy, Diane M.; Berson, Joanne F.; Marks, Michael S.

    2001-01-01

    Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA–gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted. PMID:11266471

  7. Targeting mammalian organelles with internalizing phage (iPhage) libraries

    PubMed Central

    Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

  8. Threshold-free method for three-dimensional segmentation of organelles

    NASA Astrophysics Data System (ADS)

    Chan, Yee-Hung M.; Marshall, Wallace F.

    2012-03-01

    An ongoing challenge in the field of cell biology is to how to quantify the size and shape of organelles within cells. Automated image analysis methods often utilize thresholding for segmentation, but the calculated surface of objects depends sensitively on the exact threshold value chosen, and this problem is generally worse at the upper and lower zboundaries because of the anisotropy of the point spread function. We present here a threshold-independent method for extracting the three-dimensional surface of vacuoles in budding yeast whose limiting membranes are labeled with a fluorescent fusion protein. These organelles typically exist as a clustered set of 1-10 sphere-like compartments. Vacuole compartments and center points are identified manually within z-stacks taken using a spinning disk confocal microscope. A set of rays is defined originating from each center point and radiating outwards in random directions. Intensity profiles are calculated at coordinates along these rays, and intensity maxima are taken as the points the rays cross the limiting membrane of the vacuole. These points are then fit with a weighted sum of basis functions to define the surface of the vacuole, and then parameters such as volume and surface area are calculated. This method is able to determine the volume and surface area of spherical beads (0.96 to 2 micron diameter) with less than 10% error, and validation using model convolution methods produce similar results. Thus, this method provides an accurate, automated method for measuring the size and morphology of organelles and can be generalized to measure cells and other objects on biologically relevant length-scales.

  9. Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals.

    PubMed

    Okino, Nozomu; Wakisaka, Hiroyoshi; Ishibashi, Yohei; Ito, Makoto

    2018-04-01

    Thraustochytrids are single cell marine eukaryotes that produce large amounts of polyunsaturated fatty acids such as docosahexaenoic acid. In the present study, we report the visualization of endoplasmic reticulum (ER) and mitochondria in a type strain of the thraustochytrid, Aurantiochytrium limacinum ATCC MYA-1381, using the enhanced green fluorescent protein (EGFP) with specific targeting/retaining signals. We expressed the egfp gene with ER targeting/retaining signals from A. limacinum calreticulin or BiP/GRP78 in the thraustochytrid, resulting in the distribution of EGFP signals at the perinuclear region and near lipid droplets. ER-Tracker™ Red, an authentic fluorescent probe for the visualization of ER in mammalian cells, also stained the same region. We observed small lipid droplets generated from the visualized ER in the early growth phase of cell culture. Expression of the egfp gene with the mitochondria targeting signal from A. limacinum cytochrome c oxidase resulted in the localization of EGFP near the plasma membrane. The distribution of EGFP signals coincided with that of MitoTracker® Red CMXRos, which is used to visualize mitochondria in eukaryotes. The ER and mitochondria of A. limacinum were visualized for the first time by EGFP with thraustochytrid cell organelle-specific targeting/retaining signals. These results will contribute to classification of the intracellular localization of proteins expressed in ER and mitochondria as well as analyses of these cell organelles in thraustochytrids.

  10. Expression of vesicular glutamate transporters, VGLUT1 and VGLUT2, in cholinergic spinal motoneurons.

    PubMed

    Herzog, E; Landry, M; Buhler, E; Bouali-Benazzouz, R; Legay, C; Henderson, C E; Nagy, F; Dreyfus, P; Giros, B; El Mestikawy, S

    2004-10-01

    Mammalian spinal motoneurons are cholinergic neurons that have long been suspected to use also glutamate as a neurotransmitter. We report that VGLUT1 and VGLUT2, two subtypes of vesicular glutamate transporters, are expressed in rat spinal motoneurons. Both proteins are present in somato-dendritic compartments as well as in axon terminals in primary cultures of immunopurified motoneurons and sections of spinal cord from adult rat. However, VGLUT1 and VGLUT2 are not found at neuromuscular junctions of skeletal muscles. After intracellular injection of biocytin in motoneurons, VGLUT2 is observed in anterogradely labelled terminals contacting Renshaw inhibitory interneurons. These VGLUT2- and VGLUT1-positive terminals do not express VAChT, the vesicular acetylcholine transporter. Overall, our study establishes for the first time that (i) mammalian spinal motoneurons express vesicular glutamate transporters, (ii) these motoneurons have the potential to release glutamate (in addition to acetylcholine) at terminals contacting Renshaw cells, and finally (iii) the VGLUTs are not present at neuromuscular synapses of skeletal muscles.

  11. Organization of organelles within hyphae of Ashbya gossypii revealed by electron tomography.

    PubMed

    Gibeaux, Romain; Hoepfner, Dominic; Schlatter, Ivan; Antony, Claude; Philippsen, Peter

    2013-11-01

    Ashbya gossypii grows as multinucleated and constantly elongating hyphae. Nuclei are in continuous forward and backward motion, also move during mitosis, and frequently bypass each other. Whereas these nuclear movements are well documented, comparatively little is known about the density and morphology of organelles which very likely influence these movements. To understand the three-dimensional subcellular organization of hyphae at high resolution, we performed large-scale electron tomography of the tip regions in A. gossypii. Here, we present a comprehensive space-filling model in which most membrane-limited organelles including nuclei, mitochondria, endosomes, multivesicular bodies, vacuoles, autophagosomes, peroxisomes, and vesicles are modeled. Nuclei revealed different morphologies and protrusions filled by the nucleolus. Mitochondria are very abundant and form a tubular network with a polarized spherical fraction. The organelles of the degradative pathways show a clustered organization. By analyzing vesicle-like bodies, we identified three size classes of electron-dense vesicles (∼200, ∼150, and ∼100 nm) homogeneously distributed in the cytoplasm which most likely represent peroxisomes. Finally, coated and uncoated vesicles with approximately 40-nm diameters show a polarized distribution toward the hyphal tip with the coated vesicles preferentially localizing at the hyphal periphery.

  12. 77 FR 74787 - Notice of Availability of an Evaluation of the Swine Vesicular Disease Status of Certain Regions...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-18

    ... Swine Vesicular Disease Status of Certain Regions in Italy AGENCY: Animal and Plant Health Inspection... provinces of Trento and Bolzano are free of swine vesicular disease. This determination is based on our... United States in order to prevent the introduction of various communicable diseases, including swine...

  13. Identification of a vesicular-arbuscular mycorrhizal fungus by using monoclonal antibodies in an enzyme-linked immunosorbent assay.

    PubMed

    Wright, S F; Morton, J B; Sworobuk, J E

    1987-09-01

    Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

  14. Resonance Raman Probes for Organelle-Specific Labeling in Live Cells

    NASA Astrophysics Data System (ADS)

    Kuzmin, Andrey N.; Pliss, Artem; Lim, Chang-Keun; Heo, Jeongyun; Kim, Sehoon; Rzhevskii, Alexander; Gu, Bobo; Yong, Ken-Tye; Wen, Shangchun; Prasad, Paras N.

    2016-06-01

    Raman microspectroscopy provides for high-resolution non-invasive molecular analysis of biological samples and has a breakthrough potential for dissection of cellular molecular composition at a single organelle level. However, the potential of Raman microspectroscopy can be fully realized only when novel types of molecular probes distinguishable in the Raman spectroscopy modality are developed for labeling of specific cellular domains to guide spectrochemical spatial imaging. Here we report on the design of a next generation Raman probe, based on BlackBerry Quencher 650 compound, which provides unprecedentedly high signal intensity through the Resonance Raman (RR) enhancement mechanism. Remarkably, RR enhancement occurs with low-toxic red light, which is close to maximum transparency in the biological optical window. The utility of proposed RR probes was validated for targeting lysosomes in live cultured cells, which enabled identification and subsequent monitoring of dynamic changes in this organelle by Raman imaging.

  15. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    DOE PAGES

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut; ...

    2014-12-02

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

  16. Leading-process actomyosin coordinates organelle positioning and adhesion receptor dynamics in radially migrating cerebellar granule neurons

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Trivedi, Niraj; Ramahi, Joseph S.; Karakaya, Mahmut

    During brain development, neurons migrate from germinal zones to their final positions to assemble neural circuits. A unique saltatory cadence involving cyclical organelle movement (e.g., centrosome motility) and leading-process actomyosin enrichment prior to nucleokinesis organizes neuronal migration. While functional evidence suggests that leading-process actomyosin is essential for centrosome motility, the role of the actin-enriched leading process in globally organizing organelle transport or traction forces remains unexplored. Our results show that myosin ii motors and F-actin dynamics are required for Golgi apparatus positioning before nucleokinesis in cerebellar granule neurons (CGNs) migrating along glial fibers. Moreover, we show that primary cilia aremore » motile organelles, localized to the leading-process F-actin-rich domain and immobilized by pharmacological inhibition of myosin ii and F-actin dynamics. Finally, leading process adhesion dynamics are dependent on myosin ii and F-actin. In conclusion, we propose that actomyosin coordinates the overall polarity of migrating CGNs by controlling asymmetric organelle positioning and cell-cell contacts as these cells move along their glial guides.« less

  17. Curvature of Double-Membrane Organelles Generated by Changes in Membrane Size and Composition

    PubMed Central

    Knorr, Roland L.; Dimova, Rumiana; Lipowsky, Reinhard

    2012-01-01

    Transient double-membrane organelles are key players in cellular processes such as autophagy, reproduction, and viral infection. These organelles are formed by the bending and closure of flat, double-membrane sheets. Proteins are believed to be important in these morphological transitions but the underlying mechanism of curvature generation is poorly understood. Here, we describe a novel mechanism for this curvature generation which depends primarily on three membrane properties: the lateral size of the double-membrane sheets, the molecular composition of their highly curved rims, and a possible asymmetry between the two flat faces of the sheets. This mechanism is evolutionary advantageous since it does not require active processes and is readily available even when resources within the cell are restricted as during starvation, which can induce autophagy and sporulation. We identify pathways for protein-assisted regulation of curvature generation, organelle size, direction of bending, and morphology. Our theory also provides a mechanism for the stabilization of large double-membrane sheet-like structures found in the endoplasmic reticulum and in the Golgi cisternae. PMID:22427874

  18. Prokaryotic cells: structural organisation of the cytoskeleton and organelles.

    PubMed

    Souza, Wanderley de

    2012-05-01

    For many years, prokaryotic cells were distinguished from eukaryotic cells based on the simplicity of their cytoplasm, in which the presence of organelles and cytoskeletal structures had not been discovered. Based on current knowledge, this review describes the complex components of the prokaryotic cell cytoskeleton, including (i) tubulin homologues composed of FtsZ, BtuA, BtuB and several associated proteins, which play a fundamental role in cell division, (ii) actin-like homologues, such as MreB and Mb1, which are involved in controlling cell width and cell length, and (iii) intermediate filament homologues, including crescentin and CfpA, which localise on the concave side of a bacterium and along its inner curvature and associate with its membrane. Some prokaryotes exhibit specialised membrane-bound organelles in the cytoplasm, such as magnetosomes and acidocalcisomes, as well as protein complexes, such as carboxysomes. This review also examines recent data on the presence of nanotubes, which are structures that are well characterised in mammalian cells that allow direct contact and communication between cells.

  19. 78 FR 24670 - Notice of a Determination Regarding the Swine Vesicular Disease Status of Certain Regions in Italy

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-26

    .... APHIS-2012-0094] Notice of a Determination Regarding the Swine Vesicular Disease Status of Certain...-Romagna, Veneto, and Piemonte and the autonomous provinces of Trento and Bolzano are free of swine... introducing swine vesicular disease into the United States. This determination is based on our review of the...

  20. Outbreaks of Vesicular stomatitis Alagoas virus in horses and cattle in northeastern Brazil.

    PubMed

    Cargnelutti, Juliana F; Olinda, Roberio G; Maia, Lisanka A; de Aguiar, Gildeni M N; Neto, Eldinê G M; Simões, Sara V D; de Lima, Tatiane G; Dantas, Antônio F M; Weiblen, Rudi; Flores, Eduardo F; Riet-Correa, Franklin

    2014-11-01

    The current article describes outbreaks of vesicular stomatitis (VS) in horses and cattle in Paraiba and Rio Grande do Norte states, northeastern Brazil, between June and August 2013. The reported cases affected 15-20 horses and 6 cattle distributed over 6 small farms in 4 municipalities, but additional data indicated the involvement of a large number of animals on several farms. The disease was characterized by blisters; eruptive lesions in coronary bands, lips, mouth, and muzzle; salivation; claudication and loss of condition. Swollen lower limbs and lips, and ulcerated and erosive areas in the lips and muzzle were observed in some horses. A necrotizing vesiculopustular dermatitis and stomatitis was observed histologically. Vesicular stomatitis virus was isolated from the vesicular fluid of a horse lesion and shown to be serologically related to the VS Indiana serogroup (VSIV) by virus neutralization. Convalescent sera of affected horses and cattle, and from healthy contacts, harbored high levels of neutralizing antibodies against the isolated virus (named VSIV-3 2013SaoBento/ParaibaE). Genomic sequences of VSIV subtype 3 (Vesicular stomatitis Alagoas virus) were amplified by reverse transcription polymerase chain reaction out of clinical specimens from a cow and a horse from different farms. Nucleotide sequencing and phylogenetic analysis of the phosphoprotein gene indicated that the 2 isolates were derived from the same virus and clustered them in VSIV-3, along with VS viruses identified in southeastern and northeastern Brazil in the last decades. Thus, the present report demonstrates the circulation of VSIV-3 in northeastern Brazil and urges for more effective diagnosis and surveillance. © 2014 The Author(s).

  1. SPLICS: a split green fluorescent protein-based contact site sensor for narrow and wide heterotypic organelle juxtaposition.

    PubMed

    Cieri, Domenico; Vicario, Mattia; Giacomello, Marta; Vallese, Francesca; Filadi, Riccardo; Wagner, Tina; Pozzan, Tullio; Pizzo, Paola; Scorrano, Luca; Brini, Marisa; Calì, Tito

    2018-06-01

    Contact sites are discrete areas of organelle proximity that coordinate essential physiological processes across membranes, including Ca 2+ signaling, lipid biosynthesis, apoptosis, and autophagy. However, tools to easily image inter-organelle proximity over a range of distances in living cells and in vivo are lacking. Here we report a split-GFP-based contact site sensor (SPLICS) engineered to fluoresce when organelles are in proximity. Two SPLICS versions efficiently measured narrow (8-10 nm) and wide (40-50 nm) juxtapositions between endoplasmic reticulum and mitochondria, documenting the existence of at least two types of contact sites in human cells. Narrow and wide ER-mitochondria contact sites responded differently to starvation, ER stress, mitochondrial shape modifications, and changes in the levels of modulators of ER-mitochondria juxtaposition. SPLICS detected contact sites in soma and axons of D. rerio Rohon Beard (RB) sensory neurons in vivo, extending its use to analyses of organelle juxtaposition in the whole animal.

  2. Identification of a vesicular ATP release inhibitor for the treatment of neuropathic and inflammatory pain.

    PubMed

    Kato, Yuri; Hiasa, Miki; Ichikawa, Reiko; Hasuzawa, Nao; Kadowaki, Atsushi; Iwatsuki, Ken; Shima, Kazuhiro; Endo, Yasuo; Kitahara, Yoshiro; Inoue, Tsuyoshi; Nomura, Masatoshi; Omote, Hiroshi; Moriyama, Yoshinori; Miyaji, Takaaki

    2017-07-18

    Despite the high incidence of neuropathic and inflammatory pain worldwide, effective drugs with few side effects are currently unavailable for the treatment of chronic pain. Recently, researchers have proposed that inhibitors of purinergic chemical transmission, which plays a key role in the pathological pain response, may allow for targeted treatment of pathological neuropathic and inflammatory pain. However, such therapeutic analgesic agents have yet to be developed. In the present study, we demonstrated that clodronate, a first-generation bisphosphonate with comparatively fewer side effects than traditional treatments, significantly attenuates neuropathic and inflammatory pain unrelated to bone abnormalities via inhibition of vesicular nucleotide transporter (VNUT), a key molecule for the initiation of purinergic chemical transmission. In vitro analyses indicated that clodronate inhibits VNUT at a half-maximal inhibitory concentration of 15.6 nM without affecting other vesicular neurotransmitter transporters, acting as an allosteric modulator through competition with Cl - A low concentration of clodronate impaired vesicular ATP release from neurons, microglia, and immune cells. In vivo analyses revealed that clodronate is more effective than other therapeutic agents in attenuating neuropathic and inflammatory pain, as well as the accompanying inflammation, in wild-type but not VNUT -/- mice, without affecting basal nociception. These findings indicate that clodronate may represent a unique treatment strategy for chronic neuropathic and inflammatory pain via inhibition of vesicular ATP release.

  3. Identification and staining of distinct populations of secretory organelles in astrocytes.

    PubMed

    Bezzi, Paola; Volterra, Andrea

    2014-05-01

    Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

  4. Cytopathogenesis of Vesicular Stomatitis virus is regulated by the PSAP motif of M protein in a species-dependent manner

    USDA-ARS?s Scientific Manuscript database

    Vesicular stomatitis virus (VSV) is an important vector-borne pathogen of bovine and equine species, causing a reportable vesicular disease. The matrix (M) protein of VSV is multifunctional and plays a key role in cytopathogenesis, apoptosis, host protein shut-off, and virion assembly/budding. Our ...

  5. Vesicular neurotransmitter transporters in Huntington's disease: initial observations and comparison with traditional synaptic markers.

    PubMed

    Suzuki, M; Desmond, T J; Albin, R L; Frey, K A

    2001-09-15

    Markers of identified neuronal populations have previously suggested selective degeneration of projection neurons in Huntington's disease (HD) striatum. Interpretations are, however, limited by effects of compensatory regulation and atrophy. Studies of the vesicular monoamine transporter type-2 (VMAT2) and of the vesicular acetylcholine transporter (VAChT) in experimental animals indicate that they are robust markers of presynaptic integrity and are not subject to regulation. We measured dopamine and acetylcholine vesicular transporters to characterize the selectivity of degeneration in HD striatum. Brains were obtained at autopsy from four HD patients and five controls. Autoradiography was used to quantify radioligand binding to VMAT2, VAChT, the dopamine plasmalemmal transporter (DAT), benzodiazepine (BZ) binding sites, and D2-type dopamine receptors. The activity of choline acetyltransferase (ChAT) was determined as an additional marker of cholinergic neurons. Autoradiograms were analyzed by video-assisted densitometry and assessment of atrophy was made from regional structural areas in the coronal projection. Striatal VMAT2, DAT, and VAChT concentrations were unchanged or increased, while D2 and BZ binding and ChAT activity were decreased in HD. After atrophy correction, all striatal binding sites were decreased. However, the decrease in ChAT activity was 3-fold greater than that of VAChT binding. In addition to degeneration of striatal projection neurons, there are losses of extrinsic nigrostriatal projections and of striatal cholinergic interneurons in HD on the basis of vesicular transporter measures. There is also markedly reduced expression of ChAT by surviving cholinergic striatal interneurons. Copyright 2001 Wiley-Liss, Inc.

  6. The distribution of vesicular-arbuscular mycorrhizal fungi in India.

    PubMed

    Rani, R; Mukerji, K G

    1990-01-01

    Vesicular-arbuscular mycorrhizal fungi are widely distributed throughout the area studied including different altitudes ranging from sea level to 2500 ft above sea level. VAM fungi were recorded from 88% of the sites examined with Glomus fasciculatum and Glomus macrocarpum being the most commonly recorded. Mean species diversity was found to be maximum in the areas thickly vegetated and undisturbed.

  7. Nucleotide sequence analysis of the L gene of Newcastle disease virus: homologies with Sendai and vesicular stomatitis viruses.

    PubMed Central

    Yusoff, K; Millar, N S; Chambers, P; Emmerson, P T

    1987-01-01

    The nucleotide sequence of the L gene of the Beaudette C strain of Newcastle disease virus (NDV) has been determined. The L gene is 6704 nucleotides long and encodes a protein of 2204 amino acids with a calculated molecular weight of 248822. Mung bean nuclease mapping of the 5' terminus of the L gene mRNA indicates that the transcription of the L gene is initiated 11 nucleotides upstream of the translational start site. Comparison with the amino acid sequences of the L genes of Sendai virus and vesicular stomatitis virus (VSV) suggests that there are several regions of homology between the sequences. These data provide further evidence for an evolutionary relationship between the Paramyxoviridae and the Rhabdoviridae. A non-coding sequence of 46 nucleotides downstream of the presumed polyadenylation site of the L gene may be part of a negative strand leader RNA. Images PMID:3035486

  8. Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay †

    PubMed Central

    Wright, Sara F.; Morton, Joseph B.; Sworobuk, Janis E.

    1987-01-01

    Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains. PMID:16347441

  9. Real-time Raman spectroscopy of optically trapped living cells and organelles

    NASA Astrophysics Data System (ADS)

    Xie, Changan; Goodman, Charles; Dinno, Mumtaz A.; Li, Yong-Qing

    2004-12-01

    We report on real-time Raman spectroscopic studies of optically trapped living cells and organelles using an inverted confocal laser-tweezers-Raman-spectroscopy (LTRS) system. The LTRS system was used to hold a single living cell in a physiological solution or to hold a functional organelle within a living cell and consequently measured its Raman spectra. We have measured the changes in Raman spectra of a trapped yeast cell as the function of the temperature of the bathing solution and studied the irreversible cell degeneration during the heat denaturation. In addition, we measured the in-vitro Raman spectra of the nuclei within living pine cells and B. sporeformer, Strep. salivarius, and E. coli bacteria suspended in solution and showed the possibility of using LTRS system as a sensor for rapid identification of microbes in a fluid.

  10. ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion.

    PubMed

    Cater, Michael A; La Fontaine, Sharon; Shield, Kristy; Deal, Yolanda; Mercer, Julian F B

    2006-02-01

    The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.

  11. Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein

    NASA Astrophysics Data System (ADS)

    Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Arimura, Shin-ichi; Tsutsumi, Nobuhiro; Fukui, Kiichi; Itoh, Kazuyoshi

    2008-02-01

    We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

  12. Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

    PubMed

    Neuman, Benjamin W

    2016-11-01

    Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Membranes and mammalian glycolipid transferring proteins.

    PubMed

    Tuuf, Jessica; Mattjus, Peter

    2014-02-01

    Glycolipids are synthesized in and on various organelles throughout the cell. Their trafficking inside the cell is complex and involves both vesicular and protein-mediated machineries. Most important for the bulk lipid transport is the vesicular system, however, lipids moved by transfer proteins are also becoming more characterized. Here we review the latest advances in the glycolipid transfer protein (GLTP) and the phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) field, from a membrane point of view. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Immunocytochemical localization of three vesicular glutamate transporters in the cat retina.

    PubMed

    Fyk-Kolodziej, Bozena; Dzhagaryan, Arturik; Qin, Pu; Pourcho, Roberta G

    2004-08-02

    Vesicular transporters play an essential role in the packaging of glutamate for synaptic release and so are of particular importance in the retina, where glutamate serves as the neurotransmitter for photoreceptors, bipolar cells, and ganglion cells. In the present study, we have examined the distribution of the three known isoforms of vesicular glutamate transporter (VGLUT) in the cat retina. VGLUT1 was localized to all photoreceptor and bipolar cells, whereas VGLUT2 was found in ganglion cells. This basic pattern of complementary distribution for the two transporters among known populations of glutamatergic cells is similar to previous findings in the brain and spinal cord. However, the axon terminals of S-cone photoreceptors were found to express both VGLUT1 and VGLUT2 and some ganglion cells labeled for both VGLUT2 and VGLUT3. Such colocalizations suggest the existence of dual modes of regulation of vesicular glutamate transport in these neurons. Staining for VGLUT2 was also present in a small number of varicose processes, which were seen to ramify throughout the inner plexiform layer. These fibers may represent axon collaterals of ganglion cells. The most prominent site of VGLUT3 immunoreactivity was in a population of amacrine cells; the axon terminals of B-type horizontal cells were also labeled at their contacts with rod spherules. The presence of the VGLUT3 transporter at sites not otherwise implicated in glutamate release may indicate novel modes of glutamate signaling or additional roles for the transporter molecule. Copyright 2004 Wiley-Liss, Inc.

  15. Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna

    Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combinedmore » with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).« less

  16. Multiple marker abundance profiling: combining selected reaction monitoring and data-dependent acquisition for rapid estimation of organelle abundance in subcellular samples

    DOE PAGES

    Hooper, Cornelia M.; Stevens, Tim J.; Saukkonen, Anna; ...

    2017-10-12

    Measuring changes in protein or organelle abundance in the cell is an essential, but challenging aspect of cell biology. Frequently-used methods for determining organelle abundance typically rely on detection of a very few marker proteins, so are unsatisfactory. In silico estimates of protein abundances from publicly available protein spectra can provide useful standard abundance values but contain only data from tissue proteomes, and are not coupled to organelle localization data. A new protein abundance score, the normalized protein abundance scale (NPAS), expands on the number of scored proteins and the scoring accuracy of lower-abundance proteins in Arabidopsis. NPAS was combinedmore » with subcellular protein localization data, facilitating quantitative estimations of organelle abundance during routine experimental procedures. A suite of targeted proteomics markers for subcellular compartment markers was developed, enabling independent verification of in silico estimates for relative organelle abundance. Estimation of relative organelle abundance was found to be reproducible and consistent over a range of tissues and growth conditions. In silico abundance estimations and localization data have been combined into an online tool, multiple marker abundance profiling, available in the SUBA4 toolbox (http://suba.live).« less

  17. Genome sequences of nine vesicular stomatitis virus isolates from South America

    USDA-ARS?s Scientific Manuscript database

    We report nine full-genome sequences of vesicular stomatitis virus obtrained by Illumina next-generation sequencing of RNA, isolated from either cattle epithelial suspensions or cell culture supernatants. Seven of these viral genomes belonged to the New Jersey serotype/species, clade III, while two...

  18. Cholesterol transfer at endosomal-organelle membrane contact sites.

    PubMed

    Ridgway, Neale D; Zhao, Kexin

    2018-06-01

    Cholesterol is delivered to the limiting membrane of late endosomes by Niemann-Pick Type C1 and C2 proteins. This review summarizes recent evidence that cholesterol transfer from endosomes to the endoplasmic reticulum and other organelles is mediated by lipid-binding proteins that localize to membrane contact sites (MCS). LDL-cholesterol in the late endosomal/lysosomes is exported to the plasma membrane, where most cholesterol resides, and the endoplasmic reticulum, which harbors the regulatory complexes and enzymes that control the synthesis and esterification of cholesterol. A major advance in dissecting these cholesterol transport pathways was identification of frequent and dynamic MCS between endosomes and the endoplasmic reticulum, peroxisomes and plasma membrane. Positioned at these MCS are members of the oxysterol-binding protein (OSBP) and steroidogenic acute regulatory protein-related lipid-transfer family of lipid transfer proteins that bridge the opposing membranes and directly or indirectly mediate cholesterol transfer. OSBP-related protein 1L (ORP1L), ORP5 and ORP6 mediate cholesterol transfer to the endoplasmic reticulum that regulates cholesterol homeostasis. ORP1L and STARD3 also move cholesterol from the endoplasmic reticulum-to-late endosomal/lysosomes under low-cholesterol conditions to facilitate intraluminal vesicle formation. Cholesterol transport also occurs at MCS with peroxisomes and possibly the plasma membrane. Frequent contacts between organelles and the endo-lysosomal vesicles are sites for bidirectional transfer of cholesterol.

  19. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.

    2008-05-10

    The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganizationmore » of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.« less

  20. Index-of-refraction-dependent subcellular light scattering observed with organelle-specific dyes.

    PubMed

    Wilson, Jeremy D; Cottrell, William J; Foster, Thomas H

    2007-01-01

    Angularly resolved light scattering and wavelength-resolved darkfield scattering spectroscopy measurements were performed on intact, control EMT6 cells and cells stained with high-extinction lysosomal- or mitochondrial-localizing dyes. In the presence of the lysosomal-localizing dye NPe6, we observe changes in the details of light scattering from stained and unstained cells, which have both wavelength- and angular-dependent features. Analysis of measurements performed at several wavelengths reveals a reduced scattering cross section near the absorption maximum of the lysosomal-localizing dye. When identical measurements are made with cells loaded with a similar mitochondrial-localizing dye, HPPH, we find no evidence that staining mitochondria had any effect on the light scattering. Changes in the scattering properties of candidate populations of organelles induced by the addition of an absorber are modeled with Mie theory, and we find that any absorber-induced scattering response is very sensitive to the inherent refractive index of the organelle population. Our measurements and modeling are consistent with EMT6-cell-mitochondria having refractive indices close to those reported in the literature for organelles, approximately 1.4. The reduction in scattering cross section induced by NPe6 constrains the refractive index of lysosomes to be significantly higher. We estimate the refractive index of lysosomes in EMT6 cells to be approximately 1.6.

  1. AQUATIC PLANT SPECIATION AFFECTED BY DIVERSIFYING SELECTION OF ORGANELLE DNA REGIONS(1).

    PubMed

    Kato, Syou; Misawa, Kazuharu; Takahashi, Fumio; Sakayama, Hidetoshi; Sano, Satomi; Kosuge, Keiko; Kasai, Fumie; Watanabe, Makoto M; Tanaka, Jiro; Nozaki, Hisayoshi

    2011-10-01

    Many of the genes that control photosynthesis are carried in the chloroplast. These genes differ among species. However, evidence has yet to be reported revealing the involvement of organelle genes in the initial stages of plant speciation. To elucidate the molecular basis of aquatic plant speciation, we focused on the unique plant species Chara braunii C. C. Gmel. that inhabits both shallow and deep freshwater habitats and exhibits habitat-based dimorphism of chloroplast DNA (cpDNA). Here, we examined the "shallow" and "deep" subpopulations of C. braunii using two nuclear DNA (nDNA) markers and cpDNA. Genetic differentiation between the two subpopulations was measured in both nDNA and cpDNA regions, although phylogenetic analyses suggested nuclear gene flow between subpopulations. Neutrality tests based on Tajima's D demonstrated diversifying selection acting on organelle DNA regions. Furthermore, both "shallow" and "deep" haplotypes of cpDNA detected in cultures originating from bottom soils of three deep environments suggested that migration of oospores (dormant zygotes) between the two habitats occurs irrespective of the complete habitat-based dimorphism of cpDNA from field-collected vegetative thalli. Therefore, the two subpopulations are highly selected by their different aquatic habitats and show prezygotic isolation, which represents an initial process of speciation affected by ecologically based divergent selection of organelle genes. © 2011 Phycological Society of America.

  2. Diversity and origins of anaerobic metabolism in mitochondria and related organelles

    PubMed Central

    Stairs, Courtney W.; Leger, Michelle M.; Roger, Andrew J.

    2015-01-01

    Across the diversity of life, organisms have evolved different strategies to thrive in hypoxic environments, and microbial eukaryotes (protists) are no exception. Protists that experience hypoxia often possess metabolically distinct mitochondria called mitochondrion-related organelles (MROs). While there are some common metabolic features shared between the MROs of distantly related protists, these organelles have evolved independently multiple times across the breadth of eukaryotic diversity. Until recently, much of our knowledge regarding the metabolic potential of different MROs was limited to studies in parasitic lineages. Over the past decade, deep-sequencing studies of free-living anaerobic protists have revealed novel configurations of metabolic pathways that have been co-opted for life in low oxygen environments. Here, we provide recent examples of anaerobic metabolism in the MROs of free-living protists and their parasitic relatives. Additionally, we outline evolutionary scenarios to explain the origins of these anaerobic pathways in eukaryotes. PMID:26323757

  3. Optical tweezers for single molecule force spectroscopy on bacterial adhesion organelles

    NASA Astrophysics Data System (ADS)

    Andersson, Magnus; Axner, Ove; Uhlin, Bernt Eric; Fällman, Erik

    2006-08-01

    Instrumentation and methodologies for single molecule force spectroscopy on bacterial adhesion organelles by the use of force measuring optical tweezers have been developed. A thorough study of the biomechanical properties of fimbrial adhesion organelles expressed by uropathogenic E. coli, so-called pili, is presented. Steady-state as well as dynamic force measurements on P pili, expressed by E. coli causing pyelonephritis, have revealed, among other things, various unfolding and refolding properties of the helical structure of P pili, the PapA rod. Based on these properties an energy landscape model has been constructed by which specific biophysical properties of the PapA rod have been extracted, e.g. the number of subunits, the length of a single pilus, bond lengths and activation energies for bond opening and closure. Moreover, long time repetitive measurements have shown that the rod can be unfolded and refolded repetitive times without losing its intrinsic properties. These properties are believed to be of importance for the bacteria's ability to maintain close contact with host cells during initial infections. The results presented are considered to be of importance for the field of biopolymers in general and the development of new pharmaceuticals towards urinary tract infections in particular. The results show furthermore that the methodology can be used to gain knowledge of the intrinsic biomechanical function of adhesion organelles. The instrumentation is currently used for characterization of type 1 pili, expressed by E. coli causing cystitis, i.e. infections in the bladder. The first force spectrometry investigations of these pili will be presented.

  4. Vesicular zinc promotes presynaptic and inhibits postsynaptic long term potentiation of mossy fiber-CA3 synapse

    PubMed Central

    Pan, Enhui; Zhang, Xiao-an; Huang, Zhen; Krezel, Artur; Zhao, Min; Tin-berg, Christine E.; Lippard, Stephen J.; McNamara, James O.

    2011-01-01

    The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits a novel form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions of vesicular zinc is critical to proper function of hippocampal circuitry in health and disease. PMID:21943607

  5. Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics.

    PubMed

    Reumann, Sigrun; Bartel, Bonnie

    2016-12-01

    Peroxisomes are essential for life in plants. These organelles house a variety of metabolic processes that generate and inactivate reactive oxygen species. Our knowledge of pathways and mechanisms that depend on peroxisomes and their constituent enzymes continues to grow, and in this review we highlight recent advances in understanding the identity and biological functions of peroxisomal enzymes and metabolic processes. We also review how peroxisomal matrix and membrane proteins enter the organelle from their sites of synthesis. Peroxisome homeostasis is regulated by specific degradation mechanisms, and we discuss the contributions of specialized autophagy and a peroxisomal protease to the degradation of entire peroxisomes and peroxisomal enzymes that are damaged or superfluous. Finally, we review how peroxisomes can flexibly change their morphology to facilitate inter-organellar contacts. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Subaqueous non-vesicular to poorly-vesicular shards: hydroclastic fragmentation on seamounts and summit calderas

    NASA Astrophysics Data System (ADS)

    Mueller, W. U.; Dingwell, D. B.; Downey, W. S.; Mastin, L. G.

    2008-12-01

    Recognizing pyroclastic deposits that originate directly from magmatic and phreatomagmatic explosions in a subaqueous setting is based upon sedimentary structures, such as massive, stratified, and graded beds as well as (pyro)clast size. Ideally such deposits form ordered fining-and thinning-upward sequences. Pumice, scoria, glass shards, euhedral and broken crystals, and lithic fragments are constituents that support an explosive heritage. Recent deep-sea ROV and submersible dives have retrieved non-vesicular to vesicle- poor, mm-scale, mafic shards in 5-15 cm-thick massive and/or graded (stratified) deposits, for which a subaqueous explosive origin has been inferred. These sheet hyaloclastites with variable shard shapes were first documented on Seamount 6 as deep-sea Limu O Pele at water depths > 1000 m. We identified in Seamount 6 samples equant to blocky shards with angular to subrounded terminations, but also subordinate hair-like and contorted glassy filaments, warped shards and irregular shards. Shards display internal laminations (flow-banding?) and have local perlitic fractures. Bubble wall shards derived from scoria burst were rare. In combination with all the above and a poor shard vesicularity (< 2%), a magmatic explosive origin seems improbable. Such small-volume deposits have been reported from seamounts and summit calderas associated with subaqueous drainage tubes and ponded magma in depths > 1000 m. We envision that hydrostatic pressure commensurate with water depth played a significant role. The deposits can be readily explained by a hydroclastic process whereby fragmentation occurred at the milli-second (Limu) to second scale (hyaloclastite). Hence, hyperquenched glass shards or thread-like glass filaments need not require magmatic explosivity. Constant surface interaction between aphyric, low-viscosity, high temperature, magma-lava at depth with seawater causes fragmentation (granulation) that can generate such delicate shards. The transfer of

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

    PubMed

    Ellis, Kathryn; Bagwell, Jennifer; Bagnat, Michel

    2013-03-04

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

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

    PubMed Central

    Ellis, Kathryn; Bagwell, Jennifer

    2013-01-01

    The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H+-ATPase–dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development. PMID:23460678

  9. THE INFLUENCE OF X RAYS ON ORGANELLE INDUCTION AND DIFFERENTIATION IN GRASSHOPPER SPERMATOGENESIS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tahmisian, T.N.; Devine, R.L.

    1961-01-01

    The effects or x radiation on grasshopper spermatogenesis were studied by light and electron microscopy. The insects were irradiated at the second instar prior to the presence of maturation stages and observed at the last instar and imago stages. Dosages of 100 to 800 r retarded the differentiation of the nucleus and mitochondrial nebenkern in spermatids. Irradiation caused a curtailment and disorganization in the differentiation of the nebenkern from mitochondria. It also induced the formation of supernumerary centrioles, flagellar filaments, and acrosomes; nuclear disorganization as well as pycnosis and fragmentation also occurred. The nucleus appeared to be drawn toward eachmore » radioinduced supernumerary acrosome, with consequent multipolarity of the nucleus. Induction of a set of flagellar filaments was seen only where the centriolar structure was in contact with the nucleus. Details are given of an organelle that is composed of anastomosed and interwoven cytoplasmic strands. The results show that radioinduced changes in cell morphology are preceded by either alteration or curtailment of organelle differentiation. Furthermore, organelle differentiation during spermatogenesis was altered by doses that did not visibly affect the antecedent mitotic, maturation, or meiotic divisions; grasshoppers irradiated during the third instar showed the first effects of radiation at the last instar. (P.C.H.)« less

  10. Clofibric acid increases the formation of oleic acid in endoplasmic reticulum of the liver of rats.

    PubMed

    Hirose, Akihiko; Yamazaki, Tohru; Sakamoto, Takeshi; Sunaga, Katsuyoshi; Tsuda, Tadashi; Mitsumoto, Atsushi; Kudo, Naomi; Kawashima, Yoichi

    2011-01-01

    The effects of 2-(4-chlorophenoxy)-2-methylpropionic acid (clofibric acid) on the formation of oleic acid (18:1) from stearic acid (18:0) and utilization of the 18:1 formed for phosphatidylcholine (PC) formation in endoplasmic reticulum in the liver of rats were studied in vivo. [¹⁴C]18:0 was intravenously injected into control Wistar male rats and rats that had been fed on a diet containing 0.5% (w/w) clofibric acid for 7 days; and the distribution of radiolabeled fatty acids among subcellular organelles, microsomes, peroxisomes, and mitochondria, was estimated on the basis of correction utilizing the yields from homogenates of marker enzymes for these organelles. The radioactivity was mostly localized in microsomes and the radiolabeled fatty acids present in microsomes were significantly increased by the treatment of rats with clofibric acid. The formation of radiolabeled 18:1 in microsomes markedly increased and incorporations of the formed [¹⁴C]18:1 into PC and phosphatidylethanolamine in microsomes were augmented in response to clofibric acid. The [¹⁴C]18:1 incorporated into PC was mostly located at the C-2 position, but not the C-1 position, of PC, and the radioactivity in 18:1 at the C-2 position of PC was strikingly increased by clofibric acid. These results obtained from the in vivo experiments directly link the findings that clofibric acid treatment induces microsomal stearoyl-CoA desaturase and 1-acylglycerophosphocholine acyltransferase in the liver and the findings that the treatment with the drug elevated absolute mass and mass proportion of 18:1 at the C-2 position, but not the C-1 position, of PC in the liver together.

  11. Organelle relationships in cultured 3T3-L1 preadipocytes

    PubMed Central

    1980-01-01

    In differentiating 3T3-L1 cells, lipid spheres, the endoplasmic reticulum (ER), microperoxisomes, and mitochondria form "constellations" that may reflect the interplay of lipid metabolizing enzymes in these organelles. ER cisternae are also situated very close to "rosettes,"plasmalemmal specializations found in mature adipocytes in vivo. As in hepatocytes and absorptive cells of the intestine, this spatial relationship of ER and plasmalemma suggests a role for rosettes in the uptake of exogenous lipid precursors. The morphological differentiation of 3T3-L1 preadipocytes includes the loss of "stress fibers" and the appearance of microfilament like structures that encase, in a complex manner, the cytosolic lipid spheres that appear during differentiation. Other features described for the first time in 3T3-L1 preadipocytes include: (a) the presence of an extensive acid phosphatase (AcPase) positive GERL from which coated vesicles apparently arise (these coated vesicles display AcPase activity and are much smaller and far more numerous than the coated vesicles that seem to arise from the plasmalemmal coated pits); (b) the abundance of AcPase-positive autophagic vacuoles; and (c) a high level of alpha- naphthyl-acetate-esterase activity which, by light microscopy cytochemistry, appears to be localized in the cytosol. PMID:7191426

  12. Ammonia Assimilation in Zea mays L. Infected with a Vesicular-Arbuscular Mycorrhizal Fungus Glomus fasciculatum.

    PubMed

    Cliquet, J. B.; Stewart, G. R.

    1993-03-01

    To investigate nitrogen assimilation and translocation in Zea mays L. colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum (Thax. sensu Gerd.), we measured key enzyme activities, 15N incorporation into free amino acids, and 15N translocation from roots to shoots. Glutamine synthetase and nitrate reductase activities were increased in both roots and shoots compared with control plants, and glutamate dehydrogenase activity increased in roots only. In the presence of [15N]ammonium, glutamine amide was the most heavily labeled product. More label was incorporated into amino acids in VAM plants. The kinetics of 15N labeling and effects of methionine sulfoximine on distribution of 15N-labeled products were entirely consistent with the operation of the glutamate synthase cycle. No evidence was found for ammonium assimilation via glutamate dehydrogenase. 15N translocation from roots to shoots through the xylem was higher in VAM plants compared with control plants. These results establish that, in maize, VAM fungi increase ammonium assimilation, glutamine production, and xylem nitrogen translocation. Unlike some ectomycorrhizal fungi, VAM fungi do not appear to alter the pathway of ammonium assimilation in roots of their hosts.

  13. Vesicular komatiites, 3.5-Ga Komati Formation, Barberton Greenstone Belt, South Africa: inflation of submarine lavas and origin of spinifex zones

    NASA Astrophysics Data System (ADS)

    Dann, Jesse

    2001-08-01

    Komatiites of the 3.5-Ga Komati Formation are ultramafic lavas (>23% MgO) erupted in a submarine, lava plain environment. Newly discovered vesicular komatiites have vesicular upper crusts disrupted by synvolcanic structures that are similar to inflation-related structures of modern lava flows. Detailed outcrop maps reveal flows with upper vesicular zones, 2-15 m thick, which were (1) rotated by differential inflation, (2) intruded by dikes from the interior of the flow, (3) extended, forming a flooded graben, and/or (4) entirely engulfed. The largest inflated structure is a tumulus with 20 m of surface relief, which was covered by a compound flow unit of spinifex flow lobes. The lava that inflated and rotated the upper vesicular crust did not vesiculate, but crystallized as a thick spinifex zone with fist-size skeletal olivine. Instead of representing rapidly cooled lava, the spinifex zone cooled slowly beneath an insulating upper crust during inflation. Overpressure of the inflating lava may have inhibited vesiculation. This work describes the oldest vesicular komatiites known, illustrates the first field evidence for inflated structures in komatiite flows, proposes a new factor in the development of spinifex zones, and concludes that the inflation model is useful for understanding the evolution of komatiite submarine flow fields.

  14. Off to the Organelles - Killing Cancer Cells with Targeted Gold Nanoparticles

    PubMed Central

    Kodiha, Mohamed; Wang, Yi Meng; Hutter, Eliza; Maysinger, Dusica; Stochaj, Ursula

    2015-01-01

    Gold nanoparticles (AuNPs) are excellent tools for cancer cell imaging and basic research. However, they have yet to reach their full potential in the clinic. At present, we are only beginning to understand the molecular mechanisms that underlie the biological effects of AuNPs, including the structural and functional changes of cancer cells. This knowledge is critical for two aspects of nanomedicine. First, it will define the AuNP-induced events at the subcellular and molecular level, thereby possibly identifying new targets for cancer treatment. Second, it could provide new strategies to improve AuNP-dependent cancer diagnosis and treatment. Our review summarizes the impact of AuNPs on selected subcellular organelles that are relevant to cancer therapy. We focus on the nucleus, its subcompartments, and mitochondria, because they are intimately linked to cancer cell survival, growth, proliferation and death. While non-targeted AuNPs can damage tumor cells, concentrating AuNPs in particular subcellular locations will likely improve tumor cell killing. Thus, it will increase cancer cell damage by photothermal ablation, mechanical injury or localized drug delivery. This concept is promising, but AuNPs have to overcome multiple hurdles to perform these tasks. AuNP size, morphology and surface modification are critical parameters for their delivery to organelles. Recent strategies explored all of these variables, and surface functionalization has become crucial to concentrate AuNPs in subcellular compartments. Here, we highlight the use of AuNPs to damage cancer cells and their organelles. We discuss current limitations of AuNP-based cancer research and conclude with future directions for AuNP-dependent cancer treatment. PMID:25699096

  15. Mitochondria: An Organelle of Bacterial Origin Controlling Inflammation

    PubMed Central

    Meyer, Alain; Laverny, Gilles; Bernardi, Livio; Charles, Anne Laure; Alsaleh, Ghada; Pottecher, Julien; Sibilia, Jean; Geny, Bernard

    2018-01-01

    Inflammation is a cellular and molecular response to infection and/or tissues injury. While a suited inflammatory response in intensity and time allows for killing pathogens, clearing necrotic tissue, and healing injury; an excessive inflammatory response drives various diseases in which inflammation and tissues damages/stress self-sustain each other. Microbes have been poorly implied in non-resolving inflammation, emphasizing the importance of endogenous regulation of inflammation. Mitochondria have been historically identified as the main source of cellular energy, by coupling the oxidation of fatty acids and pyruvate with the production of high amount of adenosine triphosphate by the electron transport chain. Mitochondria are also the main source of reactive oxygen species. Interestingly, research in the last decade has highlighted that since its integration in eukaryote cells, this organelle of bacterial origin has not only been tolerated by immunity, but has also been placed as a central regulator of cell defense. In intact cells, mitochondria regulate cell responses to critical innate immune receptors engagement. Downstream intracellular signaling pathways interact with mitochondrial proteins and are tuned by mitochondrial functioning. Moreover, upon cell stress or damages, mitochondrial components are released into the cytoplasm or the extra cellular milieu, where they act as danger signals when recognized by innate immune receptors. Finally, by regulating the energetic state of immunological synapse between dendritic cells and lymphocytes, mitochondria regulate the inflammation fate toward immunotolerance or immunogenicity. As dysregulations of these processes have been recently involved in various diseases, the identification of the underlying mechanisms might open new avenues to modulate inflammation. PMID:29725325

  16. Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

    PubMed

    Fermie, Job; Liv, Nalan; Ten Brink, Corlinda; van Donselaar, Elly G; Müller, Wally H; Schieber, Nicole L; Schwab, Yannick; Gerritsen, Hans C; Klumperman, Judith

    2018-05-01

    Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity

    PubMed Central

    Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

    2017-01-01

    A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the “core” nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin–NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR–ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops. PMID:28193863

  18. Modular electron-transport chains from eukaryotic organelles function to support nitrogenase activity.

    PubMed

    Yang, Jianguo; Xie, Xiaqing; Yang, Mingxuan; Dixon, Ray; Wang, Yi-Ping

    2017-03-21

    A large number of genes are necessary for the biosynthesis and activity of the enzyme nitrogenase to carry out the process of biological nitrogen fixation (BNF), which requires large amounts of ATP and reducing power. The multiplicity of the genes involved, the oxygen sensitivity of nitrogenase, plus the demand for energy and reducing power, are thought to be major obstacles to engineering BNF into cereal crops. Genes required for nitrogen fixation can be considered as three functional modules encoding electron-transport components (ETCs), proteins required for metal cluster biosynthesis, and the "core" nitrogenase apoenzyme, respectively. Among these modules, the ETC is important for the supply of reducing power. In this work, we have used Escherichia coli as a chassis to study the compatibility between molybdenum and the iron-only nitrogenases with ETC modules from target plant organelles, including chloroplasts, root plastids, and mitochondria. We have replaced an ETC module present in diazotrophic bacteria with genes encoding ferredoxin-NADPH oxidoreductases (FNRs) and their cognate ferredoxin counterparts from plant organelles. We observe that the FNR-ferredoxin module from chloroplasts and root plastids can support the activities of both types of nitrogenase. In contrast, an analogous ETC module from mitochondria could not function in electron transfer to nitrogenase. However, this incompatibility could be overcome with hybrid modules comprising mitochondrial NADPH-dependent adrenodoxin oxidoreductase and the Anabaena ferredoxins FdxH or FdxB. We pinpoint endogenous ETCs from plant organelles as power supplies to support nitrogenase for future engineering of diazotrophy in cereal crops.

  19. Scanning ion images; analysis of pharmaceutical drugs at organelle levels

    NASA Astrophysics Data System (ADS)

    Larras-Regard, E.; Mony, M.-C.

    1995-05-01

    With the ion analyser IMS 4F used in microprobe mode, it is possible to obtain images of fields of 10 × 10 [mu]m2, corresponding to an effective magnification of 7000 with lateral resolution of 250 nm, technical characteristics that are appropriate for the size of cell organelles. It is possible to characterize organelles by their relative CN-, P- and S- intensities when the tissues are prepared by freeze fixation and freeze substitution. The recognition of organelles enables correlation of the tissue distribution of ebselen, a pharmaceutical drug containing selenium. The various metabolites characterized in plasma, bile and urine during biotransformation of ebselen all contain selenium, so the presence of the drug and its metabolites can be followed by images of Se. We were also able to detect the endogenous content of Se in tissue, due to the increased sensitivity of ion analysis in microprobe mode. Our results show a natural occurrence of Se in the border corresponding to the basal lamina of cells of proximal but not distal tubules of the kidney. After treatment of rats with ebselen, an additional site of Se is found in the lysosomes. We suggest that in addition to direct elimination of ebselen and its metabolites by glomerular filtration and urinary elimination, a second process of elimination may occur: Se compounds reaching the epithelial cells via the basal lamina accumulate in lysosomes prior to excretion into the tubular fluid. The technical developments of using the IMS 4F instrument in the microprobe mode and the improvement in preparation of samples by freeze fixation and substitution further extend the limit of ion analysis in biology. Direct imaging of trace elements and molecules marked with a tracer make it possible to determine their targets by comparison with images of subcellular structures. This is a promising advance in the study of pathways of compounds within tissues, cells and the whole organism.

  20. Preimplantation bovine embryos: Pathobiology of Haemophilus somnus exposure and resistance mechanisms to vesicular stomatitis virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Thomson, M.S.

    1988-01-01

    Preimplantation bovine embryos were exposed in vitro to H. somnus to determine if the bacteria would adhere to zona pellucida-intact (ZP-I) embryos or adhere to or infect ZP-free embryos. The effect of H. somnus on embryonic development in vitro was also investigated. Electrophoretic comparisons of outer membrane proteins of H. somnus revealed 2 major protein bands common to 10 H. somnus isolates. A monoclonal antibody produced against the outer membrane proteins reacted to one of the major protein bands. The sensitivity of a nucleic acid probe for detection of vesicular stomatitis virus (VSV) was validated in cells in culture andmore » used to determine if the synthetic double-stranded complex of polyriboinosinic and polyribocytidylic acids (poly I:C) would induce viral resistance in cultured bovine embryos. Two {sup 32}P-nick translated probes of high specific activity prepared from plasmids containing nucleic acid sequences of VSV virus were employed for viral mRNA detection in the tissue culture cells using a DNA-hybridization dot-blot technique. Using one of the probes, the technique was applied to detect differences in viral replication between four groups of bovine embryos (nonexposed, exposed to VSV virus, poly I:C-treated, and poly I:C-treated and exposed to VSV). The nucleic acid probe was sufficiently sensitive to detect differences in quantities of VSV mRNA among embryo treatment groups, resulting in the demonstration that resistance to viral infection was induced in day 9 bovine embryos.« less

  1. SAD-B kinase regulates pre-synaptic vesicular dynamics at hippocampal Schaffer collateral synapses and affects contextual fear memory.

    PubMed

    Watabe, Ayako M; Nagase, Masashi; Hagiwara, Akari; Hida, Yamato; Tsuji, Megumi; Ochiai, Toshitaka; Kato, Fusao; Ohtsuka, Toshihisa

    2016-01-01

    Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, but their roles in mature brains were only partially known. Here, we demonstrated, at mature pre-synaptic terminals, that SAD-B regulates vesicular release probability and synaptic plasticity. Moreover, hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice, suggesting that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. © 2015 International

  2. Genetic modifiers of abnormal organelle biogenesis in a Drosophila model of BLOC-1 deficiency

    PubMed Central

    Cheli, Verónica T.; Daniels, Richard W.; Godoy, Ruth; Hoyle, Diego J.; Kandachar, Vasundhara; Starcevic, Marta; Martinez-Agosto, Julian A.; Poole, Stephen; DiAntonio, Aaron; Lloyd, Vett K.; Chang, Henry C.; Krantz, David E.; Dell'Angelica, Esteban C.

    2010-01-01

    Biogenesis of lysosome-related organelles complex 1 (BLOC-1) is a protein complex formed by the products of eight distinct genes. Loss-of-function mutations in two of these genes, DTNBP1 and BLOC1S3, cause Hermansky–Pudlak syndrome, a human disorder characterized by defective biogenesis of lysosome-related organelles. In addition, haplotype variants within the same two genes have been postulated to increase the risk of developing schizophrenia. However, the molecular function of BLOC-1 remains unknown. Here, we have generated a fly model of BLOC-1 deficiency. Mutant flies lacking the conserved Blos1 subunit displayed eye pigmentation defects due to abnormal pigment granules, which are lysosome-related organelles, as well as abnormal glutamatergic transmission and behavior. Epistatic analyses revealed that BLOC-1 function in pigment granule biogenesis requires the activities of BLOC-2 and a putative Rab guanine-nucleotide-exchange factor named Claret. The eye pigmentation phenotype was modified by misexpression of proteins involved in intracellular protein trafficking; in particular, the phenotype was partially ameliorated by Rab11 and strongly enhanced by the clathrin-disassembly factor, Auxilin. These observations validate Drosophila melanogaster as a powerful model for the study of BLOC-1 function and its interactions with modifier genes. PMID:20015953

  3. Distribution of vesicular glutamate transporters in the human brain

    PubMed Central

    Vigneault, Érika; Poirel, Odile; Riad, Mustapha; Prud'homme, Josée; Dumas, Sylvie; Turecki, Gustavo; Fasano, Caroline; Mechawar, Naguib; El Mestikawy, Salah

    2015-01-01

    Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3) are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe) while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains. PMID:25798091

  4. Distribution of vesicular glutamate transporters in the human brain.

    PubMed

    Vigneault, Érika; Poirel, Odile; Riad, Mustapha; Prud'homme, Josée; Dumas, Sylvie; Turecki, Gustavo; Fasano, Caroline; Mechawar, Naguib; El Mestikawy, Salah

    2015-01-01

    Glutamate is the major excitatory transmitter in the brain. Vesicular glutamate transporters (VGLUT1-3) are responsible for uploading glutamate into synaptic vesicles. VGLUT1 and VGLUT2 are considered as specific markers of canonical glutamatergic neurons, while VGLUT3 is found in neurons previously shown to use other neurotransmitters than glutamate. Although there exists a rich literature on the localization of these glutamatergic markers in the rodent brain, little is currently known about the distribution of VGLUT1-3 in the human brain. In the present study, using subtype specific probes and antisera, we examined the localization of the three vesicular glutamate transporters in the human brain by in situ hybridization, immunoautoradiography and immunohistochemistry. We found that the VGLUT1 transcript was highly expressed in the cerebral cortex, hippocampus and cerebellum, whereas VGLUT2 mRNA was mainly found in the thalamus and brainstem. VGLUT3 mRNA was localized in scarce neurons within the cerebral cortex, hippocampus, striatum and raphe nuclei. Following immunoautoradiographic labeling, intense VGLUT1- and VGLUT2-immunoreactivities were observed in all regions investigated (cerebral cortex, hippocampus, caudate-putamen, cerebellum, thalamus, amygdala, substantia nigra, raphe) while VGLUT3 was absent from the thalamus and cerebellum. This extensive mapping of VGLUT1-3 in human brain reveals distributions that correspond for the most part to those previously described in rodent brains.

  5. A predicted physicochemically distinct sub-proteome associated with the intracellular organelle of the anammox bacterium Kuenenia stuttgartiensis.

    PubMed

    Medema, Marnix H; Zhou, Miaomiao; van Hijum, Sacha A F T; Gloerich, Jolein; Wessels, Hans J C T; Siezen, Roland J; Strous, Marc

    2010-05-12

    Anaerobic ammonium-oxidizing (anammox) bacteria perform a key step in global nitrogen cycling. These bacteria make use of an organelle to oxidize ammonia anaerobically to nitrogen (N2) and so contribute approximately 50% of the nitrogen in the atmosphere. It is currently unknown which proteins constitute the organellar proteome and how anammox bacteria are able to specifically target organellar and cell-envelope proteins to their correct final destinations. Experimental approaches are complicated by the absence of pure cultures and genetic accessibility. However, the genome of the anammox bacterium Candidatus "Kuenenia stuttgartiensis" has recently been sequenced. Here, we make use of these genome data to predict the organellar sub-proteome and address the molecular basis of protein sorting in anammox bacteria. Two training sets representing organellar (30 proteins) and cell envelope (59 proteins) proteins were constructed based on previous experimental evidence and comparative genomics. Random forest (RF) classifiers trained on these two sets could differentiate between organellar and cell envelope proteins with ~89% accuracy using 400 features consisting of frequencies of two adjacent amino acid combinations. A physicochemically distinct organellar sub-proteome containing 562 proteins was predicted with the best RF classifier. This set included almost all catabolic and respiratory factors encoded in the genome. Apparently, the cytoplasmic membrane performs no catabolic functions. We predict that the Tat-translocation system is located exclusively in the organellar membrane, whereas the Sec-translocation system is located on both the organellar and cytoplasmic membranes. Canonical signal peptides were predicted and validated experimentally, but a specific (N- or C-terminal) signal that could be used for protein targeting to the organelle remained elusive. A physicochemically distinct organellar sub-proteome was predicted from the genome of the anammox bacterium K

  6. The (in)complete organelle genome: exploring the use and nonuse of available technologies for characterizing mitochondrial and plastid chromosomes.

    PubMed

    Sanitá Lima, Matheus; Woods, Laura C; Cartwright, Matthew W; Smith, David Roy

    2016-11-01

    Not long ago, scientists paid dearly in time, money and skill for every nucleotide that they sequenced. Today, DNA sequencing technologies epitomize the slogan 'faster, easier, cheaper and more', and in many ways, sequencing an entire genome has become routine, even for the smallest laboratory groups. This is especially true for mitochondrial and plastid genomes. Given their relatively small sizes and high copy numbers per cell, organelle DNAs are currently among the most highly sequenced kind of chromosome. But accurately characterizing an organelle genome and the information it encodes can require much more than DNA sequencing and bioinformatics analyses. Organelle genomes can be surprisingly complex and can exhibit convoluted and unconventional modes of gene expression. Unravelling this complexity can demand a wide assortment of experiments, from pulsed-field gel electrophoresis to Southern and Northern blots to RNA analyses. Here, we show that it is exactly these types of 'complementary' analyses that are often lacking from contemporary organelle genome papers, particularly short 'genome announcement' articles. Consequently, crucial and interesting features of organelle chromosomes are going undescribed, which could ultimately lead to a poor understanding and even a misrepresentation of these genomes and the genes they express. High-throughput sequencing and bioinformatics have made it easy to sequence and assemble entire chromosomes, but they should not be used as a substitute for or at the expense of other types of genomic characterization methods. © 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

  7. An Atypical Local Vesicular Reaction to the Yellow Fever Vaccine.

    PubMed

    Wauters, Robert H; Hernandez, Camellia L; Petersen, Maureen M

    2017-09-19

    Yellow fever vaccine is a live attenuated viral inoculation indicated for patients traveling to endemic areas. The vaccine is generally well tolerated with minimal adverse effects. Typical side effects include malaise, pain at the injection site, and, albeit rarely, immediate hypersensitivity reactions. We present a case of a rare adverse reaction to yellow fever vaccine in which a patient developed vesicular lesions resulting in bullae and circumferential hyperpigmentation.

  8. Membrane fusion activity of vesicular stomatitis virus glycoprotein G is induced by low pH but not by heat or denaturant.

    PubMed

    Yao, Yi; Ghosh, Kakoli; Epand, Raquel F; Epand, Richard M; Ghosh, Hara P

    2003-06-05

    The fusogenic envelope glycoprotein G of the rhabdovirus vesicular stomatitis virus (VSV) induces membrane fusion at acidic pH. At acidic pH the G protein undergoes a major structural reorganization leading to the fusogenic conformation. However, unlike other viral fusion proteins, the low-pH-induced conformational change of VSV G is completely reversible. As well, the presence of an alpha-helical coiled-coil motif required for fusion by a number of viral and cellular fusion proteins was not predicted in VSV G protein by using a number of algorithms. Results of pH dependence of the thermal stability of G protein as determined by intrinsic Trp fluorescence and circular dichroism (CD) spectroscopy show that the G protein is equally stable at neutral or acidic pH. Destabilization of G structure at neutral pH with either heat or urea did not induce membrane fusion or conformational change(s) leading to membrane fusion. Taken together, these data suggest that the mechanism of VSV G-induced fusion is distinct from the fusion mechanism of fusion proteins that involve a coiled-coil motif.

  9. Phase separation and the formation of the pyrenoid, a carbon-fixing organelle

    NASA Astrophysics Data System (ADS)

    Xu, Bin; Freeman Rosenzweig, Elizabeth; Mackinder, Luke; Jonikas, Martin; Wingreen, Ned S.

    In the chloroplasts of most algae, the carbon-fixing enzyme Rubisco is concentrated in a non-membrane-bound structure called the pyrenoid, which enables more efficient carbon capture than that of most land plants. In contrast to the long-held assumptions of the field, the pyrenoid matrix is not a solid crystal, but behaves as a phase-separated, liquid-like organelle. In this system, the linker protein EPYC1 is thought to form multivalent specific bonds with Rubisco, and the formation of the pyrenoid occurs via the phase separation of these two associating proteins. Through analytical and numerical studies, we determine a phase diagram for this system. We also show how the length of the linker protein can affect the formation and dissolution of the pyrenoid in an unexpected manner. This new view of the pyrenoid matrix provides important insights into the structure, regulation, and inheritance of pyrenoid. More broadly, our findings give insights into fundamental principles of the architecture and inheritance of liquid-phase organelles.

  10. Organelle sedimentation in gravitropic roots of Limnobium is restricted to the elongation zone

    NASA Technical Reports Server (NTRS)

    Sack, F. D.; Kim, D.; Stein, B.

    1994-01-01

    Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud. were evaluated by light and electron microscopy to determine the distribution of organelle sedimentation towards gravity. Roots of Limnobium are strongly gravitropic. The rootcap consists of only two layers of cells. Although small amyloplasts are present in the central cap cells, no sedimentation of any organelle, including amyloplasts, was found. In contrast, both amyloplasts and nuclei sediment consistently and completely in cells of the elongation zone. Sedimentation occurs in one cell layer of the cortex just outside the endodermis. Sedimentation of both amyloplasts and nuclei begins in cells that are in their initial stages of elongation and persists at least to the level of the root where root hairs emerge. This is the first modern report of the presence of sedimentation away from, but not in, the rootcap. It shows that sedimentation in the rootcap is not necessary for gravitropic sensing in at least one angiosperm. If amyloplast sedimentation is responsible for gravitropic sensing, then the site of sensing in Limnobium roots is the elongation zone and not the rootcap. These data do not necessarily conflict with the hypothesis that sensing occurs in the cap in other roots, since Limnobium roots are exceptional in rootcap origin and structure, as well as in the distribution of organelle sedimentation. Similarly, if nuclear sedimentation is involved in gravitropic sensing, then nuclear mass would function in addition to, not instead of, that of amyloplasts.

  11. Partitioning of Intermediary Carbon Metabolism in Vesicular-Arbuscular Mycorrhizal Leek.

    PubMed

    Shachar-Hill, Y.; Pfeffer, P. E.; Douds, D.; Osman, S. F.; Doner, L. W.; Ratcliffe, R. G.

    1995-05-01

    Vesicular-arbuscular mycorrhizal fungi are symbionts for a large variety of crop plants; however, the form in which they take up carbon from the host is not established. To trace the course of carbon metabolism, we have used nuclear magnetic resonance spectroscopy with [13C]glucose labeling in vivo and in extracts to examine leek (Allium porrum) roots colonized by Glomus etunicatum (and uncolonized controls) as well as germinating spores. These studies implicate glucose as a likely substrate for vesicular-arbuscular mycorrhizal fungi in the symbiotic state. Root feeding of 0.6 mM 1-[13C]glucose labeled only the fungal metabolites trehalose and glycogen. The time course of this labeling was dependent on the status of the host. Incubation with 50 mM 1-[13C]glucose caused labeling of sucrose (in addition to fungal metabolites) with twice as much labeling in uncolonized plants. There was no detectable scrambling of the label from C1 glucose to the C6 position of glucose moieties in trehalose or glycogen. Labeling of mannitol C1,6 in the colonized root tissue was much less than in axenically germinating spores. Thus, carbohydrate metabolism of host and fungus are significantly altered in the symbiotic state.

  12. The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans

    PubMed Central

    Yang, Li; Ding, Yunfeng; Chen, Yong; Zhang, Shuyan; Huo, Chaoxing; Wang, Yang; Yu, Jinhai; Zhang, Peng; Na, Huimin; Zhang, Huina; Ma, Yanbin; Liu, Pingsheng

    2012-01-01

    Lipid droplets are cellular organelles that consists of a neutral lipid core covered by a monolayer of phospholipids and many proteins. They are thought to function in the storage, transport, and metabolism of lipids, in signaling, and as a specialized microenvironment for metabolism in most types of cells from prokaryotic to eukaryotic organisms. Lipid droplets have received a lot of attention in the last 10 years as they are linked to the progression of many metabolic diseases and hold great potential for the development of neutral lipid-derived products, such as biofuels, food supplements, hormones, and medicines. Proteomic analysis of lipid droplets has yielded a comprehensive catalog of lipid droplet proteins, shedding light on the function of this organelle and providing evidence that its function is conserved from bacteria to man. This review summarizes many of the proteomic studies on lipid droplets from a wide range of organisms, providing an evolutionary perspective on this organelle. PMID:22534641

  13. Correlation of changes of (non)exfoliated endometrial organelles and expressions of Musashi-1 and β-catenin with endometriosis in menstrual period.

    PubMed

    Yu, Cong-Xiang; Song, Jing-Hui; Liang, Lei

    2014-01-01

    This study aims to investigate the correlation of structural changes of endometrial organelles and expressions of Musashi-1 (Msi-1) and β-catenin with the endometriosis (EMs) in the menstrual period. The structural changes of exfoliated and nonexfoliated endometrial organelles in the experimental group and the control group were observed by the transmission electron microscopy (TEM) on the first and fifth day of menstruation. (1) TEM: compared with the control group, the exfoliated endometrial organelles in the experimental group on the first day were rich, with irregular nucleus, the bi-nucleolus could be seen, with rich chromatin; while the shapes of epithelial secretory cells in the nonexfoliated endometrial gland were irregular, with abundant organelles, the basal film varied in width, with abnormal curvature, and a lot of intercellular collagen fibers could be seen. (2) The expressions of Msi-1 and β-catenin in the exfoliated and nonexfoliated endometrium of the experimental group were higher than those of the control group and exhibited positively correlation, while no correlation could be found within the control group. (1) The organelles' structural changes might cause the changes of endometrial cellular functions. (2) Msi-1 might participate in the formation of EMs through activating the Wnt/β-catenin signaling pathway.

  14. Action potentials and amphetamine release antipsychotic drug from dopamine neuron synaptic VMAT vesicles.

    PubMed

    Tucker, Kristal R; Block, Ethan R; Levitan, Edwin S

    2015-08-11

    Based on lysotracker red imaging in cultured hippocampal neurons, antipsychotic drugs (APDs) were proposed to accumulate in synaptic vesicles by acidic trapping and to be released in response to action potentials. Because many APDs are dopamine (DA) D2 receptor (D2R) antagonists, such a mechanism would be particularly interesting if it operated in midbrain DA neurons. Here, the APD cyamemazine (CYAM) is visualized directly by two-photon microscopy in substantia nigra and striatum brain slices. CYAM accumulated slowly into puncta based on vacuolar H(+)-ATPase activity and dispersed rapidly upon dissipating organelle pH gradients. Thus, CYAM is subject to acidic trapping and released upon deprotonation. In the striatum, Ca(2+)-dependent reduction of the CYAM punctate signal was induced by depolarization or action potentials. Striatal CYAM overlapped with the dopamine transporter (DAT). Furthermore, parachloroamphetamine (pCA), acting via vesicular monoamine transporter (VMAT), and a charged VMAT, substrate 1-methyl-4-phenylpyridinium (MPP(+)), reduced striatal CYAM. In vivo CYAM administration and in vitro experiments confirmed that clinically relevant CYAM concentrations result in vesicular accumulation and pCA-dependent release. These results show that some CYAM is in DA neuron VMAT vesicles and suggests a new drug interaction in which amphetamine induces CYAM deprotonation and release as a consequence of the H(+) countertransport by VMAT that accompanies vesicular uptake, but not by inducing exchange or acting as a weak base. Therefore, in the striatum, APDs are released with DA in response to action potentials and an amphetamine. This synaptic corelease is expected to enhance APD antagonism of D2Rs where and when dopaminergic transmission occurs.

  15. Protective actions of the vesicular monoamine transporter 2 (VMAT2) in monoaminergic neurons.

    PubMed

    Guillot, Thomas S; Miller, Gary W

    2009-04-01

    Vesicular monoamine transporters (VMATs) are responsible for the packaging of neurotransmitters such as dopamine, serotonin, norepinephrine, and epinephrine into synaptic vesicles. These proteins evolved from precursors in the major facilitator superfamily of transporters and are among the members of the toxin extruding antiporter family. While the primary function of VMATs is to sequester neurotransmitters within vesicles, they can also translocate toxicants away from cytosolic sites of action. In the case of dopamine, this dual role of VMAT2 is combined-dopamine is more readily oxidized in the cytosol where it can cause oxidative stress so packaging into vesicles serves two purposes: neurotransmission and neuroprotection. Furthermore, the deleterious effects of exogenous toxicants on dopamine neurons, such as MPTP, can be attenuated by VMAT2 activity. The active metabolite of MPTP can be kept within vesicles and prevented from disrupting mitochondrial function thereby sparing the dopamine neuron. The highly addictive drug methamphetamine is also neurotoxic to dopamine neurons by using dopamine itself to destroy the axon terminals. Methamphetamine interferes with vesicular sequestration and increases the production of dopamine, escalating the amount in the cytosol and leading to oxidative damage of terminal components. Vesicular transport seems to resist this process by sequestering much of the excess dopamine, which is illustrated by the enhanced methamphetamine neurotoxicity in VMAT2-deficient mice. It is increasingly evident that VMAT2 provides neuroprotection from both endogenous and exogenous toxicants and that while VMAT2 has been adapted by eukaryotes for synaptic transmission, it is derived from phylogenetically ancient proteins that originally evolved for the purpose of cellular protection.

  16. Activity-dependent formation of a vesicular inhibitory amino acid transporter gradient in the superior olivary complex of NMRI mice.

    PubMed

    Ebbers, Lena; Weber, Maren; Nothwang, Hans Gerd

    2017-10-26

    In the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem. Immunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14 -/- mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient. Our results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular

  17. Geometric modeling of subcellular structures, organelles, and multiprotein complexes

    PubMed Central

    Feng, Xin; Xia, Kelin; Tong, Yiying; Wei, Guo-Wei

    2013-01-01

    SUMMARY Recently, the structure, function, stability, and dynamics of subcellular structures, organelles, and multi-protein complexes have emerged as a leading interest in structural biology. Geometric modeling not only provides visualizations of shapes for large biomolecular complexes but also fills the gap between structural information and theoretical modeling, and enables the understanding of function, stability, and dynamics. This paper introduces a suite of computational tools for volumetric data processing, information extraction, surface mesh rendering, geometric measurement, and curvature estimation of biomolecular complexes. Particular emphasis is given to the modeling of cryo-electron microscopy data. Lagrangian-triangle meshes are employed for the surface presentation. On the basis of this representation, algorithms are developed for surface area and surface-enclosed volume calculation, and curvature estimation. Methods for volumetric meshing have also been presented. Because the technological development in computer science and mathematics has led to multiple choices at each stage of the geometric modeling, we discuss the rationales in the design and selection of various algorithms. Analytical models are designed to test the computational accuracy and convergence of proposed algorithms. Finally, we select a set of six cryo-electron microscopy data representing typical subcellular complexes to demonstrate the efficacy of the proposed algorithms in handling biomolecular surfaces and explore their capability of geometric characterization of binding targets. This paper offers a comprehensive protocol for the geometric modeling of subcellular structures, organelles, and multiprotein complexes. PMID:23212797

  18. C-Terminal Truncation of α-COP Affects Functioning of Secretory Organelles and Calcium Homeostasis in Hansenula polymorpha

    PubMed Central

    Chechenova, Maria B.; Romanova, Nina V.; Deev, Alexander V.; Packeiser, Anna N.; Smirnov, Vladimir N.; Agaphonov, Michael O.; Ter-Avanesyan, Michael D.

    2004-01-01

    In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding α-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated α-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca2+ ions, whereas a lack of α-COP expression was lethal. The α-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca2+-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca2+ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca2+ homeostasis is discussed. PMID:14871936

  19. Development of a reverse transcription loop-mediated isothermal amplification assay for the detection of vesicular stomatitis New Jersey virus: use of rapid molecular assay to differentiate between vesicular disease viruses

    USDA-ARS?s Scientific Manuscript database

    Vesicular stomatitis (VS) is endemic in Central America and northern regions of South America. Sporadic outbreaks of VS can occur in cattle and pigs where the clinical presentation can be similar to foot-and-mouth disease (FMD). There is therefore a pressing need for rapid, sensitive and specific d...

  20. Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines

    USDA-ARS?s Scientific Manuscript database

    L-Glutamate (Glu) is the major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system, including the gastrointestinal tract (GIT) of cattle. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50 mM) Glu (Km = 1 to 4 mM) into synaptic vesicles (S...

  1. Topography of the Human Papillomavirus Minor Capsid Protein L2 during Vesicular Trafficking of Infectious Entry.

    PubMed

    DiGiuseppe, Stephen; Keiffer, Timothy R; Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Guion, Lucile G M; Müller, Martin; Sapp, Martin

    2015-10-01

    The human papillomavirus (HPV) capsid is composed of the major capsid protein L1 and the minor capsid protein L2. During entry, the HPV capsid undergoes numerous conformational changes that result in endosomal uptake and subsequent trafficking of the L2 protein in complex with the viral DNA to the trans-Golgi network. To facilitate this transport, the L2 protein harbors a number of putative motifs that, if capable of direct interaction, would interact with cytosolic host cell factors. These data imply that a portion of L2 becomes cytosolic during infection. Using a low concentration of digitonin to selectively permeabilize the plasma membrane of infected cells, we mapped the topography of the L2 protein during infection. We observed that epitopes within amino acid residues 64 to 81 and 163 to 170 and a C-terminal tag of HPV16 L2 are exposed on the cytosolic side of intracellular membranes, whereas an epitope within residues 20 to 38, which are upstream of a putative transmembrane region, is luminal. Corroborating these findings, we also found that L2 protein is sensitive to trypsin digestion during infection. These data demonstrate that the majority of the L2 protein becomes accessible on the cytosolic side of intracellular membranes in order to interact with cytosolic factors to facilitate vesicular trafficking. In order to complete infectious entry, nonenveloped viruses have to pass cellular membranes. This is often achieved through the viral capsid protein associating with or integrating into intracellular membrane. Here, we determine the topography of HPV L2 protein in the endocytic vesicular compartment, suggesting that L2 becomes a transmembrane protein with a short luminal portion and with the majority facing the cytosolic side for interaction with host cell transport factors. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. A plausible mechanism of biosorption in dual symbioses by vesicular-arbuscular mycorrhizal in plants.

    PubMed

    Azmat, Rafia; Hamid, Neelofer

    2015-03-01

    Dual symbioses of vesicular-arbuscular mycorrhizal (VAM) fungi with growth of Momordica charantia were elucidated in terms of plausible mechanism of biosorption in this article. The experiment was conducted in green house and mixed inoculum of the VAM fungi was used in the three replicates. Results demonstrated that the starch contents were the main source of C for the VAM to builds their hyphae. The increased plant height and leaves surface area were explained in relation with an increase in the photosynthetic rates to produce rapid sugar contents for the survival of plants. A decreased in protein, and amino acid contents and increased proline and protease activity in VAM plants suggested that these contents were the main bio-indicators of the plants under biotic stress. The decline in protein may be due to the degradation of these contents, which later on converted into dextrose where it can easily be absorbed by for the period of symbioses. A mechanism of C chemisorption in relation with physiology and morphology of plant was discussed.

  3. Exclusion of close linkage between the synaptic vesicular monoamine transporter locus and schizophrenia spectrum disorders

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Persico, A.M.; Uhl, G.R.; Wang, Zhe Wu

    The principal brain synaptic vesicular monoamine transporter (VMAT2) is responsible for the reuptake of serotonin, dopamine, norepinephrine, epinephrine, and histamine from the cytoplasm into synaptic vesicles, thus contributing to determination of the size of releasable neurotransmitter vesicular pools. Potential involvement of VMAT2 gene variants in the etiology of schizophrenia and related disorders was tested using polymorphic VMAT2 gene markers in 156 subjects from 16 multiplex pedigrees with schizophrenia, schizophreniform, schizoaffective, and schizotypal disorders and mood incongruent psychotic depression. Assuming genetic homogeneity, complete ({theta} = 0.0) linkage to the schizophrenia spectrum was excluded under both dominant and recessive models. Allelic variantsmore » at the VMAT2 locus do not appear to provide major genetic contributions to the etiology of schizophrenia spectrum disorders in these pedigrees. 16 refs.« less

  4. The GC-rich mitochondrial and plastid genomes of the green alga Coccomyxa give insight into the evolution of organelle DNA nucleotide landscape

    DOE PAGES

    Smith, David Roy; Burki, Fabien; Yamada, Takashi; ...

    2011-08-26

    Here, most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features ofmore » this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

  5. The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smith, David Roy; Burki, Fabien; Yamada, Takashi

    2011-05-13

    Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of thismore » species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

  6. New Insights Into Roles of Ubiquitin Modification in Regulating Plastids and Other Endosymbiotic Organelles.

    PubMed

    Broad, W; Ling, Q; Jarvis, P

    2016-01-01

    Recent findings have revealed important and diverse roles for the ubiquitin modification of proteins in the regulation of endosymbiotic organelles, which include the primary plastids of plants as well as complex plastids: the secondary endosymbiotic organelles of cryptophytes, alveolates, stramenopiles, and haptophytes. Ubiquitin modifications have a variety of potential consequences, both to the modified protein itself and to cellular regulation. The ubiquitin-proteasome system (UPS) can target individual proteins for selective degradation by the cytosolic 26S proteasome. Ubiquitin modifications can also signal the removal of whole endosymbiotic organelles, for example, via autophagy as has been well characterized in mitochondria. As plastids must import over 90% of their proteins from the cytosol, the observation that the UPS selectively targets the plastid protein import machinery is particularly significant. In this way, the UPS may influence the development and interconversions of different plastid types, as well as plastid responses to stress, by reconfiguring the organellar proteome. In complex plastids, the Symbiont-derived ERAD-Like Machinery (SELMA) has coopted the protein transport capabilities of the ER-Associated Degradation (ERAD) system, whereby misfolded proteins are retrotranslocated from ER for proteasomal degradation, uncoupling them from proteolysis: SELMA components have been retargeted to the second outermost plastid membrane to mediate protein import. In spite of this wealth of new information, there still remain a large number of unanswered questions and a need to define the roles of ubiquitin modification further in the regulation of plastids. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Nonspecific Organelle-Targeting Strategy with Core-Shell Nanoparticles of Varied Lipid Components/Ratios.

    PubMed

    Zhang, Lu; Sun, Jiashu; Wang, Yilian; Wang, Jiancheng; Shi, Xinghua; Hu, Guoqing

    2016-07-19

    We report a nonspecific organelle-targeting strategy through one-step microfluidic fabrication and screening of a library of surface charge- and lipid components/ratios-varied lipid shell-polymer core nanoparticles. Different from the common strategy relying on the use of organelle-targeted moieties conjugated onto the surface of nanoparticles, here, we program the distribution of hybrid nanoparticles in lysosomes or mitochondria by tuning the lipid components/ratios in shell. Hybrid nanoparticles with 60% 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 20% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) can intracellularly target mitochondria in both in vitro and in vivo models. While replacing DOPE with the same amount of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the nanoparticles do not show mitochondrial targeting, indicating an incremental effect of cationic and fusogenic lipids on lysosomal escape which is further studied by molecular dynamics simulations. This work unveils the lipid-regulated subcellular distribution of hybrid nanoparticles in which target moieties and complex synthetic steps are avoided.

  8. Biochemistry of ungerminated and germinated spores of the vesicular-arbuscular mycorrhizal fungus, Glomus caledonius: changes in neutral and polar lipids.

    PubMed

    Beilby, J P; Kidby, D K

    1980-08-01

    Neutral and polar spore lipids of the vesicular-arbuscular (VA) endophyte Glomus caledonius, were identified and quantitatively determined during spore germination, germ tube growth, and germ tube senescence. There are no previous reports detailing the spore lipid components of any member of the Endogenaceae, which is in the Zygomycotina. The fungus contained 45 to 72% total lipid depending upon its stage of growth. The concentration of neutral lipids decreased during germination while the polar lipids increased. Triacylglycerides were the most abundant neutral lipid, and lesser amounts of diacylglycerides, monoacylglycerides, free fatty acids, bound fatty acids, hydrocarbons, and sterols. The major fatty acids identified by gas--liquid chromatography and mass spectrometry were 16:1, 16:0, and 18:1. The minor fatty acids identified were n-3 and n-6 polyunsaturates. The n-3 polyunsaturated fatty acids have not been reported before in Zygomycetes. The fatty acid composition of the individual lipid classes was examined. The major phospholipids were phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine, with smaller amounts of diphosphatidylglycerol and phosphatidic acid. The free sterol fraction was in greater quantity than sterol esters during germination and germ tube elongation. The capacity to synthesize sterols was demonstrated. Approximate net rates of change in the different lipid components were calculated. During spore germination and early germ tube growth, there was a net synthesis of lipids, with a large production of free fatty acids, in the germinating spore. Later in the growth period there was a net degradation of lipid, characterized by a large conversion of free fatty acids to unidentified compounds. During this period net free sterol synthesis ceased and sterol ester synthesis continued using the existing free sterol.

  9. Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

    PubMed Central

    Li, Xiang; Li, Xiang; Wang, Jiaxiong; Ye, Zaiyuan; Li, Ji-Cheng

    2012-01-01

    Background: Oridonin (ORI) could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood. Methods: Human prostate cancer (HPC) cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM). Chemical staining with acridine orange (AO), MDC or DAPI was used to detect acidic vesicular organelles (AVOs) and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS. Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC) cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs) were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA) reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%). Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy. Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand the anti

  10. Effects of inhibitors on early protein, RNA, and lipid synthesis in germinating vesicular-arbuscular mycorrhizal fungal spores of Glomus caledonium.

    PubMed

    Beilby, J P

    1983-05-01

    The incorporation of isotopically labelled precursors, [1-14C]leucine, [2-14C]uracil and [1-14C]acetate, by germinating spores of the vesicular-arbuscular mycorrhizal fungus Glomus caledonium was investigated after prior incubation with several metabolic inhibitors. Inhibition of isotope incorporation was greatest with ethidium bromide and cycloheximide and least with chloramphenicol and actinomycin D. The incorporation of [1-14C]leucine into protein was reduced by 92% within 60 min of incubation in either ethidium bromide at 5 micrograms/mL or cycloheximide at 100 micrograms/mL. For these studies time zero was coincident with dry quiescent spores being first wet. Lipid synthesis during germination of the spores was detected at 120 min by following the incorporation of [1-14C]acetate. At 51 h, incorporation was greater in the triacylglycerides and diacylglycerides than in the free sterols, phospholipids, or free fatty acid. Spores exposed to cycloheximide for 60 min after imbibition showed very little phospholipid synthesis and no free fatty acid synthesis. The synthesis of RNA after spore imbibition is also discussed.

  11. Dextran vesicular carriers for dual encapsulation of hydrophilic and hydrophobic molecules and delivery into cells.

    PubMed

    Pramod, P S; Takamura, Kathryn; Chaphekar, Sonali; Balasubramanian, Nagaraj; Jayakannan, M

    2012-11-12

    Dextran vesicular nanoscaffolds were developed based on polysaccharide and renewable resource alkyl tail for dual encapsulation of hydrophilic and hydrophobic molecules (or drugs) and delivery into cells. The roles of the hydrophobic segments on the molecular self-organization of dextran backbone into vesicles or nanoparticles were investigated in detail. Dextran vesicles were found to be a unique dual carrier in which water-soluble molecules (like Rhodamine-B, Rh-B) and polyaromatic anticancer drug (camptothecin, CPT) were selectively encapsulated in the hydrophilic core and hydrophobic layer, respectively. The dextran vesicles were capable of protecting the plasma-sensitive CPT lactone pharmacophore against the hydrolysis by 10× better than the CPT alone in PBS. The aliphatic ester linkage connecting the hydrophobic tail with dextran was found to be cleaved by esterase under physiological conditions for fast releasing of CPT or Rh-B. Cytotoxicity of the dextran vesicle and its drug conjugate were tested on mouse embryonic fibroblast cells (MEFs) using MTT assay. The dextran vesicular scaffold was found to be nontoxic to living cells. CPT loaded vesicles were found to be 2.5-fold more effective in killing fibroblasts compared to that of CPT alone in PBS. Confocal microscopic images confirmed that both Rh-B and CPT loaded vesicles to be taken up by fibroblasts compared to CPT alone, showing a distinctly perinuclear localization in cells. The custom designed dextran vesicular provides new research opportunities for dual loading and delivering of hydrophilic and hydrophobic drug molecules.

  12. Populus euphratica APYRASE2 Enhances Cold Tolerance by Modulating Vesicular Trafficking and Extracellular ATP in Arabidopsis Plants.

    PubMed

    Deng, Shurong; Sun, Jian; Zhao, Rui; Ding, Mingquan; Zhang, Yinan; Sun, Yuanling; Wang, Wei; Tan, Yeqing; Liu, Dandan; Ma, Xujun; Hou, Peichen; Wang, Meijuan; Lu, Cunfu; Shen, Xin; Chen, Shaoliang

    2015-09-01

    Apyrase and extracellular ATP play crucial roles in mediating plant growth and defense responses. In the cold-tolerant poplar, Populus euphratica, low temperatures up-regulate APYRASE2 (PeAPY2) expression in callus cells. We investigated the biochemical characteristics of PeAPY2 and its role in cold tolerance. We found that PeAPY2 predominantly localized to the plasma membrane, but punctate signals also appeared in the endoplasmic reticulum and Golgi apparatus. PeAPY2 exhibited broad substrate specificity, but it most efficiently hydrolyzed purine nucleotides, particularly ATP. PeAPY2 preferred Mg(2+) as a cofactor, and it was insensitive to various, specific ATPase inhibitors. When PeAPY2 was ectopically expressed in Arabidopsis (Arabidopsis thaliana), cold tolerance was enhanced, based on root growth measurements and survival rates. Moreover, under cold stress, PeAPY2-transgenic plants maintained plasma membrane integrity and showed reduced cold-elicited electrolyte leakage compared with wild-type plants. These responses probably resulted from efficient plasma membrane repair via vesicular trafficking. Indeed, transgenic plants showed accelerated endocytosis and exocytosis during cold stress and recovery. We found that low doses of extracellular ATP accelerated vesicular trafficking, but high extracellular ATP inhibited trafficking and reduced cell viability. Cold stress caused significant increases in root medium extracellular ATP. However, under these conditions, PeAPY2-transgenic lines showed greater control of extracellular ATP levels than wild-type plants. We conclude that Arabidopsis plants that overexpressed PeAPY2 could increase membrane repair by accelerating vesicular trafficking and hydrolyzing extracellular ATP to avoid excessive, cold-elicited ATP accumulation in the root medium and, thus, reduced ATP-induced inhibition of vesicular trafficking. © 2015 American Society of Plant Biologists. All Rights Reserved.

  13. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... vesicular disease is maintained on the APHIS Web site at http://www.aphis.usda.gov/import_export/animals... 260 °C for approximately 210 minutes after which they must be cooked in hot oil (deep-fried) at a...

  14. Immunolocalization of the vesicular acetylcholine transporter in larval and adult Drosophila neurons.

    PubMed

    Boppana, Sridhar; Kendall, Natalie; Akinrinsola, Opeyemi; White, Daniel; Patel, Krushali; Lawal, Hakeem

    2017-03-16

    Vesicular acetylcholine transporter (VAChT) function is essential for organismal survival, mediating the packaging of acetylcholine (ACh) for exocytotic release. However, its expression pattern in the Drosophila brain has not been fully elucidated. To investigate the localization of VAChT, we developed an antibody against the C terminal region of the protein and we show that this antibody recognizes a 65KDa protein corresponding to VAChT on an immunoblot in both Drosophila head homogenates and in Schneider 2 cells. Further, we report for the first time the expression of VAChT in the antennal lobe and ventral nerve cord of Drosophila larva; and we independently confirm the expression of the protein in mushroom bodies and optic lobes of adult Drosophila. Importantly, we show that VAChT co-localizes with a synaptic vesicle marker in vivo, confirming previous reports of the localization of VAChT to synaptic terminals. Together, these findings help establish the vesicular localization of VAChT in cholinergic neurons in Drosophila and present an important molecular tool with which to dissect the function of the transporter in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Stimulation of vesicular-arbuscular mycorrhizal fungi by mycotrophic and nonmycotrophic plant root systems.

    PubMed

    Schreiner, R P; Koide, R T

    1993-08-01

    Transformed root cultures of three nonmycotrophic and one mycotrophic plant species stimulated germination and hyphal growth of the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum (Becker & Gerd.) in a gel medium. However, only roots of the mycotrophic species (carrot) supported continued hyphal exploration after 3 to 4 weeks and promoted appressoria formation by G. etunicatum.

  16. Microbial arms race: Ballistic "nematocysts" in dinoflagellates represent a new extreme in organelle complexity.

    PubMed

    Gavelis, Gregory S; Wakeman, Kevin C; Tillmann, Urban; Ripken, Christina; Mitarai, Satoshi; Herranz, Maria; Özbek, Suat; Holstein, Thomas; Keeling, Patrick J; Leander, Brian S

    2017-03-01

    We examine the origin of harpoon-like secretory organelles (nematocysts) in dinoflagellate protists. These ballistic organelles have been hypothesized to be homologous to similarly complex structures in animals (cnidarians); but we show, using structural, functional, and phylogenomic data, that nematocysts evolved independently in both lineages. We also recorded the first high-resolution videos of nematocyst discharge in dinoflagellates. Unexpectedly, our data suggest that different types of dinoflagellate nematocysts use two fundamentally different types of ballistic mechanisms: one type relies on a single pressurized capsule for propulsion, whereas the other type launches 11 to 15 projectiles from an arrangement similar to a Gatling gun. Despite their radical structural differences, these nematocysts share a single origin within dinoflagellates and both potentially use a contraction-based mechanism to generate ballistic force. The diversity of traits in dinoflagellate nematocysts demonstrates a stepwise route by which simple secretory structures diversified to yield elaborate subcellular weaponry.

  17. Presynaptic mechanisms of lead neurotoxicity: effects on vesicular release, vesicle clustering and mitochondria number.

    PubMed

    Zhang, Xiao-Lei; Guariglia, Sara R; McGlothan, Jennifer L; Stansfield, Kirstie H; Stanton, Patric K; Guilarte, Tomás R

    2015-01-01

    Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development.

  18. Student creativity in creating cell organelles as media for learning

    NASA Astrophysics Data System (ADS)

    Fatmawati, B.

    2018-04-01

    Creativity is not formed by itself but it is influenced by some others factors. Creativity is a . person’s ability to create / generate an idea embodied in the form of a product to solve problems which is accepted socially, spiritually, artificially, scientifically, and technologically. Learning media is a means of communication to deliver learning materials. There are three kinds of learning media produced by students such as books story, playdough, and the utilization of inorganic waste. The focus of this research is to know the students’ creativity in producing learnning media to understand an Abstract material especially on topic of cell organelles of animal and plant cell. Data analysis is using two ways that calculate the score of mastery in terms of concepts and creativity. The results showed the score of students’ understanding was increasing from 15 (average score of pre-test) to 31.1 (average score of post-test). It was categorized into three level, that are, high level with 21.4% of participants, medium with 64.3%, and low with 14.3%). Seven groups of students make learning media made of waste, playdough, and waste made in story form. The assessment of creativity involved four aspects, namely, color combinations, stringing, tidiness, and make (the accuracy of the concept with the form). Thus, it can be argued that self-created learning media helps in understanding the Abstract concepts of cell organelles.

  19. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... has declared free of swine vesicular disease is maintained on the APHIS Web site at http://www.aphis... which they must be cooked in hot oil (deep-fried) at a minimum of 104 °C for an additional 150 minutes...

  20. Continuous Microfluidic Self-Assembly of Hybrid Janus-Like Vesicular Motors: Autonomous Propulsion and Controlled Release.

    PubMed

    Wang, Lei; Liu, Yijing; He, Jie; Hourwitz, Matthew J; Yang, Yunlong; Fourkas, John T; Han, Xiaojun; Nie, Zhihong

    2015-08-01

    A microfluidic strategy is developed for the continuous fabrication of hybrid Janus vesicular motors that uniquely combine the capability of autonomous propulsion and externally controlled delivery of encapsulated payload. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Strongly Acidic Auxin Indole-3-Methanesulfonic Acid

    PubMed Central

    Cohen, Jerry D.; Baldi, Bruce G.; Bialek, Krystyna

    1985-01-01

    A radiochemical synthesis is described for [14C]indole-3-methanesulfonic acid (IMS), a strongly acidic auxin analog. Techniques were developed for fractionation and purification of IMS using normal and reverse phase chromatography. In addition, the utility of both Fourier transform infrared spectrometry and fast atom bombardment mass spectrometry for analysis of IMS has been demonstrated. IMS was shown to be an active auxin, stimulating soybean hypocotyl elongation, bean first internode curvature, and ethylene production. IMS uptake by thin sections of soybean hypocotyl was essentially independent of solution pH and, when applied at a 100 micromolar concentration, IMS exhibited a basipetal polarity in its transport in both corn coleoptile and soybean hypocotyl sections. [14C]IMS should, therefore, be a useful compound to study fundamental processes related to the movement of auxins in plant tissues and organelles. PMID:16664007

  2. Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

    NASA Astrophysics Data System (ADS)

    Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

    2005-04-01

    Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

  3. Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

    PubMed Central

    Qiu, Shihong; Ogino, Minako; Luo, Ming

    2015-01-01

    ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the

  4. Stimulation of Vesicular-Arbuscular Mycorrhizal Fungi by Mycotrophic and Nonmycotrophic Plant Root Systems

    PubMed Central

    Schreiner, R. Paul; Koide, Roger T.

    1993-01-01

    Transformed root cultures of three nonmycotrophic and one mycotrophic plant species stimulated germination and hyphal growth of the vesicular-arbuscular mycorrhizal fungus Glomus etunicatum (Becker & Gerd.) in a gel medium. However, only roots of the mycotrophic species (carrot) supported continued hyphal exploration after 3 to 4 weeks and promoted appressoria formation by G. etunicatum. Images PMID:16349030

  5. Actin-based vesicular transport in the first 20 min after dusk is crucial for daily rhabdom synthesis in the compound eye of the grapsid crab Hemigrapsus sanguineus.

    PubMed

    Matsushita, A; Arikawa, K

    1997-09-01

    In the crab Hemigrapsus sanguineus, maintained under a 12 h:12 h light:dark cycle, the amount of vesicular smooth endoplasmic reticulum (vesicular sER) in the photoreceptor cell body increases after the light is turned off. This paper demonstrates that actin filaments in the photoreceptor cell body are involved in the transport of vesicular sER towards the rhabdom. To specify the time of actin contribution to rhabdom synthesis, we disrupted the organization of actin filaments in the cell body with cytochalasin D at various time around dusk. We then measured the rhabdom size and also examined the ultrastructure of the photoreceptor cell body 3 h after extinguishing the light. When cytochalasin D was applied from either 1 h before or immediately after extinguishing the light, the rhabdom size did not increase, whereas vesicular sER accumulated in the cell body. In contrast, cytochalasin D applied to the eyes from 20 min after turning the light off did not inhibit rhabdom synthesis. These results indicate that the first 20 min after the light is turned off is particularly important for the transport of vesicular sER towards the rhabdom by the cell body actin filaments.

  6. Phase transitions and size scaling of membrane-less organelles

    PubMed Central

    2013-01-01

    The coordinated growth of cells and their organelles is a fundamental and poorly understood problem, with implications for processes ranging from embryonic development to oncogenesis. Recent experiments have shed light on the cell size–dependent assembly of membrane-less cytoplasmic and nucleoplasmic structures, including ribonucleoprotein (RNP) granules and other intracellular bodies. Many of these structures behave as condensed liquid-like phases of the cytoplasm/nucleoplasm. The phase transitions that appear to govern their assembly exhibit an intrinsic dependence on cell size, and may explain the size scaling reported for a number of structures. This size scaling could, in turn, play a role in cell growth and size control. PMID:24368804

  7. Ligand-directed profiling of organelles with internalizing phage libraries

    PubMed Central

    Dobroff, Andrey S.; Rangel, Roberto; Guzman-Roja, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Bologa, Cristian G.; Oprea, Tudor I.; Brinker, C. Jeffrey; Pasqualini, Renata; Arap, Wadih

    2015-01-01

    Phage display is a resourceful tool to, in an unbiased manner, discover and characterize functional protein-protein interactions, to create vaccines, and to engineer peptides, antibodies, and other proteins as targeted diagnostic and/or therapeutic agents. Recently, our group has developed a new class of internalizing phage (iPhage) for ligand-directed targeting of organelles and/or to identify molecular pathways within live cells. This unique technology is suitable for applications ranging from fundamental cell biology to drug development. Here we describe the method for generating and screening the iPhage display system, and explain how to select and validate candidate internalizing homing peptide. PMID:25640897

  8. A divalent interaction between HPS1 and HPS4 is required for the formation of the biogenesis of lysosome-related organelle complex-3 (BLOC-3).

    PubMed

    Carmona-Rivera, Carmelo; Simeonov, Dimitre R; Cardillo, Nicholas D; Gahl, William A; Cadilla, Carmen L

    2013-03-01

    Hermansky-Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. Nine HPS-causing genes have been identified in humans. HPS-1 is the most severe subtype with a prevalence of ~1/1800 in northwest Puerto Rico due to a founder mutation in the HPS1 gene. Mutations in HPS genes affect the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and platelet dense granules. Two of these genes (HPS1 and HPS4) encode the HPS1 and HPS4 proteins, which assemble to form a complex known as Biogenesis of Lysosome-related Organelle Complex 3 (BLOC-3). We report the identification of the interacting regions in HPS1 and HPS4 required for the formation of this complex. Two regions in HPS1, spanning amino acids 1-249 and 506-700 are required for binding to HPS4; the middle portion of HPS1 (residues 250-505) is not required for this interaction. Further interaction studies showed that the N-termini of HPS1 and HPS4 interact with each other and that a discrete region of HPS4 (residues 340-528) interacts with both the N- and C-termini of the HPS1 protein. Several missense mutations found in HPS-1 patients did not affect interaction with HPS4, but some mutations involving regions interacting with HPS4 caused instability of HPS1. These observations extend our understanding of BLOC-3 assembly and represent an important first step in the identification of domains responsible for the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

    PubMed Central

    Patwardhan, Anand; Bardin, Sabine; Miserey-Lenkei, Stéphanie; Larue, Lionel; Goud, Bruno; Raposo, Graça; Delevoye, Cédric

    2017-01-01

    Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function. PMID:28607494

  10. Active diffusion and microtubule-based transport oppose myosin forces to position organelles in cells

    PubMed Central

    Lin, Congping; Schuster, Martin; Guimaraes, Sofia Cunha; Ashwin, Peter; Schrader, Michael; Metz, Jeremy; Hacker, Christian; Gurr, Sarah Jane; Steinberg, Gero

    2016-01-01

    Even distribution of peroxisomes (POs) and lipid droplets (LDs) is critical to their role in lipid and reactive oxygen species homeostasis. How even distribution is achieved remains elusive, but diffusive motion and directed motility may play a role. Here we show that in the fungus Ustilago maydis ∼95% of POs and LDs undergo diffusive motions. These movements require ATP and involve bidirectional early endosome motility, indicating that microtubule-associated membrane trafficking enhances diffusion of organelles. When early endosome transport is abolished, POs and LDs drift slowly towards the growing cell end. This pole-ward drift is facilitated by anterograde delivery of secretory cargo to the cell tip by myosin-5. Modelling reveals that microtubule-based directed transport and active diffusion support distribution, mobility and mixing of POs. In mammalian COS-7 cells, microtubules and F-actin also counteract each other to distribute POs. This highlights the importance of opposing cytoskeletal forces in organelle positioning in eukaryotes. PMID:27251117

  11. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    PubMed Central

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-01-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions. PMID:24957323

  12. Genetic and antigenic relationships of vesicular stomatitis viruses from South America.

    PubMed

    Pauszek, Steven J; Barrera, Jose Del C; Goldberg, Tony; Allende, Rossana; Rodriguez, Luis L

    2011-11-01

    Vesicular stomatitis (VS) viruses have been classified into two serotypes: New Jersey (VSNJV) and Indiana (VSIV). Here, we have characterized field isolates causing vesicular stomatitis in Brazil and Argentina over a 35-year span. Cluster analysis based on either serological relatedness, as inferred from virus neutralization and complement fixation assays, or nucleotide sequences of two separate genes (phosphoprotein or glycoprotein) grouped the field isolates into two distinct monophyletic groups within the Indiana serogroup. One group included seven viruses from Brazil and Argentina that were serologically classified as Indiana-2 and Cocal virus (COCV). The other group contained three viruses from Brazil that were serologically classified as Indiana-3 and the prototype of this group, Alagoas virus (VSAV). Interestingly, two vesiculoviruses that were isolated from insects but do not cause disease in animals, one from Brazil (Maraba virus; MARAV) and the other from Colombia (CoAr 171638), grouped into two separate genetic lineages within the Indiana serotype. Our data provide support for the classification of viruses causing clinical VS in livestock in Brazil and Argentina into two distinct groups: Indiana-2 (VSIV-2) and Indiana-3 (VSIV-3). We suggest using nomenclature for these viruses that includes the serotype, year and place of occurrence, and affected host. This nomenclature is consistent with that currently utilized to describe field isolates of VSNJV or VSIV in scientific literature.

  13. Ectopic vesicular glutamate release at the optic nerve head and axon loss in mouse experimental glaucoma.

    PubMed

    Fu, Christine T; Sretavan, David W

    2012-11-07

    Although clinical and experimental observations indicate that the optic nerve head (ONH) is a major site of axon degeneration in glaucoma, the mechanisms by which local retinal ganglion cell (RGC) axons are injured and damage spreads among axons remain poorly defined. Using a laser-induced ocular hypertension (LIOH) mouse model of glaucoma, we found that within 48 h of intraocular pressure elevation, RGC axon segments within the ONH exhibited ectopic accumulation and colocalization of multiple components of the glutamatergic presynaptic machinery including the vesicular glutamate transporter VGLUT2, several synaptic vesicle marker proteins, glutamate, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and active zone cytomatrix components, as well as ultrastructurally identified, synaptophysin-containing vesicles. Ectopic vesicle exocytosis and glutamate release were detected in acute preparations of the LIOH ONH. Immunolocalization and analysis using the ionotropic receptor channel-permeant cation agmatine indicated that ONH axon segments and glia expressed glutamate receptors, and these receptors were more active after LIOH compared with controls. Pharmacological antagonism of glutamate receptors and neuronal activity resulted in increased RGC axon sparing in vivo. Furthermore, in vivo RGC-specific genetic disruption of the vesicular glutamate transporter VGLUT2 or the obligatory NMDA receptor subunit NR1 promoted axon survival in experimental glaucoma. As the inhibition of ectopic glutamate vesicular release or glutamate receptivity can independently modify the severity of RGC axon loss, synaptic release mechanisms may provide useful therapeutic entry points into glaucomatous axon degeneration.

  14. Effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell function and viability.

    PubMed

    McDermott, Catherine; Chess-Williams, Russ; Grant, Gary D; Perkins, Anthony V; McFarland, Amelia J; Davey, Andrew K; Anoopkumar-Dukie, Shailendra

    2012-03-01

    We determined the effects of Pseudomonas aeruginosa virulence factor pyocyanin on human urothelial cell viability and function in vitro. RT4 urothelial cells were treated with pyocyanin (1 to 100 μM) for 24 hours. After exposure the treatment effects were measured according to certain end points, including changes in urothelial cell viability, reactive oxygen species formation, caspase-3 activity, basal and stimulated adenosine triphosphate release, SA-β-gal activity and detection of acidic vesicular organelles. The 24-hour pyocyanin treatment resulted in a concentration dependent decrease in cell viability at concentrations of 25 μM or greater, and increases in reactive oxygen species formation and caspase-3 activity at 25 μM or greater. Basal adenosine triphosphate release was significantly decreased at all tested pyocyanin concentrations while stimulated adenosine triphosphate release was significantly inhibited at pyocyanin concentrations of 12.5 μM or greater with no significant stimulated release at 100 μM. Pyocyanin treated RT4 cells showed morphological characteristics associated with cellular senescence, including SA-β-gal expression. This effect was not evident at 100 μM pyocyanin and may have been due to apoptotic cell death, as indicated by increased caspase-3 activity. An increase in acridine orange stained vesicular-like organelles was observed in RT4 urothelial cells after pyocyanin treatment. Exposure to pyocyanin alters urothelial cell viability, reactive oxygen species production and caspase-3 activity. Treatment also results in cellular senescence, which may affect the ability of urothelium to repair during infection. The virulence factor depressed stimulated adenosine triphosphate release, which to our knowledge is a novel finding with implications for awareness of bladder filling in patients with P. aeruginosa urinary tract infection. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier

  15. Organelle targeting during bacterial infection: insights from Listeria.

    PubMed

    Lebreton, Alice; Stavru, Fabrizia; Cossart, Pascale

    2015-06-01

    Listeria monocytogenes, a facultative intracellular bacterium responsible for severe foodborne infections, is now recognized as a multifaceted model in infection biology. Comprehensive studies of the molecular and cellular basis of the infection have unraveled how the bacterium crosses the intestinal and feto-placental barriers, invades several cell types in which it multiplies and moves, and spreads from cell to cell. Interestingly, although Listeria does not actively invade host cell organelles, it can interfere with their function. We discuss the effect of Listeria on the endoplasmic reticulum (ER) and the mechanisms leading to the fragmentation of the mitochondrial network and its consequences, and review the strategies used by Listeria to subvert nuclear functions, more precisely to control host gene expression at the chromatin level. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Second Generation TQ-Ligation for Cell Organelle Imaging.

    PubMed

    Zhang, Xiaoyun; Dong, Ting; Li, Qiang; Liu, Xiaohui; Li, Lin; Chen, She; Lei, Xiaoguang

    2015-07-17

    Bioorthogonal ligations play a crucial role in labeling diverse types of biomolecules in living systems. Herein, we describe a novel class of ortho-quinolinone quinone methide (oQQM) precursors that show a faster kinetic rate in the "click cycloaddition" with thio-vinyl ether (TV) than the first generation TQ-ligation in both chemical and biological settings. We further demonstrate that the second generation TQ-ligation is also orthogonal to the widely used strain-promoted azide-alkyne cycloaddition (SPAAC) both in vitro and in vivo, revealing that these two types of bioorthogonal ligations could be used as an ideal reaction pair for the simultaneous tracking of multiple elements within a single system. Remarkably, the second generation TQ-ligation and SPAAC are effective for selective and simultaneous imaging of two different cell organelles in live cells.

  17. Endoplasmic Reticulum - Plasma Membrane Crosstalk Mediated by the Extended Synaptotagmins.

    PubMed

    Saheki, Yasunori

    2017-01-01

    The endoplasmic reticulum (ER) possesses multiplicity of functions including protein synthesis, membrane lipid biogenesis, and Ca 2+ storage and has broad localization throughout the cell. While the ER and most other membranous organelles are highly interconnected via vesicular traffic that relies on membrane budding and fusion reactions, the ER forms direct contacts with virtually all other membranous organelles, including the plasma membrane (PM), without membrane fusion. Growing evidence suggests that these contacts play major roles in cellular physiology, including the regulation of Ca 2+ homeostasis and signaling and control of cellular lipid homeostasis. Extended synaptotagmins (E-Syts) are evolutionarily conserved family of ER-anchored proteins that tether the ER to the PM in PM PI(4,5)P 2 -dependent and cytosolic Ca 2+ -regulated manner. In addition, E-Syts possess a cytosolically exposed lipid-harboring module that confers the ability to transfer/exchange glycerolipids between the ER and the PM at E-Syts-mediated ER-PM contacts. In this chapter, the functions of ER-PM contacts and their role in non-vesicular lipid transport with special emphasis on the crosstalk between the two bilayers mediated by E-Syts will be discussed.

  18. Development and validation of rt-qpcr for vesicular stomatitis virus detection (Alagoas vesiculovirus).

    PubMed

    de Oliveira, Anapolino Macedo; Fonseca, Antônio Augusto; Camargos, Marcelo Fernandes; Orzil, Lívia Maria; Laguardia-Nascimento, Mateus; Oliveira, Anna Gabriella Guimarães; Rodrigues, Jacqueline Gomes; Sales, Mariana Lázaro; de Oliveira, Tatiana Flávia Pinheiro; de Melo, Cristiano Barros

    2018-07-01

    Vesicular stomatitis is an infectious disease that occurs mainly in countries of the Western Hemisphere and affects cattle, swine and horses. The clinical symptoms in cattle and swine are similar to foot-and-mouth disease and include vesicular ulceration of the tongue and mouth. The disease requires a rapid and accurate differential diagnosis, aiming for immediate implementation of control measures. The objective of the present study was to develop and perform validation tests of multiplex RT-qPCR(s) for the detection of RNA from Alagoas vesiculovirus, considering the parameters of sensitivity and analytical specificity, analytical performance (repeatability and reproducibility criteria) and the uncertainty of the measurement. The threshold cycle values obtained in triplicate from each sample were evaluated by considering the variations between days, analysts and equipment in an analysis of variance aimed at determining the variances of repeatability and reproducibility. The results showed that RT-qPCRs had excellent sensitivity and specificity in the detection of RNA of the Alagoas vesiculovirus. The validation parameters showed low coefficients of variation and were equivalent to those found in other validation studies, indicating that the tests presented excellent repeatability and reproducibility. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Metabolic Control of Vesicular Glutamate Transport and Release

    PubMed Central

    Juge, Narinobu; Gray, John A.; Omote, Hiroshi; Miyaji, Takaaki; Inoue, Tsuyoshi; Hara, Chiaki; Uneyama, Hisayuki; Edwards, Robert H.; Nicoll, Roger A.; Moriyama, Yoshinori

    2010-01-01

    Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl−. Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl− acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl− at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses, and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity. PMID:20920794

  20. Laser Surgery: Organelles to Organs

    NASA Astrophysics Data System (ADS)

    Berns, Michael W. D.

    1998-03-01

    Understanding the physical mechanisms of light interaction with biological molecules and structure has resulted in the application of photons to a wide variety of biological and medical problems ranging from subcellular manipulation/surgery to the successful diagnosis and treatment of human disease. Mechanisms such as the generation and transfer of heat, light-driven chemistry (photochemistry), high peak power acoustic-mechanical effects, high photon-energy induced bond breaking, and optical induced forces through momentum transfer, are being utilized in single cells at the microscopic (submicron and micron) level as well as the macroscopic level in tissue and organs. At the subcellular level, focused laser microbeams (laser scissors and tweezers) are being used to cut and move chromosomes to study genetic function as well as to clone and sequence genes. The same laser technology is being used to manipulate a variety of cell organelles such as mitochondria, cell membranes, nucleoli, and mitochondria in order to study their functions in cell physiology. At the tissue level, lasers are being used to diagnose and treat malignancy in combination with light-activated drugs, to ablate cornea and other hard and soft tissue through ultraviolet photoablation, to selectively ablate structures within the skin under controlled heating/cooling conditions, and to differentiate normal from abnormal tissue using a variety of fluorescence detection and light scattering techniques.

  1. From the Cover: 7,8-Dihydroxyflavone Rescues Lead-Induced Impairment of Vesicular Release: A Novel Therapeutic Approach for Lead Intoxicated Children.

    PubMed

    Zhang, Xiao-Lei; McGlothan, Jennifer L; Miry, Omid; Stansfield, Kirstie H; Loth, Meredith K; Stanton, Patric K; Guilarte, Tomás R

    2018-01-01

    Childhood lead (Pb2+) intoxication is a public health problem of global proportion. Lead exposure during development produces multiple effects on the central nervous system including impaired synapse formation, altered synaptic plasticity, and learning deficits. In primary hippocampal neurons in culture and hippocampal slices, Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites, an effect associated with Pb2+ inhibition of NMDA receptor-mediated trans-synaptic Brain-Derived Neurotrophic Factor (BDNF) signaling. The objective of this study was to determine if activation of TrkB, the cognate receptor for BDNF, would rescue Pb2+-induced impairments of vesicular release. Rats were chronically exposed to Pb2+ prenatally and postnatally until 50 days of age. This chronic Pb2+ exposure paradigm enhanced paired-pulse facilitation of synaptic potentials in Schaffer collateral-CA1 synapses in the hippocampus, a phenomenon indicative of reduced vesicular release probability. Decreased vesicular release probability was confirmed by both mean-variance analysis and direct 2-photon imaging of vesicular release from hippocampal slices of rats exposed to Pb2+in vivo. We also found a Pb2+-induced impairment of calcium influx in Schaffer collateral-CA1 synaptic terminals. Intraperitoneal injections of Pb2+ rats with the TrkB receptor agonist 7,8-dihydroxyflavone (5 mg/kg) for 14-15 days starting at postnatal day 35, reversed all Pb2+-induced impairments of presynaptic transmitter release at Schaffer collateral-CA1 synapses. This study demonstrates for the first time that in vivo pharmacological activation of TrkB receptors by small molecules such as 7,8-dihydroxyflavone can reverse long-term effects of chronic Pb2+ exposure on presynaptic terminals, pointing to TrkB receptor activation as a promising therapeutic intervention in Pb2+-intoxicated children. © The Author 2017. Published by Oxford University Press on behalf of the Society of

  2. REGULATED VESICULAR TRAFFICKING OF SPECIFIC PCDH15 AND VLGR1 VARIANTS IN AUDITORY HAIR CELLS

    PubMed Central

    Zallocchi, Marisa; Delimont, Duane; Meehan, Daniel T.; Cosgrove, Dominic

    2012-01-01

    Usher syndrome is a genetically heterogeneous disorder characterized by hearing and balance dysfunction and progressive retinitis pigmentosa. Mouse models carrying mutations for the nine Usher-associated genes have splayed stereocilia and some show delayed maturation of ribbon synapses suggesting these proteins may play different roles in terminal differentiation of auditory hair cells. The presence of the Usher proteins at the basal and apical aspects of the neurosensory epithelia suggests the existence of regulated trafficking through specific transport proteins and routes. Immature mouse cochleae and UB/OC-1 cells were used in this work to address whether specific variants of PCDH15 and VLGR1 are being selectively transported to opposite poles of the hair cells. Confocal co-localization studies between apical and basal vesicular markers and the different PCDH15 and VLGR1 variants along with sucrose density gradients and the use of vesicle trafficking inhibitors show the existence of Usher protein complexes in at least two vesicular sub-pools. The apically trafficked pool co-localized with the early endosomal vesicle marker, rab5, while the basally trafficked pool associates with membrane microdomains and SNAP25. Moreover, co-immunoprecipitation experiments between SNAP25 and VLGR1 show a physical interaction of these two proteins in organ of Corti and brain. Collectively, these findings establish the existence of a differential vesicular trafficking mechanism for specific Usher protein variants in mouse cochlear hair cells, with the apical variants playing a potential role in endosomal recycling and stereocilia development/maintenance and the basolateral variants involved in vesicle docking and/or fusion through SNAP25-mediated interactions. PMID:23035094

  3. Characterization of pH-sensitive molecular switches that trigger the structural transition of vesicular stomatitis virus glycoprotein from the postfusion state toward the prefusion state.

    PubMed

    Ferlin, Anna; Raux, Hélène; Baquero, Eduard; Lepault, Jean; Gaudin, Yves

    2014-11-01

    Vesicular stomatitis virus (VSV; the prototype rhabdovirus) fusion is triggered at low pH and mediated by glycoprotein G, which undergoes a low-pH-induced structural transition. A unique feature of rhabdovirus G is that its conformational change is reversible. This allows G to recover its native prefusion state at the viral surface after its transport through the acidic Golgi compartments. The crystal structures of G pre- and postfusion states have been elucidated, leading to the identification of several acidic amino acid residues, clustered in the postfusion trimer, as potential pH-sensitive switches controlling the transition back toward the prefusion state. We mutated these residues and produced a panel of single and double mutants whose fusion properties, conformational change characteristics, and ability to pseudotype a virus lacking the glycoprotein gene were assayed. Some of these mutations were also introduced in the genome of recombinant viruses which were further characterized. We show that D268, located in the segment consisting of residues 264 to 273, which refolds into postfusion helix F during G structural transition, is the major pH sensor while D274, D395, and D393 have additional contributions. Furthermore, a single passage of recombinant virus bearing the mutation D268L (which was demonstrated to stabilize the G postfusion state) resulted in a pseudorevertant with a compensatory second mutation, L271P. This revealed that the propensity of the segment of residues 264 to 273 to refold into helix F has to be finely tuned since either an increase (mutation D268L alone) or a decrease (mutation L271P alone) of this propensity is detrimental to the virus. Vesicular stomatitis virus enters cells via endocytosis. Endosome acidification induces a structural transition of its unique glycoprotein (G), which mediates fusion between viral and endosomal membranes. G conformational change is reversible upon increases in pH. This allows G to recover its native

  4. [Vesicular and nonvesicular glutamate release in the nucleus accumbens during a forced switch in behavioral strategy].

    PubMed

    Saul'skaia, N B; Mikhaĭlova, M O

    2004-01-01

    By means of in vivo microdialysis combined with HPLC analysis, we have shown that glutamate extracellular level in the rat n. accumbens increases during a forced switch in behavioral strategy. When infused in the n. accumbens, a Na+ channel blocker tetrodotoxin (TTX, 1 microM) completely prevents this increase whereas a potent cystine/glutamate exchanger blocker (S)-4-carboxyphenylglycine ((S)-4-CPG, 5 microM) has no effect. In contrast, TT (1 microM), infused in the n. accumbens, fails to significantly alter basal level of extracellular glutamate in this region whereas (S)-4-CPG (5 microM) produced a significant decrease. Our data suggest that basal and factional glutamate releases in the n. accumbens are differently regulated. The source of basal glutamate release is a non-vesicular release via cystine/glutamate exchanger. Functional glutamate release observed during a forced switch in behavioral strategy derives from vesicular synaptic pool.

  5. 9 CFR 94.12 - Pork and pork products from regions where swine vesicular disease exists.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... not endanger the livestock of the United States. (c) Requirements for pork-filled pasta products from regions affected with swine vesicular disease. (1) Pork-filled pasta products processed for export to the... or of § 94.17. (2) The operator of the pork-filled pasta processing facility must have signed a...

  6. Isolation and characterization of a prokaryotic cell organelle from the anammox bacterium Kuenenia stuttgartiensis.

    PubMed

    Neumann, Sarah; Wessels, Hans J C T; Rijpstra, W Irene C; Sinninghe Damsté, Jaap S; Kartal, Boran; Jetten, Mike S M; van Niftrik, Laura

    2014-11-01

    Anaerobic ammonium oxidizing (anammox) bacteria oxidize ammonium with nitrite to nitrogen gas in the absence of oxygen. These microorganisms form a significant sink for fixed nitrogen in the oceans and the anammox process is applied as a cost-effective and environment-friendly nitrogen removal system from wastewater. Anammox bacteria have a compartmentalized cell plan that consists of three separate compartments. Here we report the fractionation of the anammox bacterium Kuenenia stuttgartiensis in order to isolate and analyze the innermost cell compartment called the anammoxosome. The subcellular fractions were microscopically characterized and all membranes in the anammox cell were shown to contain ladderane lipids which are unique for anammox bacteria. Proteome analyses and activity assays with the isolated anammoxosomes showed that these organelles harbor the energy metabolism in anammox cells. Together the experimental data provide the first thorough characterization of a respiratory cell organelle from a bacterium and demonstrate the essential role of the anammoxosome in the production of a major portion of the nitrogen gas in our atmosphere. © 2014 John Wiley & Sons Ltd.

  7. Identification of differentiation-associated brain-specific phosphate transporter as a second vesicular glutamate transporter (VGLUT2).

    PubMed

    Takamori, S; Rhee, J S; Rosenmund, C; Jahn, R

    2001-11-15

    Glutamate is the major excitatory neurotransmitter in mammalian CNS. In the presynaptic nerve terminal, glutamate is stored in synaptic vesicles and released by exocytosis. Previously, it has been shown that a transport protein originally identified as a brain-specific Na(+)-dependent inorganic phosphate transporter I (BNPI) functions as vesicular glutamate transporter and thus has been renamed VGLUT1. Recently, a protein highly homologous to VGLUT1, "differentiation-associated BNPI" (DNPI), has been discovered. Northern blot and in situ hybridization analyses indicate that DNPI mRNA is expressed in some brain regions in which VGLUT1 mRNA is not expressed. We now show that DNPI functions as vesicular glutamate transporter with properties very similar to VGLUT1 and propose to rename the protein VGLUT2. VGLUT2 is highly enriched in synaptic vesicles. Furthermore, VGLUT2 resides on a vesicle population that is distinct from vesicles containing the vesicular GABA transporter or VGLUT1, showing that the expression of VGLUT1 and VGLUT2 do not overlap. When VGLUT2 was expressed in BON cells, membrane fractions displayed ATP-dependent, carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive glutamate uptake. Overexpression of VGLUT2 in cultured autaptic GABAergic neurons yielded postsynaptic currents that were insensitive to the GABA(A) receptor antagonist bicuculline but blocked by the AMPA-receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[F]quinoxaline. Thus, expression of VGLUT2 suffices to cause GABAergic neurons to release glutamate in addition to GABA in a manner very similar to that reported previously for VGLUT1.

  8. Role of Intermediate Filaments in Vesicular Traffic.

    PubMed

    Margiotta, Azzurra; Bucci, Cecilia

    2016-04-25

    Intermediate filaments are an important component of the cellular cytoskeleton. The first established role attributed to intermediate filaments was the mechanical support to cells. However, it is now clear that intermediate filaments have many different roles affecting a variety of other biological functions, such as the organization of microtubules and microfilaments, the regulation of nuclear structure and activity, the control of cell cycle and the regulation of signal transduction pathways. Furthermore, a number of intermediate filament proteins have been involved in the acquisition of tumorigenic properties. Over the last years, a strong involvement of intermediate filament proteins in the regulation of several aspects of intracellular trafficking has strongly emerged. Here, we review the functions of intermediate filaments proteins focusing mainly on the recent knowledge gained from the discovery that intermediate filaments associate with key proteins of the vesicular membrane transport machinery. In particular, we analyze the current understanding of the contribution of intermediate filaments to the endocytic pathway.

  9. Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal-Like Compartments and the Golgi Complex

    PubMed Central

    Jackson, Allison J; Clucas, Caroline; Mamczur, Nicola J; Ferguson, David J; Meissner, Markus

    2013-01-01

    Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. PMID:23962112

  10. Use of the bolus tracking technique for the tomographic evaluation of the uretero-vesicular junction in dogs and assessment of dose records.

    PubMed

    Longo, Maurizio; Andreis, Maria Elena; Pettinato, Cinzia; Ravasio, Giuliano; Rabbogliatti, Vanessa; De Zani, Donatella; Di Giancamillo, Mauro; Zani, Davide Danilo

    2016-03-29

    The aim of the work is the application of a bolus tracking technique for tomographic evaluation of the uretero-vesicular junction in dogs. Ten adult dogs (8-14 years) with variable body weight (2,8-32 kg) were enrolled in the prospective study. The patients were placed in sternal recumbency with a 10° elevated pelvis and the visualization of the uretero-vesicular junction was obtained with the bolus tracking technique after intravenous administration of non-ionic contrast medium. In the post-contrast late phase a region of interest was placed within the lumen of the distal ureters and the density values were monitored before starting the helical scan. The uretero-vesicular junction was clearly visible in 100% of patients with the visualization of the endoluminal ureteral contrast enhancement and bladder washout. At the end of the tomographic study an evaluation of the dose records was performed and compared to human exposures reported in literature for the pelvic region. The effective dose estimated for each patient (37,5-138 mSv) proved to be elevated, when compared to those reported in human patients. The bolus tracking technique could be applied for the visualization of the uretero-vesicular junction in non-pathological patients, placing the region of interest in the distal ureters. The high effective doses recorded in our study support the need of specific thresholds for veterinary patients, pointing out the attention for paediatric patient's exposure also in veterinary imaging.

  11. Fluorescence recovery after photobleaching measured by confocal microscopy as a tool for the analysis of vesicular lipid transport and plasma membrane mobility

    NASA Astrophysics Data System (ADS)

    Schmitz, Gerd; Goetz, Alexandra; Orso, Evelyn; Rothe, Gregor

    1998-04-01

    The vesicular transport of lipids from the endoplasmic reticulum via the Golgi apparatus affects the composition of the plasma membrane. The purpose of our study was to develop an in vitro test system for characterization of vesicular lipid transport kinetics by using confocal microscopy and fluorescence recovery after photobleaching (FRAP). Fibroblasts from two patients homozygous for the hypercatabolic HDL deficiency syndrome Tangier disease and 4 control subjects were pulsed with the C6-NBD-ceramide for 30 minutes. Chase incubation at room temperature resulted in the metabolic accumulation of fluorescent C6-NBD-sphingolyelin and C6-NBD-glycosylceramides in the medial- and trans-Golgi region. Cells were analyzed with an inverted Leica TCS microscope. Calibration was performed through the analysis of diffusion of 50 nm microparticles embedded in media of different viscosity. An acousto optical tunable filter (AOTF) was used for the selective bleaching of the medial- and trans- Golgi region followed by analysis of the fluorescence recovery for 4 minutes. Post-bleach fluorescence recovery through the trans-Golgi-oriented transport of NBD-sphingomyelin was calculated from 2-dimensional scans. Tangier fibroblasts displayed a retarded recovery of fluorescence in the trans- Golgi region. This suggests that the vesicular transport of sphingomyelin and cholesterol is disturbed in Tangier disease confirming data from our laboratory generated with radiometabolites on whole cells. Our data suggest that FRAP analysis allows a sensitive kinetic and spatially resolved analysis of disturbances of vesicular lipid transport.

  12. Bubble formation, vesicularity and fractionation of noble gases during MORB degassing

    NASA Astrophysics Data System (ADS)

    Sator, N.; Guillot, B. B.; Aubry, G.

    2012-12-01

    The fractionation of noble gases in oceanic basalts gives information on the source region and on the transport of volatiles up to the seafloor. For instance, the large distribution (~1-1,000) of the 4He/40Ar* ratio in mid-ocean ridge basalts (MORB), is interpreted as the signature of different degassing scenarios taking place at depth. Thus, a low value of this ratio is explained by a closed system degassing whereas a high value is assigned either to an open system degassing (where vesicles are lost in a magma chamber or at depth during magma ascent) or to a kinetic disequilibrium induced by a rapid magma ascent just prior eruption. Unfortunately, CO2 has a very low solubility in basaltic melts at pressure corresponding to the seafloor and an overwhelming majority of erupted lavas have lost their pristine volatile contents. However notable exceptions are the popping rocks characterized by a large vesicularity, a high CO2 content and a 4He/40Ar* ratio compatible with the expected U/K ratio of the upper mantle. Those samples likely have experienced a CO2 exsolution at about 35 km depth in the oceanic mantle. So, the very existence of these exceptional MORB samples suggests that CO2-rich melts could be present at a greater depth. Thus, explosive eruptions near ocean spreading centers are well documented (Hekinian et al., 2000) and are associated with volcaniclastic deposits containing highly vesicular basalts, a feature which suggests that this volcanism is driven by CO2-rich magmas (Helo et al., 2011). But how much CO2-rich are these magmas, that is the question. The objective of this study is to use molecular dynamics simulation (MD) to evaluate the vesicularity and the fractionation of noble gases in a degassing MORB melt. A previous simulation study (Guillot and Sator, 2011) has shown that the solubility of CO2 in basaltic melts increases steadily with the pressure and deviates significantly from the Henry's law at high pressures. From the CO2 solubility curve

  13. The shells of BMC-type microcompartment organelles in bacteria.

    PubMed

    Yeates, Todd O; Jorda, Julien; Bobik, Thomas A

    2013-01-01

    Bacterial microcompartments are large proteinaceous structures that act as metabolic organelles in many bacterial cells. A shell or capsid, which is composed of a few thousand protein subunits, surrounds a series of sequentially acting enzymes and controls the diffusion of substrates and products into and out of the lumen. The carboxysome and the propanediol utilization microcompartment represent two well-studied systems among seven or more distinct types that can be delineated presently. Recent structural studies have highlighted a number of sophisticated mechanisms that underlie the function of bacterial microcompartment shell proteins. This review updates our understanding of bacterial microcompartment shells, how they are assembled, and how they carry out their functions in molecular transport and enzyme organization. Copyright © 2013 S. Karger AG, Basel.

  14. TPC2 mediates new mechanisms of platelet dense granule membrane dynamics through regulation of Ca2+ release

    PubMed Central

    Ambrosio, Andrea L.; Boyle, Judith A.; Di Pietro, Santiago M.

    2015-01-01

    Platelet dense granules (PDGs) are acidic calcium stores essential for normal hemostasis. They develop from late endosomal compartments upon receiving PDG-specific proteins through vesicular trafficking, but their maturation process is not well understood. Here we show that two-pore channel 2 (TPC2) is a component of the PDG membrane that regulates PDG luminal pH and the pool of releasable Ca2+. Using a genetically encoded Ca2+ biosensor and a pore mutant TPC2, we establish the function of TPC2 in Ca2+ release from PDGs and the formation of perigranular Ca2+ nanodomains. For the first time, Ca2+ spikes around PDGs—or any organelle of the endolysosome family—are visualized in real time and revealed to precisely mark organelle “kiss-and-run” events. Further, the presence of membranous tubules transiently connecting PDGs is revealed and shown to be dramatically enhanced by TPC2 in a mechanism that requires ion flux through TPC2. “Kiss-and-run” events and tubule connections mediate transfer of membrane proteins and luminal content between PDGs. The results show that PDGs use previously unknown mechanisms of membrane dynamics and content exchange that are regulated by TPC2. PMID:26202466

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

    PubMed Central

    Nolan, Sabrina J.; Luechtefeld, Thomas

    2015-01-01

    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. PMID:25750213

  16. Foot & Mouth Disease & Ulcerative/Vesicular Rule-outs: Challenges Encountered in Recent Outbreaks

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hullinger, P

    2008-01-28

    carriers. It has been shown that the African Cape buffalo are the major maintenance host of SAT serotypes. FMDV transmission can occur by either direct or indirect contact. Indirect transmission can occur via contaminated animate vectors (humans, etc.), inanimate vectors (vehicles, implements) or airborne transmission. Indirect disease transmission via animate or inanimate vectors can play a major role in disease transmission. Good biosecurity can significantly reduce this type of transmission. Airborne transmission is often debated and is known to be serotype and species specific as well as require specific environmental conditions to occur. Airborne transmission is favored in temperate zones and has been postulated to occur over distances of up to 60 km overland and 300 km by sea. Foot and mouth disease virus is an unenveloped virus which is preserved by refrigeration and freezing and progressively inactivated by temperatures above 50 C. FMDV is highly sensitive to pH change and is inactivated by pH < 6.0 or > 9.0. There are many disinfectants which are effective against FMDV including sodium hydroxide (2%), sodium carbonate (4%), and citric acid (0.2%). FMDV is resistant to iodophores, quaternary ammonium compounds, hypochlorite and phenol, especially in the presence of organic matter. The virus can survive in lymph nodes and bone marrow at neutral pH, but is destroyed in muscle when is pH < 6.0 i.e. after rigor mortis. FMDV can persist in contaminated feed/commodities and the environment for over to 1 month, depending on the temperature and pH conditions. The incubation period for FMD is 2-14 days. Animals transition through latent (infected but not infectious), subclinically infected (infectious but lacking clinical signs) clinically infected and recovered disease states. In cattle clinical signs include pyrexia, reluctance to eat, bruxism, drooling, lameness, treading or stamping of the feet and decreased milk production. Most clinical signs are related to the

  17. Metabolic control of vesicular glutamate transport and release.

    PubMed

    Juge, Narinobu; Gray, John A; Omote, Hiroshi; Miyaji, Takaaki; Inoue, Tsuyoshi; Hara, Chiaki; Uneyama, Hisayuki; Edwards, Robert H; Nicoll, Roger A; Moriyama, Yoshinori

    2010-10-06

    Fasting has been used to control epilepsy since antiquity, but the mechanism of coupling between metabolic state and excitatory neurotransmission remains unknown. Previous work has shown that the vesicular glutamate transporters (VGLUTs) required for exocytotic release of glutamate undergo an unusual form of regulation by Cl(-). Using functional reconstitution of the purified VGLUTs into proteoliposomes, we now show that Cl(-) acts as an allosteric activator, and the ketone bodies that increase with fasting inhibit glutamate release by competing with Cl(-) at the site of allosteric regulation. Consistent with these observations, acetoacetate reduced quantal size at hippocampal synapses and suppresses glutamate release and seizures evoked with 4-aminopyridine in the brain. The results indicate an unsuspected link between metabolic state and excitatory neurotransmission through anion-dependent regulation of VGLUT activity. Copyright © 2010 Elsevier Inc. All rights reserved.

  18. REDO: RNA Editing Detection in Plant Organelles Based on Variant Calling Results.

    PubMed

    Wu, Shuangyang; Liu, Wanfei; Aljohi, Hasan Awad; Alromaih, Sarah A; Alanazi, Ibrahim O; Lin, Qiang; Yu, Jun; Hu, Songnian

    2018-05-01

    RNA editing is a post-transcriptional or cotranscriptional process that changes the sequence of the precursor transcript by substitutions, insertions, or deletions. Almost all of the land plants undergo RNA editing in organelles (plastids and mitochondria). Although several software tools have been developed to identify RNA editing events, there has been a great challenge to distinguish true RNA editing events from genome variation, sequencing errors, and other factors. Here we introduce REDO, a comprehensive application tool for identifying RNA editing events in plant organelles based on variant call format files from RNA-sequencing data. REDO is a suite of Perl scripts that illustrate a bunch of attributes of RNA editing events in figures and tables. REDO can also detect RNA editing events in multiple samples simultaneously and identify the significant differential proportion of RNA editing loci. Comparing with similar tools, such as REDItools, REDO runs faster with higher accuracy, and more specificity at the cost of slightly lower sensitivity. Moreover, REDO annotates each RNA editing site in RNAs, whereas REDItools reports only possible RNA editing sites in genome, which need additional steps to obtain RNA editing profiles for RNAs. Overall, REDO can identify potential RNA editing sites easily and provide several functions such as detailed annotations, statistics, figures, and significantly differential proportion of RNA editing sites among different samples.

  19. Black fly involvement in the epidemic transmission of vesicular stomatitis New Jersey virus (Rhabdoviridae: Vesiculovirus).

    PubMed

    Mead, Daniel G; Howerth, Elizabeth W; Murphy, Molly D; Gray, Elmer W; Noblet, Raymond; Stallknecht, David E

    2004-01-01

    The transmission routes of Vesicular stomatitis New Jersey virus (VSNJV), a causative agent of vesicular stomatitis, an Office International des Epizooties List-A disease, are not completely understood. Epidemiological and entomological studies conducted during the sporadic epidemics in the western United States have identified potential virus transmission routes involving insect vectors and animal-to-animal contact. In the present study we experimentally tested the previously proposed transmission routes which were primarily based on field observations. Results obtained provide strong evidence for the following: (1) hematophagous insects acquire VSNJV by unconventional routes while blood feeding on livestock, (2) clinical course of VSNJV infection in livestock following transmission by an infected insect is related to insect bite site, (3) infection of livestock via insect bite can result in multiple transmission possibilities, including animal-to-animal contact. Taken together, these data significantly add to our understanding of the transmission routes of a causative agent of one of the oldest known infectious diseases of livestock, for which the details have remained largely unknown despite decades of research.

  20. Acid-$beta$-glycerophosphatase reaction products in the central nervous system mitochondria following x-ray irradiation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Roizin, L.; Orlovskaja, D.; Liu, J.C.

    A survey of the literature to date on the enzyme histochemistry of intracellular organelles has not yielded any reference to the presence of acid phosphatase reaction products in the mammalian mitochondria of the central nervous system. A combination of Gomori's acid phosphatase method, however, with standard electron microscopy has disclosed the presence of enzyme reaction products in the mitochondria of the central nervous system of rats from 2 hr to 22 weeks after x-ray irradiation, as well as in a cerebral biopsy performed on a patient affected by Huntington's chorea. No enzyme reaction products, on the other hand, were observedmore » in serial sections that had been incubated in substrates either containing sodium fluoride or lacking in $beta$- glycerophosphate. The abnormal mitochondrial enzyme reaction (chemical lesion) is considered to be the consequence of the pathologic process affecting the ultrastructural-chemical organization of the organelle. (auth)« less

  1. The grain size dependency of vesicular particle shapes strongly affects the drag of particles. First results from microtomography investigations of Campi Flegrei fallout deposits

    NASA Astrophysics Data System (ADS)

    Mele, Daniela; Dioguardi, Fabio

    2018-03-01

    Acknowledging the grain size dependency of shape is important in volcanology, in particular when dealing with tephra produced and emplaced during and after explosive volcanic eruptions. A systematic measurement of the tridimensional shape of vesicular pyroclasts of Campi Flegrei fallout deposits (Agnano-Monte Spina, Astroni 6 and Averno 2 eruptions) varying in size from 8.00 to 0.016 mm has been carried out by means of X-Ray Microtomography. Data show that particle shape changes with size, especially for juvenile vesicular clasts, since it is dependent on the distribution and size of vesicles that contour the external clast outline. Two drag laws that include sphericity in the formula were used for estimating the dependency of settling velocity on shape. Results demonstrate that it is not appropriate to assume a size-independent shape for vesicular particles, in contrast with the approach commonly employed when simulating the ash dispersion in the atmosphere.

  2. Myosin Ib modulates the morphology and the protein transport within multi-vesicular sorting endosomes.

    PubMed

    Salas-Cortes, Laura; Ye, Fei; Tenza, Danièle; Wilhelm, Claire; Theos, Alexander; Louvard, Daniel; Raposo, Graça; Coudrier, Evelyne

    2005-10-15

    Members of at least four classes of myosin (I, II, V and VI) have been implicated in the dynamics of a large variety of organelles. Despite their common motor domain structure, some of these myosins, however, are non processive and cannot move organelles along the actin tracks. Here, we demonstrate in the human pigmented MNT-1 cell line that, (1) the overexpression of one of these myosins, myosin 1b, or the addition of cytochalasin D affects the morphology of the sorting multivesicular endosomes; (2) the overexpression of myosin 1b delays the processing of Pmel17 (the product of murine silver locus also named GP100), which occurs in these multivesicular endosomes; (3) myosin 1b associated with endosomes coimmunoprecipitates with Pmel17. All together, these observations suggest that myosin 1b controls the traffic of protein cargo in multivesicular endosomes most probably through its ability to modulate with actin the morphology of these sorting endosomes.

  3. Dual regulation of Ca2+-dependent glutamate release from astrocytes: vesicular glutamate transporters and cytosolic glutamate levels.

    PubMed

    Ni, Yingchun; Parpura, Vladimir

    2009-09-01

    Vesicular glutamate transporters (VGLUTs) are responsible for vesicular glutamate storage and exocytotic glutamate release in neurons and astrocytes. Here, we selectively and efficiently overexpressed individual VGLUT proteins (VGLUT1, 2, or 3) in solitary astrocytes and studied their effects on mechanical stimulation-induced Ca2+-dependent glutamate release. Neither VGLUT1 nor VGLUT2 overexpression changed the amount of glutamate release, whereas overexpression of VGLUT3 significantly enhanced Ca2+-dependent glutamate release from astrocytes. None of the VGLUT overexpression affected mechanically induced intracellular Ca2+ increase. Inhibition of glutamine synthetase activity by L-methionine sulfoximine in astrocytes, which leads to increased cytosolic glutamate concentration, greatly increased their mechanically induced Ca2+-dependent glutamate release, without affecting intracellular Ca2+ dynamics. Taken together, these data indicate that both VGLUT3 and the cytosolic concentration of glutamate are key limiting factors in regulating the Ca2+-dependent release of glutamate from astrocytes.

  4. Energy dispersive X-ray analyses of organelles of NaCI-treated maize root cells

    NASA Astrophysics Data System (ADS)

    Stelzer, Ralf

    1984-04-01

    NaCl sensitive plants of Zea mays cv. ADOUR were grown in nutrient solutions with or without NaCl. Frozen, hydrated root-tip tissues were investigated by means of an ETEC scanning electron microscope fitted with a KEVEX energy dispersive X-ray analyser. Morphological details of the gently etched but non-coated surface of the cross fractured specimen were easy to identify and to analyse using an electron beam with a low intensity at 10 kV. X-ray data obtained from cell compartments and organelles as nuclei, nucleoli and mitochondria within individual cells establish typical X-ray spectra. Comparisons of these spectra support the hypothesis that Na + ions are predominantly localized in vacuoles and also to a lesser extent in the cytoplasm, e.g. in small vesicles, but not in other cell organelles. Furthermore the analysed cell compartments show differences in the distribution of Mg, P, S, Cl, K and Ca effected by the addition of NaCl to the growth medium. The X-ray data are discussed in relation to the physiological meaning of a NaCl induced redistribution of elements within individual maize root cells.

  5. A Two-Tier Golgi-Based Control of Organelle Size Underpins the Functional Plasticity of Endothelial Cells

    PubMed Central

    Ferraro, Francesco; Kriston-Vizi, Janos; Metcalf, Daniel J.; Martin-Martin, Belen; Freeman, Jamie; Burden, Jemima J.; Westmoreland, David; Dyer, Clare E.; Knight, Alex E.; Ketteler, Robin; Cutler, Daniel F.

    2014-01-01

    Summary Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures (“quanta”) of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities. PMID:24794632

  6. Detection, imaging, and kinetics of sub-micron organelles of chondrocytes by multiple beam interference microscopy

    NASA Astrophysics Data System (ADS)

    Joshi, Narahari V.; Medina, Honorio; Barboza, J. M.; Colantuoni, Gladys; Quintero, Maritza

    2004-07-01

    Chondrocytes, obtained from testosterone treated human articular cartilage, were examined by a recently developed Multiple Beam Interference Microscopy (MBIM) attached to a confocal set up, Video-enhanced differential interference microphotography and also by cinematography. In the MBIM, the intensity of the transmitted pattern is given by the Airy function which increases the contrast dramatically as the coefficient of the reflectance of the parallel plates increases. Moreover, in this configuration, the beam passes several times through a specific organelle and increases its optical path difference both because of the increase in the trajectory and refractive index (high density) of the organelle. The improved contrast enhances the resolving power of the system and makes visible several structural details of sub micron dimensions like nucleolus, retraction fibers, podia, etc. which are not possible to reveal with such a clarity by conventional techniques such as bright field, phase contrast or DIC. This technique permits to detect the oscillatory and rotational motions of unstained cilia for the first time. The frequency of oscillations was found to be 0.8 Hz.

  7. A plasma membrane localization signal in the HIV-1 envelope cytoplasmic domain prevents localization at sites of vesicular stomatitis virus budding and incorporation into VSV virions.

    PubMed

    Johnson, J E; Rodgers, W; Rose, J K

    1998-11-25

    Previous studies showed that the HIV-1 envelope (Env) protein was not incorporated into vesicular stomatitis virus (VSV) virions unless its cytoplasmic tail was replaced with that of the VSV glycoprotein (G). To determine whether the G tail provided a positive incorporation signal for Env, or if sequences in the Env tail prevented incorporation, we generated mutants of Env with its 150-amino-acid tail shortened to 29, 10, or 3 amino acids (Envtr mutants). Cells infected with VSV recombinants expressing these proteins or an Env-G tail hybrid showed similar amounts of Env protein at the surface. The Env-G tail hybrid or the Envtr3 mutant were incorporated at the highest levels into budding VSV virions. In contrast, the Envtr29 or Envtr10 mutants were incorporated poorly. These results defined a signal preventing incorporation within the 10 membrane-proximal amino acids of the Env tail. Confocal microscopy revealed that this signal functioned by causing localization of human immunodeficiency virus type 1 Env to plasma membrane domains distinct from the VSV budding sites, where VSV proteins were concentrated. Copyright 1998 Academic Press.

  8. Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain.

    PubMed

    Moechars, Diederik; Weston, Matthew C; Leo, Sandra; Callaerts-Vegh, Zsuzsanna; Goris, Ilse; Daneels, Guy; Buist, A; Cik, M; van der Spek, P; Kass, Stefan; Meert, Theo; D'Hooge, Rudi; Rosenmund, Christian; Hampson, R Mark

    2006-11-15

    Uptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

  9. Acrylamide and glycidamide impair neurite outgrowth in differentiating N1E.115 neuroblastoma without disturbing rapid bidirectional transport of organelles observed by video microscopy.

    PubMed

    Brat, D J; Brimijoin, S

    1993-06-01

    The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene-bis-acrylamide were weakly cytotoxic in a 51Cr-release assay, but only at > or = 10 mM (order of efficacy: methylene-bis-acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100-fold lower concentrations, with similar EC50 values for all three toxicants, i.e., acrylamide, 70 +/- 15 microM; methylene-bis-acrylamide, 92 +/- 31 microM; glycidamide, 120 +/- 30 microM. Only glycidamide (1 mM) caused degeneration of established neurites within a period of 48 h. Video-enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of < or = 48 h at 1 mM, neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.

  10. Loss of endocytic capacity in aging Paramecium. The importance of cytoplasmic organelles

    PubMed Central

    1976-01-01

    Aged cells have significantly fewer food vacuoles and ingest fewer bacteria than young cells. Loss of food vacuoles was explained by a decreasing difference in the food vacuole formation and excretion rates; the formation rate declined more rapidly than the excretion rate, approaching equivalence at 160 fissions, when the proportion of cells with no food vacuoles, in the presence of excess food, abruptly increased. A model for cellular aging is presented in which control of organelle numbers and cyclical interactions between the nucleus and cytoplasm may be of critical importance. PMID:993263

  11. Valine but not leucine or isoleucine supports neurotransmitter glutamate synthesis during synaptic activity in cultured cerebellar neurons.

    PubMed

    Bak, Lasse K; Johansen, Maja L; Schousboe, Arne; Waagepetersen, Helle S

    2012-09-01

    Synthesis of neuronal glutamate from α-ketoglutarate for neurotransmission necessitates an amino group nitrogen donor; however, it is not clear which amino acid(s) serves this role. Thus, the ability of the three branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, to act as amino group nitrogen donors for synthesis of vesicular neurotransmitter glutamate was investigated in cultured mouse cerebellar (primarily glutamatergic) neurons. The cultures were superfused in the presence of (15) N-labeled BCAAs, and synaptic activity was induced by pulses of N-methyl-D-aspartate (300 μM), which results in release of vesicular glutamate. At the end of the superfusion experiment, the vesicular pool of glutamate was released by treatment with α-latrotoxin (3 nM, 5 min). This experimental paradigm allows a separate analysis of the cytoplasmic and vesicular pools of glutamate. Amount and extent of (15) N labeling of intracellular amino acids plus vesicular glutamate were analyzed employing HPLC and LC-MS analysis. Only when [(15) N]valine served as precursor did the labeling of both cytoplasmic and vesicular glutamate increase after synaptic activity. In addition, only [(15) N]valine was able to maintain the amount of vesicular glutamate during synaptic activity. This indicates that, among the BCAAs, only valine supports the increased need for synthesis of vesicular glutamate. Copyright © 2012 Wiley Periodicals, Inc.

  12. Polysaccharide nano-vesicular multidrug carriers for synergistic killing of cancer cells.

    PubMed

    Pramod, P S; Shah, Ruchira; Chaphekar, Sonali; Balasubramanian, Nagaraj; Jayakannan, Manickam

    2014-10-21

    Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic efficacies against cancer cells. Here, we report dual delivery polysaccharide nano-vesicles that are capable of loading and delivering both water soluble and water insoluble drugs together in a single polymer scaffold. The selective rupture of the nano-vesicular assembly under intracellular enzyme conditions allowed the simultaneous delivery of a hydrophobic drug camptothecin (CPT) and hydrophilic drug doxorubicin (DOX) supporting their synergistic killing of breast and colon cancer cells. The polysaccharide nano-vesicles have allowed us to address a few important questions regarding the need for multiple drug administration in cancer cells including (a) the role of simultaneous drug release, (b) antagonistic versus synergistic effects of drug combinations and (c) how these are affected by the ratio of drugs. Further, evaluation of the role of caveolae in endocytosis of these polymer scaffolds was also made. The vesicular scaffolds were found to preserve and deliver DOX resulting in 50-60% better killing of cancer cells than the free drug. Additionally, dual loaded nano-vesicles when compared to drug cocktails with individual drugs in separate nano-vesicles (at comparable molar ratios) suggest the relative drug concentration following release and mode of delivery to be both important in cancer cell killing. Results from these experiments have revealed newly developed polysaccharide nano-vesicles loaded with DOX and CPT drugs as potential candidates for improved breast cancer cell killing. Thus, these custom-designed polysaccharide nano-vesicles provide a new perspective on multi-anticancer drug delivery systems and their efficacy.

  13. Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

    PubMed Central

    Okada, Seiko F.; Ribeiro, Carla M. P.; Sesma, Juliana I.; Seminario-Vidal, Lucia; Abdullah, Lubna H.; van Heusden, Catharina; Lazarowski, Eduardo R.

    2013-01-01

    ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion. PMID:23763446

  14. Extensive In Vitro Hyphal Growth of Vesicular-Arbuscular Mycorrhizal Fungi in the Presence of CO(2) and Flavonols.

    PubMed

    Bécard, G; Douds, D D; Pfeffer, P E

    1992-03-01

    Various flavonoids were tested for their ability to stimulate in vitro growth of germinated spores of vesicular-arbuscular mycorrhizal fungi. Experiments were performed in the presence of 2% CO(2), previously demonstrated to be required for growth of Gigaspora margarita (G. Bécard and Y. Piché, Appl. Environ. Microbiol. 55:2320-2325, 1989). Only the flavonols stimulated fungal growth. The flavones, flavanones, and isoflavones tested were generally inhibitory. Quercetin (10 muM) prolonged hyphal growth from germinated spores of G. margarita from 10 to 42 days. An average of more than 500 mm of hyphal growth and 13 auxiliary cells per spore were obtained. Quercetin also stimulated the growth of Glomus etunicatum. The glycosides of quercetin, rutin, and quercitrin were not stimulatory. The axenic growth of G. margarita achieved here under rigorously defined conditions is the most ever reported for a vesicular-arbuscular mycorrhizal fungus.

  15. High-throughput imaging of heterogeneous cell organelles with an X-ray laser (CXIDB ID 25)

    DOE Data Explorer

    Hantke, Max, F.

    2014-11-17

    Preprocessed detector images that were used for the paper "High-throughput imaging of heterogeneous cell organelles with an X-ray laser". The CXI file contains the entire recorded data - including both hits and blanks. It also includes down-sampled images and LCLS machine parameters. Additionally, the Cheetah configuration file is attached that was used to create the pre-processed data.

  16. A morphometric analysis of the redistribution of organelles in columella cells of horizontally-oriented roots of Zea mays

    NASA Technical Reports Server (NTRS)

    Moore, R.

    1986-01-01

    In order to determine what structural changes in graviperceptive cells are associated with onset of root gravicurvature, the redistribution of organelles in columella cells of horizontally-oriented, graviresponding roots of Zea mays has been quantified. Root gravicurvature began by 15 min after reorientation, and did not involve significant changes in the (i) volume of individual columella cells or amyloplasts, (ii) relative volume of any cellular organelle, (iii) number of amyloplasts per columella cell, or (iv) surface area of cellular location of endoplasmic reticulum. Sedimentation of amyloplasts began within 1 to 2 min after reorientation, and was characterized by an intensely staining area of cytoplasm adjacent to the sedimenting amyloplasts. By 5 min after reorientation, amyloplasts were located in the lower distal corner of columella cells, and, by 15 min after reorientation, overlaid the entire length of the lower cell wall. No consistent contact between amyloplasts and any cellular structure was detected at any stage of gravicurvature. Centrally-located nuclei initially migrated upward in columella cells of horizontally-oriented roots, after which they moved to the proximal ends of the cells by 15 min after reorientation. No significant pattern of redistribution of vacuoles, mitochondria, dictyosomes, or hyaloplasm was detected that correlated with the onset of gravicurvature. These results indicate that amyloplasts and nuclei are the only organelles whose movements correlate positively with the onset of gravicurvature by primary roots of this cultivar of Zea mays.

  17. Polysaccharide nano-vesicular multidrug carriers for synergistic killing of cancer cells

    NASA Astrophysics Data System (ADS)

    Pramod, P. S.; Shah, Ruchira; Chaphekar, Sonali; Balasubramanian, Nagaraj; Jayakannan, Manickam

    2014-09-01

    Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic efficacies against cancer cells. Here, we report dual delivery polysaccharide nano-vesicles that are capable of loading and delivering both water soluble and water insoluble drugs together in a single polymer scaffold. The selective rupture of the nano-vesicular assembly under intracellular enzyme conditions allowed the simultaneous delivery of a hydrophobic drug camptothecin (CPT) and hydrophilic drug doxorubicin (DOX) supporting their synergistic killing of breast and colon cancer cells. The polysaccharide nano-vesicles have allowed us to address a few important questions regarding the need for multiple drug administration in cancer cells including (a) the role of simultaneous drug release, (b) antagonistic versus synergistic effects of drug combinations and (c) how these are affected by the ratio of drugs. Further, evaluation of the role of caveolae in endocytosis of these polymer scaffolds was also made. The vesicular scaffolds were found to preserve and deliver DOX resulting in 50-60% better killing of cancer cells than the free drug. Additionally, dual loaded nano-vesicles when compared to drug cocktails with individual drugs in separate nano-vesicles (at comparable molar ratios) suggest the relative drug concentration following release and mode of delivery to be both important in cancer cell killing. Results from these experiments have revealed newly developed polysaccharide nano-vesicles loaded with DOX and CPT drugs as potential candidates for improved breast cancer cell killing. Thus, these custom-designed polysaccharide nano-vesicles provide a new perspective on multi-anticancer drug delivery systems and their efficacy.Multi-drug delivery based on polymer nano-scaffolds is an essential protocol to be developed for better administration of anticancer drugs to enhance their therapeutic

  18. Vesicular Monoamine Transporter 2 (VMAT2) Level Regulates MPTP Vulnerability and Clearance of Excess Dopamine in Mouse Striatal Terminals.

    PubMed

    Lohr, Kelly M; Chen, Merry; Hoffman, Carlie A; McDaniel, Miranda J; Stout, Kristen A; Dunn, Amy R; Wang, Minzheng; Bernstein, Alison I; Miller, Gary W

    2016-09-01

    The vesicular monoamine transporter 2 (VMAT2) packages neurotransmitters for release during neurotransmission and sequesters toxicants into vesicles to prevent neuronal damage. In mice, low VMAT2 levels causes catecholaminergic cell loss and behaviors resembling Parkinson's disease, while high levels of VMAT2 increase dopamine release and protect against dopaminergic toxicants. However, comparisons across these VMAT2 mouse genotypes were impossible due to the differing genetic background strains of the animals. Following back-crossing to a C57BL/6 line, we confirmed that mice with approximately 95% lower VMAT2 levels compared with wild-type (VMAT2-LO) display significantly reduced vesicular uptake, progressive dopaminergic terminal loss with aging, and exacerbated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. Conversely, VMAT2-overexpressing mice (VMAT2-HI) are protected from the loss of striatal terminals following MPTP treatment. We also provide evidence that enhanced vesicular filling in the VMAT2-HI mice modifies the handling of newly synthesized dopamine, indicated by changes in indirect measures of extracellular dopamine clearance. These results confirm the role of VMAT2 in the protection of vulnerable nigrostriatal dopamine neurons and may also provide new insight into the side effects of L-DOPA treatments in Parkinson's disease. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  19. Multiclassifier combinatorial proteomics of organelle shadows at the example of mitochondria in chromatin data.

    PubMed

    Kustatscher, Georg; Grabowski, Piotr; Rappsilber, Juri

    2016-02-01

    Subcellular localization is an important aspect of protein function, but the protein composition of many intracellular compartments is poorly characterized. For example, many nuclear bodies are challenging to isolate biochemically and thus remain inaccessible to proteomics. Here, we explore covariation in proteomics data as an alternative route to subcellular proteomes. Rather than targeting a structure of interest biochemically, we target it by machine learning. This becomes possible by taking data obtained for one organelle and searching it for traces of another organelle. As an extreme example and proof-of-concept we predict mitochondrial proteins based on their covariation in published interphase chromatin data. We detect about ⅓ of the known mitochondrial proteins in our chromatin data, presumably most as contaminants. However, these proteins are not present at random. We show covariation of mitochondrial proteins in chromatin proteomics data. We then exploit this covariation by multiclassifier combinatorial proteomics to define a list of mitochondrial proteins. This list agrees well with different databases on mitochondrial composition. This benchmark test raises the possibility that, in principle, covariation proteomics may also be applicable to structures for which no biochemical isolation procedures are available. © 2015 The Authors. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

    PubMed

    Wu, Tsung-Meng; Lin, Ke-Chun; Liau, Wei-Shiang; Chao, Yun-Yang; Yang, Ling-Hung; Chen, Szu-Yun; Lu, Chung-An; Hong, Chwan-Yang

    2016-01-01

    In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

  1. Spatial and phylogenetic analysis of the vesicular stomatitis virus epidemic in the southwestern United States in 2004-2006

    USDA-ARS?s Scientific Manuscript database

    The southwestern United States has been incidentally affected by vesicular stomatitis virus (VSV) epidemics during the last 100 years. By the time this manuscript was written, the last episodes were reported in 2004-2006. Results of space clustering and phylogenetic analysis techniques used here sug...

  2. Oncolytic recombinant vesicular stomatitis virus (VSV) is nonpathogenic and non-transmissible in pigs, a natural host of VSV

    USDA-ARS?s Scientific Manuscript database

    Vesicular stomatitis virus (VSV) is a negative stranded RNA virus that naturally causes disease in agricultural livestock including horses, cattle and pigs. The two main identified VSV strains are the New Jersey (VSNJV) and Indiana (VSIV) strains. VSV is a rapidly replicating, potently immunogenic v...

  3. APPARENT LACK OF VESICULAR-ARBUSCULAR MYCORRHIZA (VAM) IN SEAGRASSES ZOSTERA MARINA L. AND THALASSIA TESTUDIUM BANKS EX KONIG

    EPA Science Inventory

    We examined two populations of Zostera marina L. and one of Thalassia testudinum Banks ex Konig for presence of vesicular-arbuscular mycorrhiza (VAM). None of these plants showed any VAM colonization. In addition, we were unable to find any literature references on the presence o...

  4. Differential distribution of vesicular glutamate transporters in the rat cerebellar cortex.

    PubMed

    Hioki, H; Fujiyama, F; Taki, K; Tomioka, R; Furuta, T; Tamamaki, N; Kaneko, T

    2003-01-01

    The chemical organization of excitatory axon terminals in the rat cerebellar cortex was examined by immunocytochemistry and in situ hybridization histochemistry of vesicular glutamate transporters 1 and 2 (VGluT1 and VGluT2). Chemical depletion of the inferior olivary complex neurons by 3-acetylpyridine treatment almost completely removed VGluT2 immunoreactivity from the molecular layer, leaving VGluT1 immunoreactivity apparently intact. On the other hand, neuronal deprivation of the cerebellar cortex by kainic acid injection induced a large loss of VGluT1 immunoreactivity in the molecular layer. In the cerebellar granular layer, both VGluT1 and VGluT2 immunoreactivities were found in mossy fiber terminals, and the two immunoreactivities were mostly colocalized in single-axon terminals. Signals for mRNA encoding VGluT2 were found in the inferior olivary complex, and those for VGluT1 and VGluT2 mRNAs were observed in most brainstem precerebellar nuclei sending mossy fibers, such as the pontine, pontine tegmental reticular, lateral reticular and external cuneate nuclei. These results indicate that climbing and parallel fibers selectively use VGluT2 and VGluT1, respectively, whereas mossy fibers apply both VGluT1 and VGluT2 together to accumulate glutamate into synaptic vesicles. Since climbing-fiber and parallel-fiber terminals are known to make depressing and facilitating synapses, respectively, VGluT1 and VGluT2 might have distinct properties associated with those synaptic characteristics. Thus, it would be the next interesting issue to determine whether mossy-fiber terminals co-expressing VGluT1 and VGluT2 show synaptic facilitation or depression.

  5. The Multivesicular Bodies (MVBs)-Localized AAA ATPase LRD6-6 Inhibits Immunity and Cell Death Likely through Regulating MVBs-Mediated Vesicular Trafficking in Rice

    PubMed Central

    Liang, Sihui; Liang, Ruihong; Zhou, Xiaogang; Chen, Zhixiong; Zhao, Wen; Wang, Jing; Li, Weitao; He, Min; Yuan, Can; Miyamoto, Koji; Ma, Bingtian; Wang, Jichun; Qin, Peng; Chen, Weilan; Wang, Yuping; Wang, Wenming; Wu, Xianjun; Yamane, Hisakazu; Zhu, Lihuang; Li, Shigui; Chen, Xuewei

    2016-01-01

    Previous studies have shown that multivesicular bodies (MVBs)/endosomes-mediated vesicular trafficking may play key roles in plant immunity and cell death. However, the molecular regulation is poorly understood in rice. Here we report the identification and characterization of a MVBs-localized AAA ATPase LRD6-6 in rice. Disruption of LRD6-6 leads to enhanced immunity and cell death in rice. The ATPase activity and homo-dimerization of LRD6-6 is essential for its regulation on plant immunity and cell death. An ATPase inactive mutation (LRD6-6E315Q) leads to dominant-negative inhibition in plants. The LRD6-6 protein co-localizes with the MVBs marker protein RabF1/ARA6 and interacts with ESCRT-III components OsSNF7 and OsVPS2. Further analysis reveals that LRD6-6 is required for MVBs-mediated vesicular trafficking and inhibits the biosynthesis of antimicrobial compounds. Collectively, our study shows that the AAA ATPase LRD6-6 inhibits plant immunity and cell death most likely through modulating MVBs-mediated vesicular trafficking in rice. PMID:27618555

  6. Compartmentation of metals in foliage of Populus tremula grown on soils with mixed contamination. II. Zinc binding inside leaf cell organelles.

    PubMed

    Vollenweider, Pierre; Bernasconi, Petra; Gautschi, Hans-Peter; Menard, Terry; Frey, Beat; Günthardt-Goerg, Madeleine S

    2011-01-01

    The phytoextraction potential of plants for removing heavy metals from polluted soils is determined by their capacity to store contaminants in aboveground organs and complex them safely. In this study, the metal compartmentation, elemental composition of zinc deposits and zinc complexation within leaves from poplars grown on soil with mixed metal contamination was analysed combining several histochemical and microanalytical approaches. Zinc was the only heavy metal detected and was stored in several organelles in the form of globoid deposits showing β-metachromasy. It was associated to oxygen anions and different cations, noteworthy phosphorous. The deposit structure, elemental composition and element ratios indicated that zinc was chelated by phytic acid ligands. Maturation processes in vacuolar vs. cytoplasmic deposits were suggested by differences in size and amounts of complexed zinc. Hence, zinc complexation by phytate contributed to metal detoxification and accumulation in foliage but could not prevent toxicity reactions therein. Copyright © 2010 Elsevier Ltd. All rights reserved.

  7. Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes

    PubMed Central

    Medeiros, Lia Carolina Soares; Gomes, Fabio; Maciel, Luis Renato Maia; Seabra, Sergio Henrique; Docampo, Roberto; Moreno, Silvia; Plattner, Helmut; Hentschel, Joachim; Kawazoe, Urara; Barrabin, Hector; de Souza, Wanderley; DaMatta, Renato Augusto; Miranda, Kildare

    2012-01-01

    The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites. PMID:21699625

  8. Interaction of rhizosphere bacteria, fertilizer, and vesicular-arbuscular mycorrhizal fungi with sea oats.

    PubMed

    Will, M E; Sylvia, D M

    1990-07-01

    Plants must be established quickly on replenished beaches in order to stabilize the sand and begin the dune-building process. The objective of this research was to determine whether inoculation of sea oats (Uniola paniculata L.) with bacteria (indigenous rhizosphere bacteria and N(2) fixers) alone or in combination with vesicular-arbuscular mycorrhizal fungi would enhance plant growth in beach sand. At two fertilizer-N levels, Klebsiella pneumoniae and two Azospirillum spp. did not provide the plants with fixed atmospheric N; however, K. pneumoniae increased root and shoot growth. When a sparingly soluble P source (CaHPO(4)) was added to two sands, K. pneumoniae increased plant growth in sand with a high P content. The phosphorus content of shoots was not affected by bacterial inoculation, indicating that a mechanism other than bacterially enhanced P availability to plants was responsible for the growth increases. When sea oats were inoculated with either K. pneumoniae or Acaligenes denitrificans and a mixed Glomus inoculum, there was no consistent evidence of a synergistic effect on plant growth. Nonetheless, bacterial inoculation increased root colonization by vesicular-arbuscular mycorrhizal fungi when the fungal inoculum consisted of colonized roots but had no effect on colonization when the inoculum consisted of spores alone. K. pneumoniae was found to increase spore germination and hyphal growth of Glomus deserticola compared with the control. The use of bacterial inoculants to enhance establishment of pioneer dune plants warrants further study.

  9. Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle diversification mechanism and intragenus phylotyping strategy in microalgae.

    PubMed

    Wei, Li; Xin, Yi; Wang, Dongmei; Jing, Xiaoyan; Zhou, Qian; Su, Xiaoquan; Jia, Jing; Ning, Kang; Chen, Feng; Hu, Qiang; Xu, Jian

    2013-08-05

    Microalgae are promising feedstock for production of lipids, sugars, bioactive compounds and in particular biofuels, yet development of sensitive and reliable phylotyping strategies for microalgae has been hindered by the paucity of phylogenetically closely-related finished genomes. Using the oleaginous eustigmatophyte Nannochloropsis as a model, we assessed current intragenus phylotyping strategies by producing the complete plastid (pt) and mitochondrial (mt) genomes of seven strains from six Nannochloropsis species. Genes on the pt and mt genomes have been highly conserved in content, size and order, strongly negatively selected and evolving at a rate 33% and 66% of nuclear genomes respectively. Pt genome diversification was driven by asymmetric evolution of two inverted repeats (IRa and IRb): psbV and clpC in IRb are highly conserved whereas their counterparts in IRa exhibit three lineage-associated types of structural polymorphism via duplication or disruption of whole or partial genes. In the mt genomes, however, a single evolution hotspot varies in copy-number of a 3.5 Kb-long, cox1-harboring repeat. The organelle markers (e.g., cox1, cox2, psbA, rbcL and rrn16_mt) and nuclear markers (e.g., ITS2 and 18S) that are widely used for phylogenetic analysis obtained a divergent phylogeny for the seven strains, largely due to low SNP density. A new strategy for intragenus phylotyping of microalgae was thus proposed that includes (i) twelve sequence markers that are of higher sensitivity than ITS2 for interspecies phylogenetic analysis, (ii) multi-locus sequence typing based on rps11_mt-nad4, rps3_mt and cox2-rrn16_mt for intraspecies phylogenetic reconstruction and (iii) several SSR loci for identification of strains within a given species. This first comprehensive dataset of organelle genomes for a microalgal genus enabled exhaustive assessment and searches of all candidate phylogenetic markers on the organelle genomes. A new strategy for intragenus phylotyping of

  10. A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.

    PubMed

    Dolman, Nick J; Kilgore, Jason A; Davidson, Michael W

    2013-07-01

    Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image. © 2013 by John Wiley & Sons, Inc.

  11. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof.

    PubMed

    Margulis, L

    1975-01-01

    The purpose of a scientific theory is to unite apparently disparate observations into a coherent set of generalizations with predictive power. Historical theories, which necessarily treat complex irreversible events, can never be directly tested. However they certainly can lead to predictions. The 'extreme' version of the serial endosymbiotic theory argues that three classes of eukaryotic organelles had free-living ancestors: mitochondria, basal bodies/flagella/cilia [(9 + 2) homologues] and photosynthetic plastids. Many lines of evidence support this theory and can be interpreted in relation to one another on the basis of this theory. Even if this theory should eventually be proved wrong it has the real advantage of generating a large number of unique experimentally verifiable hypotheses.

  12. Heterogeneous transport of digitalis-like compounds by P-glycoprotein in vesicular and cellular assays.

    PubMed

    Gozalpour, Elnaz; Wilmer, Martijn J; Bilos, Albert; Masereeuw, Rosalinde; Russel, Frans G M; Koenderink, Jan B

    2016-04-01

    Digitalis-like compounds (DLCs), the ancient medication of heart failure and Na,K-ATPase inhibitors, are characterized by their toxicity. Drug-drug interactions (DDIs) at absorption and excretion levels play a key role in their toxicity, hence, knowledge about the transporters involved might prevent these unwanted interactions. In the present study, the transport of fourteen DLCs with human P-glycoprotein (P-gp; ABCB1) was studied using a liquid chromatography-mass spectrometry (LC-MS) quantification method. DLC transport by P-gp overexpressing Madin-Darby canine kidney (MDCK) and immortalized human renal cells (ciPTEC) was compared to vesicular DLC transport. Previously, we identified convallatoxin as a substrate using membrane vesicles overexpressing P-gp; however, we could not measure transport of other DLCs in this assay (Gozalpour et al., 2014a). Here, we showed that lipophilic digitoxin, digoxigenin, strophanthidin and proscillaridin A are P-gp substrates in cellular accumulation assays, whereas the less lipophilic convallatoxin was not. P-gp function in the cellular accumulation assays depends on the entrance of lipophilic compounds by passive diffusion, whereas the vesicular transport assay is more appropriate for hydrophilic substrates. In conclusion, we identified digitoxin, digoxigenin, strophanthidin and proscillaridin A as P-gp substrates using cellular accumulation assays and recognized lipophilicity as an important factor in selecting a suitable transport assay. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Experimental transmission of vesicular stomatitis New Jersey virus from Simulium vittatum to cattle: clinical outcome is influenced by site of insect feeding.

    PubMed

    Mead, D G; Lovett, K Rainwater; Murphy, M D; Pauszek, S J; Smoliga, G; Gray, E W; Noblet, R; Overmyer, J; Rodriguez, L L

    2009-07-01

    Vesicular stomatitis New Jersey virus (VSNJV) is an insect-transmitted Rhabdovirus causing vesicular disease in domestic livestock including cattle, horses, and pigs. Natural transmission during epidemics remains poorly understood, particularly in cattle, one of the most affected species during outbreaks. This study reports the first successful transmission of VSNJV to cattle by insect bite resulting in clinical disease. When infected black flies (Simulium vittatum Zetterstedt) fed at sites where VS lesions are usually observed (mouth, nostrils, and foot coronary band), infection occurred, characterized by local viral replication, vesicular lesions, and high neutralizing antibody titers (> 1: 256). Viral RNA was detected up to 9 d postinfection in tissues collected during necropsy from lesion sites and lymph nodes draining those sites. Interestingly, when flies were allowed to feed on flank or neck skin, viral replication was poor, lesions were not observed, and low levels of neutralizing antibodies (range, 1:8-1:32) developed. Viremia was never observed in any of the animals and infectious virus was not recovered from tissues on necropsies performed between 8 and 27 d postinfection. Demonstration that VSNJV transmission to cattle by infected black flies can result in clinical disease contributes to a better understanding of the epidemiology and potential prevention and control methods for this important disease.

  14. Optogenetic Acidification of Synaptic Vesicles and Lysosomes

    PubMed Central

    Grauel, M. Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J.; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2016-01-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes. PMID:26551543

  15. Optogenetic acidification of synaptic vesicles and lysosomes.

    PubMed

    Rost, Benjamin R; Schneider, Franziska; Grauel, M Katharina; Wozny, Christian; Bentz, Claudia; Blessing, Anja; Rosenmund, Tanja; Jentsch, Thomas J; Schmitz, Dietmar; Hegemann, Peter; Rosenmund, Christian

    2015-12-01

    Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

  16. Vesicular glutamate transporters play a role in neuronal differentiation of cultured SVZ-derived neural precursor cells

    PubMed Central

    Sánchez-Mendoza, Eduardo H.; Bellver-Landete, Victor; Arce, Carmen; Doeppner, Thorsten R.; Hermann, Dirk M.

    2017-01-01

    The role of glutamate in the regulation of neurogenesis is well-established, but the role of vesicular glutamate transporters (VGLUTs) and excitatory amino acid transporters (EAATs) in controlling adult neurogenesis is unknown. Here we investigated the implication of VGLUTs in the differentiation of subventricular zone (SVZ)-derived neural precursor cells (NPCs). Our results show that NPCs express VGLUT1-3 and EAAT1-3 both at the mRNA and protein level. Their expression increases during differentiation closely associated with the expression of marker genes. In expression analyses we show that VGLUT1 and VGLUT2 are preferentially expressed by cultured SVZ-derived doublecortin+ neuroblasts, while VGLUT3 is found on GFAP+ glial cells. In cultured NPCs, inhibition of VGLUT by Evans Blue increased the mRNA level of neuronal markers doublecortin, B3T and MAP2, elevated the number of NPCs expressing doublecortin protein and promoted the number of cells with morphological appearance of branched neurons, suggesting that VGLUT function prevents neuronal differentiation of NPCs. This survival- and differentiation-promoting effect of Evans blue was corroborated by increased AKT phosphorylation and reduced MAPK phosphorylation. Thus, under physiological conditions, VGLUT1-3 inhibition, and thus decreased glutamate exocytosis, may promote neuronal differentiation of NPCs. PMID:28493916

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

    PubMed Central

    Kumar, Rajesh; Neuser, Nicole; Tyedmers, Jens

    2017-01-01

    ABSTRACT 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 3 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 uses 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 PMID:28277942

  18. Cell type-specific Rab32 and Rab38 cooperate with the ubiquitous lysosome biogenesis machinery to synthesize specialized lysosome-related organelles

    PubMed Central

    Bultema, Jarred J.; Di Pietro, Santiago M.

    2013-01-01

    Lysosome-related organelles (LROs) exist in specialized cells to serve specific functions and typically co-exist with conventional lysosomes. The biogenesis of LROs is known to utilize much of the common protein machinery used in the transport of integral membrane proteins to lysosomes. Consequently, an outstanding question in the field has been how specific cargoes are trafficked to LROs instead of lysosomes, particularly in cells that simultaneously produce both organelles. One LRO, the melanosome, is responsible for the production of the pigment melanin and has long been used as a model system to study the formation of specialized LROs. Importantly, melanocytes, where melanosomes are synthesized, are a cell type that also produces lysosomes and must therefore segregate traffic to each organelle. Two small GTPases, Rab32 and Rab38, are key proteins in the biogenesis of melanosomes and were recently shown to redirect the ubiquitous machinery—BLOC-2, AP-1 and AP-3—to traffic specialized cargoes to melanosomes in melanocytes. In addition, the study revealed Rab32 and Rab38 have both redundant and unique roles in the trafficking of melanin-producing enzymes and overall melanosome biogenesis. Here we review these findings, integrate them with previous knowledge on melanosome biogenesis and discuss their implications for biogenesis of other LROs. PMID:23247405

  19. Vaccinia virus-free rescue of fluorescent replication-defective vesicular stomatitis virus and pseudotyping with Puumala virus glycoproteins for use in neutralization tests.

    PubMed

    Paneth Iheozor-Ejiofor, Rommel; Levanov, Lev; Hepojoki, Jussi; Strandin, Tomas; Lundkvist, Åke; Plyusnin, Alexander; Vapalahti, Olli

    2016-05-01

    Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replication-defective recombinant vesicular stomatitis virus, rVSVΔG*EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 105-108, and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (rs = 0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins.

  20. Rapid Recovery of Vesicular Dopamine Levels in Methamphetamine Users in Early Abstinence

    PubMed Central

    Boileau, Isabelle; McCluskey, Tina; Tong, Junchao; Furukawa, Yoshiaki; Houle, Sylvain; Kish, Stephen J

    2016-01-01

    We previously reported very low levels of dopamine in post-mortem striatum of chronic methamphetamine users, raising the possibility that restoration of normal dopamine levels could help in this addiction and perhaps prevent early relapse. To establish relevance of this finding to the living brain, we tested whether striatal [11C]-(+)-dihydrotetrabenazine binding, a vesicular monoamine transporter probe sensitive to changes in (stored) vesicular dopamine, is elevated in methamphetamine users. Chronic methamphetamine users underwent [11C]-(+)-dihydrotetrabenazine positron emission tomography scans during early (mean 2.6 days) and later (~10 days) abstinence. Striatal [11C]-(+)-dihydrotetrabenazine binding was elevated (suggesting low stored dopamine) in methamphetamine users (n=28; 2.6 days after last use) relative to controls (n=22) (+28%, p<0.0001) and correlated with severity and recency of drug use and with cognitive impairment and withdrawal symptoms. Mean [11C]-(+)-dihydrotetrabenazine binding levels in the subgroup of methamphetamine users who could remain abstinent ~10 days following last use (n=17) were normal at the follow-up scan. Our imaging data support post-mortem findings and suggest that chronic methamphetamine users have low brain levels of stored dopamine during very early abstinence from MA, which could contribute to behavioral and cognitive deficits. Findings also suggest a rapid recovery of stored dopamine in some methamphetamine users who become abstinent and who therefore might not benefit from dopamine replacement medication (eg, levodopa). Further study is necessary to establish whether those users who could not maintain abstinence for the second scan might have a more severe and persistent dopamine deficiency and who could benefit from this medication. PMID:26321315

  1. Rapid Recovery of Vesicular Dopamine Levels in Methamphetamine Users in Early Abstinence.

    PubMed

    Boileau, Isabelle; McCluskey, Tina; Tong, Junchao; Furukawa, Yoshiaki; Houle, Sylvain; Kish, Stephen J

    2016-03-01

    We previously reported very low levels of dopamine in post-mortem striatum of chronic methamphetamine users, raising the possibility that restoration of normal dopamine levels could help in this addiction and perhaps prevent early relapse. To establish relevance of this finding to the living brain, we tested whether striatal [(11)C]-(+)-dihydrotetrabenazine binding, a vesicular monoamine transporter probe sensitive to changes in (stored) vesicular dopamine, is elevated in methamphetamine users. Chronic methamphetamine users underwent [(11)C]-(+)-dihydrotetrabenazine positron emission tomography scans during early (mean 2.6 days) and later (~10 days) abstinence. Striatal [(11)C]-(+)-dihydrotetrabenazine binding was elevated (suggesting low stored dopamine) in methamphetamine users (n=28; 2.6 days after last use) relative to controls (n=22) (+28%, p<0.0001) and correlated with severity and recency of drug use and with cognitive impairment and withdrawal symptoms. Mean [(11)C]-(+)-dihydrotetrabenazine binding levels in the subgroup of methamphetamine users who could remain abstinent ~10 days following last use (n=17) were normal at the follow-up scan. Our imaging data support post-mortem findings and suggest that chronic methamphetamine users have low brain levels of stored dopamine during very early abstinence from MA, which could contribute to behavioral and cognitive deficits. Findings also suggest a rapid recovery of stored dopamine in some methamphetamine users who become abstinent and who therefore might not benefit from dopamine replacement medication (eg, levodopa). Further study is necessary to establish whether those users who could not maintain abstinence for the second scan might have a more severe and persistent dopamine deficiency and who could benefit from this medication.

  2. A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

    PubMed

    Mackinder, Luke C M; Meyer, Moritz T; Mettler-Altmann, Tabea; Chen, Vivian K; Mitchell, Madeline C; Caspari, Oliver; Freeman Rosenzweig, Elizabeth S; Pallesen, Leif; Reeves, Gregory; Itakura, Alan; Roth, Robyn; Sommer, Frederik; Geimer, Stefan; Mühlhaus, Timo; Schroda, Michael; Goodenough, Ursula; Stitt, Mark; Griffiths, Howard; Jonikas, Martin C

    2016-05-24

    Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

  3. Volatile concentrations in variably vesicular pyroclasts from the Rotongaio ash (181 AD Taupo eruption): did shallow magma degassing trigger exceptionally violent phreatomagmatic activity?

    NASA Astrophysics Data System (ADS)

    Tuffen, Hugh; Houghton, Bruce F.; Dingwellp, Donald B.; Pinkerton, Harry

    2010-05-01

    Measurement of dissolved volatile concentrations in pyroclasts has formed the basis of our understanding of the links between magma degassing and the explosivity of silicic eruptions[1]. To date these studies have focussed exclusively on the densest pyroclastic obsidians, which comprise on a tiny proportion of the erupted products, in order to bypass the difficulty of analysing vesicular material. As a consequence, crucial information is missing about how degassing in the densest clasts relates to the behaviour of the bulk of the magma volume. To overcome this shortcoming, the volatile content of variably vesicular pyroclasts from the Rotongaio ash has been analysed using both micro-analytical (SIMS, synchrotron FTIR) and bulk techniques (TGA-MS). The Rotongaio ash was an exceptionally violent phase of phreatomagmatic activity during the 181 AD rhyolitic eruption of Taupo (New Zealand), the most powerful worldwide in the last 5000 years. The Rotongaio phase involved opening of new vents beneath Lake Taupo and the ash is characterised by a wide range of clast vesicularities (<10 to ~80 % by volume). Volatile measurement was challenging due to the high bubble number densities and small clast sizes. The mismatch between the water content of matrix glasses measured using bulk and micro-analytical techniques reflects pervasive post-eruption hydration of vesicle walls, which is most problematic at high vesicularities. Micron-scale maps of water concentration variations around vesicles in 30-50 vol % vesicular samples were acquired using SIMS. They indicate strong hydration within ~5 microns of vesicle walls, with pockets of unhydrated glass remaining in the thickest septa. Analysis of these unhydrated domains allowed robust measurement of water contents in pyroclasts ranging from ~1 to >50 vol % vesicles. Matrix glasses had largely degassed (0.19-0.49 wt % H2O, compared with an initial concentration in melt inclusions of ~3.6 wt %). The water contents measured using SIMS

  4. Periodicity in Attachment Organelle Revealed by Electron Cryotomography Suggests Conformational Changes in Gliding Mechanism of Mycoplasma pneumoniae

    PubMed Central

    Kawamoto, Akihiro; Matsuo, Lisa; Kato, Takayuki; Yamamoto, Hiroki

    2016-01-01

    ABSTRACT Mycoplasma pneumoniae, a pathogenic bacterium, glides on host surfaces using a unique mechanism. It forms an attachment organelle at a cell pole as a protrusion comprised of knoblike surface structures and an internal core. Here, we analyzed the three-dimensional structure of the organelle in detail by electron cryotomography. On the surface, knoblike particles formed a two-dimensional array, albeit with limited regularity. Analyses using a nonbinding mutant and an antibody showed that the knoblike particles correspond to a naplike structure that has been observed by negative-staining electron microscopy and is likely to be formed as a complex of P1 adhesin, the key protein for binding and gliding. The paired thin and thick plates feature a rigid hexagonal lattice and striations with highly variable repeat distances, respectively. The combination of variable and invariant structures in the internal core and the P1 adhesin array on the surface suggest a model in which axial extension and compression of the thick plate along a rigid thin plate is coupled with attachment to and detachment from the substrate during gliding. PMID:27073090

  5. Immunolocalization of vesicular glutamate transporters 1 and 2 in the rat inferior colliculus.

    PubMed

    Altschuler, R A; Tong, L; Holt, A G; Oliver, D L

    2008-06-12

    The inferior colliculus is a major relay nucleus in the ascending auditory pathways that receives multiple glutamatergic inputs. Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) most often have complementary non-overlapping distributions and can be used to differentiate glutamatergic inputs. The present study therefore examined co-immunolabeling of VGLUT1 and VGLUT2 in three divisions of the rat inferior colliculus. Additional co-immunolabeling of microtubule-associated protein 2 and neuronal class III beta-tubulin provided visualization of neuronal soma and processes and allowed identification of axo-somatic versus axo-dendritic contacts. Results showed numerous VGLUT1 and 2 immunolabeled terminals in the central nucleus, lateral cortex and dorsal cortex. In all three divisions there was little to no co-containment of the two vesicular glutamate transporters indicating a complementary distribution. VGLUT1 made predominantly axo-dendritic connections in the neuropil, while VGLUT2 had many axo-somatic contacts in addition to axo-dendritic contacts. VGLUT2 immunolabeled terminals were numerous on the soma and proximal dendrites of many medium-to-large and large neurons in the central nucleus and medium to large neurons in the dorsal cortex. There were more VGLUT2 terminals than VGLUT1 in all divisions and more VGLUT2 terminals in dorsal and lateral cortices than in the central nucleus. This study shows that VGLUT1 and VGLUT2 differentiate complementary patterns of glutamatergic inputs into the central nucleus, lateral and dorsal cortex of the inferior colliculus with VGLUT1 endings predominantly on the dendrites and VGLUT2 on both dendrites and somas.

  6. Multiscale smeared finite element model for mass transport in biological tissue: From blood vessels to cells and cellular organelles.

    PubMed

    Kojic, M; Milosevic, M; Simic, V; Koay, E J; Kojic, N; Ziemys, A; Ferrari, M

    2018-05-21

    One of the basic and vital processes in living organisms is mass exchange, which occurs on several levels: it goes from blood vessels to cells and organelles within cells. On that path, molecules, as oxygen, metabolic products, drugs, etc. Traverse different macro and micro environments - blood, extracellular/intracellular space, and interior of organelles; and also biological barriers such as walls of blood vessels and membranes of cells and organelles. Many aspects of this mass transport remain unknown, particularly the biophysical mechanisms governing drug delivery. The main research approach relies on laboratory and clinical investigations. In parallel, considerable efforts have been directed to develop computational tools for additional insight into the intricate process of mass exchange and transport. Along these lines, we have recently formulated a composite smeared finite element (CSFE) which is composed of the smeared continuum pressure and concentration fields of the capillary and lymphatic system, and of these fields within tissue. The element offers an elegant and simple procedure which opens up new lines of inquiry and can be applied to large systems such as organs and tumors models. Here, we extend this concept to a multiscale scheme which concurrently couples domains that span from large blood vessels, capillaries and lymph, to cell cytosol and further to organelles of nanometer size. These spatial physical domains are coupled by the appropriate connectivity elements representing biological barriers. The composite finite element has "degrees of freedom" which include pressures and concentrations of all compartments of the vessels-tissue assemblage. The overall model uses the standard, measurable material properties of the continuum biological environments and biological barriers. It can be considered as a framework into which we can incorporate various additional effects (such as electrical or biochemical) for transport through membranes or within

  7. Two mechanisms of H+/OH- transport across phospholipid vesicular membrane facilitated by gramicidin A.

    PubMed Central

    Prabhananda, B S; Kombrabail, M H

    1996-01-01

    Two rate-limiting mechanisms have been proposed to explain the gramicidin channel facilitated decay of the pH difference across vesicular membrane (delta pH) in the pH region 6-8 and salt (MCI, M+ = K+, Na+) concentration range 50-300 mM. 1) At low pH conditions (approximately 6), H+ transport through the gramicidin channel predominantly limits the delta pH decay rate. 2) At higher pH conditions (approximately 7.5), transport of a deprotonated species (but not through the channel) predominantly limits the rate. The second mechanism has been suggested to be the hydroxyl ion propogation through water chains across the bilayer by hydrogen bond exchange. In both mechanisms alkali metal ion transport providing the compensating flux takes place through the gramicidin channels. Such an identification has been made from a detailed study of the delta pH decay rate as a function of 1) gramicidin concentration, 2) alkali metal ion concentration, 3) pH, 4) temperature, and 5) changes in the membrane order (by adding small amounts of chloroform to vesicle solutions). The apparent activation energy associated with the second mechanism (approximately 3.2 kcal/mol) is smaller than that associated with the first mechanism (approximately 12 kcal/mol). In these experiments, delta pH was created by temperature jump, and vesicles were prepared using soybean phospholipid or a mixture of 94% egg phosphatidylcholine and 6% phosphatidic acid. PMID:8968580

  8. Vesicular glutamate transporters in the lateral geniculate nucleus: expression of VGLUT2 by retinal terminals.

    PubMed

    Land, Peter W; Kyonka, E; Shamalla-Hannah, L

    2004-01-23

    We used immunohistochemistry to localize vesicular glutamate transporters VGLUT1 and VGLUT2 in the rat lateral geniculate nucleus. The lateral geniculate nucleus is intensely immunoreactive for both transporters. Monocular eye removal abolished staining for VGLUT2 in a pattern corresponding to the distribution of terminals from the missing eye, without affecting distribution of VGLUT1 immunoreactivity. These data indicate retinal ganglion cells are the source of VGLUT2-containing synapses in the lateral geniculate nucleus.

  9. Hybrid Markov-mass action law model for cell activation by rare binding events: Application to calcium induced vesicular release at neuronal synapses.

    PubMed

    Guerrier, Claire; Holcman, David

    2016-10-18

    Binding of molecules, ions or proteins to small target sites is a generic step of cell activation. This process relies on rare stochastic events where a particle located in a large bulk has to find small and often hidden targets. We present here a hybrid discrete-continuum model that takes into account a stochastic regime governed by rare events and a continuous regime in the bulk. The rare discrete binding events are modeled by a Markov chain for the encounter of small targets by few Brownian particles, for which the arrival time is Poissonian. The large ensemble of particles is described by mass action laws. We use this novel model to predict the time distribution of vesicular release at neuronal synapses. Vesicular release is triggered by the binding of few calcium ions that can originate either from the synaptic bulk or from the entry through calcium channels. We report here that the distribution of release time is bimodal although it is triggered by a single fast action potential. While the first peak follows a stimulation, the second corresponds to the random arrival over much longer time of ions located in the synaptic terminal to small binding vesicular targets. To conclude, the present multiscale stochastic modeling approach allows studying cellular events based on integrating discrete molecular events over several time scales.

  10. Flow Fields of the 3.5 Ga Komati Formation, South Africa: Geochemical, Stratigraphic, and Temporal relationships between Massive, Vesicular, and Spinifex flows

    NASA Astrophysics Data System (ADS)

    Dann, J. C.

    2007-12-01

    A challenge of Archean volcanology is to reconstruct submarine flow fields by mapping and analyzing vertically dipping sequences of lavas. Some flow fields are bound by sediments and/or seafloor alteration that mark clear gaps in volcanism. Flow fields in the Lower Komati Fm are defined by alternating layers of komatiite (26% MgO) and komatiitic basalt (15% MgO). Five komatiite flow fields (100-200m thick) repeat the same stratigraphic zoning of spinifex overlying massive komatiite, and each flow field has a distinct Al2O3/CaO, a ratio unaffected by olivine fractionation, consistent with the contention that each komatiite flow field represents a distinct batch of mantle melting. Although massive and spinifex komatiite form distinct stratigraphic units on a map scale, detailed outcrop mapping reveals that the change in flow type represents a transition within a single flow field. In one type of transition, thin massive flows alternate with spinifex flow lobes of a compound flow unit. In another, a vesicular flow along the boundary links the underlying massive komatiite and overlying spinifex flows in time. The vesicular flow has alternating spinifex and vesicular layers that form a distinctive crust above a thick massive interior. Locally, this crust is tilted, intruded by massive komatiite from the interior, and overlain by a thick breccia including a spinifex flow broken into blocks and rotated like dominoes by the tilting. These outcrop relations indicate that spinifex flow lobes were starting to flow over the vesicular flow before it had undergone differential inflation, a temporal link between the lower massive and upper spinifex komatiites consistent with their belonging to the same flow field. The transition in flow type may reflect 1) an overlap of proximal and distal facies of komatiite flows as eruption rates waned and/or 2) thermal maturation prior to eruption. Early, cooler, crystal-rich, massive lava, flowing out as thick sheet flows, was replaced by

  11. Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health

    PubMed Central

    Martin, William F.

    2017-01-01

    Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. PMID:28444372

  12. Lassa-Vesicular Stomatitis Chimeric Virus Safely Destroys Brain Tumors

    PubMed Central

    Wollmann, Guido; Drokhlyansky, Eugene; Davis, John N.; Cepko, Connie

    2015-01-01

    ABSTRACT High-grade tumors in the brain are among the deadliest of cancers. Here, we took a promising oncolytic virus, vesicular stomatitis virus (VSV), and tested the hypothesis that the neurotoxicity associated with the virus could be eliminated without blocking its oncolytic potential in the brain by replacing the neurotropic VSV glycoprotein with the glycoprotein from one of five different viruses, including Ebola virus, Marburg virus, lymphocytic choriomeningitis virus (LCMV), rabies virus, and Lassa virus. Based on in vitro infections of normal and tumor cells, we selected two viruses to test in vivo. Wild-type VSV was lethal when injected directly into the brain. In contrast, a novel chimeric virus (VSV-LASV-GPC) containing genes from both the Lassa virus glycoprotein precursor (GPC) and VSV showed no adverse actions within or outside the brain and targeted and completely destroyed brain cancer, including high-grade glioblastoma and melanoma, even in metastatic cancer models. When mice had two brain tumors, intratumoral VSV-LASV-GPC injection in one tumor (glioma or melanoma) led to complete tumor destruction; importantly, the virus moved contralaterally within the brain to selectively infect the second noninjected tumor. A chimeric virus combining VSV genes with the gene coding for the Ebola virus glycoprotein was safe in the brain and also selectively targeted brain tumors but was substantially less effective in destroying brain tumors and prolonging survival of tumor-bearing mice. A tropism for multiple cancer types combined with an exquisite tumor specificity opens a new door to widespread application of VSV-LASV-GPC as a safe and efficacious oncolytic chimeric virus within the brain. IMPORTANCE Many viruses have been tested for their ability to target and kill cancer cells. Vesicular stomatitis virus (VSV) has shown substantial promise, but a key problem is that if it enters the brain, it can generate adverse neurologic consequences, including death. We

  13. Photoelectrocyclization as an activation mechanism for organelle-specific live-cell imaging probes.

    PubMed

    Tran, Mai N; Chenoweth, David M

    2015-05-26

    Photoactivatable fluorophores are useful tools in live-cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle-specific live-cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water-soluble, non-cytotoxic, cell-permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre-activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single-cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single- or multi-cell labeling experiments. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Calcium homeostasis and organelle function in the pathogenesis of obesity and diabetes

    PubMed Central

    Arruda, Ana Paula; Hotamisligil, Gökhan S.

    2015-01-01

    Summary A number of chronic metabolic pathologies, including obesity, diabetes, cardiovascular disease, asthma, and cancer cluster together to present the greatest threat to human health. As research in this field has advanced, it has become clear that unresolved metabolic inflammation, organelle dysfunction, and other cellular and metabolic stresses underlie the development of these chronic metabolic diseases. However, the relationship between these systems and pathological mechanisms is poorly understood. Here, we will discuss the role of cellular Ca2+ homeostasis as a critical mechanism integrating the myriad of cellular and subcellular dysfunctional networks found in metabolic tissues such as liver and adipose tissue in the context of metabolic disease particularly in obesity and diabetes. PMID:26190652

  15. A systematic review of the asymmetric inheritance of cellular organelles in eukaryotes: A critique of basic science validity and imprecision

    PubMed Central

    Collins, Anne; Ross, Janine

    2017-01-01

    We performed a systematic review to identify all original publications describing the asymmetric inheritance of cellular organelles in normal animal eukaryotic cells and to critique the validity and imprecision of the evidence. Searches were performed in Embase, MEDLINE and Pubmed up to November 2015. Screening of titles, abstracts and full papers was performed by two independent reviewers. Data extraction and validity were performed by one reviewer and checked by a second reviewer. Study quality was assessed using the SYRCLE risk of bias tool, for animal studies and by developing validity tools for the experimental model, organelle markers and imprecision. A narrative data synthesis was performed. We identified 31 studies (34 publications) of the asymmetric inheritance of organelles after mitotic or meiotic division. Studies for the asymmetric inheritance of centrosomes (n = 9); endosomes (n = 6), P granules (n = 4), the midbody (n = 3), mitochondria (n = 3), proteosomes (n = 2), spectrosomes (n = 2), cilia (n = 2) and endoplasmic reticulum (n = 2) were identified. Asymmetry was defined and quantified by variable methods. Assessment of the statistical reliability of the results indicated only two studies (7%) were judged to have low concern, the majority of studies (77%) were 'unclear' and five (16%) were judged to have 'high concerns'; the main reasons were low technical repeats (<10). Assessment of model validity indicated that the majority of studies (61%) were judged to be valid, ten studies (32%) were unclear and two studies (7%) were judged to have 'high concerns'; both described 'stem cells' without providing experimental evidence to confirm this (pluripotency and self-renewal). Assessment of marker validity indicated that no studies had low concern, most studies were unclear (96.5%), indicating there were insufficient details to judge if the markers were appropriate. One study had high concern for marker validity due to the contradictory results of two

  16. Hypothalamic orexin (hypocretin) neurons express vesicular glutamate transporters VGLUT1 or VGLUT2.

    PubMed

    Rosin, Diane L; Weston, Matthew C; Sevigny, Charles P; Stornetta, Ruth L; Guyenet, Patrice G

    2003-10-27

    Initially recognized for their importance in control of appetite, orexins (also called hypocretins) are neuropeptides that are also involved in regulating sleep, arousal, and cardiovascular function. Loss of orexin appears to be the primary cause of narcolepsy. Cells expressing the orexins are restricted to a discrete region of the hypothalamus, but their terminal projections are widely distributed throughout the brain. With the diversity of function and broad distribution of orexin terminals, it is not known whether the orexin cells constitute a homogeneous population. Because orexins produce neuroexcitatory effects, we hypothesized that orexin-containing neurons are glutamatergic. In the present study we used digoxigenin-labeled cRNA probes for the vesicular glutamate transporters, VGLUT1 and VGLUT2, for in situ hybridization studies in combination with immunohistochemical detection of orexin cell bodies in the hypothalamus. In general, cells in the hypothalamus expressed low levels of the vesicular glutamate transporters relative to other areas of the forebrain, such as the cortex and thalamus. Light labeling for VGLUT2 mRNA was detected in about 50% of the orexin-immunoreactive neurons, and a much smaller percentage (approximately 13%) of orexin-immunoreactive cells was found to express VGLUT1. Despite the fact that intense labeling for GAD67 mRNA was found in a large number of cells throughout the hypothalamus, none of the orexin-immunoreactive cells was found to be GABAergic. These findings, showing that many of the orexin neurons are glutamatergic, are consistent with the neuroexcitatory effects of orexin but suggest that another neurochemical phenotype may define the remaining subset of orexin neurons. Copyright 2003 Wiley-Liss, Inc.

  17. Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

    PubMed Central

    2011-01-01

    Many bacterial species contain intracellular nano- and micro-compartments consisting of self-assembling proteins that form protein-only shells. These structures are built up by combinations of a reduced number of repeated elements, from 60 repeated copies of one unique structural element self-assembled in encapsulins of 24 nm to 10,000-20,000 copies of a few protein species assembled in a organelle of around 100-150 nm in cross-section. However, this apparent simplicity does not correspond to the structural and functional sophistication of some of these organelles. They package, by not yet definitely solved mechanisms, one or more enzymes involved in specific metabolic pathways, confining such reactions and sequestering or increasing the inner concentration of unstable, toxics or volatile intermediate metabolites. From a biotechnological point of view, we can use the self assembling properties of these particles for directing shell assembling and enzyme packaging, mimicking nature to design new applications in biotechnology. Upon appropriate engineering of the building blocks, they could act as a new family of self-assembled, protein-based vehicles in Nanomedicine to encapsulate, target and deliver therapeutic cargoes to specific cell types and/or tissues. This would provide a new, intriguing platform of microbial origin for drug delivery. PMID:22046962

  18. Phenol homeostasis is ensured in vanilla fruit by storage under solid form in a new chloroplast-derived organelle, the phenyloplast

    PubMed Central

    Conéjéro, Geneviève

    2014-01-01

    A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a ‘phenyloplast’. PMID:24683183

  19. Potential fossil endoliths in vesicular pillow basalt, Coral Patch Seamount, eastern North Atlantic Ocean.

    PubMed

    Cavalazzi, Barbara; Westall, Frances; Cady, Sherry L; Barbieri, Roberto; Foucher, Frédéric

    2011-09-01

    The chilled rinds of pillow basalt from the Ampère-Coral Patch Seamounts in the eastern North Atlantic were studied as a potential habitat of microbial life. A variety of putative biogenic structures, which include filamentous and spherical microfossil-like structures, were detected in K-phillipsite-filled amygdules within the chilled rinds. The filamentous structures (∼2.5 μm in diameter) occur as K-phillipsite tubules surrounded by an Fe-oxyhydroxide (lepidocrocite) rich membranous structure, whereas the spherical structures (from 4 to 2 μm in diameter) are associated with Ti oxide (anatase) and carbonaceous matter. Several lines of evidence indicate that the microfossil-like structures in the pillow basalt are the fossilized remains of microorganisms. Possible biosignatures include the carbonaceous nature of the spherical structures, their size distributions and morphology, the presence and distribution of native fluorescence, mineralogical and chemical composition, and environmental context. When taken together, the suite of possible biosignatures supports the hypothesis that the fossil-like structures are of biological origin. The vesicular microhabitat of the rock matrix is likely to have hosted a cryptoendolithic microbial community. This study documents a variety of evidence for past microbial life in a hitherto poorly investigated and underestimated microenvironment, as represented by the amygdules in the chilled pillow basalt rinds. This kind of endolithic volcanic habitat would have been common on the early rocky planets in our Solar System, such as Earth and Mars. This study provides a framework for evaluating traces of past life in vesicular pillow basalts, regardless of whether they occur on early Earth or Mars.

  20. Topical vesicular formulations of Curcuma longa extract on recuperating the ultraviolet radiation-damaged skin.

    PubMed

    Kaur, Chanchal Deep; Saraf, Swarnlata

    2011-12-01

      Ultraviolet radiations generate reactive oxygen species, leading to adverse effects on skin properties. Botanical extracts are multifunctional in nature having various properties like photoprotection, anti-aging, moisturizing, antioxidant, astringent, anti-irritant, and antimicrobial activity.   The aim of this study was to formulate creams having Curcuma longa extract loaded novel vesicular systems (liposomes, ethosomes, and transfersomes) and study their photoprotective effect by assessment of skin hydration (Cutometer) and sebum content (Sebumeter).   The alcoholic C. longa extract loaded liposomes, ethosomes, and transfersomes having 0.5-2.0% w/w extract were prepared, evaluated for size, entrapment efficiency, and incorporated into the cream. Their long-term interaction with skin (6 weeks) was compared in terms of their effects on skin hydration and sebum content.   Vesicular size obtained was in the range 167.3 ± 3.0 to 262.4 ± 2.4 nm with low polydispersity index (0.2-0.3) and high entrapment efficiency. The efficacy was in the order C. longa extract loaded transfersomal creams > C. longa extract loaded ethosomal creams > C. longa extract loaded liposomal creams > C. longa extract loaded creams > Empty transfersome loaded cream > Empty ethosome loaded cream > Empty liposome loaded cream > Base cream.   The photoprotective properties of the constituents of C. longa extract and hydrant, moisturizing lipid components of nano vesicles with better skin penetration resulted in improvement in skin properties like skin hydration and sebum content. The herbal extract loaded nano vesicles incorporated in cream could be used as photoprotective formulations. © 2011 Wiley Periodicals, Inc.

  1. Quantifying immunogold labelling patterns of cellular compartments when they comprise mixtures of membranes (surface-occupying) and organelles (volume-occupying).

    PubMed

    Mayhew, Terry M; Lucocq, John M

    2008-03-01

    In quantitative immunoelectron microscopy, subcellular compartments that are preferentially labelled with colloidal gold particles can be identified by estimating labelling densities (LDs) and relative labelling indices (RLIs). Hitherto, this approach has been limited to compartments which are either surface occupying (membranes) or volume occupying (organelles) but not a mixture of both (membranes and organelles). However, some antigens are known to translocate between membrane and organelle compartments and the problem then arises of expressing gold particle LDs in a consistent manner (e.g., as number per compartment profile area). Here, we present one possible solution to tackle this problem. With this method, each membrane is treated as a volume-occupying compartment and this is achieved by creating an acceptance zone at a fixed distance on each side of membrane images. Gold signal intensity is then expressed as an LD within the membrane profile area so created and this LD can be compared to LDs found in volume-occupying compartments. Acceptance zone width is determined largely by the expected dispersion of gold labelling. In some cases, the zone can be applied to all visible membrane images but there is a potential problem when image loss occurs due to the fact that membranes are not cut orthogonal to their surface but are tilted within the section. The solution presented here is to select a subset of clear images representing orthogonally sectioned membranes (so-called local vertical windows, LVWs). The fraction of membrane images forming LVWs can be estimated in two ways: goniometrically (by determining the angle at which images become unclear) or stereologically (by counting intersections with test lines). The fraction obtained by either method can then be used to calculate a factor correcting for membrane image loss. In turn, this factor is used to estimate the total gold labelling associated with the acceptance zone of the entire (volume

  2. Crystal Structure of Swine Vesicular Disease Virus and Implications for Host Adaptation

    PubMed Central

    Fry, Elizabeth E.; Knowles, Nick J.; Newman, John W. I.; Wilsden, Ginette; Rao, Zihe; King, Andrew M. Q.; Stuart, David I.

    2003-01-01

    Swine vesicular disease virus (SVDV) is an Enterovirus of the family Picornaviridae that causes symptoms indistinguishable from those of foot-and-mouth disease virus. Phylogenetic studies suggest that it is a recently evolved genetic sublineage of the important human pathogen coxsackievirus B5 (CBV5), and in agreement with this, it has been shown to utilize the coxsackie and adenovirus receptor (CAR) for cell entry. The 3.0-Å crystal structure of strain UK/27/72 SVDV (highly virulent) reveals the expected similarity in core structure to those of other picornaviruses, showing most similarity to the closest available structure to CBV5, that of coxsackievirus B3 (CBV3). Features that help to cement together and rigidify the protein subunits are extended in this virus, perhaps explaining its extreme tolerance of environmental factors. Using the large number of capsid sequences available for both SVDV and CBV5, we have mapped the amino acid substitutions that may have occurred during the supposed adaptation of SVDV to a new host onto the structure of SVDV and a model of the SVDV/CAR complex generated by reference to the cryo-electron microscopy-visualized complex of CBV3 and CAR. The changes fall into three clusters as follows: one lines the fivefold pore, a second maps to the CAR-binding site and partially overlaps the site for decay accelerating factor (DAF) to bind to echovirus 7 (ECHO7), and the third lies close to the fivefold axis, where the low-density lipoprotein receptor binds to the minor group of rhinoviruses. Later changes in SVDV (post-1971) map to the first two clusters and may, by optimizing recognition of a pig CAR and/or DAF homologue, have improved the adaptation of the virus to pigs. PMID:12692248

  3. Synthesis of Non-Toxic Silica Particles Stabilized by Molecular Complex Oleic-Acid/Sodium Oleate

    PubMed Central

    Spataru, Catalin Ilie; Ianchis, Raluca; Petcu, Cristian; Nistor, Cristina Lavinia; Purcar, Violeta; Trica, Bogdan; Nitu, Sabina Georgiana; Somoghi, Raluca; Alexandrescu, Elvira; Oancea, Florin; Donescu, Dan

    2016-01-01

    The present work is focused on the preparation of biocompatible silica particles from sodium silicate, stabilized by a vesicular system containing oleic acid (OLA) and its alkaline salt (OLANa). Silica nanoparticles were generated by the partial neutralization of oleic acid (OLA), with the sodium cation present in the aqueous solutions of sodium silicate. At the molar ratio OLA/Na+ = 2:1, the molar ratio (OLA/OLANa = 1:1) required to form vesicles, in which the carboxyl and carboxylate groups have equal concentrations, was achieved. In order to obtain hydrophobically modified silica particles, octadecyltriethoxysilane (ODTES) was added in a sodium silicate sol–gel mixture at different molar ratios. The interactions between the octadecyl groups from the modified silica and the oleyl chains from the OLA/OLANa stabilizing system were investigated via simultaneous thermogravimetry (TG) and differential scanning calorimetry (DSC) (TG-DSC) analyses.A significant decrease in vaporization enthalpy and an increase in amount of ODTES were observed. Additionally, that the hydrophobic interaction between OLA and ODTES has a strong impact on the hybrids’ final morphology and on their textural characteristics was revealed. The highest hydrodynamic average diameter and the most negative ζ potential were recorded for the hybrid in which the ODTES/sodium silicate molar ratio was 1:5. The obtained mesoporous silica particles, stabilized by the OLA/OLANa vesicular system, may find application as carriers for hydrophobic bioactive molecules. PMID:27869768

  4. Lesões vesiculares e prurido intenso em paciente com sífilis secundária:manifestação clínica atípica.

    PubMed

    Duarte Bezerra Pinto, Roberta; Rubim Bellott Batista Nascimento, Thiago; Pacheco Bicalho de Andrade, Jhéssica; Policarpo, Fernanda; Ribeiro Estrella, Rogério; Guedes Vilar, Enoi

    2016-06-15

    Syphilis is an infectious disease with mucocutaneous lesions that are characteristic of their stage (primary, secondary, or tertiary). These are not always typical and may have other morphologies and unusual symptoms, making diagnosis difficult. The report herein is of a healthy woman with vesicular lesions associated with intense itching.  The diagnosis of secondary syphilis was confirmed with VDRL 1/512 and FTA Abs positive.  Histopathology showed spongiosis in the epidermis and infiltrated lymphocytes and plasma cell in the dermis.  Clinical improvement occurred after the first dose of benzathine penicillin. In the literature, few cases of vesicular lesions in syphilis have been found and itching is mentioned as uncharacteristic . Histology showing spongiosis is also unusual.

  5. Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Response of Potato to Phosphorus Deficiency.

    PubMed

    McArthur, DAJ.; Knowles, N. R.

    1993-01-01

    Morphological and biochemical interactions between a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus fasciculatum [Thaxt. sensu Gerdemann] Gerdemann and Trappe) and potato (Solanum tuberosum L.) plants during the development of P deficiency were characterized. Nonmycorrhizal (NM) plants grown for 63 d with low abiotic P supply (0.5 mM) produced 34, 52, and 73% less root, shoot, and tuber dry matter, respectively, than plants grown with high P (2.5 mM). The total leaf area and the leaf area:plant dry weight ratio of low-P plants were substantially lower than those of high-P plants. Moreover, a lower shoot:root dry weight ratio and tuber:plant dry weight ratio in low-P plants than in high-P plants characterized a major effect of P deficiency stress on dry matter partitioning. In addition to a slower rate of growth, low-P plants accumulated nonreducing sugars and nitrate. Furthermore, root respiration and leaf nitrate reductase activity were lower in low-P plants than in high-P plants. Low abiotic P supply also induced physiological changes that contributed to the greater efficiency of P acquisition by low-P plants than by high-P plants. For example, allocation of dry matter and P to root growth was less restricted by P deficiency stress than to shoot and tuber growth. Also, the specific activities of root acid phosphatases and vanadate-sensitive microsomal ATPases were enhanced in P-deficient plants. The establishment of a VAM symbiosis by low-P plants was essential for efficient P acquisition, and a greater root infection level for P-stressed plants indicated increased compatibility to the VAM fungus. By 63 d after planting, low-P VAM plants had recovered 42% more of the available soil P than low-P NM plants. However, the VAM fungus only partially alleviated P deficiency stress and did not completely compensate for inadequate abiotic P supply. Although the specific activities of acid phosphatases and microsomal ATPases were only marginally influenced by VAM

  6. Airstream fractionation of vesicular-arbuscular mycorrhizal fungi: concentration and enumeration of propagules.

    PubMed

    Tommerup, I C

    1982-09-01

    Spores and fragments of vesicular-arbuscular mycorrhizal fungi in dry soils were concentrated up to 100-fold when the soils were partitioned by fluidization and elutriation with a series of upward airstreams at progressively increasing velocities. The propagules were transported with the finer soil particles according to their equivalent spherical diameters. The system was used to predict the transport of propagules by wind. Concentrated propagules were rapidly separated from the soil particles in each soil fraction by an aqueous flotation method. The technique is proposed as a quantitative method for estimating the numbers of spores and fragments of mycorrhizae. The scheme includes a viability test that was used to differentiate between potentially infective propagules and those that were either dormant or incapable of regrowth.

  7. Vesicular glutamate release from central axons contributes to myelin damage.

    PubMed

    Doyle, Sean; Hansen, Daniel Bloch; Vella, Jasmine; Bond, Peter; Harper, Glenn; Zammit, Christian; Valentino, Mario; Fern, Robert

    2018-03-12

    The axon myelin sheath is prone to injury associated with N-methyl-D-aspartate (NMDA)-type glutamate receptor activation but the source of glutamate in this context is unknown. Myelin damage results in permanent action potential loss and severe functional deficit in the white matter of the CNS, for example in ischemic stroke. Here, we show that in rats and mice, ischemic conditions trigger activation of myelinic NMDA receptors incorporating GluN2C/D subunits following release of axonal vesicular glutamate into the peri-axonal space under the myelin sheath. Glial sources of glutamate such as reverse transport did not contribute significantly to this phenomenon. We demonstrate selective myelin uptake and retention of a GluN2C/D NMDA receptor negative allosteric modulator that shields myelin from ischemic injury. The findings potentially support a rational approach toward a low-impact prophylactic therapy to protect patients at risk of stroke and other forms of excitotoxic injury.

  8. Epizootic vesicular stomatitis in Colorado, 1982: epidemiologic and entomologic studies.

    PubMed

    Walton, T E; Webb, P A; Kramer, W L; Smith, G C; Davis, T; Holbrook, F R; Moore, C G; Schiefer, T J; Jones, R H; Janney, G C

    1987-01-01

    An epizootic of vesicular stomatitis (VS) caused by the New Jersey serotype of VS virus affected livestock and humans in 14 western states in 1982-1983. Epidemiological observations were made on at least 10% of the cattle in 4 dairy herds that were located in the vicinity of Grand Junction, Colorado. High rates of neutralizing antibody to the New Jersey serotype were seen in all cattle regardless of whether livestock in the dairy had clinical VS or a decrease in mild production. Antibody titers remained high in these cattle for as long as 2 years after the epizootic. No virus isolations were made from 32 humans with clinical signs compatible with viral disease. Entomological information was obtained during the epizootic from 23 premises in northwestern Colorado. Insect collections yielded 4 isolates from Culicoides spp. midges, 2 from C. variipennis, and 1 each from C. stellifer and C. (Selfia) spp. This is the first report of VS virus isolations from field-collected Culicoides.

  9. Regulation of autophagy by amino acids and MTOR-dependent signal transduction.

    PubMed

    Meijer, Alfred J; Lorin, Séverine; Blommaart, Edward F; Codogno, Patrice

    2015-10-01

    Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.

  10. Phenol homeostasis is ensured in vanilla fruit by storage under solid form in a new chloroplast-derived organelle, the phenyloplast.

    PubMed

    Brillouet, Jean-Marc; Verdeil, Jean-Luc; Odoux, Eric; Lartaud, Marc; Grisoni, Michel; Conéjéro, Geneviève

    2014-06-01

    A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a 'phenyloplast'. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  11. UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons

    PubMed Central

    Edwards, Stacey L.; Morrison, Logan M.; Yorks, Rosalina M.; Hoover, Christopher M.; Boominathan, Soorajnath; Miller, Kenneth G.

    2015-01-01

    The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16’s organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(−) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(−) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(−) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16’s organelle transport regulatory function. PMID:26354976

  12. ExPPNing how acetylcholine improves gait in Parkinson's disease: An Editorial Highlight for 'Deletion of the Vesicular Acetylcholine Transporter from Pedunculopontine/laterodorsal tegmental neurons modifies gait'.

    PubMed

    Falkenburger, Björn

    2017-03-01

    Read the highlighted article 'Deletion of the Vesicular Acetylcholine Transporter from Pedunculopontine/laterodorsal tegmental neurons modifies gait' on page 787. © 2017 International Society for Neurochemistry.

  13. Retargeting Vesicular Stomatitis Virus Glycoprotein Pseudotyped Lentiviral Vectors with Enhanced Stability by In Situ Synthesized Polymer Shell

    PubMed Central

    Liang, Min; Yan, Ming; Lu, Yunfeng

    2013-01-01

    Abstract The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus “targeting nanovirus.” The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes. PMID:23327104

  14. Not All the Organelles of Living Cells Are Equal! Or Are They? Engaging Students in Deep Learning and Conceptual Change

    ERIC Educational Resources Information Center

    Cherif, Abour H.; Siuda, JoElla Eaglin; Jedlicka, Dianne M.; Bondoc, Jasper Marc; Movahedzadeh, Farahnaz

    2016-01-01

    The cell is the fundamental basis for understanding biology much like the atom is the fundamental basis for understanding physics. Understanding biology requires the understanding of the fundamental functions performed by components within each cell. These components, or organelles, responsible for both maintenance and functioning of the cell…

  15. Magnetic nanoparticles to recover cellular organelles and study the time resolved nanoparticle-cell interactome throughout uptake.

    PubMed

    Bertoli, Filippo; Davies, Gemma-Louise; Monopoli, Marco P; Moloney, Micheal; Gun'ko, Yurii K; Salvati, Anna; Dawson, Kenneth A

    2014-08-27

    Nanoparticles in contact with cells and living organisms generate quite novel interactions at the interface between the nanoparticle surface and the surrounding biological environment. However, a detailed time resolved molecular level description of the evolving interactions as nanoparticles are internalized and trafficked within the cellular environment is still missing and will certainly be required for the emerging arena of nanoparticle-cell interactions to mature. In this paper promising methodologies to map out the time resolved nanoparticle-cell interactome for nanoparticle uptake are discussed. Thus silica coated magnetite nanoparticles are presented to cells and their magnetic properties used to isolate, in a time resolved manner, the organelles containing the nanoparticles. Characterization of the recovered fractions shows that different cell compartments are isolated at different times, in agreement with imaging results on nanoparticle intracellular location. Subsequently the internalized nanoparticles can be further isolated from the recovered organelles, allowing the study of the most tightly nanoparticle-bound biomolecules, analogous to the 'hard corona' that so far has mostly been characterized in extracellular environments. Preliminary data on the recovered nanoparticles suggest that significant portion of the original corona (derived from the serum in which particles are presented to the cells) is preserved as nanoparticles are trafficked through the cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Evaluation of the Protective Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against Marburg Hemorrhagic Fever in Nonhuman Primate Models

    DTIC Science & Technology

    2007-01-19

    fever in Nonhuman Primate Models" Date d?JO )oi Date )&*7 Date Dissertation and Abstract Approved: Robert Friedm ,M.D. Department of Pathology Committee...thesis manuscript entitled: "Evaluation of the Protective Efficacy of Recombinant Vesicular Stomatitis Virus Vectors Against Marburg Hemorrhagic fever ...stomatitis virus vectors against Marburg hemorrhagic fever in nonhuman primate models By Kathleen Daddario-DiCaprio Dissertation

  17. Pathogenic mechanisms in lysosomal disease: a reappraisal of the role of the lysosome.

    PubMed

    Walkley, Steven U

    2007-04-01

    The view that lysosomes simply represent end organelles in the serial degradation of polymeric molecules derived from the cell surface and its interior has led to major misconceptions about the nature of lysosomal storage diseases and the pathogenic cascades that characterize them. Accordingly, lysosomal storage bodies are often considered 'inert', inducing cell dysfunction and death primarily through mechanical overcrowding of normal organelles or by other non-specific means leading to generalized cytotoxicity. However, modern studies of lysosomes and their component proteins provide evidence to support a far greater role for these organelles in cell metabolism. In intimate association with endosomal, autophagosomal and related vesicular systems, the greater lysosomal system can be conceptualized as a vital recycling centre that serves as a central metabolic coordinator, influencing literally every aspect of the cell, from signal transduction to regulation of gene expression. This broader view of the role of lysosomes in cells not only provides insight into how single gene defects impacting on lysosomal function can result in the plethora of complex cellular transformations characteristic of these diseases, but also suggests new and innovative therapies that may hold considerable promise for ameliorating disease progression.

  18. Inositol 1,4,5-trisphosphate-induced calcium release in the organelle layers of the stratified, intact egg of Xenopus laevis

    PubMed Central

    1990-01-01

    Using double-barreled, Ca2(+)-sensitive microelectrodes, we have examined the characteristics of the Ca2+ release by inositol 1,4,5- trisphosphate (Ins(1,4,5)P3) in the various layers of Xenopus laevis eggs in which the organelles had been stratified by centrifugation. Centrifugation of living eggs stratifies the organelles yet retains them in the normal cytoplasmic milieu. The local increase in intracellular free Ca2+ in each layer was directly measured under physiological conditions using theta-tubing, double-barreled, Ca2(+)- sensitive microelectrodes in which one barrel was filled with the Ca2+ sensor and the other was filled with Ins(1,4,5)P3 for microinjection. The two tips of these electrodes were very close to each other (3 microns apart) enabling us to measure the kinetics of both the highly localized intracellular Ca2+ release and its subsequent removal in response to Ins(1,4,5)P3 injection. Upon Ins(1,4,5)P3 injection, the ER- enriched layer exhibited the largest release of Ca2+ in a dosage- dependent manner, whereas the other layers, mitochondria, lipid, and yolk, released 10-fold less Ca2+ in a dosage-independent manner. The removal of released Ca2+ took place within approximately 1 min. The sensitivity to Ins(1,4,5)P3 and the time course of intracellular Ca2+ release in the unstratified (unactivated) egg is nearly identical to that observed in the ER layer of the stratified egg. Our data suggest that the ER is the major organelle of the Ins(1,4,5)P3-sensitive Ca2+ store in the egg of Xenopus laevis. PMID:2324195

  19. Delivery of drugs to intracellular organelles using drug delivery systems: Analysis of research trends and targeting efficiencies.

    PubMed

    Maity, Amit Ranjan; Stepensky, David

    2015-12-30

    Targeting of drug delivery systems (DDSs) to specific intracellular organelles (i.e., subcellular targeting) has been investigated in numerous publications, but targeting efficiency of these systems is seldom reported. We searched scientific publications in the subcellular DDS targeting field and analyzed targeting efficiency and major formulation parameters that affect it. We identified 77 scientific publications that matched the search criteria. In the majority of these studies nanoparticle-based DDSs were applied, while liposomes, quantum dots and conjugates were used less frequently. The nucleus was the most common intracellular target, followed by mitochondrion, endoplasmic reticulum and Golgi apparatus. In 65% of the publications, DDSs surface was decorated with specific targeting residues, but the efficiency of this surface decoration was not analyzed in predominant majority of the studies. Moreover, only 23% of the analyzed publications contained quantitative data on DDSs subcellular targeting efficiency, while the majority of publications reported qualitative results only. From the analysis of publications in the subcellular targeting field, it appears that insufficient efforts are devoted to quantitative analysis of the major formulation parameters and of the DDSs' intracellular fate. Based on these findings, we provide recommendations for future studies in the field of organelle-specific drug delivery and targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Statistical organelle dissection of Arabidopsis guard cells using image database LIPS.

    PubMed

    Higaki, Takumi; Kutsuna, Natsumaro; Hosokawa, Yoichiroh; Akita, Kae; Ebine, Kazuo; Ueda, Takashi; Kondo, Noriaki; Hasezawa, Seiichiro

    2012-01-01

    To comprehensively grasp cell biological events in plant stomatal movement, we have captured microscopic images of guard cells with various organelles markers. The 28,530 serial optical sections of 930 pairs of Arabidopsis guard cells have been released as a new image database, named Live Images of Plant Stomata (LIPS). We visualized the average organellar distributions in guard cells using probabilistic mapping and image clustering techniques. The results indicated that actin microfilaments and endoplasmic reticulum (ER) are mainly localized to the dorsal side and connection regions of guard cells. Subtractive images of open and closed stomata showed distribution changes in intracellular structures, including the ER, during stomatal movement. Time-lapse imaging showed that similar ER distribution changes occurred during stomatal opening induced by light irradiation or femtosecond laser shots on neighboring epidermal cells, indicating that our image analysis approach has identified a novel ER relocation in stomatal opening.

  1. A melanosomal two-pore sodium channel regulates pigmentation

    PubMed Central

    Bellono, Nicholas W.; Escobar, Iliana E.; Oancea, Elena

    2016-01-01

    Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation. PMID:27231233

  2. Age-dependent methamphetamine-induced alterations in vesicular monoamine transporter-2 function: implications for neurotoxicity.

    PubMed

    Truong, Jannine G; Wilkins, Diana G; Baudys, Jakub; Crouch, Dennis J; Johnson-Davis, Kamisha L; Gibb, James W; Hanson, Glen R; Fleckenstein, Annette E

    2005-09-01

    Tens of thousands of adolescents and young adults have used illicit methamphetamine. This is of concern since its high-dose administration causes persistent dopaminergic deficits in adult animal models. The effects in adolescents are less studied. In adult rodents, toxic effects of methamphetamine may result partly from aberrant cytosolic dopamine accumulation and subsequent reactive oxygen species formation. The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine into synaptic vesicles for storage and perhaps protection against dopamine-associated oxidative consequences. Accordingly, aberrant VMAT-2 function may contribute to the methamphetamine-induced persistent dopaminergic deficits. Hence, this study examined effects of methamphetamine on VMAT-2 in adolescent (postnatal day 40) and young adult (postnatal day 90) rats. Results revealed that high-dose methamphetamine treatment caused greater acute (within 1 h) decreases in vesicular dopamine uptake in postnatal day 90 versus 40 rats, as determined in a nonmembrane-associated subcellular fraction. Greater basal levels of VMAT-2 at postnatal day 90 versus 40 in this purified fraction seemed to contribute to the larger effect. Basal tissue dopamine content was also greater in postnatal day 90 versus 40 rats. In addition, postnatal day 90 rats were more susceptible to methamphetamine-induced persistent dopaminergic deficits as assessed by measuring VMAT-2 activity and dopamine content 7 days after treatment, even if drug doses were adjusted for age-related pharmacokinetic differences. Together, these data demonstrate dynamic changes in VMAT-2 susceptibility to methamphetamine as a function of development. Implications with regard to methamphetamine-induced dopaminergic deficits, as well as dopamine-associated neurodegenerative disorders such as Parkinson's disease, are discussed.

  3. Two-photon imaging of formaldehyde in live cells and animals utilizing a lysosome-targetable and acidic pH-activatable fluorescent probe.

    PubMed

    Xie, Xilei; Tang, Fuyan; Shangguan, Xiaoyan; Che, Shiyi; Niu, Jinye; Xiao, Yongsheng; Wang, Xu; Tang, Bo

    2017-06-13

    Lyso-TPFP presents lysosomal targetability and an acidic pH-activatable response toward formaldehyde. Thus, it exclusively visualizes lysosomal formaldehyde and is immune against it in neutral cytosol and other organelles. In addition, two-photon fluorescence imaging endows Lyso-TPFP with the capability of in situ tracking formaldehyde in live cells and animals.

  4. Increased sporulation of vesicular-arbuscular mycorrhizal fungi by manipulation of nutrient regimens.

    PubMed

    Douds, D D; Schenck, N C

    1990-02-01

    Adjustment of pot culture nutrient solutions increased root colonization and sporulation of vesicular-arbuscular mycorrhizal (VAM) fungi. Paspalum notatum Flugge and VAM fungi were grown in a sandy soil low in N and available P. Hoagland nutrient solution without P enhanced sporulation in soil and root colonization of Acaulospora longula, Scutellospora heterogama, Gigaspora margarita, and a wide range of other VAM fungi over levels produced by a tap water control or nutrient solutions containing P. However, Glomus intraradices produced significantly more spores in plant roots in the tap water control treatment. The effect of the nutrient solutions was not due solely to N nutrition, because the addition of NH(4)NO(3) decreased both colonization and sporulation by G. margarita relative to levels produced by Hoagland solution without P.

  5. tRNA travels from the cytoplasm to organelles

    PubMed Central

    Rubio, Mary Anne T.; Hopper, Anita K.

    2011-01-01

    Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitchondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles. PMID:21976284

  6. Quantifying the Number of Independent Organelle DNA Insertions in Genome Evolution and Human Health.

    PubMed

    Hazkani-Covo, Einat; Martin, William F

    2017-05-01

    Fragments of organelle genomes are often found as insertions in nuclear DNA. These fragments of mitochondrial DNA (numts) and plastid DNA (nupts) are ubiquitous components of eukaryotic genomes. They are, however, often edited out during the genome assembly process, leading to systematic underestimation of their frequency. Numts and nupts, once inserted, can become further fragmented through subsequent insertion of mobile elements or other recombinational events that disrupt the continuity of the inserted sequence relative to the genuine organelle DNA copy. Because numts and nupts are typically identified through sequence comparison tools such as BLAST, disruption of insertions into smaller fragments can lead to systematic overestimation of numt and nupt frequencies. Accurate identification of numts and nupts is important, however, both for better understanding of their role during evolution, and for monitoring their increasingly evident role in human disease. Human populations are polymorphic for 141 numt loci, five numts are causal to genetic disease, and cancer genomic studies are revealing an abundance of numts associated with tumor progression. Here, we report investigation of salient parameters involved in obtaining accurate estimates of numt and nupt numbers in genome sequence data. Numts and nupts from 44 sequenced eukaryotic genomes reveal lineage-specific differences in the number, relative age and frequency of insertional events as well as lineage-specific dynamics of their postinsertional fragmentation. Our findings outline the main technical parameters influencing accurate identification and frequency estimation of numts in genomic studies pertinent to both evolution and human health. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  7. Enhanced N-Transfer from a Soybean to Maize by Vesicular Arbuscular Mycorrhizal (VAM) Fungi.

    PubMed

    van Kessel, C; Singleton, P W; Hoben, H J

    1985-10-01

    Using a split-root technique, roots of soybean plants were divided between two pots. In one of the two pots, two maize plants were grown and half of those pots were inoculated with the vesicular arbuscular mycorrhizal (VAM) fungus, Glomus fasciculatus. Fifty-two days after planting, (15)N-labeled ammonium sulfate was applied to the pots which contained only soybean roots. Forty-eight hours after application, significantly higher values for atom per cent (15)N excess were found in roots and leaves of VAM-infected maize plants as compared with the non-VAM-infected maize plants. Results indicated that VAM fungi did enhance N transfer from one plant to another.

  8. Propeptides are sufficient to regulate organelle-specific pH-dependent activation of furin and proprotein convertase 1/3.

    PubMed

    Dillon, Stephanie L; Williamson, Danielle M; Elferich, Johannes; Radler, David; Joshi, Rajendra; Thomas, Gary; Shinde, Ujwal

    2012-10-12

    The proprotein convertases (PCs) furin and proprotein convertase 1/3 (PC1) cleave substrates at dibasic residues along the eukaryotic secretory/endocytic pathway. PCs are evolutionarily related to bacterial subtilisin and are synthesized as zymogens. They contain N-terminal propeptides (PRO) that function as dedicated catalysts that facilitate folding and regulate activation of cognate proteases through multiple-ordered cleavages. Previous studies identified a histidine residue (His69) that functions as a pH sensor in the propeptide of furin (PRO(FUR)), which regulates furin activation at pH~6.5 within the trans-Golgi network. Although this residue is conserved in the PC1 propeptide (PRO(PC1)), PC1 nonetheless activates at pH~5.5 within the dense core secretory granules. Here, we analyze the mechanism by which PRO(FUR) regulates furin activation and examine why PRO(FUR) and PRO(PC1) differ in their pH-dependent activation. Sequence analyses establish that while both PRO(FUR) and PRO(PC1) are enriched in histidines when compared with cognate catalytic domains and prokaryotic orthologs, histidine content in PRO(FUR) is ~2-fold greater than that in PRO(PC1), which may augment its pH sensitivity. Spectroscopy and molecular dynamics establish that histidine protonation significantly unfolds PRO(FUR) when compared to PRO(PC1) to enhance autoproteolysis. We further demonstrate that PRO(FUR) and PRO(PC1) are sufficient to confer organelle sensing on folding and activation of their cognate proteases. Swapping propeptides between furin and PC1 transfers pH-dependent protease activation in a propeptide-dictated manner in vitro and in cells. Since prokaryotes lack organelles and eukaryotic PCs evolved from propeptide-dependent, not propeptide-independent prokaryotic subtilases, our results suggest that histidine enrichment may have enabled propeptides to evolve to exploit pH gradients to activate within specific organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

    PubMed

    Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

    2010-02-16

    Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

  10. BIOCHEMICAL CHARACTERIZATION OF NATIVE USHER PROTEIN COMPLEXES FROM A VESICULAR SUBFRACTION OF TRACHEAL EPITHELIAL CELLS†

    PubMed Central

    Zallocchi, Marisa; Sisson, Joseph H.; Cosgrove, Dominic

    2010-01-01

    Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells, and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in non-detergent buffer, and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15 and VLGR-1, and a different one at the top of the gradient that included all the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100–200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins co-sediment into the gradient at a sedimentation coefficient of approximately 50S, correlating with a predicted molecular mass of 2 × 106 Daltons. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher proteins complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors. PMID:20058854

  11. Regulation of traffic and organelle architecture of the ER-Golgi interface by signal transduction.

    PubMed

    Tillmann, Kerstin D; Millarte, Valentina; Farhan, Hesso

    2013-09-01

    The components that control trafficking between organelles of the secretory pathway as well as their architecture were uncovered to a reasonable extent in the past decades. However, only recently did we begin to explore the regulation of the secretory pathway by cellular signaling. In the current review, we focus on trafficking between the endoplasmic reticulum and the Golgi apparatus. We highlight recent advances that have been made toward a better understanding of how the secretory pathway is regulated by signaling and discuss how this knowledge is important to obtain an integrative view of secretion in the context of other homeostatic processes such as growth and proliferation.

  12. Mitochondria on the move: emerging paradigms of organelle trafficking in tumour plasticity and metastasis.

    PubMed

    Altieri, Dario C

    2017-07-25

    There is now a resurgent interest in the role of mitochondria in cancer. Long considered controversial or outright unimportant, mitochondrial biology is now increasingly recognised as an important tumour driver. The underlying mechanisms remain to be fully elucidated. But recent studies have uncovered a complex landscape where reprogramming of mitochondrial homoeostasis, including organelle dynamics, metabolic output, apoptosis control and redox status converge to promote tumour adaptation to an unfavourable microenvironment and inject new traits of aggressive disease. In particular, mechanisms of subcellular mitochondrial trafficking have unexpectedly emerged as central regulators of metastatic competence in disparate tumours. Some of these pathways are druggable, opening fresh therapeutic opportunities for advanced and disseminated disease.

  13. Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

    PubMed Central

    Chakraborty, Paramita; Bjork, Per; Källberg, Eva; Olsson, Anders; Riva, Matteo; Mörgelin, Matthias; Liberg, David; Ivars, Fredrik; Leanderson, Tomas

    2015-01-01

    We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway. PMID:26661255

  14. Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria.

    PubMed

    Onfelt, Björn; Nedvetzki, Shlomo; Benninger, Richard K P; Purbhoo, Marco A; Sowinski, Stefanie; Hume, Alistair N; Seabra, Miguel C; Neil, Mark A A; French, Paul M W; Davis, Daniel M

    2006-12-15

    We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

  15. A vesicular glutamate transporter in lampreys: cDNA cloning and early expression in the nervous system.

    PubMed

    Villar-Cerviño, Verona; Rocancourt, Claire; Menuet, Arnaud; Da Silva, Corinne; Wincker, Patrick; Anadón, Ramón; Mazan, Sylvie; Rodicio, Maria Celina

    2010-09-01

    Vesicular glutamate transporters (VGLUTs) accumulate glutamate into synaptic vesicles of glutamatergic neurons, and thus are considered to define the phenotype of these neurons. Glutamate also appears to play a role in the development of the nervous system of vertebrates. Here we report the characterization of a vesicular glutamate transporter of lamprey (lVGluT), a novel member of the VGluT gene family. Phylogenetic analysis indicates that lVGLUT cannot be assigned to any of the three VGLUT isoforms characterized in teleosts and mammals, suggesting that these classes may have been fixed after the splitting between cyclostomes and gnathostomes. Expression pattern analysis during lamprey embryogenesis and prolarval stages shows that lVGluT expression is restricted to the nervous system. The first structure to express lVGluT was the olfactory epithelium of late embryos. In the brain of early prolarvae, lVGluT was expressed in most of the neuronal populations that generate the early axonal scaffold. lVGluT expression was also observed in neuronal populations of the rhombencephalon and spinal cord and in ganglia of the branchiomeric, octaval and posterior lateral line nerves. In the rhombencephalon, lVGluT expression appears to be spatially restricted in dorsal and ventral longitudinal domains. Comparison of the early expression of VGluT genes between the lamprey and some anamniotan gnathostomes (frog, zebrafish) reveals a conserved expression pattern, likely to reflect ancestral vertebrate characteristics. 2010 Elsevier B.V. All rights reserved.

  16. Effect of phospholipids and their molecular species on cholesterol solubility and nucleation in human and model biles.

    PubMed Central

    Halpern, Z; Moshkowitz, M; Laufer, H; Peled, Y; Gilat, T

    1993-01-01

    Much research in the pathophysiology of gall stones has been devoted to various molecular species of bile salts. Recent findings have shown the importance of phospholipids in biliary pathophysiology. In the present study the addition of increasing doses of egg lecithin to human and model biles progressively prolonged the nucleation time. Concurrently biliary cholesterol was shifted from the vesicular to the non-vesicular carrier(s) while the cholesterol/phospholipid ratio of the remaining vesicles was progressively lowered. Model bile solutions of identical lipid concentration were prepared using phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine as the only phospholipid. With phosphatidylethanolamine most of the cholesterol was shifted to the vesicular carrier while phosphatidylserine shifted most of the cholesterol to the non-vesicular carrier(s). With phosphatidylcholine the cholesterol was distributed in both carriers. Phosphatidyl choline species composed of various acyl fatty acids in the sn-1 and sn-2 positions were used as the sole phospholipid in otherwise identical model bile solutions. With palmitic acid in the sn-1 position and arachidonic acid in the sn-2 position most of the cholesterol was found in the non-vesicular carrier. When stearic acid was used in sn-2 position instead of arachidonic acid most of the cholesterol was found in the vesicular carrier. These and other variations in phospholipid molecular species shifted cholesterol among its carriers and also modified the nucleation time of model biles. Most of these effects were also found upon addition of the various phospholipid species to human biles. These findings show the importance of phospholipid species in biliary pathophysiology and may be useful when trying to manipulate cholesterol carriers and solubility in bile. PMID:8432440

  17. Primary cilium suppression by SREBP1c involves distortion of vesicular trafficking by PLA2G3

    PubMed Central

    Gijs, Hannah Laura; Willemarck, Nicolas; Vanderhoydonc, Frank; Khan, Niamat Ali; Dehairs, Jonas; Derua, Rita; Waelkens, Etienne; Taketomi, Yoshitaka; Murakami, Makoto; Agostinis, Patrizia; Annaert, Wim; Swinnen, Johannes V.

    2015-01-01

    Distortion of primary cilium formation is increasingly recognized as a key event in many human pathologies. One of the underlying mechanisms involves aberrant activation of the lipogenic transcription factor sterol regulatory element–binding protein 1c (SREBP1c), as observed in cancer cells. To gain more insight into the molecular pathways by which SREBP1c suppresses primary ciliogenesis, we searched for overlap between known ciliogenesis regulators and targets of SREBP1. One of the candidate genes that was consistently up-regulated in cellular models of SREBP1c-induced cilium repression was phospholipase A2 group III (PLA2G3), a phospholipase that hydrolyzes the sn-2 position of glycerophospholipids. Use of RNA interference and a chemical inhibitor of PLA2G3 rescued SREBP1c-induced cilium repression. Cilium repression by SREBP1c and PLA2G3 involved alterations in endosomal recycling and vesicular transport toward the cilium, as revealed by aberrant transferrin and Rab11 localization, and was largely mediated by an increase in lysophosphatidylcholine and lysophosphatidylethanolamine levels. Together these findings indicate that aberrant activation of SREBP1c suppresses primary ciliogenesis by PLA2G3-mediated distortion of vesicular trafficking and suggest that PLA2G3 is a novel potential target to normalize ciliogenesis in SREBP1c-overexpressing cells, including cancer cells. PMID:25904332

  18. Measurement of Instantaneous Velocity Vectors of Organelle Transport: Mitochondrial Transport and Bioenergetics in Hippocampal Neurons

    PubMed Central

    Gerencser, Akos A.; Nicholls, David G.

    2008-01-01

    Impaired transport of mitochondria, in dendrites and axons of neurons, and bioenergetic deficit are increasingly recognized to be of pathological importance in neurodegenerative diseases. To study the relationship between transport and bioenergetics, we have developed what to our knowledge is a novel technique to quantify organelle velocity in cultured cells. The aim was to combine measurement of motion and bioenergetic parameters while minimizing photodynamic oxidative artifacts evoked by fluorescence excitation. Velocity determination from sequential fluorescence images is not trivial, and here we describe an application of “optical flow”, the flow of gray values in grayscale images, to this problem. Based on the principles of photon shot noise occurring in low light level fluorescence microscopy, we describe and validate here an optical flow-based, robust method to measure velocity vectors for organelles expressing fluorescent proteins. This method features instantaneous velocity determination from a pair of images by detecting motion of edges, with no assumptions about the separation or shapes of the objects in the image. Optical flow was used in combination with single mitochondrion assay of mitochondrial thiol redox status by mitochondrially targeted redox-sensitive green fluorescent protein and measurement of mitochondrial membrane potential by tetramethylrhodamine methyl ester. Mitochondrial populations of resting cultured hippocampal neurons were analyzed. It was found that mitochondria with more oxidized thiol redox status have lower membrane potentials and are smaller in size. These mitochondria are more motile than the average; however, mitochondrial motility is only slightly dependent on the observed bioenergetic parameters and is correlated the best to the size of the mitochondria. PMID:18757564

  19. The Cytoskeleton-Autophagy Connection

    PubMed Central

    Kast, David J.; Dominguez, Roberto

    2017-01-01

    Summary Actin cytoskeleton dynamics plays vital roles in most forms of intracellular trafficking by promoting the biogenesis and transport of vesicular cargoes. Mounting evidence indicates that actin dynamics and membrane-cytoskeleton scaffolds also play essential roles in macroautophagy, the process by which cellular waste is isolated inside specialized vesicles called autophagosomes for recycling and degradation. Thus, branched-actin polymerization is necessary for the biogenesis of autophagosomes from the endoplasmic reticulum (ER) membrane. Actomyosin-based transport is then used to feed the growing phagophore with pre-selected cargoes and debris derived from different membranous organelles inside the cell. Mature autophagosomes then detach from the ER membrane by an unknown mechanism, and are transported and fused with lysosomes, endosomes and multi-vesicular bodies through mechanisms that involve actin- and microtubule-based motility, cytoskeleton-membrane scaffolds and signaling proteins. In this minireview, we highlight the considerable progress made recently towards understanding the diverse roles of the cytoskeleton in autophagy. PMID:28441569

  20. Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling

    PubMed Central

    Karpinska, Barbara; Alomrani, Sarah Owdah

    2017-01-01

    Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction–oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1, WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’. PMID:28808105

  1. The Vesicular Arbuscular Mycorrhiza Associated with Three Cultivars of Rice (Oryza sativa L.).

    PubMed

    Bhattacharjee, Sujata; Sharma, G D

    2011-07-01

    The present study deals with the occurrence of vesicular arbuscular mycorrhizal fungi in three cultivars of rice in Barak valley. Three cultivars of rice were Pankaj, Malati and Ranjit. The results revealed the association of VAM fungi in all the cultivars of rice. The association was maximum in Pankaj cultivar followed by Malati, and Ranjit, respectively, in all the three sampling phases. All the three cultivars of rice crop showed maximum soil spore population and number of VAM fungal species at the harvesting phase (135 DAS) and minimum at the phase of maturation (90 DAS). Glomus species were found dominating followed by Acaulospora species. Glomus microcarpum, Glomus claroideum, Glomus mosseae and Acaulospora scrobiculata were found in all the three fields.

  2. A polymethoxyflavone mixture extracted from orange peels, mainly containing nobiletin, 3,3',4',5,6,7,8-heptamethoxyflavone and tangeretin, suppresses melanogenesis through the acidification of cell organelles, including melanosomes.

    PubMed

    Yoshizaki, Norihiro; Hashizume, Ron; Masaki, Hitoshi

    2017-10-01

    Skin color is determined by melanin contents and its distribution. Melanin is synthesized in melanosomes of melanocytes, catalyzed by tyrosinase, melanogenic enzymes. Regarding the process of melanin synthesis, melanosomal pH is considered to play an important role, because it has been reported to differ between Caucasian and Black melanocytes. Although polymethoxyflavone (PMF) has many beneficial effects, it has not been reported which PMF suppresses melanogenesis. In this study, we identified the mechanism underlying the effect of PMF on melanogenesis METHODS: We determined the effects of a PMF mixture extracted from orange peels on melanogenesis, on tyrosinase expression, on the localization of tyrosinase and on the acidification of organelles, including melanosomes, in HM3KO human melanoma cells. RESULTS TREATMENT: with the PMF mixture elicited the suppression of melanogenesis, the degradation of tyrosinase in lysosomes and the mislocalization of tyrosinase associated with the acidification of intracellular organelles, including melanosomes. The neutralization of cell organelle pH by ammonium chloride restored melanogenesis and the correct localization of tyrosinase to melanosomes, which had been suppressed by the PMF mixture. These results suggest that the PMF mixture suppresses the localization of tyrosinase to melanosomes and consequently inhibits melanogenesis due to the acidification of cell organelles, including melanosomes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  3. Three-Dimensional Geometric Modeling of Membrane-bound Organelles in Ventricular Myocytes: Bridging the Gap between Microscopic Imaging and Mathematical Simulation

    PubMed Central

    Yu, Zeyun; Holst, Michael J.; Hayashi, Takeharu; Bajaj, Chandrajit L.; Ellisman, Mark H.; McCammon, J. Andrew; Hoshijima, Masahiko

    2009-01-01

    A general framework of image-based geometric processing is presented to bridge the gap between three-dimensional (3D) imaging that provides structural details of a biological system and mathematical simulation where high-quality surface or volumetric meshes are required. A 3D density map is processed in the order of image pre-processing (contrast enhancement and anisotropic filtering), feature extraction (boundary segmentation and skeletonization), and high-quality and realistic surface (triangular) and volumetric (tetrahedral) mesh generation. While the tool-chain described is applicable to general types of 3D imaging data, the performance is demonstrated specifically on membrane-bound organelles in ventricular myocytes that are imaged and reconstructed with electron microscopic (EM) tomography and two-photon microscopy (T-PM). Of particular interest in this study are two types of membrane-bound Ca2+-handling organelles, namely, transverse tubules (T-tubules) and junctional sarcoplasmic reticulum (jSR), both of which play an important role in regulating the excitation-contraction (E-C) coupling through dynamic Ca2+ mobilization in cardiomyocytes. PMID:18835449

  4. Three-dimensional geometric modeling of membrane-bound organelles in ventricular myocytes: bridging the gap between microscopic imaging and mathematical simulation.

    PubMed

    Yu, Zeyun; Holst, Michael J; Hayashi, Takeharu; Bajaj, Chandrajit L; Ellisman, Mark H; McCammon, J Andrew; Hoshijima, Masahiko

    2008-12-01

    A general framework of image-based geometric processing is presented to bridge the gap between three-dimensional (3D) imaging that provides structural details of a biological system and mathematical simulation where high-quality surface or volumetric meshes are required. A 3D density map is processed in the order of image pre-processing (contrast enhancement and anisotropic filtering), feature extraction (boundary segmentation and skeletonization), and high-quality and realistic surface (triangular) and volumetric (tetrahedral) mesh generation. While the tool-chain described is applicable to general types of 3D imaging data, the performance is demonstrated specifically on membrane-bound organelles in ventricular myocytes that are imaged and reconstructed with electron microscopic (EM) tomography and two-photon microscopy (T-PM). Of particular interest in this study are two types of membrane-bound Ca(2+)-handling organelles, namely, transverse tubules (T-tubules) and junctional sarcoplasmic reticulum (jSR), both of which play an important role in regulating the excitation-contraction (E-C) coupling through dynamic Ca(2+) mobilization in cardiomyocytes.

  5. An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

    PubMed Central

    Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

    2017-01-01

    The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

  6. Use of Oral Fluids for Detection of Virus and Antibodies in Pigs Infected with Swine Vesicular Disease Virus.

    PubMed

    Senthilkumaran, C; Bittner, H; Ambagala, A; Lung, O; Babiuk, S; Yang, M; Zimmerman, J; Giménez-Lirola, L G; Nfon, C

    2017-12-01

    The use of swine oral fluid (OF) for the detection of nucleic acids and antibodies is gaining significant popularity. Assays have been developed for this purpose for endemic and foreign animal diseases of swine. Here, we report the use of OF for the detection of virus and antibodies in pigs experimentally infected with swine vesicular disease virus (SVDV), a virus that causes a disease clinically indistinguishable from the economically devastating foot-and-mouth disease. Viral genome was detected in OF by real-time reverse transcription polymerase chain reaction (RRT-PCR) from 1 day post-infection (DPI) to 21 DPI. Virus isolation from OF was also successful at 1-5 DPI. An adapted competitive ELISA based on the monoclonal antibodies 5B7 detected antibodies to SVDV in OF starting at DPI 6. Additionally, using isotype-specific indirect ELISAs, SVDV-specific IgM and IgA were evaluated in OF. IgM response started at DPI 6, peaking at DPI 7 or 14 and declining sharply at DPI 21, while IgA response started at DPI 7, peaked at DPI 14 and remained high until the end of the experiment. These results confirm the potential use of OF for SVD surveillance using both established and partially validated assays in this study. © 2016 Her Majesty the Queen in Right of Canada.

  7. Intracellular Mannose Binding Lectin Mediates Subcellular Trafficking of HIV-1 gp120 in Neurons

    PubMed Central

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, CL; Kaul, M; Singh, KK

    2014-01-01

    Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. PMID:24825317

  8. Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

    PubMed Central

    Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona

    2017-01-01

    The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF4− and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles. PMID:28462015

  9. Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum.

    PubMed

    Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona; Patankar, Swati

    2017-01-01

    The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPx Gl ) and inhibitors of vesicular transport. As expected, the G protein-dependent vesicular fusion inhibitor AlF 4 - and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPx Gl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G protein-dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPx Gl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPx Gl ; second, trafficking of apicoplast luminal proteins appear to be independent of G protein-coupled vesicles.

  10. Recombinant vesicular stomatitis virus-based vaccines against Ebola and Marburg virus infections.

    PubMed

    Geisbert, Thomas W; Feldmann, Heinz

    2011-11-01

    The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections.

  11. Vesicular erythema migrans: an atypical and easily misdiagnosed form of Lyme disease.

    PubMed

    Mazori, Daniel R; Orme, Charisse M; Mir, Adnan; Meehan, Shane A; Neimann, Andrea L

    2015-08-15

    Erythema migrans is the initial sign in the majority of patients infected with Borrelia, the genus of spirochetes that causes Lyme disease. Early identification and treatment decrease the risk of progression to later stages of disease. Although a "bull's eye" appearance owing to lesional clearing is considered classic for erythema migrans, this feature is surprisingly often lacking among patients in the United States. Furthermore, cutaneous Lyme disease can exhibit a wide range of morphologic variability in a minority of patients. Herein, we describe the case of a patient with Lyme disease in which the presence of atypical vesicular features, in conjunction with the initial absence of clearing, resulted in multiple misdiagnoses and delayed treatment. We also review the literature on the epidemiology and management of erythema migrans for cases in which the diagnosis may pose a challenge.

  12. Recombinant Vesicular Stomatitis Virus–Based Vaccines Against Ebola and Marburg Virus Infections

    PubMed Central

    Feldmann, Heinz

    2011-01-01

    The filoviruses, Marburg virus and Ebola virus, cause severe hemorrhagic fever with a high mortality rate in humans and nonhuman primates. Among the most-promising filovirus vaccines under development is a system based on recombinant vesicular stomatitis virus (rVSV) that expresses a single filovirus glycoprotein (GP) in place of the VSV glycoprotein (G). Importantly, a single injection of blended rVSV-based filovirus vaccines was shown to completely protect nonhuman primates against Marburg virus and 3 different species of Ebola virus. These rVSV-based vaccines have also shown utility when administered as a postexposure treatment against filovirus infections, and a rVSV-based Ebola virus vaccine was recently used to treat a potential laboratory exposure. Here, we review the history of rVSV-based vaccines and pivotal animal studies showing their utility in combating Ebola and Marburg virus infections. PMID:21987744

  13. Swine vesicular disease in northern Italy: diffusion through densely populated pig areas.

    PubMed

    Bellini, S; Alborali, L; Zanardi, G; Bonazza, V; Brocchi, E

    2010-12-01

    At the end of 2006, a recrudescence of swine vesicular disease (SVD) was recorded in Italy and the disease spread widely throughout the northern regions. Lombardy, a densely populated pig area, was most affected and the presence of the disease caused heavy economic losses to the entire pig industry. Although SVD is considered only moderately contagious, the epidemic in the north was characterised by a rapid spread of the condition. Numerous difficulties were encountered in eradicating it. Over the past decade, there has been a significant increase in the population of pigs in Lombardy, concentrated mainly in a few areas which were the most severely affected during the 2006 to 2007 SVD epidemic. Increases in both the pig population and animal movements, combined with weak biosecurity measures, increased the spread rate of the disease and hampered eradication activities.

  14. Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

    PubMed

    Matsui, T; Nakayama, H; Yoshida, K; Shinmyo, A

    2003-10-01

    Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

  15. Differential localization of vesicular glutamate transporters and peptides in corneal afferents to trigeminal nucleus caudalis.

    PubMed

    Hegarty, Deborah M; Tonsfeldt, Karen; Hermes, Sam M; Helfand, Helen; Aicher, Sue A

    2010-09-01

    Trigeminal afferents convey nociceptive information from the corneal surface of the eye to the trigeminal subnucleus caudalis (Vc). Trigeminal afferents, like other nociceptors, are thought to use glutamate and neuropeptides as neurotransmitters. The current studies examined whether corneal afferents contain both neuropeptides and vesicular glutamate transporters. Corneal afferents to the Vc were identified by using cholera toxin B (CTb). Corneal afferents project in two clusters to the rostral and caudal borders of the Vc, regions that contain functionally distinct nociceptive neurons. Thus, corneal afferents projecting to these two regions were examined separately. Dual immunocytochemical studies combined CTb with either calcitonin gene-related peptide (CGRP), substance P (SP), vesicular glutamate transporter 1 (VGluT1), or VGluT2. Corneal afferents were more likely to contain CGRP than SP, and corneal afferents projecting to the rostral region were more likely to contain CGRP than afferents projecting caudally. Overall, corneal afferents were equally likely to contain VGluT1 or VGluT2. Together, 61% of corneal afferents contained either VGluT1 or VGluT2, suggesting that some afferents lack a VGluT. Caudal corneal afferents were more likely to contain VGluT2 than VGluT1, whereas rostral corneal afferents were more likely to contain VGluT1 than VGluT2. Triple-labeling studies combining CTb, CGRP, and VGluT2 showed that very few corneal afferents contain both CGRP and VGluT2, caudally (1%) and rostrally (2%). These results suggest that most corneal afferents contain a peptide or a VGluT, but rarely both. Our results are consistent with a growing literature suggesting that glutamatergic and peptidergic sensory afferents may be distinct populations.

  16. Biliary secretion of fluid-phase markers by the isolated perfused rat liver. Role of transcellular vesicular transport.

    PubMed Central

    Lake, J R; Licko, V; Van Dyke, R W; Scharschmidt, B F

    1985-01-01

    In these studies, we have used several approaches to systematically explore the contribution of transcellular vesicular transport (transcytosis) to the blood-to-bile movement of inert fluid-phase markers of widely varying molecular weight. First, under steady-state conditions, the perfused rat liver secreted even large markers in appreciable amounts. The bile-to-plasma (B/P) ratio of these different markers, including microperoxidase (B/P ratio = 0.06; mol wt = 1,879), insulin (B/P ratio = 0.09, mol wt = 5,000), horseradish peroxidase (B/P ratio = 0.04, mol wt = 40,000), and dextran (B/P ratio = 0.09, mol wt = 70,000), exhibited no clear ordering based on size alone, and when dextrans of two different sizes (40,000 and 70,000 mol wt) were studied simultaneously, the relative amounts of the two dextran species in bile were the same as in perfusate. Taurocholate administration produced a 71% increase in bile flow but little or no (0-20%) increase in the output of horseradish peroxidase, microperoxidase, inulin, and dextran. Second, under nonsteady-state conditions in which the appearance in or disappearance from bile of selected markers was studied after their abrupt addition to or removal from perfusate, erythritol reached a B/P ratio of 1 within 2 min. Microperoxidase and dextran appeared in bile only after a lag period of approximately 12 min and then slowly approached maximal values, whereas sucrose exhibited kinetically intermediate behavior. A similar pattern was observed after removal of greater than 95% of the marker from the perfusate. Erythritol rapidly reapproached a B/P ratio of 1, whereas the B/P ratio for sucrose, dextran, and microperoxidase fell much more slowly and exceeded 1 for a full 30 min after perfusate washout. Finally, electron microscopy and fluorescence microscopy of cultured hepatocytes demonstrated the presence of horseradish peroxidase and fluorescein-dextran, respectively, in intracellular vesicles, and fractionation of perfused liver

  17. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions.

    PubMed

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  18. The Autophagoproteasome a Novel Cell Clearing Organelle in Baseline and Stimulated Conditions

    PubMed Central

    Lenzi, Paola; Lazzeri, Gloria; Biagioni, Francesca; Busceti, Carla L.; Gambardella, Stefano; Salvetti, Alessandra; Fornai, Francesco

    2016-01-01

    Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be

  19. [Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation].

    PubMed

    Samartsev, V N; Rybakova, S R; Dubinin, M V

    2013-01-01

    The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.

  20. Expression and immunological analysis of capsid protein precursor of swine vesicular disease virus HK/70.

    PubMed

    Tian, Hong; Wu, Jing-yan; Shang, You-jun; Ying, Shuang-hui; Zheng, Hai-xue; Liu, Xiang-tao

    2010-06-01

    VP1, a capsid protein of swine vesicular disease virus, was cloned from the SVDV HK/70 strain and inserted into retroviral vector pBABE puro, and expressed in PK15 cells by an retroviral expression system. The ability of the VP1 protein to induce an immune response was then evaluated in guinea pigs. Western blot and ELISA results indicated that the VP1 protein can be recognized by SVDV positive serum, Furthermore, anti-SVDV specific antibodies and lymphocyte proliferation were elicited and increased by VP1 protein after vaccination. These results encourage further work towards the development of a vaccine against SVDV infection.

  1. [Methods of substances and organelles introduction in living cell for cell engineering technologies].

    PubMed

    Nikitin, V A

    2007-01-01

    We have presented the classification of more than 40 methods of genetic material, substances and organelles introduction into a living cell. Each of them has its characteristic advantages, disadvantages and limitations with respect to cell viability, transfer efficiency, general applicability, and technical requirements. It this article we have enlarged on the description of our developments of several new and improved approaches, methods and devices of the direct microinjection into a single cell and cell microsurgery with the help of glass micropipettes. The problem of low efficiency of mammalian cloning is discussed with emphasis on the necessity of expertizing of each step of single cell reconstruction to begin with microsurgical manipulations and necessity of the development of such methods of single cell resonstruction that could minimize the possible damage of the cell.

  2. Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

    PubMed

    Tücking, Katrin-Stephanie; Grützner, Verena; Unger, Ronald E; Schönherr, Holger

    2015-07-01

    The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Dynamics of vesicular-arbuscular mycorrhizae during old field succession.

    PubMed

    Johnson, Nancy Collins; Zak, Donald R; Tilman, David; Pfleger, F L

    1991-05-01

    The species composition of vesicular-arbuscular mycorrhizal (VAM) fungal communities changed during secondary succession of abandoned fields based on a field to forest chronosequence. Twenty-five VAM fungal species were identified. Seven species were clearly early successional and five species were clearly late successional. The total number of VAM fungal species did not increase with successional time, but diversity as measured by the Shannon-Wiener index tended to increase, primarily because the community became more even as a single species, Glomus aggregatum, became less dominant in the older sites. Diversity of the VAM fungal community was positively correlated with soil C and N. The density of VAM fungi, as measured by infectivity and total spore count, first increased with time since abandonment and then decreased in the late successional forest sites. Within 12 abandoned fields, VAM fungal density increased with increasing soil pH, H 2 O soluble soil C, and root biomass, but was inversely related to extractable soil P and percent cover of non-host plant species. The lower abundance of VAM fungi in the forest sites compared with the field sites agrees with the findings of other workers and corresponds with a shift in the dominant vegetation from herbaceous VAM hosts to woody ectomycorrhizal hosts.

  4. Cleavage of the vesicular glutamate transporters under excitotoxic conditions.

    PubMed

    Lobo, Andrea C; Gomes, João R; Catarino, Tatiana; Mele, Miranda; Fernandez, Pedro; Inácio, Ana R; Bahr, Ben A; Santos, Armanda E; Wieloch, Tadeusz; Carvalho, Ana Luísa; Duarte, Carlos B

    2011-12-01

    Glutamate is loaded into synaptic vesicles by vesicular glutamate transporters (VGLUTs), and alterations in the transporters expression directly regulate neurotransmitter release. We investigated changes in VGLUT1 and VGLUT2 protein levels after ischemic and excitotoxic insults. The results show that VGLUT2 is cleaved by calpains after excitotoxic stimulation of hippocampal neurons with glutamate, whereas VGLUT1 is downregulated to a lower extent. VGLUT2 was also cleaved by calpains after oxygen/glucose deprivation (OGD), and downregulated after middle cerebral artery occlusion (MCAO) and intrahippocampal injection of kainate. In contrast, VGLUT1 was not affected after OGD. Incubation of isolated synaptic vesicles with recombinant calpain also induced VGLUT2 cleavage, with a little effect observed for VGLUT1. N-terminal sequencing analysis showed that calpain cleaves VGLUT2 in the C-terminus, at Asn(534) and Lys(542). The truncated GFP-VGLUT2 forms were found to a great extent in non-synaptic regions along neurites, when compared to GFP-VGLUT2. These findings show that excitotoxic and ischemic insults downregulate VGLUT2, which is likely to affect glutamatergic transmission and cell death, especially in the neonatal period when the transporter is expressed at higher levels. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Functional genetic variants in the vesicular monoamine transporter 1 modulate emotion processing.

    PubMed

    Lohoff, F W; Hodge, R; Narasimhan, S; Nall, A; Ferraro, T N; Mickey, B J; Heitzeg, M M; Langenecker, S A; Zubieta, J-K; Bogdan, R; Nikolova, Y S; Drabant, E; Hariri, A R; Bevilacqua, L; Goldman, D; Doyle, G A

    2014-01-01

    Emotional behavior is in part heritable and often disrupted in psychopathology. Identification of specific genetic variants that drive this heritability may provide important new insight into molecular and neurobiological mechanisms involved in emotionality. Our results demonstrate that the presynaptic vesicular monoamine transporter 1 (VMAT1) Thr136Ile (rs1390938) polymorphism is functional in vitro, with the Ile allele leading to increased monoamine transport into presynaptic vesicles. Moreover, we show that the Thr136Ile variant predicts differential responses in emotional brain circuits consistent with its effects in vitro. Lastly, deep sequencing of bipolar disorder (BPD) patients and controls identified several rare novel VMAT1 variants. The variant Phe84Ser was only present in individuals with BPD and leads to marked increase monoamine transport in vitro. Taken together, our data show that VMAT1 polymorphisms influence monoamine signaling, the functional response of emotional brain circuits and risk for psychopathology.

  6. Organelles Contribute Differentially to Reactive Oxygen Species-Related Events during Extended Darkness1[C][W][OA

    PubMed Central

    Rosenwasser, Shilo; Rot, Ilona; Sollner, Evelyn; Meyer, Andreas J.; Smith, Yoav; Leviatan, Noam; Fluhr, Robert; Friedman, Haya

    2011-01-01

    Treatment of Arabidopsis (Arabidopsis thaliana) leaves by extended darkness generates a genetically activated senescence program that culminates in cell death. The transcriptome of leaves subjected to extended darkness was found to contain a variety of reactive oxygen species (ROS)-specific signatures. The levels of transcripts constituting the transcriptome footprints of chloroplasts and cytoplasm ROS stresses decreased in leaves, as early as the second day of darkness. In contrast, an increase was detected in transcripts associated with mitochondrial and peroxisomal ROS stresses. The sequential changes in the redox state of the organelles during darkness were examined by redox-sensitive green fluorescent protein probes (roGFP) that were targeted to specific organelles. In plastids, roGFP showed a decreased level of oxidation as early as the first day of darkness, followed by a gradual increase to starting levels. However, in mitochondria, the level of oxidation of roGFP rapidly increased as early as the first day of darkness, followed by an increase in the peroxisomal level of oxidation of roGFP on the second day. No changes in the probe oxidation were observed in the cytoplasm until the third day. The increase in mitochondrial roGFP degree of oxidation was abolished by sucrose treatment, implying that oxidation is caused by energy deprivation. The dynamic redox state visualized by roGFP probes and the analysis of microarray results are consistent with a scenario in which ROS stresses emanating from the mitochondria and peroxisomes occur early during darkness at a presymptomatic stage and jointly contribute to the senescence program. PMID:21372201

  7. Characterization of a Planctomycetal Organelle: a Novel Bacterial Microcompartment for the Aerobic Degradation of Plant Saccharides

    PubMed Central

    Erbilgin, Onur; McDonald, Kent L.

    2014-01-01

    Bacterial microcompartments (BMCs) are organelles that encapsulate functionally linked enzymes within a proteinaceous shell. The prototypical example is the carboxysome, which functions in carbon fixation in cyanobacteria and some chemoautotrophs. It is increasingly apparent that diverse heterotrophic bacteria contain BMCs that are involved in catabolic reactions, and many of the BMCs are predicted to have novel functions. However, most of these putative organelles have not been experimentally characterized. In this study, we sought to discover the function of a conserved BMC gene cluster encoded in the majority of the sequenced planctomycete genomes. This BMC is especially notable for its relatively simple genetic composition, its remote phylogenetic position relative to characterized BMCs, and its apparent exclusivity to the enigmatic Verrucomicrobia and Planctomycetes. Members of the phylum Planctomycetes are known for their morphological dissimilarity to the rest of the bacterial domain: internal membranes, reproduction by budding, and lack of peptidoglycan. As a result, they are ripe for many discoveries, but currently the tools for genetic studies are very limited. We expanded the genetic toolbox for the planctomycetes and generated directed gene knockouts of BMC-related genes in Planctomyces limnophilus. A metabolic activity screen revealed that BMC gene products are involved in the degradation of a number of plant and algal cell wall sugars. Among these sugars, we confirmed that BMCs are formed and required for growth on l-fucose and l-rhamnose. Our results shed light on the functional diversity of BMCs as well as their ecological role in the planctomycetes, which are commonly associated with algae. PMID:24487526

  8. The Role of Teak Leaves (Tectona grandis), Rhizobium, and Vesicular-Arbuscular Mycorrhizae on Improving Soil Structure and Soil Nutrition

    NASA Astrophysics Data System (ADS)

    Yuliani; Rahayu, Y. S.

    2018-01-01

    Calcium is the largest mineral in calcareous soils. High levels of calcium carbonate lead to phosphate deposition. Nutrient deficiencies in calcareous soil (mainly Phosphate and Nitrogen) resulted only certain crops with a wide range of tolerances that can grow. Meanwhile, dynamics nutrient in calcareous soils also depend on the topography and decomposition of the litter in the growing vegetation. The purpose of this study was to describe the pattern of nutrient enhancement and soil-texture structures on calcareous soils after littering the teak leaves, Rhizobium and Vesicular Arbuscular Mycorrhiza. The research parameters were the concentration of N, P, K; C/N ratio, humid acid content, and soil structure, which measured at days 30, 60, and 85 of soil decomposition process. The results showed that at days 30, the texture and structure of the soil tend to be stable (porosity 31.2, DMR 1.93, moisture content 0.36, sandy clay) while at days 85 has been very stable (porosity 49.8; Water content 0.28, sandy clay). While C and N organic, N and K concentration at days 30 showed low value (C organic 1.03, N 0.12, K 0.49, C / N ratio 9). This condition is almost unchanged at days 85. While the P value shows very high value (60.53) at days 30 although after 60 days the P content showed a decrease.

  9. Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation

    PubMed Central

    Kantor, Gal; Cheishvili, David; Even, Aviel; Birger, Anastasya; Turetsky, Tikva; Gil, Yaniv; Even-Ram, Sharona; Aizenman, Einat; Bashir, Nibal; Maayan, Channa; Razin, Aharon; Reubinoff, Benjamim E.; Weil, Miguel

    2015-01-01

    A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD. PMID:26437462

  10. Pharmacological factors in the saliva of blood-feeding insects. Implications for vesicular stomatitis epidemiology.

    PubMed

    Tabachnick, W J

    2000-01-01

    Vesicular stomatitis (VS) epizootics in the Western United States have caused substantial economic losses to U.S. livestock industries in 1995, 1997, and 1998. The role of arthropods in transmitting VS to U.S. livestock is unclear. In particular, the impact of arthropod salivary gland factors in VS infections in livestock needs study. Pharmacological effects of arthropod salivary gland factors on animals are reviewed. The potential effects of arthropod saliva on the transmission and spread of VS virus to livestock in the Western U.S. is presented with emphasis on the biting midge, Culicoides sonorensis. Information is discussed with attention to vector potential of C. sonorensis, and its use as a model for evaluating insect salivary gland pharmacology on livestock response to VS.

  11. Organelle Redox of CF and CFTR-Corrected Airway Epithelia

    PubMed Central

    Schwarzer, Christian; Illek, Beate; Suh, Jung H.; Remington, S. James; Fischer, Horst; Machen, Terry E.

    2014-01-01

    In cystic fibrosis reduced CFTR function may alter redox properties of airway epithelial cells. Redox-sensitive GFP (roGFP1) and imaging microscopy were used to measure redox potentials of cytosol, ER, mitochondria and cell surface of cystic fibrosis nasal epithelial cells and CFTR-corrected cells. We also measured glutathione and cysteine thiol redox states in cell lysates and apical fluids to provide coverage over a range of redox potentials and environments that might be affected by CFTR. As measured with roGFP1, redox potentials at the cell surface (~ -207 ±8 mV) and in the ER (~ -217 ±1 mV) and rates of regulation of the apical fluid and ER lumen following DTT treatment were similar for CF and CFTR-corrected cells. CF and CFTR-corrected cells had similar redox potentials in mitochondria (-344 ±9 mV) and cytosol (-322 ±7 mV). Oxidation of carboxy-dichlorodihydrofluoresceindiacetate and of apical Amplex Red occurred at equal rates in CF and CFTR-corrected cells. Glutathione and cysteine redox couples in cell lysates and apical fluid were equal in CF and CFTR-corrected cells. These quantitative estimates of organelle redox potentials combined with apical and cell measurements using small molecule couples confirmed there were no differences in redox properties of CF and CFTR-corrected cells. PMID:17603939

  12. Isolation and characterization of multivesicular bodies from rat hepatocytes: an organelle distinct from secretory vesicles of the Golgi apparatus

    PubMed Central

    1985-01-01

    Hepatocytes of estradiol-treated rats, which express many low density lipoprotein receptors, rapidly accumulate intravenously injected low density lipoprotein in multivesicular bodies (MVBs). We have isolated MVBs and Golgi apparatus fractions from livers of estradiol-treated rats. MVB fractions were composed mainly of large vesicles, approximately 0.55 micron diam, filled with remnantlike very low density lipoproteins, known to be taken up into hepatocytes by receptor- mediated endocytosis. MVBs also contained numerous small vesicles, 0.05- 0.07 micron in diameter, and had two types of appendages: one fingerlike and electron dense and the other saclike and electron lucent. MVBs contained little galactosyltransferase or arylsulfatase activity, and content lipoproteins were largely intact. Very low density lipoproteins from Golgi fractions, which are derived to a large extent from secretory vesicles, were larger than those of MVB fractions and contained newly synthesized triglycerides. Membranes of MVBs contained much more cholesterol and less protein than did Golgi membranes. We conclude that two distinct lipoprotein-filled organelles are located in the bile canalicular pole of hepatocytes. MVBs, a major prelysosomal organelle of low density in the endocytic pathway, contain remnants of triglyceride-rich lipoproteins, whereas secretory vesicles of the Golgi apparatus contain nascent very low density lipoproteins. PMID:3988801

  13. Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation.

    PubMed

    Ide, Takashi; Iwase, Haruka; Amano, Saaya; Sunahara, Saki; Tachihara, Ayuka; Yagi, Minako; Watanabe, Tsuyoshi

    2017-03-01

    Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Ultrastructure of the dikaryotic form of Cyathus bulleri Brodie.

    PubMed

    Kozar, F; MacDonald, B R; Weijer, J

    1977-01-01

    The fine structure of the dikaryotic form of Cyathus bulleri Brodie was generally found to be similar to that of other hyphal forms of the Basidiomycetes. The nuclear walls were doubled, porous and in some cases connected to the endoplasmic reticulum. The presence of typical as well as filamentous and U-shaped mitochondria was confirmed. Other cellular structures and organelles, among them vacuoles, vesicular and myelinoid-like bodies, often associated with the cell membranes, glycogen and ribosomes were also observed in the cytoplasm. The presence of the dolipore/parenthesome apparatus and clamp connections typical of the Basidiomycetes was established.

  15. Structures and mechanisms of vesicle coat components and multisubunit tethering complexes

    PubMed Central

    Jackson, Lauren P; Kümmel, Daniel; Reinisch, Karin M; Owen, David J

    2012-01-01

    Eukaryotic cells face a logistical challenge in ensuring prompt and precise delivery of vesicular cargo to specific organelles within the cell. Coat protein complexes select cargo and initiate vesicle formation, while multisubunit tethering complexes participate in the delivery of vesicles to target membranes. Understanding these macromolecular assemblies has greatly benefited from their structural characterization. Recent structural data highlight principles in coat recruitment and uncoating in both the endocytic and retrograde pathways, and studies on the architecture of tethering complexes provide a framework for how they might link vesicles to the respective acceptor compartments and the fusion machinery. PMID:22728063

  16. Formation of vesicles through solvent assisted self-assembly of hydrophobic pentapeptides: encapsulation and pH responsive release of dyes by the vesicles.

    PubMed

    Kar, Sudeshna; Drew, Michael G B; Pramanik, Animesh

    2011-09-01

    In the biomimetic design two hydrophobic pentapetides Boc-Ile-Aib-Leu-Phe-Ala-OMe (I) and Boc-Gly-Ile-Aib-Leu-Phe-OMe (II) (Aib: α-aminoisobutyric acid) containing one Aib each are found to undergo solvent assisted self-assembly in methanol/water to form vesicular structures, which can be disrupted by simple addition of acid. The nanovesicles are found to encapsulate dye molecules that can be released by the addition of acid as confirmed by fluorescence microscopy and UV studies. The influence of solvent polarity on the morphology of the materials generated from the peptides has been examined systematically, and shows that fibrillar structures are formed in less polar chloroform/petroleum ether mixture and vesicular structures are formed in more polar methanol/water. Single crystal X-ray diffraction studies reveal that while β-sheet mediated self-assembly leads to the formation of fibrillar structures, the solvated β-sheet structure leads to the formation of vesicular structures. The results demonstrate that even hydrophobic peptides can generate vesicular structures from polar solvent which may be employed in model studies of complex biological phenomena.

  17. Hyperforin inhibits vesicular uptake of monoamines by dissipating pH gradient across synaptic vesicle membrane.

    PubMed

    Roz, Netta; Rehavi, Moshe

    2003-06-13

    Extracts of Hypericum perforatum (St. John's wort) have antidepressant properties in depressed patients and exert antidepressant-like action in laboratory animals. The phloroglucinol derivative hyperforin has become a topic of interest, as this Hypericum component is a potent inhibitor of monoamines reuptake. The molecular mechanism by which hyperforin inhibits monoamines uptake is yet unclear. In the present study we try to clarify the mechanism by which hyperforin inhibits the synaptic vesicle transport of monoamines. The pH gradient across the synaptic vesicle membrane, induced by vacuolar type H(+)-ATPase, is the major driving force for vesicular monoamines uptake and storage. We suggest that hyperforin, like the protonophore FCCP, dissipates an existing Delta pH generated by an efflux of inwardly pumped protons. Proton transport was measured by acridine orange fluorescence quenching. Adding Mg-ATP to a medium containing 130 mM KCl and synaptic vesicles caused an immediate decrease in fluorescence of acridine orange and the addition of 1 microM FCCP abolished this effect. H(+)-ATPase dependent proton pumping was inhibited by hyperforin in a dose dependent manner (IC(50) = 1.9 x 10(-7) M). Hyperforin acted similarly to the protonophore FCCP, abolishing the ATP induced fluorescence quenching (IC(50) = 4.3 x 10(-7) M). Hyperforin and FCCP had similar potencies for inhibiting rat brain synaptosomal uptake of [3H]monoamines as well as vesicular monoamine uptake. The efflux of [3H]5HT from synaptic vesicles was sensitive to both drugs, thus 50% of preloaded [3H]5HT was released in the presence of 2.1 x 10(-7) M FCCP and 4 x 10(-7) M hyperforin. The effect of hyperforin on the pH gradient in synaptic vesicle membrane may explain its inhibitory effect on monoamines uptake, but could only partially explain its antidepressant properties.

  18. Selective molecular transport through the protein shell of a bacterial microcompartment organelle

    DOE PAGES

    Chowdhury, Chiranjit; Chun, Sunny; Pang, Allan; ...

    2015-02-23

    Bacterial microcompartments are widespread prokaryotic organelles that have important and diverse roles ranging from carbon fixation to enteric pathogenesis. Current models for microcompartment function propose that their outer protein shell is selectively permeable to small molecules, but whether a protein shell can mediate selective permeability and how this occurs are unresolved questions. In this paper, biochemical and physiological studies of structure-guided mutants are used to show that the hexameric PduA shell protein of the 1,2-propanediol utilization (Pdu) microcompartment forms a selectively permeable pore tailored for the influx of 1,2-propanediol (the substrate of the Pdu microcompartment) while restricting the efflux ofmore » propionaldehyde, a toxic intermediate of 1,2-propanediol catabolism. Crystal structures of various PduA mutants provide a foundation for interpreting the observed biochemical and phenotypic data in terms of molecular diffusion across the shell. Finally and overall, these studies provide a basis for understanding a class of selectively permeable channels formed by nonmembrane proteins.« less

  19. Increased expression of the Drosophila vesicular glutamate transporter leads to excess glutamate release and a compensatory decrease in quantal content.

    PubMed

    Daniels, Richard W; Collins, Catherine A; Gelfand, Maria V; Dant, Jaime; Brooks, Elizabeth S; Krantz, David E; DiAntonio, Aaron

    2004-11-17

    Quantal size is a fundamental parameter controlling the strength of synaptic transmission. The transmitter content of synaptic vesicles is one mechanism that can affect the physiological response to the release of a single vesicle. At glutamatergic synapses, vesicular glutamate transporters (VGLUTs) are responsible for filling synaptic vesicles with glutamate. To investigate how VGLUT expression can regulate synaptic strength in vivo, we have identified the Drosophila vesicular glutamate transporter, which we name DVGLUT. DVGLUT mRNA is expressed in glutamatergic motoneurons and a large number of interneurons in the Drosophila CNS. DVGLUT protein resides on synaptic vesicles and localizes to the presynaptic terminals of all known glutamatergic neuromuscular junctions as well as to synapses throughout the CNS neuropil. Increasing the expression of DVGLUT in motoneurons leads to an increase in quantal size that is accompanied by an increase in synaptic vesicle volume. At synapses confronted with increased glutamate release from each vesicle, there is a compensatory decrease in the number of synaptic vesicles released that maintains normal levels of synaptic excitation. These results demonstrate that (1) expression of DVGLUT determines the size and glutamate content of synaptic vesicles and (2) homeostatic mechanisms exist to attenuate the excitatory effects of excess glutamate release.

  20. Rare earth element mobility in vesicular lava during low-grade metamorphism

    NASA Astrophysics Data System (ADS)

    Nyström, Jan Olov

    1984-12-01

    A geochemical comparison of basaltic relicts and spilitic domains from two burial metamorphosed flows in central Chile, of similar original composition and rich and poor in amygdules, respectively, demonstrates a relationship between initial vesicularity and rare earth element (REE) mobility. During spilitization the REE were partly leached from permeable parts of the flows and precipitated in voids, now amygdules and veinlets. The REE (excluding Eu) moved coherently in the highly amygdaloidal flow: spilitic domains and amygdules inherited the basaltic REE pattern. Besides being characterized by a positive Eu anomaly, epidotes separated from amygdules have a REE distribution which mimics that of the basalt; the absolute contents range widely, suggesting local and/or temporal REE variations in the metamorphic fluids. Pumpellyite differs by being strongly enriched in heavy REE. Similar ratios of Th, Hf and Ta in samples as contrasting as relict basalt and a geode are consistent with coherent leaching. Coherent mobility, when established for a rock system, can be used to elucidate, for example, whether minerals in cross-cutting veins were formed by local redistribution or from introduced material.

  1. Vesicular-arbuscular mycorrhizae of Easter lily in the northwestern United States.

    PubMed

    Ames, R N; Linderman, R G

    1977-12-01

    The vesicular-arbuscular (VA) mycorrhizal fungi of commercially grown Easter lily (Lilium longiflorum Thunb.) were studied. Soil and root samples were collected monthly from March through September 1975 from five fields in the coastal area of southern Oregon and northern California. Soil seivings were inoculated onto clover, onion, and lily to cause infections resulting in the production of many new mycorrhizal spores facilitating identification. Four VA mycorrhizal species were found: Acaulospora trappei, A. elegans, Glomus monosporus, and G. fasciculatus. All four VA species infected Easter lily, clover, and onion. Acaulospora trappei and G. fasciculatus were the most commonly isolated species from all five fields. Mycorrhizal infections in roots of field-grown lilies were sparse and presumably young in March and gradually increased in size and number until September when bulbs were harvested. Over 75% of each root system became infected with mycorrhizae in fields with all four fungal species, and those levels were reached by July. In fields with only two mycorrhizal species, usually 50% or less of each root system was infected, even by the end of the growing season.

  2. Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells

    PubMed Central

    Moss, Lacy R.; Mulik, Rohit S.; Van Treuren, Tim; Kim, Soo Young; Corbin, Ian R.

    2016-01-01

    Background Recent studies have shown that low density lipoproteins reconstituted with the natural omega 3 fatty acid docosahexaenoic acid (LDL-DHA) is selectively cytotoxic to liver cancer cells over normal hepatocytes. To date, little is known about the subcellular events which transpire following LDL-DHA treatment. Methods Herein, murine noncancer and cancer liver cells, TIB-73 and TIB-75 respectively, were investigated utilizing confocal microscopy, flow cytometry and viability assays to demonstrate differential actions of LDL-DHA nanoparticles in normal versus malignant cells. Results Our studies first showed that basal levels of oxidative stress are significantly higher in the malignant TIB-75 cells compared to the normal TIB-73 cells. As such, upon entry of LDL-DHA into the malignant TIB-75 cells, DHA is rapidly oxidized precipitating global and lysosomal lipid peroxidation along with increased lysosomal permeability. This leakage of lysosomal contents and lipid peroxidation products trigger subsequent mitochondrial dysfunction and nuclear injury. The cascade of LDL-DHA mediated lipid peroxidation and organelle damage was partially reversed by the administration of the antioxidant, N-acetylcysteine, or the iron-chelator, deferoxamine. LDL-DHA treatment in the normal TIB-73 cells was well tolerated and did not elicit any cell or organelle injury. Conclusion These studies have shown that LDL-DHA is selectively cytotoxic to liver cancer cells and that increased levels of ROS and iron catalyzed reactions promote the peroxidation of DHA which lead to organelle dysfunction and ultimately the demise of the cancer cell. General significance LDL-DHA selectively disrupts lysosomal, mitochondrial and nuclear function in cancer cells as a novel pathway for eliminating cancer cells. PMID:27418237

  3. The Drosophila vesicular monoamine transporter reduces pesticide-induced loss of dopaminergic neurons

    PubMed Central

    Lawal, Hakeem O.; Chang, Hui-Yun; Terrell, Ashley N.; Brooks, Elizabeth S.; Pulido, Dianne; Simon, Anne F.; Krantz, David E.

    2010-01-01

    Dopamine is cytotoxic and may play a role in the development of Parkinson’s disease. However, its interaction with environmental risk factors such as pesticides remains poorly understood. The vesicular monoamine transporter (VMAT) regulates intracellular dopamine content, and we have tested the neuroprotective effects of VMAT in vivo using the model organism Drosophila melanogaster. We find that Drosophila VMAT (dVMAT) mutants contain fewer dopaminergic neurons than wild type, consistent with a developmental effect, and that dopaminergic cell loss in the mutant is exacerbated by the pesticides rotenone and paraquat. Over-expression of DVMAT protein does not increase the survival of animals exposed to rotenone, but blocks the loss of dopaminergic neurons caused by this pesticide. These results are the first to demonstrate an interaction between a VMAT and pesticides in vivo, and provide an important model to investigate the mechanisms by which pesticides and cellular DA may interact to kill dopaminergic cells. PMID:20472063

  4. MicroRNAs in the intracellular space, regulation of organelle specific pathways in health and disease.

    PubMed

    Nguyen, Thao T; Brenu, Ekua W; Staines, Don R; Marshall-Gradisnik, Sonya M

    2014-01-01

    MicroRNAs (miRNA) are small (~22 nucleotide] non-coding RNA molecules originally characterised as nonsense or junk DNA. Emerging research suggests that these molecules have diverse regulatory roles in an array of molecular, cellular and physiological processes. MiRNAs are versatile and highly stable molecules, therefore, they are able to exist as intracellular or extracellular miRNAs. The purpose of this paper is to review the function and role of miRNAs in the intracellular space with specific focus on the interactions between miRNAs and organelles such as the mitochondria and the rough endoplasmic reticulum. Understanding the role of miRNAs in the intracellular space may be vital in understanding the mechanism of certain diseases.

  5. Amino Acid Neurotransmitters; Mechanisms of Their Uptake into Synaptic Vesicles

    DTIC Science & Technology

    1991-08-01

    4.1.1.15), is localized in specific GABAergic nerve terminals (Fonnum et al, 1970). The subcortical telencephalon , which contains among others the...ratio between the vesicular uptake of GABA and glycine is similar in cerebral cortex, subcortical telencephalon , whole brain, and spinal cord. This is...regions, cerebral cortex, cerebellum, medulla and subcortical telencephalon (i.e. forebrain after removal of cortex). The vesicular uptake is low and

  6. Dopamine transporter and vesicular monoamine transporter knockout mice : implications for Parkinson's disease.

    PubMed

    Miller, G W; Wang, Y M; Gainetdinov, R R; Caron, M G

    2001-01-01

    One of the most valuable methods for understanding the function of a particular protein is the generation of animals that have had the gene encoding for the protein of interest disrupted, commonly known as a "quo;knockout"quo; or null mutant. By incorporating a sequence of DNA (typically encoding antibiotic resistance to aid in the selection of the mutant gene) into embryonic stem cells by homologous recombination, the normal transcription of the gene is effectively blocked (Fig. 1). Since a particular protein is encoded by two copies of a gene, it is necessary to have the gene on both alleles "quo;knocked out."quo; This is performed by cross-breeding animals with one affected allele (heterozygote) to generate offspring that have inherited two mutant alleles (homozygote). This procedure has been used to generate animals lacking either the plasma membrane dopamine transporter (DAT; Fig. 2) or the vesicular monoamine transporter (VMAT2; Fig. 3). Both DAT and VMAT2 are essential for dopamine homeostasis and are thought to participate in the pathogenesis of Parkinson's disease (1-5). Fig. 1. Maps of the targeting vector and the mock construct. The mouse genomic fragment (clone 11) was isolated from a Stratagene 129 SvJ library by standard colony hybridization using a PCR probe from the 5' end of rat cDNA. The restriction site abbreviations are as follows: H, HindIII; N, NotI; Sc, SacI; Sn, SnaI; X, XbaI; and Xh, XhoI. The region between HindIII and SnaI on clone 11 containing the coding sequence from transmembrane domains 3 and 4 of VMAT2 was deleted and replaced with PGK-neo. The 3' fragment of clone 11 was reserved as an external probe for Southern analysis. To facilitate PCR screening of embryonic stem cell clones, a mock construct containing the SnaI/XbaI fragment and part of the Neo cassette was generated as a positive control. pPNT and pGEM4Z were used to construct knockout and mock vectors, respectively. (Reproduced with permission from ref. 1). Fig. 2. DAT and

  7. Polyacetal Carboxylic Acids: a New Group of Antiviral Polyanions

    PubMed Central

    Claes, P.; Billiau, A.; De Clercq, E.; Desmyter, J.; Schonne, E.; Vanderhaeghe, H.; De Somer, P.

    1970-01-01

    Chlorite-oxidized oxypolysaccharides are polyacetal carboxylic acids. They inhibited the cytopathic effect of vesicular stomatitis virus in mouse embryo cell cultures challenged at low input multiplicity. After intraperitoneal injection of these compounds in mice, interferon appeared in the circulation. The compounds also protected mice against lethal mengovirus infection and against the development of experimental pox lesions on the tail. Chlorite-oxidized oxyamylose was antiviral only when at least 64% of the glucopyranose units were oxidized, an observation which suggested a correlation between charge density and antiviral effect. The antiviral activity was also influenced by the molecular weight, as demonstrated by the fact that chlorite-oxidized dextrans which had a high intrinsic viscosity were more active than those with low intrinsic viscosity. PMID:4314553

  8. Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

    PubMed

    Thomas, D; Newcomb, W W; Brown, J C; Wall, J S; Hainfeld, J F; Trus, B L; Steven, A C

    1985-05-01

    Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to

  9. Mass and molecular composition of vesicular stomatitis virus: a scanning transmission electron microscopy analysis.

    PubMed Central

    Thomas, D; Newcomb, W W; Brown, J C; Wall, J S; Hainfeld, J F; Trus, B L; Steven, A C

    1985-01-01

    Dark-field scanning transmission electron microscopy was used to perform mass analyses of purified vesicular stomatitis virions, pronase-treated virions, and nucleocapsids, leading to a complete self-consistent account of the molecular composition of vesicular stomatitis virus. The masses obtained were 265.6 +/- 13.3 megadaltons (MDa) for the native virion, 197.5 +/- 8.4 MDa for the pronase-treated virion, and 69.4 +/- 4.9 MDa for the nucleocapsid. The reduction in mass effected by pronase treatment, which corresponds to excision of the external domains (spikes) of G protein, leads to an average of 1,205 molecules of G protein per virion. The nucleocapsid mass, after compensation for the RNA (3.7 MDa) and residual amounts of other proteins, yielded a complement of 1,258 copies of N protein. Calibration of the amounts of M, NS, and L proteins relative to N protein by biochemical quantitation yielded values of 1,826, 466, and 50 molecules, respectively, per virion. Assuming that the remaining virion mass is contributed by lipids in the viral envelope, we obtained a value of 56.1 MDa for its lipid content. In addition, four different electron microscopy procedures were applied to determine the nucleocapsid length, which we conclude to be 3.5 to 3.7 micron. The nucleocapsid comprises a strand of repeating units which have a center-to-center spacing of 3.3 nm as measured along the middle of the strand. We show that these repeating units represent monomers of N protein, each of which is associated with 9 +/- 1 bases of single-stranded RNA. From scanning transmission electron microscopy images of negatively stained nucleocapsids, we inferred that N protein has a wedge-shaped, bilobed structure with dimensions of approximately 9.0 nm (length), approximately 5.0 nm (depth), and approximately 3.3 nm (width, at the midpoint of its long axis). In the coiled configuration of the in situ nucleocapsid, the long axis of N protein is directed radially, and its depth corresponds to

  10. Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

    PubMed Central

    Felt, Sébastien A.; Droby, Gaith N.

    2017-01-01

    ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against

  11. Intersporal Genetic Variation of Gigaspora margarita, a Vesicular Arbuscular Mycorrhizal Fungus, Revealed by M13 Minisatellite-Primed PCR.

    PubMed

    Zeze, A; Sulistyowati, E; Ophel-Keller, K; Barker, S; Smith, S

    1997-02-01

    Spores of vesicular arbuscular mycorrhizal (VAM) fungi contain thousands of nuclei. In order to understand the karyotic structure of a VAM fungus spore, the genetic variation of the first generation of spores from a VAM fungus (Gigaspora margarita) was examined. Spores originating from both single- and multispore inoculations of the species G. margarita were analyzed by M13 minisatellite-primed PCR. In both cases, different fingerprints were obtained from individual spores with few spores exhibiting similar fingerprints. These results can be explained only by a heterokaryotic status of the nuclear population within a spore.

  12. A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots.

    PubMed

    Volpin, H.; Elkind, Y.; Okon, Y.; Kapulnik, Y.

    1994-02-01

    Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.

  13. Dietary unsaturated fatty acids differently affect catecholamine handling by adrenal chromaffin cells.

    PubMed

    Gomes, Andreia; Correia, Gustavo; Coelho, Marisa; Araújo, João Ricardo; Pinho, Maria João; Teixeira, Ana Luisa; Medeiros, Rui; Ribeiro, Laura

    2015-05-01

    Catecholamines (CA) play an important role in cardiovascular (CDV) disease risk. Namely, noradrenaline (NA) levels positively correlate whereas adrenaline (AD) levels negatively correlate with obesity and/or CDV disease. Western diets, which are tipically rich in Ω-6 fatty acids (FAs) and deficient in Ω-3 FAs, may contribute to the development of obesity, type 2 diabetes and/or coronary artery disease. Taking this into consideration and the fact that our group has already described that saturated FAs affect catecholamine handling by adrenal chromaffin cells, this work aimed to investigate the effect of unsaturated FAs upon catecholamine handling in the same model. Our results showed that chronic exposure to unsaturated FAs differently modulated CA cellular content and release, regardless of both FA series and number of carbon atoms. Namely, the Ω-6 arachidonic and linoleic acids, based on their effect on CA release and cellular content, seemed to impair NA and AD vesicular transport, whereas γ-linolenic acid selectively impaired AD synthesis and release. Within the Ω-9 FAs, oleic acid was devoid of effect, and elaidic acid behaved similarly to γ-linolenic acid. Eicosapentaenoic and docosahexaenoic acids (Ω-3 series) impaired the synthesis and release of both NA and AD. These results deserve attention and future development, namely, in what concerns the mechanisms involved and correlative effects in vivo. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Demonstration of vesicular glutamate transporter-1 in corticotroph cells in the anterior pituitary of the rat.

    PubMed

    Kocsis, Zsuzsa S; Molnár, Csilla S; Watanabe, Masahiko; Daneels, Guy; Moechars, Dieder; Liposits, Zsolt; Hrabovszky, Erik

    2010-02-01

    Recent immunohistochemical studies of the rat adenohypophysis identified type-2 vesicular glutamate transporter (VGLUT2), a marker for glutamatergic neuronal phenotype, in high percentages of adenohypophysial gonadotrophs and thyrotrophs. The presence and molecular identity of amino acid neurotransmitters in the remaining hormone producing cell types are unknown. In the present study we addressed the putative synthesis of another glutamatergic marker, VGLUT1 by adenohypophysial cells. Immunohistochemical studies revealed VGLUT1 immunoreactivity in a small subset of polygonal medium-sized cells in the anterior lobe. Western blot analysis revealed a single major 60 kDa protein band in the adenohypophysis. Furthermore, the expression of VGLUT1 mRNA was confirmed by reverse transcription-polymerase chain reaction followed by sequence analysis of the amplicon. In contrast with rats which only showed VGLUT1 signal in the anterior lobe of the pituitary, mice contained high levels of VGLUT1 immunoreactivity in the intermediate, in addition to the anterior lobe. No signal was present in VGLUT1-knockout mice, providing evidence for specificity. In rats, results of colocalization studies with dual-immunofluorescent labeling provided evidence for VGLUT1 immunoreactivity in 45.9% of corticotrophs and 7.7% of luteinizing hormone beta-immunopositive gonadotrophs. Cells of the other peptide hormone phenotypes were devoid of VGLUT1 signal. A few cells in the adenohypophysis expressed both VGLUT1 and VGLUT2 immunoreactivities. The presence of the glutamate markers VGLUT1 and VGLUT2 in distinct populations of peptide hormone-secreting hypophysial cells highly indicates the involvement of endogenous glutamate release in autocrine/paracrine regulatory mechanisms. The biological function of adenohypophysial glutamate will require clarification. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  15. Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons.

    PubMed

    Teodorof, C; Divakar, S; Soontornniyomkij, B; Achim, C L; Kaul, M; Singh, K K

    2014-09-01

    Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons. Published by Elsevier Inc.

  16. Sheared-root inocula of vesicular-arbuscular mycorrhizal fungi.

    PubMed

    Sylvia, D M; Jarstfer, A G

    1992-01-01

    For efficient handling, vesicular-arbuscular mycorrhizal fungi should be processed into small and uniform inocula; however, processing can reduce the inoculum density. In this article we describe the preparation and use of sheared-root inocula of Glomus spp. in which inoculum densities were increased during processing. Our objectives were to determine inoculum viability and density after shearing and to ascertain if the sheared inocula could be pelletized or used with a gel carrier. Root samples were harvested from aeroponic cultures, blotted dry, cut into 1-cm lengths, and sheared in a food processor for up to 80 s. After shearing, the inoculum was washed over sieves, and the propagule density in each fraction was determined. Sheared inocula were also encapsulated in carrageenan or used in a gel carrier. Shearing aeroponically produced root inocula reduced particle size. Propagule density increased with decreasing size fraction down to a size of 63 mum, after which propagule density decreased. The weighted-average propagule density of the inoculum was 135,380 propagules g (dry weight) of sheared root material. Sheared roots were encapsulated successfully in carrageenan, and the gel served as an effective carrier. Aeroponic root inoculum was stored dry at 4 degrees C for 23 months without significant reduction in propagule density; however, this material was not appropriate for shearing. Moist roots, useful for shearing, began to lose propagule density after 1 month of storage. Shearing proved to be an excellent method to prepare viable root inocula of small and uniform size, allowing for more efficient and effective use of limited inoculum supplies.

  17. Asymmetrical Transmembrane Potential in Intracellular Organelles of Adrenal Chromatin Cells Exposed to Nanosecond Electric Pulses

    NASA Astrophysics Data System (ADS)

    Aramendia Zabaleta, Guillermo Jose

    In our research on exploring the effects of 5 ns, high intensity electric pulses on neurosecretory adrenal chromaffin cells, cell modeling has played an important role in understanding and explaining the experimental results. Externally applied nanosecond-duration electric pulses (NEPs) can affect cells by creating nanopores in the cell and intracellular organelle membranes, making these membranes permeable to certain ions. A chromaffin cell contains, at a minimum, 7000 secretory granules plus other organelles such as mitochondria and the endoplasmic reticulum. In all the biological cell models constructed in the literature, there is no evidence of asymmetrical Transmembrane Potential (TMP) distribution in the intracellular membranes. However, these models do not include a realistic number of intracellular organelles. The goal of this research was to construct a more realistic cell model that incorporates a large number of secretory granules in the cytosol. To this end, a beta-version of the real-valued unstructured mesh Finite Element Method (FEM) electro-quasi-static module in Sim4life (SPEAG, Switzerland) has been used to model a chromaffin cell in which 1000 secretory granules are included in the cytosol. The model is, we believe, the most detailed geometrical cell model developed. It includes a spherical chromaffin cell (radius 8 mum), nucleus (radius 2.5 mum) located off-center, 500 granules (radius 200 nm) randomly located within a distance of 2 mum from the surface of the nucleus, and additional 500 granules randomly located in the remaining region of the cytosol. Cell and granule membrane thickness was set to 5 nm and nuclear membrane thickness to 10 nm. Dielectric properties of all constituents of the model were obtained from the literature or measured. Because the FEM Low Frequency solver is a quasi-static solver and not capable of accepting a time-varying pulse as input, all computations have been performed at single frequencies in the range DC to 60

  18. Effects of vesicular-arbuscular mycorrhizal (VAM) fungi on the seedling growth of three Pistacia species.

    PubMed

    Caglar, S; Akgun, A

    2006-07-01

    The experiment was undertaken to test the efficiency of inoculation of vesicular-arbuscular mycorrhizal (VAM) fungi on the seedling growth of three Pistacia species used as rootstocks. The stratified Pistacia seeds were inoculated with VAM fungi. The highest rate of inoculated roots was 96.7% in P. khinjuck seedlings with G. clarum and G. etunicatum, 83.3% in P. vera seedlings with G. caledonium and 73.3% in P. terebinthus seedlings with G. caledonium. Mycorrhizal inoculations improved seedling height only in P. terebinthus. Certain mycorrhizal inoculations increased the leaf N, but not P and K contents. Seedlings inoculated with G. caledonium had higher reducing sugar contents. It was concluded that pre-inoculated Pistacia seedlings could have a better growth in the harsh field conditions.

  19. Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

    PubMed

    Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

    2017-10-06

    Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Evolution of Fitness in Experimental Populations of Vesicular Stomatitis Virus

    PubMed Central

    Elena, S. F.; Gonzalez-Candelas, F.; Novella, I. S.; Duarte, E. A.; Clarke, D. K.; Domingo, E.; Holland, J. J.; Moya, A.

    1996-01-01

    The evolution of fitness in experimental clonal populations of vesicular stomatitis virus (VSV) has been compared under different genetic (fitness of initial clone) and demographic (population dynamics) regimes. In spite of the high genetic heterogeneity among replicates within experiments, there is a clear effect of population dynamics on the evolution of fitness. Those populations that went through strong periodic bottlenecks showed a decreased fitness in competition experiments with wild type. Conversely, mutant populations that were transferred under the dynamics of continuous population expansions increased their fitness when compared with the same wild type. The magnitude of the observed effect depended on the fitness of the original viral clone. Thus, high fitness clones showed a larger reduction in fitness than low fitness clones under dynamics with included periodic bottleneck. In contrast, the gain in fitness was larger the lower the initial fitness of the viral clone. The quantitative genetic analysis of the trait ``fitness'' in the resulting populations shows that genetic variation for the trait is positively correlated with the magnitude of the change in the same trait. The results are interpreted in terms of the operation of MULLER's ratchet and genetic drift as opposed to the appearance of beneficial mutations. PMID:8849878

  1. An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

    PubMed

    Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P

    2017-06-01

    The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1 -knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. © 2017 Kaur et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. Synaptically evoked Ca2+ release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones.

    PubMed

    Evstratova, Alesya; Tóth, Katalin

    2011-12-01

    The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca(2+) wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca(2+) waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca(2+) signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca(2+) release from CA3 pyramidal cell internal stores.

  3. Synaptically evoked Ca2+ release from intracellular stores is not influenced by vesicular zinc in CA3 hippocampal pyramidal neurones

    PubMed Central

    Evstratova, Alesya; Tóth, Katalin

    2011-01-01

    Abstract The co-release of neuromodulatory substances in combination with classic neurotransmitters such as glutamate and GABA from individual presynaptic nerve terminals has the capacity to dramatically influence synaptic efficacy and plasticity. At hippocampal mossy fibre synapses vesicular zinc is suggested to serve as a cotransmitter capable of regulating calcium release from internal stores in postsynaptic CA3 pyramidal cells. Here we investigated this possibility using combined intracellular ratiometric calcium imaging and patch-clamp recording techniques. In acute hippocampal slices a brief train of mossy fibre stimulation produced a large, delayed postsynaptic Ca2+ wave that was spatially restricted to the proximal apical dendrites of CA3 pyramidal cells within stratum lucidum. This calcium increase was sensitive to intracellularly applied heparin indicating reliance upon release from internal stores and was triggered by activation of both group I metabotropic glutamate and NMDA receptors. Importantly, treatment of slices with the membrane-impermeant zinc chelator CaEDTA did not influence the synaptically evoked postsynaptic Ca2+ waves. Moreover, mossy fibre stimulus evoked postsynaptic Ca2+ signals were not significantly different between wild-type and zinc transporter 3 (ZnT3) knock-out animals. Considered together our data do not support a role for vesicular zinc in regulating mossy fibre evoked Ca2+ release from CA3 pyramidal cell internal stores. PMID:21986206

  4. Toward noninvasive microspectrofluorometry of skin lesions for diagnostic and prognostic evaluation of cell metabolism and organelle interactions

    NASA Astrophysics Data System (ADS)

    Hirschberg, Joseph G.; Schachtschabel, Astrid; Kohen, Elli; Kohen, Cahide; Schachtschabel, Dietrich O.

    1995-02-01

    The basic principle of this approach relies on microspectrofluorometric observations of upheavals in the cell's energy metabolism and cell-to-cell metabolic communication in human and mouse melanoma cells. A striking feature is the definition of a highly active nuclear energy metabolism in M8255 human melanoma cells which is characterized by an intense fluorescence response associated with NAD(P) reduction by substrates of glycolysis or the hexose monophosphate shunt. Changes are also expected in the steady state levels of reduced/oxidized NAD(P) in the nuclear, cytoplasmic and mitochondrial compartments, which are probably dependent on ATP levels and distribution (as determined by cell metabolism and eventually the presence of ATP traps). A topographic scanning of skin lesions, either under metabolic steady state conditions or in the presence of permeating substrates, can lead to the recognition of characteristic patterns associated with pigmented and nonpigmented, malignant and nonmalignant skin lesions. The method is, in a way, an extension of microscopic transillumination techniques which have led to the identification of specific patterns associated with such lesions. However, here, a new dimension is added by introduction of fluorescence evaluations. This can represent the first step in a multiparameter approach to the non-invasive in situ fluorescence scan of dermatological lesions by inclusion of: (1) fluorescence excitation and emission spectra; (2) new fluorescence probes of cytoplasmic organelles and nuclear components. Primary emphasis should be placed on the highly active nuclear energy metabolism, which can be triggered to maximum levels when the role of mitochondria as the `cells's policeman' with regard to metabolic control is suppressed by use of topically cytotoxic agents such as the `antipsoriatic' anthralin and dicarboxylic acids used in the local treatment of melanoma. Fluorescence excitation spectroscopy may be of particular advantage in

  5. Engineered Ferritin for Magnetogenetic Manipulation of Proteins and Organelles Inside Living Cells.

    PubMed

    Liße, Domenik; Monzel, Cornelia; Vicario, Chiara; Manzi, John; Maurin, Isabelle; Coppey, Mathieu; Piehler, Jacob; Dahan, Maxime

    2017-11-01

    Magnetogenetics is emerging as a novel approach for remote-controlled manipulation of cellular functions in tissues and organisms with high spatial and temporal resolution. A critical, still challenging issue for these techniques is to conjugate target proteins with magnetic probes that can satisfy multiple colloidal and biofunctional constraints. Here, semisynthetic magnetic nanoparticles are tailored based on human ferritin coupled to monomeric enhanced green fluorescent protein (mEGFP) for magnetic manipulation of proteins inside living cells. This study demonstrates efficient delivery, intracellular stealth properties, and rapid subcellular targeting of those magnetic nanoparticles via GFP-nanobody interactions. By means of magnetic field gradients, rapid spatial reorganization in the cytosol of proteins captured to the nanoparticle surface is achieved. Moreover, exploiting efficient nanoparticle targeting to intracellular membranes, remote-controlled arrest of mitochondrial dynamics using magnetic fields is demonstrated. The studies establish subcellular control of proteins and organelles with unprecedented spatial and temporal resolution, thus opening new prospects for magnetogenetic applications in fundamental cell biology and nanomedicine. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Fatty acids are required for epidermal permeability barrier function.

    PubMed

    Mao-Qiang, M; Elias, P M; Feingold, K R

    1993-08-01

    The permeability barrier is mediated by a mixture of ceramides, sterols, and free fatty acids arranged as extracellular lamellar bilayers in the stratum corneum. Whereas prior studies have shown that cholesterol and ceramides are required for normal barrier function, definitive evidence for the importance of nonessential fatty acids is not available. To determine whether epidermal fatty acid synthesis also is required for barrier homeostasis, we applied 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an inhibitor of acetyl CoA carboxylase, after disruption of the barrier by acetone or tape stripping. TOFA inhibits epidermal fatty acid by approximately 50% and significantly delays barrier recovery. Moreover, coadministration of palmitate with TOFA normalizes barrier recovery, indicating that the delay is due to a deficiency in bulk fatty acids. Furthermore, TOFA treatment also delays the return of lipids to the stratum corneum and results in abnormalities in the structure of lamellar bodies, the organelle which delivers lipid to the stratum corneum. In addition, the organization of secreted lamellar body material into lamellar bilayers within the stratum corneum interstices is disrupted by TOFA treatment. Finally, these abnormalities in lamellar body and stratum corneum membrane structure are corrected by coapplication of palmitate with TOFA. These results demonstrate a requirement for bulk fatty acids in barrier homeostasis. Thus, inhibiting the epidermal synthesis of any of the three key lipids that form the extracellular, lipid-enriched membranes of the stratum corneum results in an impairment in barrier homeostasis.

  7. Fatty acids are required for epidermal permeability barrier function.

    PubMed Central

    Mao-Qiang, M; Elias, P M; Feingold, K R

    1993-01-01

    The permeability barrier is mediated by a mixture of ceramides, sterols, and free fatty acids arranged as extracellular lamellar bilayers in the stratum corneum. Whereas prior studies have shown that cholesterol and ceramides are required for normal barrier function, definitive evidence for the importance of nonessential fatty acids is not available. To determine whether epidermal fatty acid synthesis also is required for barrier homeostasis, we applied 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA), an inhibitor of acetyl CoA carboxylase, after disruption of the barrier by acetone or tape stripping. TOFA inhibits epidermal fatty acid by approximately 50% and significantly delays barrier recovery. Moreover, coadministration of palmitate with TOFA normalizes barrier recovery, indicating that the delay is due to a deficiency in bulk fatty acids. Furthermore, TOFA treatment also delays the return of lipids to the stratum corneum and results in abnormalities in the structure of lamellar bodies, the organelle which delivers lipid to the stratum corneum. In addition, the organization of secreted lamellar body material into lamellar bilayers within the stratum corneum interstices is disrupted by TOFA treatment. Finally, these abnormalities in lamellar body and stratum corneum membrane structure are corrected by coapplication of palmitate with TOFA. These results demonstrate a requirement for bulk fatty acids in barrier homeostasis. Thus, inhibiting the epidermal synthesis of any of the three key lipids that form the extracellular, lipid-enriched membranes of the stratum corneum results in an impairment in barrier homeostasis. Images PMID:8102380

  8. Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.

    PubMed

    Carroll, Bernadette; Korolchuk, Viktor I; Sarkar, Sovan

    2015-10-01

    Maintenance of amino acid homeostasis is important for healthy cellular function, metabolism and growth. Intracellular amino acid concentrations are dynamic; the high demand for protein synthesis must be met with constant dietary intake, followed by cellular influx, utilization and recycling of nutrients. Autophagy is a catabolic process via which superfluous or damaged proteins and organelles are delivered to the lysosome and degraded to release free amino acids into the cytoplasm. Furthermore, autophagy is specifically activated in response to amino acid starvation via two key signaling cascades: the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and the general control nonderepressible 2 (GCN2) pathways. These pathways are key regulators of the integration between anabolic (amino acid depleting) and catabolic (such as autophagy which is amino acid replenishing) processes to ensure intracellular amino acid homeostasis. Here, we discuss the key roles that amino acids, along with energy (ATP, glucose) and oxygen, are playing in cellular growth and proliferation. We further explore how sophisticated methods are employed by cells to sense intracellular amino acid concentrations, how amino acids can act as a switch to dictate the temporal and spatial activation of anabolic and catabolic processes and how autophagy contributes to the replenishment of free amino acids, all to ensure cell survival. Relevance of these molecular processes to cellular and organismal physiology and pathology is also discussed.

  9. Regulation of vesicular trafficking and leukocyte function by Rab27 GTPases and their effectors

    PubMed Central

    Catz, Sergio Daniel

    2013-01-01

    The Rab27 family of GTPases regulates the efficiency and specificity of exocytosis in hematopoietic cells, including neutrophils, CTLs, NK cells, and mast cells. However, the mechanisms regulated by Rab27 GTPases are cell-specific, as they depend on the differential expression and function of particular effector molecules that are recruited by the GTPases. In addition, Rab27 GTPases participate in multiple steps of the regulation of the secretory process, including priming, tethering, docking, and fusion through sequential interaction with multiple effector molecules. Finally, recent reports suggest that Rab27 GTPases and their effectors regulate vesicular trafficking mechanisms other than exocytosis, including endocytosis and phagocytosis. This review focuses on the latest discoveries on the function of Rab27 GTPases and their effectors Munc13-4 and Slp1 in neutrophil function comparatively to their functions in other leukocytes. PMID:23378593

  10. The Role of Organelle Stresses in Diabetes Mellitus and Obesity: Implication for Treatment

    PubMed Central

    Chang, Yi-Cheng; Hee, Siow-Wey; Hsieh, Meng-Lun; Chuang, Lee-Ming

    2015-01-01

    The type 2 diabetes pandemic in recent decades is a huge global health threat. This pandemic is primarily attributed to the surplus of nutrients and the increased prevalence of obesity worldwide. In contrast, calorie restriction and weight reduction can drastically prevent type 2 diabetes, indicating a central role of nutrient excess in the development of diabetes. Recently, the molecular links between excessive nutrients, organelle stress, and development of metabolic disease have been extensively studied. Specifically, excessive nutrients trigger endoplasmic reticulum stress and increase the production of mitochondrial reactive oxygen species, leading to activation of stress signaling pathway, inflammatory response, lipogenesis, and pancreatic beta-cell death. Autophagy is required for clearance of hepatic lipid clearance, alleviation of pancreatic beta-cell stress, and white adipocyte differentiation. ROS scavengers, chemical chaperones, and autophagy activators have demonstrated promising effects for the treatment of insulin resistance and diabetes in preclinical models. Further results from clinical trials are eagerly awaited. PMID:26613076

  11. Assembly and Architecture of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)*

    PubMed Central

    Lee, Hyung Ho; Nemecek, Daniel; Schindler, Christina; Smith, William J.; Ghirlando, Rodolfo; Steven, Alasdair C.; Bonifacino, Juan S.; Hurley, James H.

    2012-01-01

    BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. We show that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Å long and ∼30 Å in diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45°. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting. PMID:22203680

  12. Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles

    PubMed Central

    Moeschler, Sarah; Locher, Samira; Conzelmann, Karl-Klaus; Krämer, Beate; Zimmer, Gert

    2016-01-01

    Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses. PMID:27649230

  13. Quantification of Lyssavirus-Neutralizing Antibodies Using Vesicular Stomatitis Virus Pseudotype Particles.

    PubMed

    Moeschler, Sarah; Locher, Samira; Conzelmann, Karl-Klaus; Krämer, Beate; Zimmer, Gert

    2016-09-16

    Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet, 14 serologically and genetically diverse lyssaviruses have been identified, mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations, virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work, a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly, pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together, a pseudotype virus system has been successfully developed which allows the safe, fast, and sensitive detection of neutralizing antibodies directed against different lyssaviruses.

  14. Sensorineural Deafness and Seizures in Mice Lacking Vesicular Glutamate Transporter 3

    PubMed Central

    Seal, Rebecca P.; Akil, Omar; Yi, Eunyoung; Weber, Christopher M.; Grant, Lisa; Yoo, Jong; Clause, Amanda; Kandler, Karl; Noebels, Jeffrey L; Glowatzki, Elisabeth; Lustig, Lawrence R.; Edwards, Robert H.

    2008-01-01

    Summary The expression of unconventional vesicular glutamate transporter VGLUT3 by neurons known to release a different classical transmitter has suggested novel roles for signaling by glutamate. However, this distribution, along with the localization of VGLUT3 to dendrites and its occurrence outside the nervous system, has raised questions about whether the protein actually contributes to glutamate release. We now report that mice lacking VGLUT3 are profoundly deaf due to the absence of glutamate release from hair cells at the first synapse in the auditory pathway. Inner hair cells of the cochlea start to express VGLUT3 shortly before birth, and the early degeneration of some cochlear ganglion neurons in knock-out mice indicates an important developmental role for the glutamate released by hair cells before the onset of hearing. In addition, the mice exhibit primary, generalized epilepsy that is accompanied by remarkably little or no change in ongoing motor behavior. VGLUT3 thus contributes to the exocytotic release of glutamate, and the glutamate released has an essential role in both function and development of the auditory pathway, as well as in the control of cortical excitability. PMID:18215623

  15. Quantitative Comparison Of Vesicular Glutamate Transporters in rat Deep Cerebellar Nuclei.

    PubMed

    Mao, Haian; Hamodeh, Salah; Sultan, Fahad

    2018-04-15

    The excitatory synapses of the rat deep cerebellar nuclei (DCN) were quantitatively analyzed by vesicular glutamate transporter 1 and 2 (vGluT1 and vGluT2) immunolabeling. We calculated the number and sizes of the labeled boutons and compared them between lateral/dentate nucleus (LN/DN), posterior interposed nucleus (PIN), anterior interposed nucleus (AIN), and medial nucleus (MN). The density of vGluT1+ boutons differs significantly within these nuclei. In contrast, the vGluT2+ bouton density is more similar between different nuclei. The phylogenetically newer DCN (LN/DN and PIN) have a 39% higher density of vGluT1+ boutons than the phylogenetically older DCN (AIN and MN). The volume of vGluT1+ boutons does not differ between the DCN, however the average volume of vGluT2+ boutons is larger in MN. In summary, our current results confirm and extend our previous findings showing that the increase in dendritic and axonal wiring in phylogenetically newer DCN is associated with an increase in vGluT1+ bouton density. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

  16. Influence of nonsystemic transmission on the epidemiology of insect borne arboviruses: a case study of vesicular stomatitis epidemiology in the western United States.

    PubMed

    Lord, Cynthia C; Tabachnick, Walter J

    2002-05-01

    Nonsystemic transmission, where a pathogen is transmitted between infected and uninfected vectors without the vertebrate host becoming viremic, may provide an explanation for transmission in systems where the vertebrate hosts have been difficult to identify. This transmission pathway had been previously demonstrated for tick-borne viruses and bacteria, but the recent demonstration for Simulium and vesicular stomatitis virus is the first for a blood-feeding insect. The epidemiology of vesicular stomatitis viruses has been difficult to understand, and nonsystemic transmission may be important. We use mathematical formulations of the basic reproduction number, R(0), to compare systemic and nonsystemic transmission. The absence of a latent period before host infectiousness in nonsystemic transmission may allow a more rapid increase in prevalence in the biting flies early in the development of a new outbreak. Aggregation of flies between hosts and at favored feeding sites on hosts will be important, but further data on nonsystemic transmission as a function of space and time are required to fully assess this pathway. The data needed to compare the two pathways and their relative roles in virus epidemiology are discussed.

  17. The CoRR hypothesis for genes in organelles.

    PubMed

    Allen, John F

    2017-12-07

    Chloroplasts and mitochondria perform energy transduction in photosynthesis and respiration. These processes can be described in physico-chemical terms with no obvious requirement for co-located genetic systems, separat from those of the rest of the cell. Accordingly, biochemists once tended to regard endosymbiosis as untestable evolutionary speculation. Lynn Sagan's seminal 1967 paper "On the Origin of Mitosing Cells" outlined the evolution of eukaryotic cells by endosymbiosis of prokaryotes. The endosymbiont hypothesis is consistent with presence of DNA in chloroplasts and mitochondria, but does not assign it a function. Biochemistry and molecular biology now show that Sagan's proposal has an explanatory reach far beyond that originally envisaged. Prokaryotic origins of photosynthetic and respiratory mechanisms are apparent in protein structural insights into energy coupling. Genome sequencing confirms the underlying, prokaryotic architecture of chloroplasts and mitochondria and illustrates the profound influence of the original mergers of their ancestors' genes and proteins with those of their host cells. Peter Mitchell's 1961 chemiosmotic hypothesis applied the concept of vectorial catalysis that underlies biological energy transduction and cell structure, function, and origins. Continuity of electrical charge separation and membrane sidedness requires compartments within compartments, together with intricate mechanisms for transport within and between them. I suggest that the reason for the persistence of distinct genetic systems within bioenergetic organelles is the selective advantage of subcellular co-location of specific genes with their gene products. Co-location for Redox Regulation - CoRR - provides for a dialogue between chemical reduction-oxidation and the action of genes encoding its protein catalysts. These genes and their protein products are in intimate contact, and cannot be isolated from each other without loss of an essential mechanism of

  18. Interactions of Vesicular-Arbuscular Mycorrhizal Fungi, Phosphorus, and Heterodera glycines on Soybean.

    PubMed

    Tylka, G L; Hussey, R S; Roncadori, R W

    1991-01-01

    Effects of vesicular-arbuscular mycorrhizal (VAM) fungi and soil phosphorus (P) fertility on parasitism of soybean cultivars Bragg and Wright by soybean cyst nematode (SCN) were investigated in field micropiot and greenhouse experiments. VAM fungi increased height of both cultivars and yield of Wright in microplot studies in 1986 and 1987. Conversely, yield of mycorrhizal and nonmycorrhizal plants of both cultivars was suppressed by SCN. Soil population densities of SCN were unaffected by VAM fungi in 1986 but were greater in microplots infested with VAM fungi than in control microplots in 1987. Growth of Wright soybean was stimulated by VAM fungi and suppressed by SCN in greenhouse experiments. The effect of VAM fungi on SCN varied with time. Numbers of SCN in roots and soil were decreased by VAM fungi by as much as 73% at the highest SCN inoculum level through 49 days after planting. Later, however, SCN numbers were usually comparable on mycorrhizal and nonmycorrhizal plants. Soil P fertility generally had no effect on SCN. Results of a split-root experiment indicated that VAM fungal suppression of SCN was not systemic.

  19. Soil sealing and vesicular layer formation as initial structure development and its effect on infiltration

    NASA Astrophysics Data System (ADS)

    Badorreck, A.; Gerke, H. H.; Weller, U.; Vontobel, P.

    2009-04-01

    In the Lusatia mining district (NE-Germany) an artificial catchment was constructed to study initial ecosystem development and runoff generation. As a key process in this early stage, we investigate the surface structure dynamics as it strongly influences erosion, infiltration, matter dynamics, and vegetation establishment. The presented work focuses on observations of soil pore structure formation at the surface at five sites in the catchment and in an adjacent "younger" area composed of comparable sediments. Moreover we've conducted infiltration experiments in the lab and field to relate the soil pore structure to the hydraulic properties. The surface soil was sampled in cylindrical rings (10 cm³) down to 2 cm depth from which bulk density profiles were obtained using X-ray computed tomography (CT) (at UFZ- Halle, Germany) with a resolution of 0.084 mm. The influence of structure on infiltration was investigated using neutron radiography (at the NEUTRA facility of the Paul-Scherrer-Institut, Villigen, Switzerland) to visualise two-dimensional (2D) infiltration patterns. The slab-type samples were equilibrated to different initial water contents and then exposed to drip irrigation (to simulate rainfall) while a series of neutron radiographs were taken. In addition, field measurements with a miniature tension infiltrometer were conduced. The micro-tomographies exhibit formation of surface sealing whose thickness and intensity vary with silt and clay content. The CT images show several coarser- and finer-textured micro-layers at the sample surfaces that were formed as a consequence of repeated washing in of finer particles in underlying coarser sediment. In micro-depressions, the uppermost layers consist of sorted fine sand and silt due to wind erosion. Similar as for desert pavements, a vesicular pore structure developed in these sediments on top, but also scattered in fine sand- and silt-enriched micro-layers. The infiltration rates were severely affected by the

  20. Entry and Exit Mechanisms at the cis-Face of the Golgi Complex

    PubMed Central

    Lorente-Rodríguez, Andrés; Barlowe, Charles

    2011-01-01

    Vesicular transport of protein and lipid cargo from the endoplasmic reticulum (ER) to cis-Golgi compartments depends on coat protein complexes, Rab GTPases, tethering factors, and membrane fusion catalysts. ER-derived vesicles deliver cargo to an ER-Golgi intermediate compartment (ERGIC) that then fuses with and/or matures into cis-Golgi compartments. The forward transport pathway to cis-Golgi compartments is balanced by a retrograde directed pathway that recycles transport machinery back to the ER. How trafficking through the ERGIC and cis-Golgi is coordinated to maintain organelle structure and function is poorly understood and highlights central questions regarding trafficking routes and organization of the early secretory pathway. PMID:21482742

  1. Autophagy inhibition enhances silibinin-induced apoptosis by regulating reactive oxygen species production in human prostate cancer PC-3 cells.

    PubMed

    Kim, Sang-Hun; Kim, Kwang-Youn; Yu, Sun-Nyoung; Park, Seul-Ki; Choi, Hyeun-Deok; Ji, Jae-Hoon; Ahn, Soon-Cheol

    Silibinin is a major bioactive component of silymarin and has anticancer effects on cancer cell line and has been used as a supportive therapy for chronic inflammatory liver condition. These anticancer effects of silibinin have been demonstrated both in vitro and in vivo cancer models. Although various evidences showed apoptosis signaling pathways by silibinin, there is no report to address the clearly mechanism of silibinin-induced autophagy in prostate cancer PC-3 cells. Our study showed that silibinin triggered autophagy through up-regulation of microtubule-associated protein 1 light chain 3 (LC3)-II, formation of acidic vesicular organelles (AVO) and punctuate of GFP-LC3, which was inhibited by 3-methyladenine (3-MA), an inhibitor of specific autophagy. In addition, silibinin induced autophagy through production of reactive oxygen species (ROS). Inhibition of ROS with diphenyleneiodonium (DPI), a ROS inhibitor, attenuated silibinin-triggered autophagy. Inhibition of autophagy with 3-MA enhanced the silibinin-induced apoptosis through the regulation of caspase-3 and PARP. These results suggested that silibinin induced autophagy by regulating ROS and its mechanism played a protective role against apoptosis in PC-3 cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. An improved procedure, involving mass spectrometry, for N-terminal amino acid sequence determination of proteins which are N alpha-blocked.

    PubMed Central

    Rose, K; Kocher, H P; Blumberg, B M; Kolakofsky, D

    1984-01-01

    A modification to a previously described procedure [Gray & del Valle (1970) Biochemistry 9, 2134-2137; Rose, Simona & Offord (1983) Biochem. J. 215, 261-272] for mass-spectral identification of the N-terminal regions of proteins is shown to be useful in cases where the N-terminus is blocked. Three proteins were studied: vesicular-stomatitis-virus N protein, Sendai-virus NP protein, and a rabbit immunoglobulin lambda-light chain. These proteins, found to be blocked at the N-terminus with either the acetyl group or a pyroglutamic acid residue, had all failed to yield to attempted Edman degradation, in one case even after attempted enzymic removal of the pyroglutamic acid residue. The N-terminal regions of all three proteins were sequenced by using the new procedure. PMID:6421284

  3. Intracellular origin and ultrastructure of platelet-derived microparticles.

    PubMed

    Ponomareva, A A; Nevzorova, T A; Mordakhanova, E R; Andrianova, I A; Rauova, L; Litvinov, R I; Weisel, J W

    2017-08-01

    Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles. Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific

  4. Elicitation of anti-vesicular stomatitis virus cytotoxic T lymphocytes by using purified viral and cellular antigens incorporated into phospholipid vesicles.

    PubMed

    Ruebush, M J; Hale, A H; Harris, D T

    1981-05-01

    We evaluated the minimal molecular and cellular requirements for elicitation of anti-vesicular stomatitis virus (VSV) cytotoxic T lymphocytes (CTL). The results indicated that lipid vesicles containing the purified major surface glyco-protein of VSV (G protein) and purified H-2K(k) glycoproteins elicited specific H-2K(k)-restricted anti-VSV CTL. These antiviral CTL were shown to be Ly 1(-),2(+). However, both Ly 1(+),2(-) and Ly1(-),2(+) T-cell subpopulations were shown to be required for elicitation of these CTL.

  5. The tannosome is an organelle forming condensed tannins in the chlorophyllous organs of Tracheophyta.

    PubMed

    Brillouet, Jean-Marc; Romieu, Charles; Schoefs, Benoît; Solymosi, Katalin; Cheynier, Véronique; Fulcrand, Hélène; Verdeil, Jean-Luc; Conéjéro, Geneviève

    2013-10-01

    Condensed tannins (also called proanthocyanidins) are widespread polymers of catechins and are essential for the defence mechanisms of vascular plants (Tracheophyta). A large body of evidence argues for the synthesis of monomeric epicatechin on the cytosolic face of the endoplasmic reticulum and its transport to the vacuole, although the site of its polymerization into tannins remains to be elucidated. The aim of the study was to re-examine the cellular frame of tannin polymerization in various representatives of the Tracheophyta. Light microscopy epifluorescence, confocal microscopy, transmission electron microscopy (TEM), chemical analysis of tannins following cell fractionation, and immunocytochemistry were used as independent methods on tannin-rich samples from various organs from Cycadophyta, Ginkgophyta, Equisetophyta, Pteridophyta, Coniferophyta and Magnoliophyta. Tissues were fixed in a caffeine-glutaraldehyde mixture and examined by TEM. Other fresh samples were incubated with primary antibodies against proteins from both chloroplastic envelopes and a thylakoidal chlorophyll-carrying protein; they were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Coupled spectral analyses of chlorophyll and tannins were carried out by confocal microscopy on fresh tissues and tannin-rich accretions obtained through cell fractionation; chemical analyses of tannins and chlorophylls were also performed on the accretions. The presence of the three different chloroplast membranes inside vacuolar accretions that constitute the typical form of tannin storage in vascular plants was established in fresh tissues as well as in purified organelles, using several independent methods. Tannins are polymerized in a new chloroplast-derived organelle, the tannosome. These are formed by pearling of the thylakoids into 30 nm spheres, which are then encapsulated in a tannosome shuttle formed by budding from the chloroplast and bound by a membrane

  6. The tannosome is an organelle forming condensed tannins in the chlorophyllous organs of Tracheophyta

    PubMed Central

    Brillouet, Jean-Marc; Romieu, Charles; Schoefs, Benoît; Solymosi, Katalin; Cheynier, Véronique; Fulcrand, Hélène; Verdeil, Jean-Luc; Conéjéro, Geneviève

    2013-01-01

    Background and Aims Condensed tannins (also called proanthocyanidins) are widespread polymers of catechins and are essential for the defence mechanisms of vascular plants (Tracheophyta). A large body of evidence argues for the synthesis of monomeric epicatechin on the cytosolic face of the endoplasmic reticulum and its transport to the vacuole, although the site of its polymerization into tannins remains to be elucidated. The aim of the study was to re-examine the cellular frame of tannin polymerization in various representatives of the Tracheophyta. Methods Light microscopy epifluorescence, confocal microscopy, transmission electron microscopy (TEM), chemical analysis of tannins following cell fractionation, and immunocytochemistry were used as independent methods on tannin-rich samples from various organs from Cycadophyta, Ginkgophyta, Equisetophyta, Pteridophyta, Coniferophyta and Magnoliophyta. Tissues were fixed in a caffeine–glutaraldehyde mixture and examined by TEM. Other fresh samples were incubated with primary antibodies against proteins from both chloroplastic envelopes and a thylakoidal chlorophyll-carrying protein; they were also incubated with gelatin–Oregon Green, a fluorescent marker of condensed tannins. Coupled spectral analyses of chlorophyll and tannins were carried out by confocal microscopy on fresh tissues and tannin-rich accretions obtained through cell fractionation; chemical analyses of tannins and chlorophylls were also performed on the accretions. Key Results and Conclusions The presence of the three different chloroplast membranes inside vacuolar accretions that constitute the typical form of tannin storage in vascular plants was established in fresh tissues as well as in purified organelles, using several independent methods. Tannins are polymerized in a new chloroplast-derived organelle, the tannosome. These are formed by pearling of the thylakoids into 30 nm spheres, which are then encapsulated in a tannosome shuttle formed by

  7. A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gumpper, Ryan H.; Li, Weike; Castañeda, Carlos H.

    Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, wasmore » found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth

  8. A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid.

    PubMed

    Gumpper, Ryan H; Li, Weike; Castañeda, Carlos H; Scuderi, M José; Bashkin, James K; Luo, Ming

    2018-04-15

    Polyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus. IMPORTANCE Negative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The

  9. Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

    PubMed Central

    McLelland, Gian-Luca; Soubannier, Vincent; Chen, Carol X; McBride, Heidi M; Fon, Edward A

    2014-01-01

    Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD. PMID:24446486

  10. Differential maturation of vesicular glutamate and GABA transporter expression in the mouse auditory forebrain during the first weeks of hearing.

    PubMed

    Hackett, Troy A; Clause, Amanda R; Takahata, Toru; Hackett, Nicholas J; Polley, Daniel B

    2016-06-01

    Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11-P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1 (+) and VGluT2 (+) transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT (+) transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, perisomatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the

  11. Differential maturation of vesicular glutamate and GABA transporter expression in the mouse auditory forebrain during the first weeks of hearing

    PubMed Central

    Hackett, Troy A.; Clause, Amanda R.; Takahata, Toru; Hackett, Nicholas J.; Polley, Daniel B.

    2015-01-01

    Vesicular transporter proteins are an essential component of the presynaptic machinery that regulates neurotransmitter storage and release. They also provide a key point of control for homeostatic signaling pathways that maintain balanced excitation and inhibition following changes in activity levels, including the onset of sensory experience. To advance understanding of their roles in the developing auditory forebrain, we tracked the expression of the vesicular transporters of glutamate (VGluT1, VGluT2) and GABA (VGAT) in primary auditory cortex (A1) and medial geniculate body (MGB) of developing mice (P7, P11, P14, P21, adult) before and after ear canal opening (~P11–P13). RNA sequencing, in situ hybridization, and immunohistochemistry were combined to track changes in transporter expression and document regional patterns of transcript and protein localization. Overall, vesicular transporter expression changed the most between P7 and P21. The expression patterns and maturational trajectories of each marker varied by brain region, cortical layer, and MGB subdivision. VGluT1 expression was highest in A1, moderate in MGB, and increased with age in both regions. VGluT2 mRNA levels were low in A1 at all ages, but high in MGB, where adult levels were reached by P14. VGluT2 immunoreactivity was prominent in both regions. VGluT1+ and VGluT2+ transcripts were co-expressed in MGB and A1 somata, but co-localization of immunoreactive puncta was not detected. In A1, VGAT mRNA levels were relatively stable from P7 to adult, while immunoreactivity increased steadily. VGAT+ transcripts were rare in MGB neurons, whereas VGAT immunoreactivity was robust at all ages. Morphological changes in immunoreactive puncta were found in two regions after ear canal opening. In the ventral MGB, a decrease in VGluT2 puncta density was accompanied by an increase in puncta size. In A1, peri-somatic VGAT and VGluT1 terminals became prominent around the neuronal somata. Overall, the observed

  12. Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy.

    PubMed

    Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N; Nayak, Debasis

    2018-02-23

    Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment.

  13. Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy

    PubMed Central

    Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N.; Nayak, Debasis

    2018-01-01

    Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment. PMID:29473868

  14. Mechanisms by Which Dietary Fatty Acids Regulate Mitochondrial Structure-Function in Health and Disease.

    PubMed

    Sullivan, E Madison; Pennington, Edward Ross; Green, William D; Beck, Melinda A; Brown, David A; Shaikh, Saame Raza

    2018-05-01

    Mitochondria are the energy-producing organelles within a cell. Furthermore, mitochondria have a role in maintaining cellular homeostasis and proper calcium concentrations, building critical components of hormones and other signaling molecules, and controlling apoptosis. Structurally, mitochondria are unique because they have 2 membranes that allow for compartmentalization. The composition and molecular organization of these membranes are crucial to the maintenance and function of mitochondria. In this review, we first present a general overview of mitochondrial membrane biochemistry and biophysics followed by the role of different dietary saturated and unsaturated fatty acids in modulating mitochondrial membrane structure-function. We focus extensively on long-chain n-3 (ω-3) polyunsaturated fatty acids and their underlying mechanisms of action. Finally, we discuss implications of understanding molecular mechanisms by which dietary n-3 fatty acids target mitochondrial structure-function in metabolic diseases such as obesity, cardiac-ischemia reperfusion injury, obesity, type 2 diabetes, nonalcoholic fatty liver disease, and select cancers.

  15. Phospholipase D Signaling Pathways and Phosphatidic Acid as Therapeutic Targets in Cancer

    PubMed Central

    Bruntz, Ronald C.; Lindsley, Craig W.

    2014-01-01

    Phospholipase D is a ubiquitous class of enzymes that generates phosphatidic acid as an intracellular signaling species. The phospholipase D superfamily plays a central role in a variety of functions in prokaryotes, viruses, yeast, fungi, plants, and eukaryotic species. In mammalian cells, the pathways modulating catalytic activity involve a variety of cellular signaling components, including G protein–coupled receptors, receptor tyrosine kinases, polyphosphatidylinositol lipids, Ras/Rho/ADP-ribosylation factor GTPases, and conventional isoforms of protein kinase C, among others. Recent findings have shown that phosphatidic acid generated by phospholipase D plays roles in numerous essential cellular functions, such as vesicular trafficking, exocytosis, autophagy, regulation of cellular metabolism, and tumorigenesis. Many of these cellular events are modulated by the actions of phosphatidic acid, and identification of two targets (mammalian target of rapamycin and Akt kinase) has especially highlighted a role for phospholipase D in the regulation of cellular metabolism. Phospholipase D is a regulator of intercellular signaling and metabolic pathways, particularly in cells that are under stress conditions. This review provides a comprehensive overview of the regulation of phospholipase D activity and its modulation of cellular signaling pathways and functions. PMID:25244928

  16. Structure, Function, and Assembly of Adhesive Organelles by Uropathogenic Bacteria

    PubMed Central

    Chahales, Peter; Thanassi, David G.

    2015-01-01

    Bacteria assemble a wide range of adhesive proteins, termed adhesins, to mediate binding to receptors and colonization of surfaces. For pathogenic bacteria, adhesins are critical for early stages of infection, allowing the bacteria to initiate contact with host cells, colonize different tissues, and establish a foothold within the host. The adhesins expressed by a pathogen are also critical for bacterial-bacterial interactions and the formation of bacterial communities such as biofilms. The ability to adhere to host tissues is particularly important for bacteria that colonize sites such as the urinary tract, where the flow of urine functions to maintain sterility by washing away non-adherent pathogens. Adhesins vary from monomeric proteins that are directly anchored to the bacterial surface to polymeric, hairlike fibers that extend out from the cell surface. These latter fibers are termed pili or fimbriae, and were among the first identified virulence factors of uropathogenic Escherichia coli. Studies since then have identified a range of both pilus and non-pilus adhesins that contribute to bacterial colonization of the urinary tract, and have revealed molecular details of the structures, assembly pathways, and functions of these adhesive organelles. In this review, we describe the different types of adhesins expressed by both Gram-negative and Gram-positive uropathogens, what is known about their structures, how they are assembled on the bacterial surface, and the functions of specific adhesins in the pathogenesis of urinary tract infections. PMID:26542038

  17. Bidirectional transport of organelles: unity and struggle of opposing motors.

    PubMed

    Bryantseva, Sofiya A; Zhapparova, Olga N

    2012-01-01

    Bidirectional transport along microtubules is ensured by opposing motor proteins: cytoplasmic dynein that drives cargo to the minus-ends and various kinesins that generally move to the plus-ends of microtubules. Regulation of motor proteins that are simultaneously bound to the same organelle is required to maintain directional transport and prevent pausing of cargo pulled away by motors of opposite polarity. Debates of the recent decade have been focused on two possible mechanisms of such regulation: (i) coordination, which implies that only one type of motors is active at a given time, and (ii) tug-of-war, which assumes that both motors are active at the same time and that direction of transport depends on the outcome of motor's confrontation. The initial idea of coordination has been challenged by observations of simultaneous activity of plus- and minus-end-directed motors applied to the same cargo. Analysis of the available data indicates that coordination and tug-of-war theories rather complement than contradict each other: cargo interacts with two teams of active motors, the resulting direction and the winner team are determined by coordination complexes, but the activity of the loser team is never completely inhibited and remains at some background level. Such persisting activity might enhance the overall efficiency of transport by increasing processivity or helping to overcome the obstacles on microtubule track. © The Author(s) Journal compilation © 2012 Portland Press Limited

  18. Vesicular glutamate transporters, VGluT1 and VGluT2, in the trigeminal ganglion neurons of the rat, with special reference to coexpression.

    PubMed

    Li, Jin-Lian; Xiong, Kang-Hui; Dong, Yu-Lin; Fujiyama, Fumino; Kaneko, Takeshi; Mizuno, Noboru

    2003-08-18

    Vesicular glutamate transporters are responsible for glutamate transport into synaptic vesicles. In the present study, we examined immunohistochemically the expression of vesicular glutamate transporters, VGluT1 and VGluT2, in trigeminal ganglion neurons of the rat. Immunohistochemistry for VGluT1 and VGluT2 indicated that more than 80% of trigeminal ganglion neurons express VGluT1 and/or VGluT2 in their cell bodies. It also indicated that large and small trigeminal ganglion neurons express VGluT2 more frequently than VGluT1. Dual immunofluorescence histochemistry for VGluT1 and VGluT2 indicated that trigeminal ganglion neurons express VGluT2 more frequently than VGluT1 and that more than 80% of VGluT-expressing trigeminal ganglion neurons express VGluT1 and VGluT2. Many axon terminals in the superficial layers of the medullary dorsal horn also showed VGluT1 and VGluT2 immunoreactivities. Some of these axon terminals were confirmed to form the central core of the synaptic glomerulus. These results indicated that VGluT1 and VGluT2 are coexpressed in the cell bodies and axon terminals in most trigeminal ganglion neurons. Copyright 2003 Wiley-Liss, Inc.

  19. Gamma-aminobutyric acid and related molecules in the sea fan Eunicella cavolini (Cnidaria: Octocorallia): a biochemical and immunohistochemical approach.

    PubMed

    Girosi, Laura; Ferrando, Sara; Beltrame, Francesco; Ciarcia, Gaetano; Diaspro, Alberto; Fato, Marco; Magnone, Mirko; Raiteri, Luca; Ramoino, Paola; Tagliafierro, Grazia

    2007-07-01

    The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

  20. Vesicular stomatitis virus-based Ebola vaccines with improved cross-protective efficacy.

    PubMed

    Marzi, Andrea; Ebihara, Hideki; Callison, Julie; Groseth, Allison; Williams, Kinola J; Geisbert, Thomas W; Feldmann, Heinz

    2011-11-01

    For Ebola virus (EBOV), 4 different species are known: Zaire, Sudan, Côte d'Ivoire, and Reston ebolavirus. The newly discovered Bundibugyo ebolavirus has been proposed as a 5th species. So far, no cross-neutralization among EBOV species has been described, aggravating progress toward cross-species protective vaccines. With the use of recombinant vesicular stomatitis virus (rVSV)-based vaccines, guinea pigs could be protected against Zaire ebolavirus (ZEBOV) infection only when immunized with a vector expressing the homologous, but not a heterologous, EBOV glycoprotein (GP). However, infection of guinea pigs with nonadapted wild-type strains of the different species resulted in full protection of all animals against subsequent challenge with guinea pig-adapted ZEBOV, showing that cross-species protection is possible. New vectors were generated that contain EBOV viral protein 40 (VP40) or EBOV nucleoprotein (NP) as a second antigen expressed by the same rVSV vector that encodes the heterologous GP. After applying a 2-dose immunization approach, we observed an improved cross-protection rate, with 5 of 6 guinea pigs surviving the lethal ZEBOV challenge if vaccinated with rVSV-expressing SEBOV-GP and -VP40. Our data demonstrate that cross-protection between the EBOV species can be achieved, although EBOV-GP alone cannot induce the required immune response.

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