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Sample records for modulates phagosome maturation

  1. Phagosome maturation: aging gracefully.

    PubMed Central

    Vieira, Otilia V; Botelho, Roberto J; Grinstein, Sergio

    2002-01-01

    Foreign particles and apoptotic bodies are eliminated from the body by phagocytic leucocytes. The initial stage of the elimination process is the internalization of the particles into a plasma membrane-derived vacuole known as the phagosome. Such nascent phagosomes, however, lack the ability to kill pathogens or to degrade the ingested targets. These properties are acquired during the course of phagosomal maturation, a complex sequence of reactions that result in drastic remodelling of the phagosomal membrane and contents. The determinants and consequences of the fusion and fission reactions that underlie phagosomal maturation are the topic of this review. PMID:12061891

  2. Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila.

    PubMed

    Fortier, Anne; de Chastellier, Chantal; Balor, Stéphanie; Gros, Philippe

    2007-04-01

    Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized

  3. Role of COPI in phagosome maturation.

    PubMed

    Botelho, R J; Hackam, D J; Schreiber, A D; Grinstein, S

    2000-05-26

    Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

  4. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited

    PubMed Central

    1995-01-01

    We have used the cryosection immunogold technique to study the composition of the Mycobacterium tuberculosis phagosome. We have used quantitative immunogold staining to determine the distribution of several known markers of the endosomal-lysosomal pathway in human monocytes after ingestion of either M. tuberculosis, Legionella pneumophila, or polystyrene beads. Compared with the other phagocytic particles studied, the M. tuberculosis phagosome exhibits delayed clearance of major histocompatibility complex (MHC) class I molecules, relatively intense staining for MHC class II molecules and the endosomal marker transferrin receptor, and relatively weak staining for the lysosomal membrane glycoproteins, CD63, LAMP-1, and LAMP-2 and the lysosomal acid protease, cathepsin D. In contrast to M. tuberculosis, the L. pneumophila phagosome rapidly clears MHC class I molecules and excludes all endosomal-lysosomal markers studied. In contrast to both live M. tuberculosis and L. pneumophila phagosomes, phagosomes containing either polystyrene beads or heat-killed M. tuberculosis stain intensely for lysosomal membrane glycoproteins and cathepsin D. These findings suggest that (a) M. tuberculosis retards the maturation of its phagosome along the endosomal-lysosomal pathway and resides in a compartment with endosomal, as opposed to lysosomal, characteristics; and (b) the intraphagosomal pathway, i.e., the pathway followed by several intracellular parasites that inhibit phagosome-lysosome fusion, is heterogeneous. PMID:7807006

  5. Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

    PubMed

    Mehta, Mansi; Rajmani, Raju S; Singh, Amit

    2016-02-05

    The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (EMSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in EMSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic EMSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

  6. Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence*

    PubMed Central

    Mehta, Mansi; Rajmani, Raju S.; Singh, Amit

    2016-01-01

    The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (EMSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in EMSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic EMSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis. PMID:26637353

  7. Role of JAK-STAT signaling in maturation of phagosomes containing Staphylococcus aureus

    PubMed Central

    Zhu, Fei; Zhou, Yadong; Jiang, Chunxia; Zhang, Xiaobo

    2015-01-01

    Phagocytosis is a required mechanism for the defense against pathogens. Staphylococcus aureus, an important bacterial pathogen, can promptly escape from phagosomes and proliferate within the cytoplasm of host. However, the mechanism of phagocytosis against S. aureus has not been intensively investigated. In this study, the S. aureus was engulfed by macrophages (RAW264.7 cells) but not digested by the cells, suggesting that the phagosomes did not maturate in macrophages. Further investigation revealed that peptidoglycan (PG) induced the phagosome maturation of macrophages, resulting in the eradication of S. aureus. Genome-wide analysis and quantitative real-time PCR indicated that the JAK-STAT pathway was activated by PG during the phagosome maturation of macrophages against S. aureus. This finding presented that the PG-activated JAK-STAT pathway was required for phagosome maturation. Therefore, our study contributed evidence that revealed a novel aspect of PG-triggered JAK-STAT pathway in the phagosome maturation of macrophages. PMID:26442670

  8. The HIV-1 protein Vpr impairs phagosome maturation by controlling microtubule-dependent trafficking

    PubMed Central

    Dumas, Audrey; Lê-Bury, Gabrielle; Marie-Anaïs, Florence; Herit, Floriane; Mazzolini, Julie; Guilbert, Thomas; Bourdoncle, Pierre; Russell, David G.; Benichou, Serge; Zahraoui, Ahmed

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) impairs major functions of macrophages but the molecular basis for this defect remains poorly characterized. Here, we show that macrophages infected with HIV-1 were unable to respond efficiently to phagocytic triggers and to clear bacteria. The maturation of phagosomes, defined by the presence of late endocytic markers, hydrolases, and reactive oxygen species, was perturbed in HIV-1–infected macrophages. We showed that maturation arrest occurred at the level of the EHD3/MICAL-L1 endosomal sorting machinery. Unexpectedly, we found that the regulatory viral protein (Vpr) was crucial to perturb phagosome maturation. Our data reveal that Vpr interacted with EB1, p150Glued, and dynein heavy chain and was sufficient to critically alter the microtubule plus end localization of EB1 and p150Glued, hence altering the centripetal movement of phagosomes and their maturation. Thus, we identify Vpr as a modulator of the microtubule-dependent endocytic trafficking in HIV-1–infected macrophages, leading to strong alterations in phagolysosome biogenesis. PMID:26504171

  9. Monitoring time-dependent maturation changes in purified phagosomes from Dictyostelium discoideum.

    PubMed

    Dieckmann, Régis; Gopaldass, Navin; Escalera, Caroline; Soldati, Thierry

    2008-01-01

    The amoeba Dictyostelium discoideum is an established model to study phagocytosis. The sequence of events leading to the internalization and degradation of a particle is conserved in D. discoideum compared to metazoan cells. As its small haploid genome has been sequenced, it is now amenable to genome-wide analysis including organelle proteomics. Therefore, we adapted to Dictyostelium the classical protocol to purify phagosomes formed by ingestion of latex beads particles. The pulse-chase protocol detailed here gives easy access to pure, intact, and synchronized phagosomes from representative stages of the entire process of phagosome maturation. Recently, this protocol was used to generate individual temporal profiles of proteins and lipids during phagosome maturation generating a proteomic fingerprint of six maturation stages (1). In addition, immunolabeling of phagosomes on a coverslip was developed to visualize and quantitate antigen distribution at the level of individual phagosomes.

  10. Cutting edge: FYCO1 recruitment to dectin-1 phagosomes is accelerated by light chain 3 protein and regulates phagosome maturation and reactive oxygen production.

    PubMed

    Ma, Jun; Becker, Courtney; Reyes, Christopher; Underhill, David M

    2014-02-15

    L chain 3 (LC3)-associated phagocytosis is a process in which LC3, a protein canonically involved in engulfing intracellular materials (autophagy), is recruited to traditional phagosomes during internalization of extracellular payloads. LC3's association with phagosomes has been implicated in regulating microbial killing, Ag processing, and phagosome maturation; however, the mechanism by which LC3 influences these processes has not been clear. In this study, we report that FYVE and coiled-coil domain containing 1 (FYCO1), a protein previously implicated in autophagosome trafficking, is recruited directly by LC3 to Dectin-1 phagosomes. During LC3-associated phagocytosis, FYCO1 recruitment facilitates maturation of early p40phox(+) phagosomes into late LAMP1(+) phagosomes. When FYCO1 is lacking, phagosomes stay p40phox(+) longer and produce more reactive oxygen.

  11. Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

    PubMed

    Egami, Youhei; Araki, Nobukazu

    2012-01-01

    Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

  12. Phagosome maturation during endosome interaction revealed by partial rhodopsin processing in retinal pigment epithelium

    PubMed Central

    Wavre-Shapton, Silène T.; Meschede, Ingrid P.; Seabra, Miguel C.; Futter, Clare E.

    2014-01-01

    ABSTRACT Defects in phagocytosis and degradation of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) are associated with aging and retinal disease. The daily burst of rod outer segment (ROS) phagocytosis by the RPE provides a unique opportunity to analyse phagosome processing in vivo. In mouse retinae, phagosomes containing stacked rhodopsin-rich discs were identified by immuno-electron microscopy. Early apical phagosomes stained with antibodies against both cytoplasmic and intradiscal domains of rhodopsin. During phagosome maturation, a remarkably synchronised loss of the cytoplasmic epitope coincided with movement to the cell body and preceded phagosome–lysosome fusion and disc degradation. Loss of the intradiscal rhodopsin epitope and disc digestion occurred upon fusion with cathepsin-D-positive lysosomes. The same sequential stages of phagosome maturation were identified in cultured RPE and macrophages challenged with isolated POS. Loss of the cytoplasmic rhodopsin epitope was insensitive to pH but sensitive to protease inhibition and coincided with the interaction of phagosomes with endosomes. Thus, during pre-lysosomal maturation of ROS-containing phagosomes, limited rhodopsin processing occurs upon interaction with endosomes. This potentially provides a sensitive readout of phagosome–endosome interactions that is applicable to multiple phagocytes. PMID:25074813

  13. Proteomics fingerprinting of phagosome maturation and evidence for the role of a Galpha during uptake.

    PubMed

    Gotthardt, Daniel; Blancheteau, Vincent; Bosserhoff, Armin; Ruppert, Thomas; Delorenzi, Mauro; Soldati, Thierry

    2006-12-01

    Phagocytosis, whether of food particles in protozoa or bacteria and cell remnants in the metazoan immune system, is a conserved process. The particles are taken up into phagosomes, which then undergo complex remodeling of their components, called maturation. By using two-dimensional gel electrophoresis and mass spectrometry combined with genomic data, we identified 179 phagosomal proteins in the amoeba Dictyostelium, including components of signal transduction, membrane traffic, and the cytoskeleton. By carrying out this proteomics analysis over the course of maturation, we obtained time profiles for 1,388 spots and thus generated a dynamic record of phagosomal protein composition. Clustering of the time profiles revealed five clusters and 24 functional groups that were mapped onto a flow chart of maturation. Two heterotrimeric G protein subunits, Galpha4 and Gbeta, appeared at the earliest times. We showed that mutations in the genes encoding these two proteins produce a phagocytic uptake defect in Dictyostelium. This analysis of phagosome protein dynamics provides a reference point for future genetic and functional investigations.

  14. Mycobacterium bovis Requires P27 (LprG) To Arrest Phagosome Maturation and Replicate within Bovine Macrophages

    PubMed Central

    Vázquez, Cristina Lourdes; Bianco, María Verónica; Blanco, Federico Carlos; Forrellad, Marina Andrea; Gutierrez, Maximiliano Gabriel

    2016-01-01

    ABSTRACT Mycobacterium bovis causes tuberculosis in a wide variety of mammals, with strong tropism for cattle and eventually humans. P27, also called LprG, is among the proteins involved in the mechanisms of the virulence and persistence of M. bovis and Mycobacterium tuberculosis. Here, we describe a novel function of P27 in the interaction of M. bovis with its natural host cell, the bovine macrophage. We found that a deletion in the p27-p55 operon impairs the replication of M. bovis in bovine macrophages. Importantly, we show for the first time that M. bovis arrests phagosome maturation in a process that depends on P27. This effect is P27 specific since complementation with wild-type p27 but not p55 fully restored the wild-type phenotype of the mutant strain; this indicates that P55 plays no important role during the early events of M. bovis infection. In addition, we also showed that the presence of P27 from M. smegmatis decreases the association of LAMP-3 with bead phagosomes, indicating that P27 itself blocks phagosome-lysosome fusion by modulating the traffic machinery in the cell host. PMID:28031264

  15. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

    PubMed

    Wyroba, E; Surmacz, L; Osinska, M; Wiejak, J

    2007-01-01

    Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  16. Infection of porcine bone marrow-derived macrophages by porcine respiratory and reproductive syndrome virus impairs phagosomal maturation.

    PubMed

    Chaudhuri, Sibapriya; McKenna, Neil; Balce, Dale R; Yates, Robin M

    2016-03-01

    Porcine reproductive and respiratory syndrome virus (PRRSV), a positive-sense, ssRNA virus of the genus Arterivirus, is a devastating disease of swine worldwide. Key early targets of PRRSV infection in pigs include professional phagocytes in the lung, such as alveolar and interstitial macrophages and dendritic cells, the dysfunction of which is believed to be responsible for much of the associated mortality. In order to study the effect of virus infection on phagocyte function, the development of a robust, reproducible model would be advantageous. Given the limitations of current models, we set out to develop a porcine bone marrow-derived macrophage (PBMMΦ) cell model to study phagosomal maturation and function during PRRSV infection. Derivation of PBMMΦs from marrow using cultured L929 fibroblast supernatant produced a homogeneous population of cells that exhibited macrophage-like morphology and proficiency in Fc-receptor-mediated phagocytosis and phagosomal maturation. PBMMΦs were permissive to PRRSV infection, resulting in a productive infection that peaked at 24 h. Assessment of the effect of PRRSV infection on the properties of phagosomal maturation in PBMMΦs revealed a significant decrease in phagosomal proteolysis and lowered production of reactive oxygen species, but no change in PBMMΦ viability, phagocytosis or the ability of phagosomes to acidify. In this study, we present a new model to investigate PRRSV infection of phagocytes, which demonstrates a significant effect on phagosomal maturation with the associated implications on proper macrophage function. This model can also be used to study the effect on the phagosomal microenvironment of infection by other viruses targeting porcine macrophages.

  17. Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages

    PubMed Central

    Choi, Seoung-ryoung; Britigan, Bradley E.; Moran, David M.

    2017-01-01

    New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs. PMID:28542623

  18. Gallium nanoparticles facilitate phagosome maturation and inhibit growth of virulent Mycobacterium tuberculosis in macrophages.

    PubMed

    Choi, Seoung-Ryoung; Britigan, Bradley E; Moran, David M; Narayanasamy, Prabagaran

    2017-01-01

    New treatments and novel drugs are required to counter the growing problem of drug-resistant strains of Mycobacterium tuberculosis (M.tb). Our approach against drug resistant M.tb, as well as other intracellular pathogens, is by targeted drug delivery using nanoformulations of drugs already in use, as well as drugs in development. Among the latter are gallium (III) (Ga)-based compounds. In the current work, six different types of Ga and rifampin nanoparticles were prepared in such a way as to enhance targeting of M.tb infected-macrophages. They were then tested for their ability to inhibit growth of a fully pathogenic strain (H37Rv) or a non-pathogenic strain (H37Ra) of M.tb. Encapsulating Ga in folate- or mannose-conjugated block copolymers provided sustained Ga release for 15 days and significantly inhibited M.tb growth in human monocyte-derived macrophages. Nanoformulations with dendrimers encapsulating Ga or rifampin also showed promising anti-tuberculous activity. The nanoparticles co-localized with M.tb containing phagosomes, as measured by detection of mature cathepsin D (34 kDa, lysosomal hydrogenase). They also promoted maturation of the phagosome, which would be expected to increase macrophage-mediated killing of the organism. Delivery of Ga or rifampin in the form of nanoparticles to macrophages offers a promising approach for the development of new therapeutic anti-tuberculous drugs.

  19. Cholesterol glucosylation by Helicobacter pylori delays internalization and arrests phagosome maturation in macrophages.

    PubMed

    Du, Shin-Yi; Wang, Hung-Jung; Cheng, Hsin-Hung; Chen, Sheng-De; Wang, Lily Hui-Ching; Wang, Wen-Ching

    2016-10-01

    Helicobacter pylori colonizes the human stomach and contributes to chronic inflammation of the gastric mucosa. H. pylori persistence occurs because of insufficient eradication by phagocytic cells. A key factor of H. pylori, cholesterol-α-glucosyltransferase encoded by capJ that extracts host cholesterol and converts it to cholesteryl glucosides, is important to evade host immunity. Here, we examined whether phagocytic trafficking in macrophages was perturbed by capJ-carrying H. pylori. J774A.1 cells were infected with H. pylori at a multiplicity of infection of 50. Live-cell imaging and confocal microscopic analysis were applied to monitor the phagocytic trafficking events. The viability of H. pylori inside macrophages was determined by using gentamicin colony-forming unit assay. The phagocytic routes were characterized by using trafficking-intervention compounds. Wild type (WT) H. pylori exhibited more delayed entry into macrophages and also arrested phagosome maturation more than did capJ knockout mutant. Pretreatment of genistein and LY294002 prior to H. pylori infection reduced the internalization of WT but not capJ-knockout H. pylori in macrophages. Cholesterol glucosylation by H. pylori interferes with phagosome trafficking via a lipid-raft and PI3K-dependent manner, which retards engulfment of bacteria for prolonged intracellular survival of H. pylori. Copyright © 2014. Published by Elsevier B.V.

  20. IL-10 Produced by Trophoblast Cells Inhibits Phagosome Maturation Leading to Profound Intracellular Proliferation of Salmonella enterica Typhimurium

    PubMed Central

    Nguyen, Tina; Robinson, Nirmal; Allison, Sarah E.; Coombes, Brian K; Sad, Subash; Krishnan, Lakshmi

    2013-01-01

    Introduction Salmonella enterica Typhimurium (ST) is a phagosomal pathogen that can infect placental trophoblast cells leading to abortion and severe maternal illness. It is unclear how the trophoblast cells promote profound bacterial proliferation. Methods The mechanism of internalization, intracellular growth and phagosomal biogenesis in ST-infected human epithelial (HeLa), macrophage (THP-1) and trophoblast-derived cell lines (JEG-3, BeWo and HTR-8) was studied. Specific inhibitors were used to block bacterial internalization. Phagosomal maturation was determined by confocal microscopy, western-blotting and release of lysosomal β-galactosidase by infected cells. Bacterial colony forming units were determined by plating infected cell lysates on agar plates. Results ST proliferated minimally in macrophages but replicated profoundly within trophoblast cells. The ST-∆invA (a mutant of Salmonella pathogenicity island-1 gene effector proteins) was unable to infect epithelial.cells, but was internalized by scavenger receptors on trophoblasts and macrophages. However, ST was contrastingly localized in early (Rab5+) or late (LAMP1+) phagosomes within trophoblast cells and macrophages respectively. Furthermore trophoblast cells (unlike macrophages) did not exhibit phagoso-lysosomal fusion. ST-infected macrophages produced IL-6 whereas trophoblast cells produced IL-10. Neutralizing IL-10 in JEG-3 cells accelerated phagolysomal fusion and reduced proliferation of ST. Placental bacterial burden was curtailed in vivo in anti-IL-10 antibody treated and IL-10-deficient mice. Discussion Macrophages phagocytose but curtail intracellular replication of ST in late phagosomes. In contrast, phagocytosis by trophoblast cells results in an inappropriate cytokine response and proliferation of ST in early phagosomes. Conclusion IL-10 production by trophoblast cells that delays phagosomal maturation may facilitate proliferation of pathogens in placental cells. PMID:23834952

  1. A Conserved Role for SNX9-Family Members in the Regulation of Phagosome Maturation during Engulfment of Apoptotic Cells

    PubMed Central

    Kinchen, Jason M.; Kaech, Andres; Ravichandran, Kodi S.; Hengartner, Michael O.

    2011-01-01

    Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution. PMID:21494661

  2. Attenuated Leishmania induce pro-inflammatory mediators and influence leishmanicidal activity by p38 MAPK dependent phagosome maturation in Leishmania donovani co-infected macrophages.

    PubMed

    Banerjee, Somenath; Bose, Dipayan; Chatterjee, Nabanita; Das, Subhadip; Chakraborty, Sreeparna; Das, Tanya; Saha, Krishna Das

    2016-03-01

    Promastigote form of Leishmania, an intracellular pathogen, delays phagosome maturation and resides inside macrophages. But till date limited study has been done to manipulate the phagosomal machinery of macrophages to restrict Leishmania growth. Attenuated Leishmania strain exposed RAW 264.7 cells showed a respiratory burst and enhanced production of pro-inflammatory mediators. The augmentation of pro-inflammatory activity is mostly attributed to p38 MAPK and p44/42 MAPK. In our study, these activated macrophages are found to induce phagosome maturation when infected with pathogenic Leishmania donovani. Increased co-localization of carboxyfluorescein succinimidyl ester labeled pathogenic L. donovani with Lysosome was found. Moreover, increased co-localization was observed between pathogenic L. donovani and late phagosomal markers viz. Rab7, Lysosomal Associated Membrane Protein 1, Cathepsin D, Rab9, and V-ATPase which indicate phagosome maturation. It was also observed that inhibition of V-type ATPase caused significant hindrance in attenuated Leishmania induced phagosome maturation. Finally, it was confirmed that p38 MAPK is the key player in acidification and maturation of phagosome in attenuated Leishmania strain pre-exposed macrophages. To our knowledge, this study for the first time reported an approach to induce phagosome maturation in L. donovani infected macrophages which could potentiate short-term prophylactic response in future.

  3. Mycobacterium requires an all-around closely apposing phagosome membrane to maintain the maturation block and this apposition is re-established when it rescues itself from phagolysosomes.

    PubMed

    de Chastellier, Chantal; Forquet, Frédérique; Gordon, Alon; Thilo, Lutz

    2009-08-01

    Pathogenic mycobacteria survive in macrophages of the host organism by residing in phagosomes which they prevent from undergoing maturation and fusion with lysosomes. Several molecular mechanisms have been associated with the phagosome maturation block. Here we show for Mycobacterium avium in mouse bone marrow-derived macrophages that the maturation block required an all-around close apposition between the mycobacterial surface and the phagosome membrane. When small (0.1 microm) latex beads were covalently attached to the mycobacterial surface to act as a spacer that interfered with a close apposition, phagosomes rapidly acquired lysosomal characteristics as indicators for maturation and fusion with lysosomes. As a result, several mycobacteria were delivered into single phagolysosomes. Detailed electron-microscope observations of phagosome morphology over a 7-day post-infection period showed a linear correlation between bead attachment and phagosome-lysosome fusion. After about 3 days post infection, conditions inside phagolysosomes caused a gradual release of beads. This allowed mycobacteria to re-establish a close apposition with the surrounding membrane and sequester themselves into individual, non-maturing phagosomes which had lost lysosomal characteristics. By rescuing themselves from phagolysosomes, mycobacteria remained fully viable and able to multiply at the normal rate. In order to unify the present observations and previously reported mechanisms for the maturation block, we discuss evidence that they may act synergistically to interfere with 'Phagosome Membrane Economics' by causing relative changes in incoming and outgoing endocytic membrane fluxes.

  4. The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

    PubMed

    Verma, Kuldeep; Datta, Sunando

    2017-03-24

    One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Secreted Acid Phosphatase (SapM) of Mycobacterium tuberculosis Is Indispensable for Arresting Phagosomal Maturation and Growth of the Pathogen in Guinea Pig Tissues

    PubMed Central

    Puri, Rupangi Verma; Reddy, P. Vineel; Tyagi, Anil K.

    2013-01-01

    Tuberculosis (TB) is responsible for nearly 1.4 million deaths globally every year and continues to remain a serious threat to human health. The problem is further complicated by the growing incidence of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB), emphasizing the need for the development of new drugs against this disease. Phagosomal maturation arrest is an important strategy employed by Mycobacterium tuberculosis to evade the host immune system. Secretory acid phosphatase (SapM) of M.tuberculosis is known to dephosphorylate phosphotidylinositol 3-phosphate (PI3P) present on phagosomes. However, there have been divergent reports on the involvement of SapM in phagosomal maturation arrest in mycobacteria. This study was aimed at reascertaining the involvement of SapM in phagosomal maturation arrest in M.tuberculosis. Further, for the first time, we have also studied whether SapM is essential for the pathogenesis of M.tuberculosis. By deleting the sapM gene of M.tuberculosis, we demonstrate that MtbΔsapM is defective in the arrest of phagosomal maturation as well as for growth in human THP-1 macrophages. We further show that MtbΔsapM is severely attenuated for growth in the lungs and spleen of guinea pigs and has a significantly reduced ability to cause pathological damage in the host when compared with the parental strain. Also, the guinea pigs infected with MtbΔsapM exhibited a significantly enhanced survival when compared with M.tuberculosis infected animals. The importance of SapM in phagosomal maturation arrest as well as in the pathogenesis of M.tuberculosis establishes it as an attractive target for the development of new therapeutic molecules against tuberculosis. PMID:23923000

  6. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization.

    PubMed

    Akhter, Anwari; Caution, Kyle; Abu Khweek, Arwa; Tazi, Mia; Abdulrahman, Basant A; Abdelaziz, Dalia H A; Voss, Oliver H; Doseff, Andrea I; Hassan, Hoda; Azad, Abul K; Schlesinger, Larry S; Wewers, Mark D; Gavrilin, Mikhail A; Amer, Amal O

    2012-07-27

    Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

  7. Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization

    PubMed Central

    Akhter, Anwari; Caution, Kyle; Khweek, Arwa Abu; Tazi, Mia; Abdulrahman, Basant A.; Abdelaziz, Dalia H.A.; Voss, Oliver H.; Doseff, Andrea I.; Hassan, Hoda; Azad, Abul K.; Schlesinger, Larry S.; Wewers, Mark D.; Gavrilin, Mikhail A.; Amer, Amal O.

    2012-01-01

    Summary Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins. Yet, its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and 5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila- vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing non-pathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo. PMID:22658523

  8. Naturally produced opsonizing antibodies restrict the survival of Mycobacterium tuberculosis in human macrophages by augmenting phagosome maturation

    PubMed Central

    Kumar, Shashi Kant; Singh, Padam; Sinha, Sudhir

    2015-01-01

    This study investigated the hypothesis that serum antibodies against Mycobacterium tuberculosis present in naturally infected healthy subjects of a tuberculosis (TB) endemic area could create and/or sustain the latent form of infection. All five apparently healthy Indian donors showed high titres of serum antibodies against M. tuberculosis cell membrane antigens, including lipoarabinomannan and alpha crystallin. Uptake and killing of bacilli by the donor macrophages was significantly enhanced following their opsonization with antibody-rich, heat-inactivated autologous sera. However, the capability to opsonize was apparent for antibodies against some and not other antigens. High-content cell imaging of infected macrophages revealed significantly enhanced colocalization of the phagosome maturation marker LAMP-1, though not of calmodulin, with antibody-opsonized compared with unopsonized M. tuberculosis. Key enablers of macrophage microbicidal action—proinflammatory cytokines (IFN-γ and IL-6), phagosome acidification, inducible NO synthase and nitric oxide—were also significantly enhanced following antibody opsonization. Interestingly, heat-killed M. tuberculosis also elevated these mediators to the levels comparable to, if not higher than, opsonized M. tuberculosis. Results of the study support the emerging view that an efficacious vaccine against TB should, apart from targeting cell-mediated immunity, also generate ‘protective’ antibodies. PMID:26674415

  9. Saxifragifolin D attenuates phagosome maturation arrest in Mycobacterium tuberculosis-infected macrophages via an AMPK and VPS34-dependent pathway.

    PubMed

    Zhou, Jia; Xu, Rui; Du, Xian-Zhi; Zhou, Xiang-Dong; Li, Qi

    2017-12-01

    Saxifragifolin D (SD), a traditional Chinese medicine, is a pentacyclic triterpenoid compound first isolated from Androsace umbellata. Various plant triterpenoids have been reported to exhibit antitubercular activity. In this study, THP-1-derived macrophages were infected with an attenuated M. tuberculosis (M.tb) strain, H37Ra. Intracellular replication of M.tb was evaluated by counting the colonies after 4 weeks of incubation. The results indicated that SD treatment reduced the intracellular replication of M.tb in THP-1-derived macrophages but not in A549 cells. We performed a phagosome maturation test using confocal microscopy and found that SD treatment partially attenuated the phagosome arrest induced by M.tb infection. These effects were dependent on a VPS34-associated pathway. Immunoprecipitation assays showed that SD increased intracellular UVRAG-linked VPS34, the active VPS34 complex II. However, SD had no effect on the total VPS34 pool. Moreover, the results indicated that the SD-mediated increase in VPS34 complex II activity was mediated by an AMPK-dependent pathway. Collectively, these data indicate that SD may be a promising candidate for treatment of M.tb.

  10. Cholesterol depletion in Mycobacterium avium-infected macrophages overcomes the block in phagosome maturation and leads to the reversible sequestration of viable mycobacteria in phagolysosome-derived autophagic vacuoles.

    PubMed

    de Chastellier, Chantal; Thilo, Lutz

    2006-02-01

    Phagocytic entry of mycobacteria into macrophages requires the presence of cholesterol in the plasma membrane. This suggests that pathogenic mycobacteria may require cholesterol for their subsequent intra-cellular survival in non-maturing phagosomes. Here we report on the effect of cholesterol depletion on pre-existing phagosomes in mouse bone marrow-derived macrophages infected with Mycobacterium avium. Cholesterol depletion with methyl-beta-cyclodextrin resulted in a loosening of the close apposition between the phagosome membrane and the mycobacterial surface, followed by fusion with lysosomes. The resulting phagolysosomes then autonomously executed autophagy, which did not involve the endoplasmic reticulum. After 5 h of depletion, intact mycobacteria had accumulated in large auto-phagolysosomes. Autophagy was specific for phagolysosomes that contained mycobacteria, as it did not involve latex bead-containing phagosomes in infected cells. Upon replenishment of cholesterol, mycobacteria became increasingly aligned to the lysosomal membrane, from where they were individually sequestered in phagosomes with an all-around closely apposed phagosome membrane and which no longer fused with lysosomes. These observations indicate that, cholesterol depletion (i) resulted in phagosome maturation and fusion with lysosomes and (ii) caused mycobacterium-containing phagolysosomes to autonomously undergo autophagy. Furthermore, (iii) mycobacteria were not killed in auto-phagolysosomes, and (iv) cholesterol replenishment enabled mycobacterium to rescue itself from autophagic phagolysosomes to again reside individually in phagosomes which no longer fused with lysosomes.

  11. CLIC1 regulates dendritic cell antigen processing and presentation by modulating phagosome acidification and proteolysis

    PubMed Central

    Salao, Kanin; Jiang, Lele; Li, Hui; Tsai, Vicky W.-W.; Husaini, Yasmin; Curmi, Paul M. G.; Brown, Louise J.; Brown, David A.

    2016-01-01

    ABSTRACT Intracellular chloride channel protein 1 (CLIC1) participates in inflammatory processes by regulating macrophage phagosomal functions such as pH and proteolysis. Here, we sought to determine if CLIC1 can regulate adaptive immunity by actions on dendritic cells (DCs), the key professional antigen presenting cells. To do this, we first generated bone marrow-derived DCs (BMDCs) from germline CLIC1 gene-deleted (CLIC1−/−) and wild-type (CLIC1+/+) mice, then studied them in vitro and in vivo. We found phagocytosis triggered cytoplasmic CLIC1 translocation to the phagosomal membrane where it regulated phagosomal pH and proteolysis. Phagosomes from CLIC1−/− BMDCs displayed impaired acidification and proteolysis, which could be reproduced if CLIC1+/+, but not CLIC1−/− cells, were treated with IAA94, a CLIC family ion channel blocker. CLIC1−/− BMDC displayed reduced in vitro antigen processing and presentation of full-length myelin oligodendrocyte glycoprotein (MOG) and reduced MOG-induced experimental autoimmune encephalomyelitis. These data suggest that CLIC1 regulates DC phagosomal pH to ensure optimal processing of antigen for presentation to antigen-specific T-cells. Further, they indicate that CLIC1 is a novel therapeutic target to help reduce the adaptive immune response in autoimmune diseases. PMID:27113959

  12. Identification of the Mycobacterium tuberculosis protein PE-PGRS62 as a novel effector that functions to block phagosome maturation and inhibit iNOS expression.

    PubMed

    Thi, Emily P; Hong, Chris Joon Ho; Sanghera, Gaganjit; Reiner, Neil E

    2013-05-01

    Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.

  13. Human phagosome processing of Mycobacterium tuberculosis antigens is modulated by interferon-γ and interleukin-10

    PubMed Central

    Bobadilla, Karen; Sada, Eduardo; Jaime, Maria E; González, Yolanda; Ramachandra, Lakshmi; Rojas, Roxana E; Pedraza-Sánchez, Sigifredo; Michalak, Colette; González-Noriega, Alfonso; Torres, Martha

    2013-01-01

    Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen–MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide–MHC-II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide–MHC-II complexes in M. tuberculosis-infected human monocyte-derived macrophages (MDMs) using autologous M. tuberculosis-specific CD4+ T cells. The MDMs were pre-treated with either IFN-γ or IL-10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M. tuberculosis -specific CD4+ T cells. Our results demonstrated that in MDMs pre-treated with IFN-γ, M. tuberculosis peptide–MHC-II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN-γ, the complexes were detected in the endosomal fractions. In MDMs pre-treated with IL-10, the M. tuberculosis peptide–MHC-II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA-DR at the cell surface only in the IFN-γ-treated MDMs, suggesting that IFN-γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL-10 favours the trafficking of Ag85B to vesicles that do not contain LAMP-1. Therefore, IFN-γ and IL-10 play a role in the formation and trafficking of M. tuberculosis peptide–MHC-II complexes. PMID:22924705

  14. Dectin-1 Controls TLR9 Trafficking to Phagosomes containing β-1,3 glucan123

    PubMed Central

    Khan, Nida S.; Kasperkovitz, Pia V.; Timmons, Allison K.; Mansour, Michael K.; Tam, Jenny M.; Seward, Michael W.; Reedy, Jennifer L.; Puranam, Sravanthi; Feliu, Marianela; Vyas, Jatin M.

    2016-01-01

    Dectin-1 and TLR9 play distinct roles in the recognition and induction of innate immune responses to Aspergillus fumigatus and Candida albicans. Dectin-1 is a receptor for the major fungal cell wall carbohydrate β-1,3 glucan that induces inflammatory cytokines and controls phagosomal maturation through Syk activation. TLR9 is an endosomal Toll-like receptor that also modulates the inflammatory cytokine response to fungal pathogens. In this study, we demonstrate that β-1,3 glucan beads are sufficient to induce dynamic redistribution and accumulation of cleaved TLR9 to phagosomes. Trafficking of TLR9 to A. fumigatus and C. albicans phagosomes requires Dectin-1 recognition. Inhibition of phagosomal acidification blocks TLR9 accumulation on phagosomes containing β-1,3 glucan beads. Dectin-1 mediated Syk activation is required for TLR9 trafficking to β-1,3 glucan, A. fumigatus, and C. albicans containing phagosomes. In addition, Dectin-1 regulates TLR9 dependent gene expression. Collectively, our study demonstrates that recognition of β-1,3 glucan by Dectin-1 triggers TLR9 trafficking to β-1,3 glucan-containing phagosomes, which may be critical in coordinating innate anti-fungal defense. PMID:26829985

  15. Salmonella modulation of the phagosome membrane, role of SseJ.

    PubMed

    Kolodziejek, Anna M; Miller, Samuel I

    2015-03-01

    Salmonellae have the ability to invade, persist and replicate within an intracellular phagosome termed the Salmonella-containing vacuole (SCV). Salmonellae alter lipid and protein content of the SCV membrane and manipulate cytoskeletal elements in contact with the SCV using the Salmonella pathogenicity island 1 (SPI-2) type III secretion system effectors. These modifications result in microtubular-based movement and morphological changes, which include endosomal tubulation of the SCV membrane. SseJ is a SPI-2 effector that localizes to the cytoplasmic face of the SCV and esterifies cholesterol through its glycerophospholipid : cholesterol acyltransferase activity. SseJ enzymatic activity as well as localization to the SCV are determined by binding to the small mammalian GTPase, RhoA. This review will focus on current knowledge about the role of SseJ in SCV membrane modification and will discuss how the hypothesis that a major role of SPI-2 effectors is to modify SCV protein and lipid content to promote bacterial intracellular survival.

  16. Assessing the Phagosome Proteome by Quantitative Mass Spectrometry.

    PubMed

    Peltier, Julien; Härtlova, Anetta; Trost, Matthias

    2017-01-01

    Phagocytosis is the process that engulfs particles in vesicles called phagosomes that are trafficked through a series of maturation steps, culminating in the destruction of the internalized cargo. Because phagosomes are in direct contact with the particle and undergo constant fusion and fission events with other organelles, characterization of the phagosomal proteome is a powerful tool to understand mechanisms controlling innate immunity as well as vesicle trafficking. The ability to isolate highly pure phagosomes through the use of latex beads led to an extensive use of proteomics to study phagosomes under different stimuli. Thousands of different proteins have been identified and quantified, revealing new properties and shedding new light on the dynamics and composition of maturing phagosomes and innate immunity mechanisms. In this chapter, we describe how quantitative-based proteomic methods such as label-free, dimethyl labeling or Tandem Mass Tag (TMT) labeling can be applied for the characterization of protein composition and translocation during maturation of phagosomes in macrophages.

  17. Drosophila mauve mutants reveal a role of LYST homologs late in the maturation of phagosomes and autophagosomes.

    PubMed

    Rahman, Mokhlasur; Haberman, Adam; Tracy, Charles; Ray, Sanchali; Krämer, Helmut

    2012-12-01

    Chediak-Higashi syndrome (CHS) is a lethal disease caused by mutations that inactivate the lysosomal trafficking regulator protein (LYST). Patients suffer from diverse symptoms including oculocutaneous albinism, recurrent infections, neutropenia and progressive neurodegeneration. These defects have been traced back to over-sized lysosomes and lysosome-related organelles (LROs) in different cell types. Here, we explore mutants in the Drosophila mauve gene as a new model system for CHS. The mauve gene (CG42863) encodes a large BEACH domain protein of 3535 amino acids similar to LYST. This reflects a functional homology between these proteins as mauve mutants also display enlarged LROs, such as pigment granules. This Drosophila model also replicates the enhanced susceptibility to infections and we show a defect in the cellular immune response. Early stages of phagocytosis proceed normally in mauve mutant hemocytes but, unlike in wild type, late phagosomes fuse and generate large vacuoles containing many bacteria. Autophagy is similarly affected in mauve fat bodies as starvation-induced autophagosomes grow beyond their normal size. Together these data suggest a model in which Mauve functions to restrict homotypic fusion of different pre-lysosomal organelles and LROs. © 2012 John Wiley & Sons A/S.

  18. Analysis of Phagosomal Antigen Degradation by Flow Organellocytometry

    PubMed Central

    Hoffmann, Eik; Pauwels, Anne-Marie; Alloatti, Andrés; Kotsias, Fiorella; Amigorena, Sebastian

    2017-01-01

    Professional phagocytes internalize self and non-self particles by phagocytosis to initiate innate immune responses. After internalization, the formed phagosome matures through fusion and fission events with endosomes and lysosomes to obtain a more acidic, oxidative and hydrolytic environment for the degradation of its cargo. Interestingly, phagosome maturation kinetics differ between cell types and cell activation states. This protocol allows to quantify phagosome maturation kinetics on a single organelle level in different types of phagocytes using flow cytometry. Here, ovalbumin (OVA)-coupled particles are used as phagocytosis model system in dendritic cells (DC), which are internalized by phagocytosis. After different time points, phagosome maturation parameters, such as phagosomal degradation of OVA and acquisition of lysosomal proteins (like LAMP-1), can be measured simultaneously in a highly quantitative manner by flow organellocytometry. These read-outs can be correlated to other phagosomal functions, for example antigen degradation, processing and loading in DC. PMID:28239620

  19. Recruitment of coat-protein-complex proteins on to phagosomal membranes is regulated by a brefeldin A-sensitive ADP-ribosylation factor.

    PubMed Central

    Berón, W; Mayorga, L S; Colombo, M I; Stahl, P D

    2001-01-01

    Particle internalization in macrophages is followed by a complex maturation process. We have previously observed that proteins bound to phagocytosed particles are sorted from phagosomes into a heterogeneous population of vesicles that fuse with endosomes. However, the mechanism and the protein machinery involved in the formation of these phagosome-derived vesicles are largely unknown. It has been shown that vesicles coated with coat protein complex type I (COPI) have a role in both secretion and endocytosis. To address the possibility that COPI proteins might participate in the formation of phagosome-derived vesicles we studied the recruitment of beta-COP to highly purified phagosomes. The binding of beta-COP to phagosomal membranes was regulated by nucleotides and inhibited by brefeldin A (BFA). An ADP-ribosylation factor 1 (ARF1) mutant defective in GTP hydrolysis supported the binding of beta-COP to phagosomes independently of added nucleotide. AlF(4) and Gbetagamma subunits, agents known to modulate heterotrimeric G-protein activity, were tested in the beta-COP binding assay. AlF(4) increased beta-COP association, whereas binding was inhibited by the addition of Gbetagamma subunits. Our results suggest that COP proteins are recruited to phagosomal membranes by a mechanism that involves heterotrimeric GTP-binding proteins and a BFA-sensitive ARF. In addition, our findings indicate that COPI proteins are involved in the recycling of components from phagosomes to the cell surface. PMID:11284728

  20. Infection of macrophages with Mycobacterium tuberculosis induces global modifications to phagosomal function

    PubMed Central

    Podinovskaia, Maria; Lee, Wonsik; Caldwell, Shannon; Russell, David G.

    2013-01-01

    The phagosome is a central mediator of both the homeostatic and microbicidal functions of a macrophage. Following phagocytosis, Mycobacterium tuberculosis (Mtb) is able to establish infection through arresting phagosome maturation and avoiding the consequences of delivery to the lysosome. The infection of a macrophage by Mtb leads to marked changes in the behavior of both the macrophage and the surrounding tissue as the bacterium modulates its environment to promote its survival. In this study, we use functional physiological assays to probe the biology of the phagosomal network in Mtb-infected macrophages. The resulting data demonstrate that Mtb modifies phagosomal function in a TLR2/TLR4-dependent manner, and that most of these modifications are consistent with an increase in the activation status of the cell. Specifically, superoxide burst is enhanced and lipolytic activity is decreased upon infection. There are some species- or cell type-specific differences between human and murine macrophages in the rates of acidification and the degree of proteolysis. However, the most significant modification is the marked reduction in intra-phagosomal lipolysis because this correlates with the marked increase in the retention of host lipids in the infected macrophage, which provides a potential source of nutrients that can be accessed by Mtb. PMID:23253353

  1. Membrane proteomics of phagosomes suggests a connection to autophagy

    SciTech Connect

    Shui, Wenqing; Sheu, Leslie; Liu, Jun; Smart, Brian; Petzold, Christopher J.; Hsieh, Tsung-yen; Pitcher, Austin; Keasling*, Jay D.; Bertozzi*, Carolyn R.

    2008-11-25

    Phagocytosis is the central process by which macrophage cellsinternalize and eliminate infectious microbes as well as apoptoticcells. During maturation, phagosomes containing engulfed particlesfuse with various endosomal compartments through theaction of regulatory molecules on the phagosomal membrane. Inthis study, we performed a proteomic analysis of the membranefraction from latex bead-containing (LBC) phagosomes isolatedfrom macrophages. The profile, which comprised 546 proteins,suggests diverse functions of the phagosome and potential connectionsto secretory processes, toll-like receptor signaling, andautophagy. Many identified proteins were not previously knownto reside in the phagosome. We characterized several proteins inLBC phagosomes that change in abundance on induction of autophagy,a process that has been previously implicated in the hostdefense against microbial pathogens. These observations suggestcrosstalk between autophagy and phagocytosis that may be relevantto the innate immune response of macrophages.

  2. Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

    PubMed Central

    Naroeni, Aroem; Jouy, Nicolas; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Porte, Françoise

    2001-01-01

    Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. PMID:11119541

  3. The Mycobacterium bovis Bacille Calmette-Guérin Phagosome Proteome*

    PubMed Central

    Lee, Bai-Yu; Jethwaney, Deepa; Schilling, Birgit; Clemens, Daniel L.; Gibson, Bradford W.; Horwitz, Marcus A.

    2010-01-01

    Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin (BCG) alter the maturation of their phagosomes and reside within a compartment that resists acidification and fusion with lysosomes. To define the molecular composition of this compartment, we developed a novel method for obtaining highly purified phagosomes from BCG-infected human macrophages and analyzed the phagosomes by Western immunoblotting and mass spectrometry-based proteomics. Our purification procedure revealed that BCG grown on artificial medium becomes less dense after growth in macrophages. By Western immunoblotting, LAMP-2, Niemann-Pick protein C1, and syntaxin 3 were readily detectable on the BCG phagosome but at levels that were lower than on the latex bead phagosome; flotillin-1 and the vacuolar ATPase were barely detectable on the BCG phagosome but highly enriched on the latex bead phagosome. Immunofluorescence studies confirmed the scarcity of flotillin on BCG phagosomes and demonstrated an inverse correlation between bacterial metabolic activity and flotillin on M. tuberculosis phagosomes. By mass spectrometry, 447 human host proteins were identified on BCG phagosomes, and a partially overlapping set of 289 human proteins on latex bead phagosomes was identified. Interestingly, the majority of the proteins identified consistently on BCG phagosome preparations were also identified on latex bead phagosomes, indicating a high degree of overlap in protein composition of these two compartments. It is likely that many differences in protein composition are quantitative rather than qualitative in nature. Despite the remarkable overlap in protein composition, we consistently identified a number of proteins on the BCG phagosomes that were not identified in any of our latex bead phagosome preparations, including proteins involved in membrane trafficking and signal transduction, such as Ras GTPase-activating-like protein IQGAP1, and proteins of unknown function, such as FAM3C. Our

  4. Identification of an immune-regulated phagosomal Rab cascade in macrophages

    PubMed Central

    Pei, Gang; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel

    2014-01-01

    ABSTRACT Interferon-γ (IFN-γ) has been shown to regulate phagosome trafficking and function in macrophages, but the molecular mechanisms involved are poorly understood. Here, we identify Rab20 as part of the machinery by which IFN-γ controls phagosome maturation. We found that IFN-γ stimulates the association of Rab20 with early phagosomes in macrophages. By using imaging of single phagosomes in live cells, we found that Rab20 induces an early delay in phagosome maturation and extends the time for which Rab5a and phosphatidylinositol 3-phosphate (PI3P) remain associated with phagosomes. Moreover, Rab20 depletion in macrophages abrogates the delay in phagosome maturation induced by IFN-γ. Finally, we demonstrate that Rab20 interacts with the Rab5a guanine nucleotide exchange factor Rabex-5 (also known as RABGEF1) and that Rab20 knockdown impairs the IFN-γ-dependent recruitment of Rabex-5 and Rab5a into phagosomes. Taken together, here, we uncover Rab20 as a key player in the Rab cascade by which IFN-γ induces a delay in phagosome maturation in macrophages. PMID:24569883

  5. Vacuolar ATPase in Phagosome-Lysosome Fusion

    PubMed Central

    Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

    2015-01-01

    The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

  6. Modulation of Phagosomal pH by Candida albicans Promotes Hyphal Morphogenesis and Requires Stp2p, a Regulator of Amino Acid Transport

    PubMed Central

    Vylkova, Slavena; Lorenz, Michael C.

    2014-01-01

    Candida albicans, the most important fungal pathogen of humans, has a unique interaction with macrophages in which phagocytosis induces a switch from the yeast to hyphal form, allowing it to escape by rupturing the immune cell. While a variety of factors induce this switch in vitro, including neutral pH, it is not clear what triggers morphogenesis within the macrophage where the acidic environment should inhibit this transition. In vitro, C. albicans grown in similar conditions in which amino acids are the primary carbon source generate large quantities of ammonia to raise the extracellular pH and induce the hyphal switch. We show here that C. albicans cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking STP2, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization in vitro. Phagocytosed stp2Δ mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result stp2Δ mutant cells were killed at a higher rate and caused less damage to RAW264.7 macrophages. Stp2p-regulated import leads to alkalinization of the phagosome, since the majority of the wild type cells fail to co-localize with acidophilic dyes, whereas the stp2Δ mutant cells were located in acidic phagosomes. Furthermore, stp2Δ mutant cells were able to form hyphae and escape from neutral phagosomes, indicating that the survival defect in these cells was pH dependent. Finally, these defects are reflected in an attenuation of virulence in a mouse model of disseminated candidiasis. Altogether our results suggest that C. albicans utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages. PMID:24626429

  7. Neisseria gonorrhoeae Phagosomes Delay Fusion with Primary Granules to Enhance Bacterial Survival Inside Human Neutrophils

    PubMed Central

    Johnson, M. Brittany; Criss, Alison K.

    2013-01-01

    Summary Symptomatic infection with Neisseria gonorrhoeae (Gc) promotes inflammation driven by polymorphonuclear leukocytes (PMNs, neutrophils), yet some Gc survive PMN exposure during infection. Here we report a novel mechanism of gonococcal resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary (azurophilic) granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed in gonorrheal exudates and human PMNs infected ex vivo. Delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. Ectopically increasing primary granule-phagosome fusion, by immunoglobulin opsonization or PMN treatment with lysophosphatidylcholine, reduced intracellular Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhea is by delaying primary granule-phagosome fusion, thus preventing formation of a degradative phagolysosome. PMID:23374609

  8. The Tetrahymena thermophila Phagosome Proteome▿

    PubMed Central

    Jacobs, Mary Ellen; DeSouza, Leroi V.; Samaranayake, Haresha; Pearlman, Ronald E.; Siu, K. W. Michael; Klobutcher, Lawrence A.

    2006-01-01

    In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated. PMID:17012537

  9. The Tetrahymena thermophila phagosome proteome.

    PubMed

    Jacobs, Mary Ellen; DeSouza, Leroi V; Samaranayake, Haresha; Pearlman, Ronald E; Siu, K W Michael; Klobutcher, Lawrence A

    2006-12-01

    In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.

  10. TRPC6 channel translocation into phagosomal membrane augments phagosomal function.

    PubMed

    Riazanski, Vladimir; Gabdoulkhakova, Aida G; Boynton, Lin S; Eguchi, Raphael R; Deriy, Ludmila V; Hogarth, D Kyle; Loaëc, Nadège; Oumata, Nassima; Galons, Hervé; Brown, Mary E; Shevchenko, Pavel; Gallan, Alexander J; Yoo, Sang Gune; Naren, Anjaparavanda P; Villereal, Mitchel L; Beacham, Daniel W; Bindokas, Vytautas P; Birnbaumer, Lutz; Meijer, Laurent; Nelson, Deborah J

    2015-11-24

    Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

  11. TRPC6 channel translocation into phagosomal membrane augments phagosomal function

    PubMed Central

    Riazanski, Vladimir; Gabdoulkhakova, Aida G.; Boynton, Lin S.; Eguchi, Raphael R.; Deriy, Ludmila V.; Hogarth, D. Kyle; Loaëc, Nadège; Oumata, Nassima; Galons, Hervé; Brown, Mary E.; Shevchenko, Pavel; Gallan, Alexander J.; Yoo, Sang Gune; Naren, Anjaparavanda P.; Villereal, Mitchel L.; Beacham, Daniel W.; Bindokas, Vytautas P.; Birnbaumer, Lutz; Meijer, Laurent; Nelson, Deborah J.

    2015-01-01

    Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired. PMID:26604306

  12. Two PI 3-Kinases and One PI 3-Phosphatase Together Establish the Cyclic Waves of Phagosomal PtdIns(3)P Critical for the Degradation of Apoptotic Cells

    PubMed Central

    Lu, Nan; Shen, Qian; Mahoney, Timothy R.; Neukomm, Lukas J.; Wang, Ying; Zhou, Zheng

    2012-01-01

    Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events. PMID:22272187

  13. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes

    PubMed Central

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-01-01

    Summary Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. PMID:26853472

  14. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes.

    PubMed

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-02-11

    Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Linking inflammasome activation and phagosome maturation.

    PubMed

    Lazarevic, Vanja; Martinon, Fabio

    2008-04-17

    One-third of the world's population is infected with Mycobacterium tuberculosis, one of the most effective human pathogens, whose success is attributed to the deployment of remarkably sophisticated immune evasion mechanisms. In this issue of Cell Host & Microbe, a new study unravels a novel strategy of immune evasion and enhanced bacterial intracellular survival, which is dependent on inhibition of inflammasome activation by an M. tuberculosis-encoded metalloprotease.

  16. Force dependence of phagosome trafficking in retinal pigment epithelial cells

    NASA Astrophysics Data System (ADS)

    Daniel, Rebekah; Koll, Andrew T.; Altman, David

    2014-09-01

    Retinal pigment epithelial (RPE) cells play an integral role in the renewal of photoreceptor disk membranes. As rod and cone cells shed their outer segments, they are phagocytosed and degraded by the RPE, and a failure in this process can result in retinal degeneration. We have studied the role of myosin VI in nonspecific phagocytosis in a human RPE primary cell line (ARPE-19), testing the hypothesis that this motor generates the forces required to traffic phagosomes in these cells. Experiments were conducted in the presence of forces through the use of in vivo optical trapping. Our results support a role for myosin VI in phagosome trafficking and demonstrate that applied forces modulate rates of phagosome trafficking.

  17. Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays.

    PubMed

    D'Souza, Ashwin; Sanghavi, Paulomi; Rai, Ashim; Pathak, Divya; Mallik, Roop

    2016-12-05

    We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance.

  18. Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays

    PubMed Central

    D’Souza, Ashwin; Sanghavi, Paulomi; Rai, Ashim; Pathak, Divya; Mallik, Roop

    2017-01-01

    We describe a protocol to purify latex bead phagosomes (LBPs) from Dictyostelium cells. These can be later used for various in vitro functional assays. For instance, we use these LBPs to understand the microtubule motor-driven transport on in vitro polymerized microtubules. Phagosomes are allowed to mature for defined periods inside cells before extraction for in vitro motility. These assays allow us to probe how lipids on the phagosome membrane recruit and organize motors, and also measure the motion and force generation resulting from underlying lipid-motor interactions. This provides a unique opportunity to interrogate native-like organelles using biophysical and biochemical assays, and understand the role of motor proteins in phagosome maturation and pathogen clearance. PMID:28239623

  19. Golgi-to-phagosome transport of acid sphingomyelinase and prosaposin is mediated by sortilin.

    PubMed

    Wähe, Anna; Kasmapour, Bahram; Schmaderer, Christoph; Liebl, David; Sandhoff, Konrad; Nykjaer, Anders; Griffiths, Gareth; Gutierrez, Maximiliano G

    2010-07-15

    Sortilin, also known as neurotensin receptor 3 (NTR3), is a transmembrane protein with a dual function. It acts as a receptor for neuromediators and growth factors at the plasma membrane, but it has also been implicated in binding and transport of some lysosomal proteins. However, the role of sortilin during phagosome maturation has not been investigated before. Here, we show that in macrophages, sortilin is mainly localized in the Golgi and transported to latex-bead phagosomes (LBPs). Using live-cell imaging and electron microscopy, we found that sortilin is delivered to LBPs in a manner that depends on its cytoplasmic tail. We also show that sortilin participates in the direct delivery of acid sphingomyelinase (ASM) and prosaposin (PS) to the phagosome, bypassing fusion with lysosomal compartments. Further analysis confirmed that ASM and PS are targeted to the phagosome by sortilin in a Brefeldin-A-sensitive pathway. Analysis of primary macrophages isolated from Sort1(-/-) mice indicated that the delivery of ASM and PS, but not pro-cathepsin D, to LBPs was severely impaired. We propose a pathway mediated by sortilin by which selected lysosomal proteins are transported to the phagosome along a Golgi-dependent route during the maturation of phagosomes.

  20. Pathogenic Mycobacterium avium remodels the phagosome membrane in macrophages within days after infection.

    PubMed

    de Chastellier, Chantal; Thilo, Lutz

    2002-01-01

    As part of their strategy for intracellular survival, mycobacteria prevent maturation of the phagosomes in which they reside inside macrophages. The molecular basis for this inhibition is only now beginning to emerge, by way of the molecular characterisation of the phagosome membrane when it encloses virulent mycobacteria. Our own work has shown that at 15 days after the phagocytic uptake of Mycobacterium avium by mouse bone marrow-derived macrophages, the phagosome membrane is depleted about 4-fold for cell surface-derived membrane glycoconjugates, labelled by exogalactosylation, in comparison to the membrane of early endosomes with which it continues to interact. Here we asked whether this depletion occurred at early or late stages after infection. We found that only about half of the depletion had occurred at about 5 hours after the beginning of phagocytic uptake, with the remainder becoming established thereafter, with a half-time of about 2.5 days. Phagosomes became depleted in relation to early endosomes with which they continued to exchange membrane constituents. Early endosomes themselves became gradually depleted by about 30% during the 15-day post-infection period. In contrast, late endosomes/lysosomes remained unchanged, with a concentration of surface-derived glycoconjugates between that of early endosomes and of phagosomes at day 15 post infection. In view of the slowness of the post-infection change of phagosome membrane composition, we proposed that this change did not play a role in preventing maturation immediately after phagosome formation, but rather correlated with the process of maintaining the phagosomes in an immature state.

  1. Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1.

    PubMed

    Lê-Bury, Gabrielle; Deschamps, Chantal; Dumas, Audrey; Niedergang, Florence

    2016-09-05

    Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases. The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

  2. Magnetic phagosome motion in J774A.1 macrophages: influence of cytoskeletal drugs.

    PubMed Central

    Möller, W; Nemoto, I; Matsuzaki, T; Hofer, T; Heyder, J

    2000-01-01

    mechanism is MF-associated. MT depolymerization by CoL induces an activation of the F-actin synthesis, which may induce an accelerated relaxation and an increase of stiffness. Cell mechanical properties are not modulated by MF depolymerization, whereas MT depolymerization causes a loss of viscous resistance and a loss of cell elasticity. The mean energy for stochastic phagosome transport is 5*10(-18) Joules and corresponds to a force of 7 pN on a single 1.3-microm phagosome. PMID:10920006

  3. Analysis of phagosomal proteomes: From latex-bead to bacterial phagosomes

    PubMed Central

    Li, Qingbo; Jagannath, Chinnaswamy; Rao, Prahlad K.; Singh, Christopher R.; Lostumbo, Giovanni

    2014-01-01

    Phagosomal proteome characterization has contributed significantly to the understanding of host–pathogen interaction and the mechanism of infectious diseases caused by intracellular bacteria. The latex bead-containing phagosome has been widely used as a model system to study phagosomal proteomes at a global level. In contrast, the study of bacteria-containing phagosomes at a similar level has just begun. A number of intracellular microbial species are studied for their proteomes during the invasion of a host, providing insight into their metabolic adaptation in host cells and interaction with host-cell antimicrobial environments. In this review, we attempt to summarize the most recent advancements in the proteomic study of microbial phagosomes, especially those originating from mouse or human cells. We also briefly describe the proteomics of latex bead-containing phagosomes because they are often used as model phagosomes for study. We provide descriptions on major biological and technological components in phagosomal proteome studies. We also discuss the role of phagosomal proteome study in the broader horizon of systems biology and the technological challenges in phagosomal proteome characterization. PMID:21080496

  4. Bacillus anthracis factors for phagosomal escape.

    PubMed

    Tonello, Fiorella; Zornetta, Irene

    2012-07-01

    The mechanism of phagosome escape by intracellular pathogens is an important step in the infectious cycle. During the establishment of anthrax, Bacillus anthracis undergoes a transient intracellular phase in which spores are engulfed by local phagocytes. Spores germinate inside phagosomes and grow to vegetative bacilli, which emerge from their resident intracellular compartments, replicate and eventually exit from the plasma membrane. During germination, B. anthracis secretes multiple factors that can help its resistance to the phagocytes. Here the possible role of B. anthracis toxins, phospholipases, antioxidant enzymes and capsules in the phagosomal escape and survival, is analyzed and compared with that of factors of other microbial pathogens involved in the same type of process.

  5. Microfluidic Wound Bandage: Localized Oxygen Modulation of Collagen Maturation

    PubMed Central

    Lo, Joe F.; Brennan, Martin; Merchant, Zameer; Chen, Lin; Guo, Shujuan; Eddington, David T.; DiPietro, Luisa A.

    2013-01-01

    Restoring tissue oxygenation has the potential to improve poorly healing wounds with impaired microvasculature. Compared to more established wound therapy using hyperbaric oxygen chambers, topical oxygen therapy has lower cost and better patient comfort, although topical devices have provided inconsistent results. To provide controlled topical oxygen while minimizing moisture loss, a major issue for topical oxygen, we’ve devised a novel wound bandage based on microfluidic diffusion delivery of oxygen. In addition to modulating oxygen from 0–100% in 60s rise time, the microfluidic oxygen bandage provides a conformal seal around the wound. When 100% oxygen is delivered, it penetrates wound tissues as measured in agar phantom and in vivo wounds. Using this microfluidic bandage, we applied the oxygen modulation to 8 mm excisional wounds prepared on diabetic mice. Treatment with the microfluidic bandage demonstrated improved collagen maturity in the wound bed, although only marginal differences were observed in total collagen, microvasculature, and external closure rates. Our results show that proper topical oxygen can improve wound parameters underneath the surface. Because of the ease of fabrication, the oxygen bandage represents an economical yet practical method for oxygen wound research. PMID:23438079

  6. Intracellular Survival of Brucella spp. in Human Monocytes Involves Conventional Uptake but Special Phagosomes

    PubMed Central

    Rittig, Michael G.; Alvarez-Martinez, Maria-Teresa; Porte, Françoise; Liautard, Jean-Pierre; Rouot, Bruno

    2001-01-01

    Brucella spp. are facultative intracellular parasites of various mammals, including humans, typically infecting lymphoid as well as reproductive organs. We have investigated how B. suis and B. melitensis enter human monocytes and in which compartment they survive. Peripheral blood monocytes readily internalized nonopsonized brucellae and killed most of them within 12 to 18 h. The presence of Brucella-specific antibodies (but not complement) increased the uptake of bacteria without increasing their intracellular survival, whereas adherence of the monocytes or incubation in Ca2+- and Mg2+-free medium reduced the uptake. Engulfment of all Brucella organisms (regardless of bacterial viability or virulence) initially resulted in phagosomes with tightly apposed walls (TP). Most TP were fully fusiogenic and matured to spacious phagolysosomes containing degraded bacteria, whereas some TP (more in monocyte-derived macrophages, HeLa cells, and CHO cells than in monocytes) remained tightly apposed to intact bacteria. Immediate treatment of infected host cells with the lysosomotropic base ammonium chloride caused a swelling of all phagosomes and a rise in the intraphagosomal pH, abolishing the intracellular survival of Brucella. These results indicate that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosis-promoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) brucellae are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with the phagosomal wall. PMID:11349069

  7. Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study.

    PubMed

    de Chastellier, C; Thibon, M; Rabinovitch, M

    1999-08-01

    Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.

  8. Staphylococcus aureus Strain USA300 Perturbs Acquisition of Lysosomal Enzymes and Requires Phagosomal Acidification for Survival inside Macrophages

    PubMed Central

    Tranchemontagne, Zachary R.; Camire, Ryan B.; O'Donnell, Vanessa J.; Baugh, Jessfor

    2015-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) causes invasive, drug-resistant skin and soft tissue infections. Reports that S. aureus bacteria survive inside macrophages suggest that the intramacrophage environment may be a niche for persistent infection; however, mechanisms by which the bacteria might evade macrophage phagosomal defenses are unclear. We examined the fate of the S. aureus-containing phagosome in THP-1 macrophages by evaluating bacterial intracellular survival and phagosomal acidification and maturation and by testing the impact of phagosomal conditions on bacterial viability. Multiple strains of S. aureus survived inside macrophages, and in studies using the MRSA USA300 clone, the USA300-containing phagosome acidified rapidly and acquired the late endosome and lysosome protein LAMP1. However, fewer phagosomes containing live USA300 bacteria than those containing dead bacteria associated with the lysosomal hydrolases cathepsin D and β-glucuronidase. Inhibiting lysosomal hydrolase activity had no impact on intracellular survival of USA300 or other S. aureus strains, suggesting that S. aureus perturbs acquisition of lysosomal enzymes. We examined the impact of acidification on S. aureus intramacrophage viability and found that inhibitors of phagosomal acidification significantly impaired USA300 intracellular survival. Inhibition of macrophage phagosomal acidification resulted in a 30-fold reduction in USA300 expression of the staphylococcal virulence regulator agr but had little effect on expression of sarA, saeR, or sigB. Bacterial exposure to acidic pH in vitro increased agr expression. Together, these results suggest that S. aureus survives inside macrophages by perturbing normal phagolysosome formation and that USA300 may sense phagosomal conditions and upregulate expression of a key virulence regulator that enables its intracellular survival. PMID:26502911

  9. Molecular characterization of the evolution of phagosomes

    PubMed Central

    Boulais, Jonathan; Trost, Matthias; Landry, Christian R; Dieckmann, Régis; Levy, Emmanuel D; Soldati, Thierry; Michnick, Stephen W; Thibault, Pierre; Desjardins, Michel

    2010-01-01

    Amoeba use phagocytosis to internalize bacteria as a source of nutrients, whereas multicellular organisms utilize this process as a defense mechanism to kill microbes and, in vertebrates, initiate a sustained immune response. By using a large-scale approach to identify and compare the proteome and phosphoproteome of phagosomes isolated from distant organisms, and by comparative analysis over 39 taxa, we identified an ‘ancient' core of phagosomal proteins around which the immune functions of this organelle have likely organized. Our data indicate that a larger proportion of the phagosome proteome, compared with the whole cell proteome, has been acquired through gene duplication at a period coinciding with the emergence of innate and adaptive immunity. Our study also characterizes in detail the acquisition of novel proteins and the significant remodeling of the phagosome phosphoproteome that contributed to modify the core constituents of this organelle in evolution. Our work thus provides the first thorough analysis of the changes that enabled the transformation of the phagosome from a phagotrophic compartment into an organelle fully competent for antigen presentation. PMID:20959821

  10. Mycobacterium tuberculosis and Mycobacterium avium modify the composition of the phagosomal membrane in infected macrophages by selective depletion of cell surface-derived glycoconjugates.

    PubMed

    Pietersen, Raydean; Thilo, Lutz; de Chastellier, Chantal

    2004-05-01

    The growth of pathogenic mycobacteria in phagosomes of the host cell correlates with their ability to prevent phagosome maturation. The underlying molecular mechanism remains elusive. In a previous study, we have shown that Mycobacterium avium depletes the phagosome membrane of cell surface-derived glycoconjugates (de Chastellier and Thilo, Eur. J. Cell Biol. 81, 17-25, 2002). We now extended these quantitative observations to the major human pathogen, Mycobacterium tuberculosis (H37Rv). At increasing times after infection of mouse bone marrow-derived macrophages, cell-surface glycoconjugates were labelled enzymatically with [3H]galactose. Subsequent endocytic membrane traffic resulted in a redistribution of this label from the cell surface to endocytic membranes, including phagosomes. The steady-state distribution was measured by quantitative autoradiography at the electron microscope level. Relative to early endosomes, with which phagosomes continued to fuse and rapidly exchange membrane constituents, the phagosome membrane was depleted about 3-fold, starting during infection and in the course of 9 days thereafter. These results were in quantitative agreement with our previous observations for Mycobacterium avium. For the latter case, we now showed by cell fractionation that the depletion was selective, mainly involving glycoproteins in the 110-210 kDa range. Together, these results indicated that pathogenic mycobacteria induced and maintained a bulk change in phagosome membrane composition that could be of special relevance for survival of pathogenic mycobacteria within phagosomes.

  11. Chloride transport in functionally active phagosomes isolated from Human neutrophils

    PubMed Central

    Aiken, Martha L.; Painter, Richard G.; Zhou, Yun; Wang, Guoshun

    2012-01-01

    Chloride anion is critical for hypochlorous acid (HOCl) production and microbial killing in neutrophil phagosomes. However, the molecular mechanism by which this anion is transported to the organelle is poorly understood. In this report, membrane-enclosed and functionally active phagosomes were isolated from human neutrophils by using opsonized paramagnetic latex microspheres and a rapid magnetic separation method. The phagosomes recovered were highly enriched for specific protein markers associated with this organelle such as lysosomal-associated membrane protein-1, myeloperoxidase (MPO), lactoferrin, and NADPH oxidase. When FITC–dextran was included in the phagocytosis medium, the majority of the isolated phagosomes retained the fluorescent label after isolation, indicative of intact membrane structure. Flow cytometric measurement of acridine orange, a fluorescent pH indicator, in the purified phagosomes demonstrated that the organelle in its isolated state was capable of transporting protons to the phagosomal lumen via the vacuolar-type ATPase proton pump (V-ATPase). When NADPH was supplied, the isolated phagosomes constitutively oxidized dihydrorhodamine 123, indicating their ability to produce hydrogen peroxide. The preparations also showed a robust production of HOCl within the phagosomal lumen when assayed with the HOCl-specific fluorescent probe R19-S by flow cytometry. MPO-mediated iodination of the proteins covalently conjugated to the phagocytosed beads was quantitatively measured. Phagosomal uptake of iodide and protein iodination were significantly blocked by chloride channel inhibitors, including CFTRinh-172 and NPPB. Further experiments determined that the V-ATPase-driving proton flux into the isolated phagosomes required chloride cotransport, and the cAMP-activated CFTR chloride channel was a major contributor to the chloride transport. Taken together, the data suggest that the phagosomal preparation described herein retains ion transport

  12. Link between intraphagosomal biotin and rapid phagosomal escape in Francisella

    PubMed Central

    Napier, Brooke A.; Meyer, Lena; Bina, James E.; Miller, Mark A.; Sjöstedt, Anders; Weiss, David S.

    2012-01-01

    Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15–60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens. PMID:23071317

  13. ARTICULAR CARTILAGE TENSILE INTEGRITY: MODULATION BY MATRIX DEPLETION IS MATURATION-DEPENDENT

    PubMed Central

    Asanbaeva, Anna; Tam, Johnny; Schumacher, Barbara L.; Klisch, Stephen M.; Masuda, Koichi; Sah, Robert L.

    2008-01-01

    Articular cartilage function depends on the molecular composition and structure of its extracellular matrix (ECM). The collagen network (CN) provides cartilage with tensile integrity, but must also remodel during growth. Such remodeling may depend on matrix molecules interacting with the CN to modulate the tensile behavior of cartilage. The objective of this study was to determine the effects of increasingly selective matrix depletion on tensile properties of immature and mature articular cartilage, and thereby establish a framework for identifying molecules involved in CN remodeling. Depletion of immature cartilage with guanidine, chondroitinase ABC, chondroitinase AC, and Streptomyces hyaluronidase markedly increased tensile integrity, while the integrity of mature cartilage remained unaltered after depletion with guanidine. The enhanced tensile integrity after matrix depletion suggests that certain ECM components of immature matrix serve to inhibit CN interactions and may act as modulators of physiological alterations of cartilage geometry and tensile properties during growth/maturation. PMID:18394422

  14. CFTR-mediated halide transport in phagosomes of human neutrophils

    PubMed Central

    Painter, Richard G.; Marrero, Luis; Lombard, Gisele A.; Valentine, Vincent G.; Nauseef, William M.; Wang, Guoshun

    2010-01-01

    Chloride serves as a critical component of innate host defense against infection, providing the substrate for MPO-catalyzed production of HOCl in the phagosome of human neutrophils. Here, we used halide-specific fluorescent sensors covalently coupled to zymosan particles to investigate the kinetics of chloride and iodide transport in phagosomes of human neutrophils. Using the self-ratioable fluorescent probe specific for chloride anion, we measured chloride dynamics within phagosomes in response to extracellular chloride changes by quantitative fluorescence microscopy. Under the experimental conditions used, normal neutrophils showed rapid phagosomal chloride uptake with an initial influx rate of 0.31 ± 0.04 mM/s (n=5). GlyH-101, a CFTRinh, decreased the rate of uptake in a dose-dependent manner. Neutrophils isolated from CF patients showed a significantly slower rate of chloride uptake by phagosomes, having an initial influx rate of 0.043 ± 0.012 mM/s (n=5). Interestingly, the steady-state level of chloride in CF phagosomes was ∼26 mM, significantly lower than that of the control (∼68 mM). As CFTR transports chloride as well as other halides, we conjugated an iodide-sensitive probe as an independent approach to confirm the results. The dynamics of iodide uptake by neutrophil phagosomes were monitored by flow cytometry. CFTRinh172 blocked 40–50% of the overall iodide uptake by phagosomes in normal neutrophils. In a parallel manner, the level of iodide uptake by CF phagosomes was only 20–30% of that of the control. Taken together, these results implicate CFTR in transporting halides into the phagosomal lumen. PMID:20089668

  15. Phagosomes Induced by Cytokines Function as anti-Listeria Vaccines

    PubMed Central

    Carrasco-Marín, Eugenio; Rodriguez-Del Rio, Estela; Frande-Cabanes, Elisabet; Tobes, Raquel; Pareja, Eduardo; Lecea-Cuello, M. Jesús; Ruiz-Sáez, Marta; Madrazo-Toca, Fidel; Hölscher, Christoph; Alvarez-Dominguez, Carmen

    2012-01-01

    Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91–99)/CD8+- and LLO(189–201)/CD4+-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis. PMID:22337873

  16. Mechanically Induced Actin-mediated Rocketing of Phagosomes

    PubMed Central

    Müller-Taubenberger, Annette; Anderson, Kurt I.; Engel, Ulrike; Gerisch, Günther

    2006-01-01

    Actin polymerization can be induced in Dictyostelium by compressing the cells to bring phagosomes filled with large particles into contact with the plasma membrane. Asymmetric actin assembly results in rocketing movement of the phagosomes. We show that the compression-induced assembly of actin at the cytoplasmic face of the plasma membrane involves the Arp2/3 complex. We also identify two other proteins associated with the mechanically induced actin assembly. The class I myosin MyoB accumulates at the plasma membrane–phagosome interface early during the initiation of the response, and coronin is recruited as the actin filaments are disassembling. The forces generated by rocketing phagosomes are sufficient to push the entire microtubule apparatus forward and to dislocate the nucleus. PMID:16971511

  17. Oxidized phagosomal NOX2 is replenished from lysosomes.

    PubMed

    Dingjan, Ilse; Linders, Peter T A; van den Bekerom, Luuk; Baranov, Maksim V; Halder, Partho; Ter Beest, Martin; van den Bogaart, Geert

    2017-02-15

    In dendritic cells, the NADPH oxidase 2 (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome which kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558, traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake required to initiate T cell responses.

  18. Maturation-dependent modulation of apoptosis in cultured cerebellar granule neurons by cytokines and neurotrophins.

    PubMed

    de Luca, A; Weller, M; Frei, K; Fontana, A

    1996-09-01

    Immature cerebellar granule neurons die by apoptosis within 1 week in vitro unless maintained in depolarizing (high) concentrations of potassium (25 mM K+). Neurons allowed to survive and differentiate in high K+ medium for several days in vitro are still induced to undergo apoptosis when switched back to physiological (low) concentrations of K+ (5 mM). Here we have investigated the effects of various cytokines and growth factors in these two well-defined paradigms of neuronal apoptosis. Tumour necrosis factor-alpha, leukaemia inhibitory factor, ciliary neurotrophic factor, interleukin-10 and interleukin-13 delayed apoptosis and prolonged survival of cerebellar granule neurons maintained in low K+ medium. The effect observed required continuous exposure of the cultures to the cytokines and appeared not to involve modulation of Bcl-2 protein expression. Brain-derived neurotrophic factor accelerated neuronal death in low K+ medium. In contrast, when apoptosis of the neurons was precipitated by switching mature high K+ neurons to low K+ medium, neither tumour necrosis factor-alpha, leukaemia inhibitory factor, ciliary neurotrophic factor, interleukin-10 nor interleukin-13 prevented apoptosis. When testing the cytokines and growth factors for their capacity to alter N-methyl-D-aspartate receptor-mediated excitotoxicity of differentiated cerebellar granule neurons, no significant effect was observed. These data appear to define a maturation-dependent modulation of cerebellar granule cell survival by cytokines and neurotrophic factors that are expressed in a developmental pattern in the mammalian brain.

  19. Catalase activity is modulated by calcium and calmodulin in detached mature leaves of sweet potato.

    PubMed

    Afiyanti, Mufidah; Chen, Hsien-Jung

    2014-01-15

    Catalase (CAT) functions as one of the key enzymes in the scavenging of reactive oxygen species and affects the H2O2 homeostasis in plants. In sweet potato, a major catalase isoform was detected, and total catalase activity showed the highest level in mature leaves (L3) compared to immature (L1) and completely yellow, senescent leaves (L5). The major catalase isoform as well as total enzymatic activity were strongly suppressed by ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). This inhibition could be specifically and significantly mitigated in mature L3 leaves by exogenous CaCl2, but not MgCl2 or CoCl2. EGTA also inhibited the activity of the catalase isoform in vitro. Furthermore, chlorpromazine (CPZ), a calmodulin (CAM) inhibitor, drastically suppressed the major catalase isoform as well as total enzymatic activity, and this suppression was alleviated by exogenous sweet potato calmodulin (SPCAM) fusion protein in L3 leaves. CPZ also inhibited the activity of the catalase isoform in vitro. Protein blot hybridization showed that both anti-catalase SPCAT1 and anti-calmodulin SPCAM antibodies detect a band at the same position, which corresponds to the activity of the major catalase isoform from unboiled, but not boiled crude protein extract of L3 leaves. An inverse correlation between the major catalase isoform/total enzymatic activity and the H2O2 level was also observed. These data suggest that sweet potato CAT activity is modulated by CaCl2 and SPCAM, and plays an important role in H2O2 homeostasis in mature leaves. Association of SPCAM with the major CAT isoform is required and regulates the in-gel CAT activity band. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination

    PubMed Central

    Sheedlo, Michael J.; Qiu, Jiazhang; Tan, Yunhao; Paul, Lake N.; Luo, Zhao-Qing; Das, Chittaranjan

    2015-01-01

    Manipulation of the host’s ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition. In this report, we found that members of the Legionella pneumophila SidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeADub module engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 Å2 of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8. PMID:26598703

  1. Maturation modulates caspase-1-independent responses of dendritic cells to Anthrax lethal toxin.

    PubMed

    Reig, Núria; Jiang, Aimin; Couture, Rachael; Sutterwala, Fayyaz S; Ogura, Yasunori; Flavell, Richard A; Mellman, Ira; van der Goot, F Gisou

    2008-05-01

    Anthrax lethal toxin (LT) contributes to the immune evasion strategy of Bacillus anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however, not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knockout mice, we found that depending on the mouse strain, death of bone marrow-derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1-dependent, or by a slow caspase-1-independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1-independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1-independent responses.

  2. Maturation Modulates Caspase-1 Independent Responses of Dendritic Cells to Anthrax Lethal Toxin

    PubMed Central

    Reig, Núria; Jiang, Aimin; Couture, Rachael; Sutterwala, Fayyaz S.; Ogura, Yasunori; Flavell, Richard A.; Mellman, Ira; van der Goot, F. Gisou

    2010-01-01

    Anthrax lethal toxin (LT) contributes to the immune evasion strategy of B. anthracis by impairing the function of cells of the immune system, such as macrophages and dendritic cells (DCs). Macrophages from certain inbred mice strains undergo rapid death upon LT treatment mediated by caspase-1 activation dependent on Nalp1b, an inflammasome component. Rapid LT-induced death is however not observed in macrophages from human and many mouse strains. Here, we focused on the responses of various murine DCs to LT. Using a variety of knock-out mice, we found that depending on the mouse strain, death of bone marrow derived DCs and macrophages was mediated either by a fast Nalp1b and caspase-1 dependent, or by a slow caspase-1 independent pathway that was triggered by the impairment of MEK1/2 pathways. Caspase-1 independent death was observed in cells of different genetic backgrounds and interestingly occurred only in immature DCs. Maturation, triggered by different types of stimuli, led to full protection of DCs. These studies illustrate that the cellular damage inflicted by LT depends not only on the innate responses but also on the maturation stage of the cell, which modulates the more general caspase-1 independent responses. PMID:18194483

  3. CD4+ T Cells: guardians of the phagosome.

    PubMed

    Tubo, Noah J; Jenkins, Marc K

    2014-04-01

    CD4(+) T cells are key cells of the adaptive immune system that use T cell antigen receptors to recognize peptides that are generated in endosomes or phagosomes and displayed on the host cell surface bound to major histocompatibility complex molecules. These T cells participate in immune responses that protect hosts from microbes such as Mycobacterium tuberculosis, Cryptococcus neoformans, Leishmania major, and Salmonella enterica, which have evolved to live in the phagosomes of macrophages and dendritic cells. Here, we review studies indicating that CD4(+) T cells control phagosomal infections asymptomatically in most individuals by secreting cytokines that activate the microbicidal activities of infected phagocytes but in a way that inhibits the pathogen but does not eliminate it. Indeed, we make the case that localized, controlled, persistent infection is necessary to maintain large numbers of CD4(+) effector T cells in a state of activation needed to eradicate systemic and more pathogenic forms of the infection. Finally, we posit that current vaccines for phagosomal infections fail because they do not produce this "periodic reminder" form of CD4(+) T cell-mediated immune control.

  4. Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

    PubMed

    Song, Zhi Min; Bouchab, Leïla; Hudik, Elodie; Le Bars, Romain; Nüsse, Oliver; Dupré-Crochet, Sophie

    2017-01-17

    Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40(phox) by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40(phox) and p67(phox) at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67(phox), the key cytosolic subunit of the oxidase, and p40(phox) from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67(phox) at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40(phox) and p67(phox) at the phagosomal membrane.

  5. Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation.

    PubMed

    Meley, Daniel; Héraud, Audrey; Gouilleux-Gruart, Valerie; Ivanes, Fabrice; Velge-Roussel, Florence

    2017-01-01

    Tocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14(+) monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients.

  6. Tocilizumab Contributes to the Inflammatory Status of Mature Dendritic Cells through Interleukin-6 Receptor Subunits Modulation

    PubMed Central

    Meley, Daniel; Héraud, Audrey; Gouilleux-Gruart, Valerie; Ivanes, Fabrice; Velge-Roussel, Florence

    2017-01-01

    Tocilizumab, a humanized anti-IL-6 receptor α (IL-6Rα) is widely used in the treatment of a panel of pathologies such as adult and juvenile rheumatoid arthritis (RA) and the systemic form of juvenile idiopathic arthritis in children. Its indications are expected to be largely extended to other inflammatory diseases in close future. Dendritic cells (DCs) appear to be deeply involved in the immunopathology of these diseases, yet the effects of tocilizumab on these cells were poorly studied. In this study, we explored the effect of tocilizumab on the regulation of IL-6R subunits [gp130, soluble form of IL-6Rα (sIL-6Rα), and mIL-6Rα] in human monocyte-derived DCs. Human DCs were derived from CD14+ monocytes purified with beads with IL-4 and granulocyte macrophage colony-stimulating factor. Ex vivo cultures of DCs were performed in the presence of tocilizumab. Using lipopolysaccharide (LPS) maturation of DCs, we demonstrated that tocilizumab did not inhibit IL-6 secretion, enhanced mIL-6Rα expression, and largely increased sIL-6Rα secretion. MAPK modulated STAT3 phosphorylation and surface expression of IL-6Rα in LPS-DCs. Tocilizumab had no impact on STAT3 phosphorylation in LPS-DCs while both LPS and IL-6 increased its activation. Tocilizumab modulated the regulation of IL-6R subunits leading to an inflammatory status of DCs and a massive secretion of IL-6Rα. Our results demonstrate that DCs acquire a pro-inflammatory profile following tocilizumab treatment, becoming a major source of IL-6 trans-signaling activation that might explain the poor clinical benefit in some RA patients. PMID:28861079

  7. Live Salmonella Recruits N-Ethylmaleimide–Sensitive Fusion Protein on Phagosomal Membrane and Promotes Fusion with Early Endosome

    PubMed Central

    Mukherjee, Konark; Siddiqi, Shadab A.; Hashim, Shehla; Raje, Manoj; Basu, Sandip K.; Mukhopadhyay, Amitabha

    2000-01-01

    To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPγS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, α-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)–depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPγS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of α-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes. PMID:10684255

  8. A Novel, Noncanonical BMP Pathway Modulates Synapse Maturation at the Drosophila Neuromuscular Junction

    PubMed Central

    Sulkowski, Mikolaj J.; Han, Tae Hee; Ott, Carolyn; Wang, Qi; Verheyen, Esther M.; Lippincott-Schwartz, Jennifer; Serpe, Mihaela

    2016-01-01

    At the Drosophila NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway—which modulates transcription of BMP target genes, and a noncanonical pathway—which connects local BMP/BMP receptor complexes with the cytoskeleton. Here we describe a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches we demonstrate that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP’s role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development. PMID:26815659

  9. Mature neurons modulate neurogenesis through chemical signals acting on neural stem cells.

    PubMed

    Pardal, Ricardo; López Barneo, José

    2016-06-01

    The discovery of neural stem cells has revealed a much higher structural and functional plasticity in the adult nervous system than previously anticipated. Progenitor cells are able to give rise to new neurons and glial cells when needed, thanks to their surveillance of the environment from the germinal niches. Multiple different factors define neural stem cell niches, including cellular and non-cellular components. Innervation of neurogenic centers is crucial, as it allows the functional connection between stem cell behavior and surrounding neuronal activity. Although the association between organismal behavior and neurogenesis is well documented, much less is known about the cellular and molecular mechanisms by which neurons control stem cell activity. In this review we discuss the existing data on this type of regulation from the three best characterized germinal niches in the adult nervous system: the subventricular zone, the hippocampal subgranular zone, and the carotid body. In all cases, neuronal activity modulates stem cell behavior either by neurotransmitter spillover or by synaptic-like contacts. Currently, the molecular mechanisms underlying mature neuron-stem cell interaction are being clarified. Functional consequences and potential clinical relevance of these phenomena are also discussed. © 2016 Japanese Society of Developmental Biologists.

  10. An improved flow cytometry assay to monitor phagosome acidification.

    PubMed

    Colas, Chloé; Menezes, Shinelle; Gutiérrez-Martínez, Enric; Péan, Claire B; Dionne, Marc S; Guermonprez, Pierre

    2014-10-01

    Phago-lysosome formation is important for cell-autonomous immunity to intracellular pathogens, antigen presentation and metabolism. A hallmark feature of phago-lysosomal compartments is that they undergo progressive luminal acidification controlled by the activation of vacuolar V-ATPase. Acidification is required for many enzymatic processes taking place in phago-lysosomes, like proteolysis, and supports the microbicidal activity of macrophages. Here we present a new quantitative methodology to assess phagosome acidification by flow cytometry based on the use of bi-fluorescent particles. This method relies on the use of UV polystyrene beads labelled with the acid sensor pHrodo-succinimidyl ester (pHrodo(TM) SE red) and enables us to dissociate particle association with phagocytes from their engulfment in acidified compartments. This methodology is well suited to monitor the acidification of phagosomes formed in vivo after fluorescent bead administration.

  11. Novel analysis of maturation of murine bone-marrow-derived dendritic cells induced by Ginkgo Seed Polysaccharides

    PubMed Central

    Chen, Yinghan; Meng, Yiming; Cao, Yan; Wen, Hua; Luo, Hong; Gao, Xinghua; Shan, Fengping

    2015-01-01

    Our understanding of the mechanisms of effect of Ginkgo Seed Polysaccharides (GSPs) on the immune system remains unclear. The aim of this work was to investigate the effect of GSPs on the maturation and function of bone-marrow-derived dendritic cells (BMDCs). The results demonstrate that GSP could exert positive immune modulation on the maturation and functions of BMDCs. This effect was evidenced by decreased changes of phagosome number inside BMDCs, decreased activity of acidic phosphatase (ACP), decreased phagocytosis of BMDCs, and increased changes of key membrane molecules on BMDCs. Upregulated production of cytokines IL-12 and TNF-α also was confirmed. Therefore, it can be concluded that GSPs can efficiently induce the maturation of BMDCs. Our exploration provides direct data and a rationale for potential application of GSPs as an immune enhancer in improving immunity and as a potent adjuvant in the design of DC-based vaccines. PMID:25806792

  12. Voltage-gated proton channel is expressed on phagosomes

    SciTech Connect

    Okochi, Yoshifumi; Sasaki, Mari; Iwasaki, Hirohide; Okamura, Yasushi

    2009-05-01

    Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

  13. Voltage-gated proton channel is expressed on phagosomes.

    PubMed

    Okochi, Yoshifumi; Sasaki, Mari; Iwasaki, Hirohide; Okamura, Yasushi

    2009-05-01

    Voltage-gated proton channel has been suggested to help NADPH oxidase activity during respiratory burst of phagocytes through its activities of compensating charge imbalance and regulation of pH. In phagocytes, robust production of reactive oxygen species occurs in closed membrane compartments, which are called phagosomes. However, direct evidence for the presence of voltage-gated proton channels in phagosome has been lacking. In this study, the expression of voltage-gated proton channels was studied by Western blot with the antibody specific to the voltage-sensor domain protein, VSOP/Hv1, that has recently been identified as the molecular correlate for the voltage-gated proton channel. Phagosomal membranes of neutrophils contain VSOP/Hv1 in accordance with subunits of NADPH oxidases, gp91, p22, p47 and p67. Superoxide anion production upon PMA activation was significantly reduced in neutrophils from VSOP/Hv1 knockout mice. These are consistent with the idea that voltage-gated proton channels help NADPH oxidase in phagocytes to produce reactive oxygen species.

  14. Regulation of fatty acid oxidation in mouse cumulus-oocyte complexes during maturation and modulation by PPAR agonists.

    PubMed

    Dunning, Kylie R; Anastasi, Marie R; Zhang, Voueleng J; Russell, Darryl L; Robker, Rebecca L

    2014-01-01

    Fatty acid oxidation is an important energy source for the oocyte; however, little is known about how this metabolic pathway is regulated in cumulus-oocyte complexes. Analysis of genes involved in fatty acid oxidation showed that many are regulated by the luteinizing hormone surge during in vivo maturation, including acyl-CoA synthetases, carnitine transporters, acyl-CoA dehydrogenases and acetyl-CoA transferase, but that many are dysregulated when cumulus-oocyte complexes are matured under in vitro maturation conditions using follicle stimulating hormone and epidermal growth factor. Fatty acid oxidation, measured as production of ³H₂O from [³H]palmitic acid, occurs in mouse cumulus-oocyte complexes in response to the luteinizing hormone surge but is significantly reduced in cumulus-oocyte complexes matured in vitro. Thus we sought to determine whether fatty acid oxidation in cumulus-oocyte complexes could be modulated during in vitro maturation by lipid metabolism regulators, namely peroxisome proliferator activated receptor (PPAR) agonists bezafibrate and rosiglitazone. Bezafibrate showed no effect with increasing dose, while rosiglitazone dose dependently inhibited fatty acid oxidation in cumulus-oocyte complexes during in vitro maturation. To determine the impact of rosiglitazone on oocyte developmental competence, cumulus-oocyte complexes were treated with rosiglitazone during in vitro maturation and gene expression, oocyte mitochondrial activity and embryo development following in vitro fertilization were assessed. Rosiglitazone restored Acsl1, Cpt1b and Acaa2 levels in cumulus-oocyte complexes and increased oocyte mitochondrial membrane potential yet resulted in significantly fewer embryos reaching the morula and hatching blastocyst stages. Thus fatty acid oxidation is increased in cumulus-oocyte complexes matured in vivo and deficient during in vitro maturation, a known model of poor oocyte quality. That rosiglitazone further decreased fatty acid

  15. High Content Phenotypic Cell-Based Visual Screen Identifies Mycobacterium tuberculosis Acyltrehalose-Containing Glycolipids Involved in Phagosome Remodeling

    PubMed Central

    Larrouy-Maumus, Gerald; Gilleron, Martine; Ewann, Fanny; Christophe, Thierry; Fenistein, Denis; Jang, Jichan; Jang, Mi-Seon; Park, Sei-Jin; Rauzier, Jean; Carralot, Jean-Philippe; Shrimpton, Rachel; Genovesio, Auguste; Gonzalo-Asensio, Jesus A.; Puzo, Germain; Martin, Carlos; Brosch, Roland; Stewart, Graham R.; Gicquel, Brigitte; Neyrolles, Olivier

    2010-01-01

    The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials. PMID:20844580

  16. Differential requirement of Rab22a for the recruitment of ER-derived proteins to phagosomes and endosomes in dendritic cells.

    PubMed

    Croce, Cristina; Mayorga, Luis S; Cebrian, Ignacio

    2017-09-29

    The recruitment of endoplasmic reticulum (ER) components to dendritic cell (DC) phagosomes and endosomes is a crucial event to achieve efficient cross-presentation of exogenous antigens. We have previously identified the small GTPase Rab22a as a key regulator of MHC-I trafficking and antigen cross-presentation by DCs. In this study we show that low expression of Rab22a does not prevent the normal delivery of ER-derived proteins to DC phagosomes. In contrast, the presence of these proteins was diminished in endosomes labelled with a fluid phase marker. These observations were confirmed by a functional assay that assesses the translocation of a soluble protein to the cytosol. Interestingly, we also demonstrate that early endosomal maturation is altered in Rab22a deficient DCs. Our results indicate that Rab22a plays a major role in endosomal function and highlight the importance of studying the endocytic and phagocytic pathways separately in DCs.

  17. Measuring Phagosomal pH by Fluorescence Microscopy.

    PubMed

    Canton, Johnathan; Grinstein, Sergio

    2017-01-01

    Dual wavelength ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole population methods of being able to resolve individual cells and even individual organelles. In this chapter we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.

  18. MALDI mass spectrometry reveals that cumulus cells modulate the lipid profile of in vitro-matured bovine oocytes.

    PubMed

    Vireque, Alessandra A; Tata, Alessandra; Belaz, Katia Roberta A; Grázia, João Gabriel V; Santos, Fábio N; Arnold, Daniel R; Basso, Andrea C; Eberlin, Marcos N; Silva-de-Sá, Marcos Felipe; Ferriani, Rui A; Sá Rosa-E-Silva, Ana Carolina J

    2017-04-01

    The influence of cumulus cells (CC) on the lipid profile of bovine oocytes matured in two different lipid sources was investigated. Cumulus-oocyte complexes (COC) or denuded oocytes (DO) were matured in tissue culture medium (TCM) supplemented with fetal bovine serum (FBS) or serum substitute supplement (SSS). Lipid profiles of TCM, serum supplements, immature CC and oocyte (IO), and in vitro-matured oocytes from COC and DO were then analyzed by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and submitted to partial least squares-discriminant analysis (PLS-DA). The developmental competence of such oocytes was also assessed. Differences in lipid composition were observed between two types of sera and distinctly influenced the lipid profile of CC. As revealed by PLS-DA, the abundance of specific ions corresponding to triacylglycerols (TAG) or phospholipids (PL) were higher in COC compared to DO both supplemented with FBS or SSS and to some extent affected the subsequent DO in vitro embryo development. DO exposed to SSS had however a marked diminished ability to develop to the blastocyst stage. These results indicate a modulation by CC of the oocyte TAG and PL profiles associated with a specific cell response to the serum supplement used for in vitro maturation.

  19. Junctate boosts phagocytosis by recruiting endoplasmic reticulum Ca2+ stores near phagosomes.

    PubMed

    Guido, Daniele; Demaurex, Nicolas; Nunes, Paula

    2015-11-15

    Local intracellular Ca(2+) elevations increase the efficiency of phagocytosis, a process that is essential for innate and adaptive immunity. These local Ca(2+) elevations are generated in part by the store-operated Ca(2+) entry (SOCE) sensor STIM1, which recruits endoplasmic reticulum (ER) cisternae to phagosomes and opens phagosomal Ca(2+) channels at ER-phagosome junctions. However, residual ER-phagosome contacts and periphagosomal Ca(2+) hotspots remain in Stim1(-/-) cells. Here, we tested whether junctate (also called ASPH isoform 8), a molecule that targets STIM1 to ER-plasma-membrane contacts upon Ca(2+)-store depletion, cooperates with STIM1 at phagosome junctions. Junctate expression in Stim1(-/-) and Stim1(-/-); Stim2(-/-) phagocytic fibroblasts increased phagocytosis and periphagosomal Ca(2+) elevations, yet with only a minimal impact on global SOCE. These Ca(2+) hotspots were only marginally reduced by the SOCE channel blocker lanthanum chloride (La(3+)) but were abrogated by inositol trisphosphate receptor inhibitors 2-APB and xestospongin-C, revealing that unlike STIM1-mediated hotspots, junctate-mediated Ca(2+) originates predominantly from periphagosomal Ca(2+) stores. Accordingly, junctate accumulates near phagosomes and elongates ER-phagosome junctions in Stim1(-/-) cells. Thus, junctate mediates an alternative mechanism for generating localized Ca(2+) elevations within cells, promoting Ca(2+) release from internal stores recruited to phagosomes, thereby boosting phagocytosis.

  20. The Timing for Neuronal Maturation in the Adult Hippocampus Is Modulated by Local Network Activity

    PubMed Central

    Piatti, Verónica C.; Davies-Sala, M. Georgina; Espósito, M. Soledad; Mongiat, Lucas A.; Trinchero, Mariela F.; Schinder, Alejandro F.

    2013-01-01

    The adult hippocampus continuously generates new cohorts of immature neurons with increased excitability and plasticity. The window for the expression of those unique properties in each cohort is determined by the time required to acquire a mature neuronal phenotype. Here, we show that local network activity regulates the rate of maturation of adult-born neurons along the septotemporal axis of the hippocampus. Confocal microscopy and patch-clamp recordings were combined to assess marker expression, morphological development, and functional properties in retrovirally labeled neurons over time. The septal dentate gyrus displayed higher levels of basal network activity and faster rates of newborn neuron maturation than the temporal region. Voluntary exercise enhanced network activity only in the temporal region and, in turn, accelerated neuronal development. Finally, neurons developing within a highly active environment exhibited a delayed maturation when their intrinsic electrical activity was reduced by the cell-autonomous overexpression of Kir2.1, an inward-rectifying potassium channel. Our findings reveal a novel type of activity-dependent plasticity acting on the timing of neuronal maturation and functional integration of newly generated neurons along the longitudinal axis of the adult hippocampus. PMID:21613484

  1. Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

    PubMed

    Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa

    2015-12-01

    Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis.

  2. Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells

    PubMed Central

    Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa

    2015-01-01

    Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis. PMID:26720149

  3. Opa+ Neisseria gonorrhoeae Exhibits Reduced Survival in Human Neutrophils Via Src Family Kinase-Mediated Bacterial Trafficking Into Mature Phagolysosomes

    PubMed Central

    Johnson, M. Brittany; Ball, Louise M.; Daily, Kylene P.; Martin, Jennifer N.; Columbus, Linda; Criss, Alison K.

    2015-01-01

    Summary During gonorrheal infection, there is a heterogeneous population of Neisseria gonorrhoeae (Gc) varied in their expression of opacity-associated (Opa) proteins. While Opa proteins are important for bacterial attachment and invasion of epithelial cells, Opa+ Gc has a survival defect after exposure to neutrophils. Here, we use constitutively Opa- and OpaD+ Gc in strain background FA1090 to show that Opa+ Gc is more sensitive to killing inside adherent, chemokine-treated primary human neutrophils due to increased bacterial residence in mature, degradative phagolysosomes that contain primary and secondary granule antimicrobial content. Although Opa+ Gc stimulates a potent oxidative burst, neutrophil killing of Opa+ Gc was instead attributable to non-oxidative components, particularly neutrophil proteases and the bactericidal/permeability-increasing protein. Blocking interaction of Opa+ Gc with carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) or inhibiting Src family kinase signaling, which is downstream of CEACAM activation, enhanced the survival of Opa+ Gc in neutrophils. Src family kinase signaling was required for fusion of Gc phagosomes with primary granules to generate mature phagolysosomes. Conversely, ectopic activation of Src family kinases or coinfection with Opa+ Gc resulted in decreased survival of Opa- Gc in neutrophils. From these results, we conclude that Opa protein expression is an important modulator of Gc survival characteristics in neutrophils by influencing phagosome dynamics and thus bacterial exposure to neutrophils’ full antimicrobial arsenal. PMID:25346239

  4. Modulation of oligodendrocyte differentiation and maturation by combined biochemical and mechanical cues

    PubMed Central

    Lourenço, Tânia; Paes de Faria, Joana; Bippes, Christian A.; Maia, João; Lopes-da-Silva, José A.; Relvas, João B.; Grãos, Mário

    2016-01-01

    Extracellular matrix (ECM) proteins play a key role during oligodendrogenesis. While fibronectin (FN) is involved in the maintenance and proliferation of oligodendrocyte progenitor cells (OPCs), merosin (MN) promotes differentiation into oligodendrocytes (OLs). Mechanical properties of the ECM also seem to affect OL differentiation, hence this study aimed to clarify the impact of combined biophysical and biochemical elements during oligodendrocyte differentiation and maturation using synthetic elastic polymeric ECM-like substrates. CG-4 cells presented OPC- or OL-like morphology in response to brain-compliant substrates functionalised with FN or MN, respectively. The expression of the differentiation and maturation markers myelin basic protein — MBP — and proteolipid protein — PLP — (respectively) by primary rat oligodendrocytes was enhanced in presence of MN, but only on brain-compliant conditions, considering the distribution (MBP) or amount (PLP) of the protein. It was also observed that maturation of OLs was attained earlier (by assessing PLP expression) by cells differentiated on MN-functionalised brain-compliant substrates than on standard culture conditions. Moreover, the combination of MN and substrate compliance enhanced the maturation and morphological complexity of OLs. Considering the distinct degrees of stiffness tested ranging within those of the central nervous system, our results indicate that 6.5 kPa is the most suitable rigidity for oligodendrocyte differentiation. PMID:26879561

  5. The modulation of EEG variability between internally- and externally-driven cognitive states varies with maturation and task performance

    PubMed Central

    Willatt, Stephanie E.; Cortese, Filomeno; Protzner, Andrea B.

    2017-01-01

    Increasing evidence suggests that brain signal variability is an important measure of brain function reflecting information processing capacity and functional integrity. In this study, we examined how maturation from childhood to adulthood affects the magnitude and spatial extent of state-to-state transitions in brain signal variability, and how this relates to cognitive performance. We looked at variability changes between resting-state and task (a symbol-matching task with three levels of difficulty), and within trial (fixation, post-stimulus, and post-response). We calculated variability with multiscale entropy (MSE), and additionally examined spectral power density (SPD) from electroencephalography (EEG) in children aged 8–14, and in adults aged 18–33. Our results suggest that maturation is characterized by increased local information processing (higher MSE at fine temporal scales) and decreased long-range interactions with other neural populations (lower MSE at coarse temporal scales). Children show MSE changes that are similar in magnitude, but greater in spatial extent when transitioning between internally- and externally-driven brain states. Additionally, we found that in children, greater changes in task difficulty were associated with greater magnitude of modulation in MSE. Our results suggest that the interplay between maturational and state-to-state changes in brain signal variability manifest across different spatial and temporal scales, and influence information processing capacity in the brain. PMID:28750035

  6. Kaposi's sarcoma-associated herpesvirus K7 modulates Rubicon-mediated inhibition of autophagosome maturation.

    PubMed

    Liang, Qiming; Chang, Brian; Brulois, Kevin F; Castro, Kamilah; Min, Chan-Ki; Rodgers, Mary A; Shi, Mude; Ge, Jianning; Feng, Pinghui; Oh, Byung-Ha; Jung, Jae U

    2013-11-01

    Autophagy is an important innate safeguard mechanism for protecting an organism against invasion by pathogens. We have previously discovered that Kaposi's sarcoma-associated herpesvirus (KSHV) evades this host defense through tight suppression of autophagy by targeting multiple steps of autophagy signal transduction. Here, we report that KSHV K7 protein interacts with Rubicon autophagy protein and inhibits the autophagosome maturation step by blocking Vps34 enzymatic activity, further highlighting how KSHV deregulates autophagy-mediated host immunity for its life cycle.

  7. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length

    PubMed Central

    Sydor, Tobias; Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

    2013-01-01

    Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. PMID:23078612

  8. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

    PubMed

    Sydor, Tobias; von Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

    2013-03-01

    Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. © 2012 Blackwell Publishing Ltd.

  9. WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors

    PubMed Central

    Buckley, Catherine M.; Gopaldass, Navin; Bosmani, Cristina; Johnston, Simon A.; Insall, Robert H.

    2016-01-01

    Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surface receptor levels and sustain phagocytosis. WASH functionally interacts with the retromer complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components. PMID:27647881

  10. Redox-sensitive probes for the measurement of redox chemistries within phagosomes of macrophages and dendritic cells☆

    PubMed Central

    Balce, Dale R.; Yates, Robin M.

    2013-01-01

    There is currently much interest in factors that affect redox chemistries within phagosomes of macrophages and dendritic cells. In addition to the antimicrobial role of reactive oxygen species generation within phagosomes, accumulating evidence suggests that phagosomal redox chemistries influence other phagosomal functions such as macromolecular degradation and antigen processing. Whilst the redox chemistries within many sub-cellular compartments are being heavily scrutinized with the increasing use of fluorescent probe technologies, there is a paucity of tools to assess redox conditions within phagosomes. Hence the systems that control redox homeostasis in these unique environments remain poorly defined. This review highlights current redox-sensitive probes that can measure oxidative or reductive activity in phagosomes and discusses their suitability and limitations of use. Probes that are easily targeted to the phagosome by using established approaches are emphasized. PMID:24191242

  11. Resistance mechanisms of Mycobacterium tuberculosis against phagosomal copper overload

    PubMed Central

    Rowland, Jennifer L.; Niederweis, Michael

    2012-01-01

    SUMMARY Mycobacterium tuberculosis is an important bacterial pathogen with an extremely slow growth rate, an unusual outer membrane of very low permeability and a cunning ability to survive inside the human host despite a potent immune response. A key trait of M. tuberculosis is to acquire essential nutrients while still preserving its natural resistance to toxic compounds. In this regard, copper homeostasis mechanisms are particularly interesting, because copper is an important element for bacterial growth, but copper overload is toxic. In M. tuberculosis at least two enzymes require copper as a cofactor: the Cu/Zn-superoxide dismutase SodC and the cytochrome c oxidase which is essential for growth in vitro. Mutants of M. tuberculosis lacking the copper metallothionein MymT, the efflux pump CtpV and the membrane protein MctB are more susceptible to copper indicating that these proteins are part of a multipronged system to balance intracellular copper levels. Recent evidence showed that part of copper toxicity is a reversible damage of accessible Fe-S clusters of dehydratases and the displacement of other divalent cations such as zinc and manganese as cofactors in proteins. There is accumulating evidence that macrophages use copper to poison bacteria trapped inside phagosomes. Here, we review the rapidly increasing knowledge about copper homeostasis mechanisms in M. tuberculosis and contrast those with similar mechanisms in E. coli. These findings reveal an intricate interplay between the host which aims to overload the phagosome with copper and M. tuberculosis which utilizes several mechanisms to reduce the toxic effects of excess copper. PMID:22361385

  12. Dendritic cells inhibit the progression of Listeria monocytogenes intracellular infection by retaining bacteria in major histocompatibility complex class II-rich phagosomes and by limiting cytosolic growth.

    PubMed

    Westcott, Marlena M; Henry, Curtis J; Amis, Jacqueline E; Hiltbold, Elizabeth M

    2010-07-01

    Dendritic cells (DC) provide a suboptimal niche for the growth of Listeria monocytogenes, a facultative intracellular bacterial pathogen of immunocompromised and pregnant hosts. This is due in part to a failure of large numbers of bacteria to escape to the cytosol, an essential step in the intracellular life cycle that is mediated by listeriolysin O (LLO). Here, we demonstrate that wild-type bacteria that failed to enter the cytosol of bone marrow-derived DC were retained in a LAMP2+ compartment. An isogenic L. monocytogenes strain that produces an LLO protein with reduced pore-forming activity had a severe escape and growth phenotype in DC. Few mutant bacteria entered the cytosol in the first 2 h and were instead found in LAMP2+, major histocompatibility complex class II+ (MHC-II+) H2-DM vesicles characteristic of MHC-II antigen loading compartments (MIIC). In contrast, the mutant had a minor phenotype in bone marrow-derived macrophages (BMM) despite the reduced LLO activity. In the first hour, DC phagosomes acidified to a pH that was, on average, half a point higher than that of BMM phagosomes. Unlike BMM, L. monocytogenes growth in DC was minimal after 5 h, and consequently, DC remained viable and matured late in infection. Taken together, the data are consistent with a model in which phagosomal maturation events associated with the acquisition of MHC-II molecules present a suboptimal environment for L. monocytogenes escape to the DC cytosol, possibly by limiting the activity of LLO. This, in combination with an undefined mechanism that controls bacterial growth late in infection, promotes DC survival during the critical maturation response.

  13. Dendritic Cells Inhibit the Progression of Listeria monocytogenes Intracellular Infection by Retaining Bacteria in Major Histocompatibility Complex Class II-Rich Phagosomes and by Limiting Cytosolic Growth▿ †

    PubMed Central

    Westcott, Marlena M.; Henry, Curtis J.; Amis, Jacqueline E.; Hiltbold, Elizabeth M.

    2010-01-01

    Dendritic cells (DC) provide a suboptimal niche for the growth of Listeria monocytogenes, a facultative intracellular bacterial pathogen of immunocompromised and pregnant hosts. This is due in part to a failure of large numbers of bacteria to escape to the cytosol, an essential step in the intracellular life cycle that is mediated by listeriolysin O (LLO). Here, we demonstrate that wild-type bacteria that failed to enter the cytosol of bone marrow-derived DC were retained in a LAMP2+ compartment. An isogenic L. monocytogenes strain that produces an LLO protein with reduced pore-forming activity had a severe escape and growth phenotype in DC. Few mutant bacteria entered the cytosol in the first 2 h and were instead found in LAMP2+, major histocompatibility complex class II+ (MHC-II+) H2-DM vesicles characteristic of MHC-II antigen loading compartments (MIIC). In contrast, the mutant had a minor phenotype in bone marrow-derived macrophages (BMM) despite the reduced LLO activity. In the first hour, DC phagosomes acidified to a pH that was, on average, half a point higher than that of BMM phagosomes. Unlike BMM, L. monocytogenes growth in DC was minimal after 5 h, and consequently, DC remained viable and matured late in infection. Taken together, the data are consistent with a model in which phagosomal maturation events associated with the acquisition of MHC-II molecules present a suboptimal environment for L. monocytogenes escape to the DC cytosol, possibly by limiting the activity of LLO. This, in combination with an undefined mechanism that controls bacterial growth late in infection, promotes DC survival during the critical maturation response. PMID:20404078

  14. Modulation of T-bet and Eomes during Maturation of Peripheral Blood NK Cells Does Not Depend on Licensing/Educating KIR.

    PubMed

    Pradier, Amandine; Simonetta, Federico; Waldvogel, Sophie; Bosshard, Carine; Tiercy, Jean-Marie; Roosnek, Eddy

    2016-01-01

    Peripheral natural killer (NK) cells upregulate T-bet and downregulate Eomes, the key transcription factors regulating NK cell maturation and function during the last maturation steps toward terminally differentiated effector cells. During this process, NK cells acquire killer immunoglobulin-like receptors (KIR) and effector functions, such as cytotoxicity and target cell-induced cytokine production. Inhibitory KIR are pivotal in the control of effector functions, but whether they also modulate T-bet/Eomes expression is unknown. We have measured T-bet/Eomes levels, KIR expression, and effector functions of maturing CD94(neg)CD56(dim)NK cells using CD57 as surface marker for maturation. Our cohort consisted of 23 healthy blood donors (HBD) homozygous for the KIR A haplotype that contains only inhibitory KIR2DL1 (ligand HLA-C2), KIR2DL3 (ligand HLA-C1), and KIR3DL1 (ligand HLA-Bw4). We confirm that during maturation of NK cells, the number of KIR increases, levels of T-bet/Eomes are modulated, and that cells acquire effector functions, such as cytotoxicity (CD107) and target cell-induced cytokine production (TNF-α). Because maturation was associated with the increase of the number of KIR as well as with the modulation of T-bet/Eomes, the number of KIR correlated with the extent of T-bet/Eomes modulation. However, whether the KIR were triggered by their cognate HLA ligands or not had no impact on T-bet and Eomes expression, indicating that modulation of T-box transcription factors during NK cell maturation does not depend on signals conveyed by KIR. We discuss the relevance of this finding in the context of models of NK cell maturation while cautioning that results obtained in a perhaps quite heterogeneous cohort of HBD are not necessarily conclusive.

  15. Cdx2 levels modulate intestinal epithelium maturity and Paneth cell development

    PubMed Central

    Ann Crissey, Mary; Guo, Rong-Jun; Funakoshi, Shinsuke; Kong, Jianping; Liu, Jesse; Lynch, John P

    2010-01-01

    Background & Aims Caudal-related homeobox protein 2 (Cdx2) is an intestine-specific transcription factor that is important for intestinal development and intestine-specific gene expression. Cdx2 regulates intestinal cell–cell adhesion, proliferation, and the transcriptional activities of Wnt and β-catenin in cell culture systems. We generated transgenic mice that overexpress Cdx2 in the small intestinal and colonic epithelium to investigate the role of Cdx2 in differentiation and function of the intestinal epithelium. Methods We established 4 different lines of villin–Cdx2 transgenic mice. Intestines were collected from infant, 3-month old, and wild-type mice. Genes of interest and cell lineage markers were examined by PCR and immunohistochemistry. Results Villin–Cdx2 transgenic mice had complex phenotypes that were associated with transgene expression levels. The 2 lines that had the greatest levels of transgene expression had significant, pre-weaning failure to grow and death; these were the result of early epithelial maturation and alterations in nutrient digestion and absorption. Fat malabsorption was a prominent feature. Other effects associated with the transgene expression included loss of Paneth cell markers, increases in goblet cells, and migration of proliferating, EphB2-expressing cells to the crypt base. Loss of Paneth cell markers was associated with reduced nuclear localization of β-catenin but not homeotic posteriorization of the epithelium by Cdx2. Conclusions Overexpression of Cdx2 in the small intestine is associated with reduced post-natal growth, early epithelial maturation, alterations in crypt base organization, and changes in Paneth and goblet cell lineages. Cdx2 is a critical regulator not only of intestine-specific genes, but also processes that determine epithelial maturity and function, PMID:21081128

  16. TwinFocus, a concentrated photovoltaic module based on mature technologies

    NASA Astrophysics Data System (ADS)

    Antonini, Piergiorgio; Centro, Sandro; Golfetto, Stelvio; Saccà, Alessandro

    2014-12-01

    Among solar power generation, concentrated photovoltaics (CPV) based on multijunction (MJ) solar cells, is one of the most promising technology for hot climates. The fact that multijunction solar cells based on direct band gap semiconductors demonstrate lower dependence on temperature than silicon solar cells boosted their use in concentrated photovoltaics modules. Departing from the mainstream design of Fresnel lenses, the CPV module based on TwinFocus design with off-axis quasi parabolic mirrors differentiates itself for its compactness and the possibility of easy integration also in roof-top applications. A detailed description of the module and of the systems will be given together with measured performances, and expectations for the next release.

  17. Aminophylline modulation of the mouse respiratory network changes during postnatal maturation.

    PubMed

    Wilken, B; Ramirez, J M; Hanefeld, F; Richter, D W

    2000-11-01

    Aminophylline is a respiratory stimulant commonly used for the treatment of central apnea. Experiences from clinical practice, however, revealed that aminophylline is not reliably effective in preterm infants, whereas it is normally effective in infants and mature patients. In an established animal model for postnatal development of respiratory control mechanisms, we therefore examined the hypothesis that the clinical observations reflect a developmental change in the sensitivity of the central respiratory network to methylxanthines. The medullary respiratory network was isolated at different postnatal ages (postnatal days 1-13; P1-P13) in a transverse mouse brain stem slice preparation. This preparation contains the pre-Bötzinger complex (PBC), a region that is critical for generation of respiratory rhythm. Spontaneous rhythmic respiratory activity was recorded from the hypoglossal (XII) rootlets and from neurons in the PBC by using the whole cell patch clamp technique. Bath-applied aminophylline [20 microM] increased the frequency (+41%) in neonatal animals (P1-P6) without affecting the amplitude of respiratory burst activity in XII rootlets. The same concentration of aminophylline did not have any significant effect on the frequency of respiratory XII bursts but increased the amplitude (+31%) in juvenile animals (P7-P13). In the same age group, aminophylline also augmented the amplitude and the duration of respiratory synaptic drive currents in respiratory PBC neurons. The data demonstrate that augmentation of the respiratory output is due to direct enhancement of central respiratory network activity and increase of synaptic drive of hypoglossal motoneurons in juvenile, but not neonatal, animals. This indicates a developmental change in the efficacy of aminophylline to reinforce central respiratory network activity. Therefore, we believe that the variable success in treating respiratory disturbances in premature infants reflects maturational changes in the

  18. Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

    PubMed Central

    1983-01-01

    We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC- RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH approximately 6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH approximately 5.6 in less than 10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of approximately 6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to approximately 5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba

  19. Bovine whey protein concentrate supplementation modulates maturation of immune system in suckling rats.

    PubMed

    Pérez-Cano, Francisco J; Marín-Gallén, Silvia; Castell, Margarida; Rodríguez-Palmero, María; Rivero, Montserrat; Franch, Angels; Castellote, Cristina

    2007-10-01

    During neonatal life, challenges from breast milk and microbial flora promote immune system maturation. Immunonutrition in these stages may become an important way to increase natural defence systems. The aim of this study was to determine the effect of a daily bovine milk whey protein concentrate (WPC) supplement on the intestinal and systemic immune systems in suckling rats. The composition of intraepithelial and lamina propria lymphocytes (IEL and LPL) was analysed by flow cytometry. Systemic and intestinal humoral immune responses were determined by sera Ig levels and Ig-secreting cell quantification by ELISA and ELISPOT, respectively. From birth, suckling Wistar rats were supplemented with WPC or standard infant formula (SIF). The WPC group showed the same proportion of most of the main mucosal cell subsets as the reference animals. However, in the first days of life WPC enhanced the innate immunity by increasing the NK cell proportion in both epithelial and lamina propria (LP) compartments. A rise in intestinal CD8alphaalpha+ IEL was also induced by WPC supplementation. A time-course of sera Ig levels and spontaneous IgA, IgM and IgG production by LPL and mononuclear cells from blood and spleen, in the WPC group, exhibited a similar pattern to those pups fed only by dam's milk. In summary, the present results show the effects of WPC on enhancing mucosal innate immunity during early life.

  20. The Cell Wall Lipid PDIM Contributes to Phagosomal Escape and Host Cell Exit of Mycobacterium tuberculosis

    PubMed Central

    Quigley, Jeff; Hughitt, V. Keith; Velikovsky, Carlos A.; Mariuzza, Roy A.

    2017-01-01

    ABSTRACT The cell wall of Mycobacterium tuberculosis is composed of unique lipids that are important for pathogenesis. Indeed, the first-ever genetic screen in M. tuberculosis identified genes involved in the biosynthesis and transport of the cell wall lipid PDIM (phthiocerol dimycocerosates) as crucial for the survival of M. tuberculosis in mice. Here we show evidence for a novel molecular mechanism of the PDIM-mediated virulence in M. tuberculosis. We characterized the DNA interaction and the regulon of Rv3167c, a transcriptional repressor that is involved in virulence regulation of M. tuberculosis, and discovered that it controls the PDIM operon. A loss-of-function genetic approach showed that PDIM levels directly correlate with the capacity of M. tuberculosis to escape the phagosome and induce host cell necrosis and macroautophagy. In conclusion, our study attributes a novel role of the cell wall lipid PDIM in intracellular host cell modulation, which is important for host cell exit and dissemination of M. tuberculosis. PMID:28270579

  1. Modulation of paired-pulse responses in the dentate gyrus: effects of normal maturation and vigilance state.

    PubMed

    Blaise, J H; Bronzino, J D

    2000-01-01

    This study examined the effect of normal development and vigilance state on the modulation of dentate granule cell activity in the freely moving rat at 15, 30, and 90 days of age across three vigilance states: quiet waking, slow-wave sleep, and rapid eye movement sleep. Using paired-pulse stimulation, the paired-pulse index (PPI) was obtained for the dentate evoked field potentials elicited by the stimulation of the medial perforant path. Although significant differences in PPI values were observed during development, no significant vigilance state related changes were obtained. Preweaning infant rats, i.e., 15-day old, exhibited significantly less early (interpulse intervals, IPI= 20-50 ms) and late (IPI = 300-1,000 ms) inhibition, and less facilitation (IPI = 50-150 ms) when compared to the 90-day old adult rats during all three vigilance states. PPI values obtained from the 30-day old group fell intermediate between the 15- and 90-day old animals. These changes in PPI values provide a quantitative measure of changes in the modulation of dentate granule cell excitability during normal maturation. They can now can be used to evaluate the impact of various insults, such as prenatal protein malnutrition or neonatal stress, on hippocampal development.

  2. Cognitive flexibility modulates maturation and music-training-related changes in neural sound discrimination.

    PubMed

    Saarikivi, Katri; Putkinen, Vesa; Tervaniemi, Mari; Huotilainen, Minna

    2016-07-01

    Previous research has demonstrated that musicians show superior neural sound discrimination when compared to non-musicians, and that these changes emerge with accumulation of training. Our aim was to investigate whether individual differences in executive functions predict training-related changes in neural sound discrimination. We measured event-related potentials induced by sound changes coupled with tests for executive functions in musically trained and non-trained children aged 9-11 years and 13-15 years. High performance in a set-shifting task, indexing cognitive flexibility, was linked to enhanced maturation of neural sound discrimination in both musically trained and non-trained children. Specifically, well-performing musically trained children already showed large mismatch negativity (MMN) responses at a young age as well as at an older age, indicating accurate sound discrimination. In contrast, the musically trained low-performing children still showed an increase in MMN amplitude with age, suggesting that they were behind their high-performing peers in the development of sound discrimination. In the non-trained group, in turn, only the high-performing children showed evidence of an age-related increase in MMN amplitude, and the low-performing children showed a small MMN with no age-related change. These latter results suggest an advantage in MMN development also for high-performing non-trained individuals. For the P3a amplitude, there was an age-related increase only in the children who performed well in the set-shifting task, irrespective of music training, indicating enhanced attention-related processes in these children. Thus, the current study provides the first evidence that, in children, cognitive flexibility may influence age-related and training-related plasticity of neural sound discrimination.

  3. Temperature-dependent sex determination modulates cardiovascular maturation in embryonic snapping turtles Chelydra serpentina.

    PubMed

    Alvine, Travis; Rhen, Turk; Crossley, Dane A

    2013-03-01

    We investigated sex differences in cardiovascular maturation in embryos of the snapping turtle Chelydra serpentina, a species with temperature-dependent sex determination. One group of eggs was incubated at 26.5°C to produce males. Another group of eggs was incubated at 26.5°C until embryos reached stage 17; eggs were then shifted to 31°C for 6 days to produce females, and returned to 26.5°C for the rest of embryogenesis. Thus, males and females were at the same temperature when autonomic tone was determined and for most of development. Cholinergic blockade increased resting blood pressure (P(m)) and heart rate (f(H)) in both sexes at 75% and 90% of incubation. However, the magnitude of the f(H) response was enhanced in males compared with females at 90% of incubation. β-adrenergic blockade increased P(m) at 75% of incubation in both sexes but had no effect at 90% of incubation. β-adrenergic blockade reduced f(H) at both time points but produced a stronger response at 90% versus 75% of incubation. We found that α-adrenergic blockade decreased P(m) in both sexes at 75% and 90% of incubation and decreased f(H) at 75% of incubation in both sexes. At 90% of incubation, f(H) decreased in females but not males. Although these data clearly demonstrate sexual dimorphism in the autonomic regulation of cardiovascular physiology in embryos, further studies are needed to test whether differences are caused by endocrine signals from gonads or by a hormone-independent temperature effect.

  4. Hormonal Modulation of Dendritic Cells Differentiation, Maturation and Function: Implications for the Initiation and Progress of Systemic Autoimmunity.

    PubMed

    Mackern-Oberti, Juan Pablo; Jara, Evelyn L; Riedel, Claudia A; Kalergis, Alexis M

    2017-04-01

    Hormonal homeostasis is crucial for keeping a competent and healthy immune function. Several hormones can modulate the function of various immune cells such as dendritic cells (DCs) by influencing the initiation of the immune response and the maintenance of peripheral tolerance to self-antigens. Hormones, such as estrogens, prolactin, progesterone and glucocorticoids may profoundly affect DCs differentiation, maturation and function leading to either a pro-inflammatory or an anti-inflammatory (or tolerogenic) phenotype. If not properly regulated, these processes can contribute to the pathogenesis of autoimmune disease. An unbalanced hormonal status may affect the production of pro-inflammatory cytokines, the expression of activating/inhibitory receptors and co-stimulatory molecules on conventional and plasmacytoid DCs (pDCs), conferring susceptibility to develop autoimmunity. Estrogen receptor (ER)-α signaling in conventional DCs can promote IFN-α and IL-6 production and induce the expression of CD40, CD86 and MHCII molecules. Furthermore, estrogen modulates the pDCs response to Toll-like receptor ligands enhancing T cell priming. During lupus pathogenesis, ER-α deficiency decreased the expression of MHC II on pDCs from the spleen. In contrast, estradiol administration to lupus-prone female mice increased the expression of co-stimulatory molecules, enhanced the immunogenicity and produced large amounts of IL-6, IL-12 and TNF-α by bone marrow-derived DCs. These data suggest that estradiol/ER signaling may play an active role during lupus pathology. Similarly, understanding hormonal modulation of DCs may favor the design of new therapeutic strategies based on autologous tolerogenic DCs transfer, especially in sex-biased systemic autoimmune diseases. In this review, we discuss recent data relative to the role of different hormones (estrogen, prolactin, progesterone and glucocorticoids) in DC function during systemic autoimmune pathogenesis.

  5. Anaplasma marginale Actively Modulates Vacuolar Maturation during Intracellular Infection of Its Tick Vector, Dermacentor andersoni

    PubMed Central

    Thompson, Chelsea Wright; Schneider, David A.; Noh, Susan M.

    2016-01-01

    ABSTRACT Tick-borne transmission of bacterial pathogens in the order Rickettsiales is responsible for diverse infectious diseases, many of them severe, in humans and animals. Transmission dynamics differ among these pathogens and are reflected in the pathogen-vector interaction. Anaplasma marginale has been shown to establish and maintain infectivity within Dermacentor spp. for weeks to months while escaping the complex network of vacuolar peptidases that are responsible for digestion of the tick blood meal. How this prolonged maintenance of infectivity in a potentially hostile environment is achieved has been unknown. Using the natural vector Dermacentor andersoni, we demonstrated that A. marginale-infected tick vacuoles (AmVs) concurrently recruit markers of the early endosome (Rab5), recycling endosome (Rab4 and Rab11), and late endosome (Rab7), are maintained near neutral pH, do not fuse with lysosomes, exclude the protease cathepsin L, and engage the endoplasmic reticulum and Golgi apparatus for up to 21 days postinfection. Maintenance of this safe vacuolar niche requires active A. marginale protein synthesis; in its absence, the AmVs mature into acidic, protease-active phagolysosomes. Identification of this bacterially directed modeling of the tick midgut endosome provides a mechanistic basis for examination of the differences in transmission efficiency observed among A. marginale strains and among vector populations. IMPORTANCE Ticks transmit a variety of intracellular bacterial pathogens that cause significant diseases in humans and animals. For successful transmission, these bacterial pathogens must first gain entry into the tick midgut digestive cells, avoid digestion, and establish a replicative niche without harming the tick vector. Little is known about how this replicative niche is established and maintained. Using the ruminant pathogen A. marginale and its natural tick vector, D. andersoni, this study characterized the features of the A. marginale

  6. Developmental competence of mature yak vitrified-warmed oocytes is enhanced by IGF-I via modulation of CIRP during in vitro maturation.

    PubMed

    Pan, Yangyang; Cui, Yan; He, Honghong; Baloch, Abdul Rasheed; Fan, Jiangfeng; Xu, Gengquan; He, Junfeng; Yang, Kun; Li, Guyue; Yu, Sijiu

    2015-12-01

    The objective of this study was to investigate whether developmental competence of mature vitrified-warmed yak (Bos grunniens) oocytes can be enhanced by supplemented insulin-like growth factor I (IGF-1) during in vitro maturation (IVM), and its relationship with the expression of cold-inducible RNA-binding protein (CIRP). In experiment 1, immature yak oocytes were divided into four groups, and IVM supplemented with 0, 50, 100 and 200 ng/mL IGF-1 was evaluated; the mRNA and protein expression levels of CIRP in mature oocytes in the four groups were evaluated using quantitative real-time PCR and western blotting analyses. In experiment 2, the mature yak oocytes in the four groups were cryopreserved using the Cryotop (CT) method, followed by chemical activation and in vitro culture for two days and eight days to determine cleavage, blastocyst rates, and total cell number in the blastocysts. Mature yak oocytes without vitrification served as a control group. The outcomes were as following: (1) the expression of CIRP in the matured oocytes was up-regulated in the IGF-1 groups and was highest expression was observed in the 100 ng/mL IGF-1 treatment group. (2) In the vitrified-warmed groups, the rates of cleavage and blastocyst were also highest in the 100 ng/mL IGF-1 treatment group (81.04 ± 1.06%% and 32.16 ± 1.01%), which were close to the rates observed in groups without vitrification (83.25 ± 0.85% and 32.54 ± 0.34%). The rates of cleavage and blastocyst in the other vitrified-warmed groups were 70.92 ± 1.32% and 27.33 ± 1.31% (0 ng/mL); 72.73 ± 0.74% and 29.41 ± 0.84% (50 ng/mL); 72.43 ± 0.61% and 27.61 ± 0.59% (200 ng/mL), respectively. There was no significant difference in the total cell number per blastocysts between the vitrified-warmed groups and group without vitrification. Thus, we conclude that the enhancement in developmental competence of mature yak vitrified-warmed oocytes after the addition of IGF-1 during IVM might result from the regulation

  7. Morphine Modulates Adult Neurogenesis and Contextual Memory by Impeding the Maturation of Neural Progenitors

    PubMed Central

    Zhang, Yue; Xu, Chi; Zheng, Hui; Loh, Horace H.; Law, Ping-Yee

    2016-01-01

    The regulation of adult neurogenesis by opiates has been implicated in modulating different addiction cycles. At which neurogenesis stage opiates exert their action remains unresolved. We attempt to define the temporal window of morphine’s inhibition effect on adult neurogenesis by using the POMC-EGFP mouse model, in which newborn granular cells (GCs) can be visualized between days 3–28 post-mitotic. The POMC-EGFP mice were trained under the 3-chambers conditioned place preference (CPP) paradigm with either saline or morphine. We observed after 4 days of CPP training with saline, the number of EGFP-labeled newborn GCs in sub-granular zone (SGZ) hippocampus significantly increased compared to mice injected with saline in their homecage. CPP training with morphine significantly decreased the number of EGFP-labeled GCs, whereas no significant difference in the number of EGFP-labeled GCs was observed with the homecage mice injected with the same dose of morphine. Using cell-type selective markers, we observed that morphine reduced the number of late stage progenitors and immature neurons such as Doublecortin (DCX) and βIII Tubulin (TuJ1) positive cells in the SGZ but did not reduce the number of early progenitors such as Nestin, SOX2, or neurogenic differentiation-1 (NeuroD1) positive cells. Analysis of co-localization between different cell markers shows that morphine reduced the number of adult-born GCs by interfering with differentiation of early progenitors, but not by inducing apoptosis. In addition, when NeuroD1 was over-expressed in DG by stereotaxic injection of lentivirus, it rescued the loss of immature neurons and prolonged the extinction of morphine-trained CPP. These results suggest that under the condition of CPP training paradigm, morphine affects the transition of neural progenitor/stem cells to immature neurons via a mechanism involving NeuroD1. PMID:27078155

  8. Morphine Modulates Adult Neurogenesis and Contextual Memory by Impeding the Maturation of Neural Progenitors.

    PubMed

    Zhang, Yue; Xu, Chi; Zheng, Hui; Loh, Horace H; Law, Ping-Yee

    2016-01-01

    The regulation of adult neurogenesis by opiates has been implicated in modulating different addiction cycles. At which neurogenesis stage opiates exert their action remains unresolved. We attempt to define the temporal window of morphine's inhibition effect on adult neurogenesis by using the POMC-EGFP mouse model, in which newborn granular cells (GCs) can be visualized between days 3-28 post-mitotic. The POMC-EGFP mice were trained under the 3-chambers conditioned place preference (CPP) paradigm with either saline or morphine. We observed after 4 days of CPP training with saline, the number of EGFP-labeled newborn GCs in sub-granular zone (SGZ) hippocampus significantly increased compared to mice injected with saline in their homecage. CPP training with morphine significantly decreased the number of EGFP-labeled GCs, whereas no significant difference in the number of EGFP-labeled GCs was observed with the homecage mice injected with the same dose of morphine. Using cell-type selective markers, we observed that morphine reduced the number of late stage progenitors and immature neurons such as Doublecortin (DCX) and βIII Tubulin (TuJ1) positive cells in the SGZ but did not reduce the number of early progenitors such as Nestin, SOX2, or neurogenic differentiation-1 (NeuroD1) positive cells. Analysis of co-localization between different cell markers shows that morphine reduced the number of adult-born GCs by interfering with differentiation of early progenitors, but not by inducing apoptosis. In addition, when NeuroD1 was over-expressed in DG by stereotaxic injection of lentivirus, it rescued the loss of immature neurons and prolonged the extinction of morphine-trained CPP. These results suggest that under the condition of CPP training paradigm, morphine affects the transition of neural progenitor/stem cells to immature neurons via a mechanism involving NeuroD1.

  9. Mature leaf concentrate of Sri Lankan wild type Carica papaya Linn. modulates nonfunctional and functional immune responses of rats.

    PubMed

    Jayasinghe, Chanika Dilumi; Gunasekera, Dinara S; De Silva, Nuwan; Jayawardena, Kithmini Kawya Mandakini; Udagama, Preethi Vidya

    2017-04-26

    with significant modulation of cytokines. Chemical profile of the MLCC revealed the presence of several immunomodulatory compounds. The oral gavage of the MLCC was found to be safe in terms of both hepatic and renal toxicities. The present study established that the mature leaf concentrate (MLCC) of Carica papaya Sri Lankan wild type cultivar is orally active, safe and effectively modulates nonfunctional and functional immunological parameters of rats that unequivocally corroborate the traditional medical claims.

  10. Zoonotic intestinal helminths interact with the canine immune system by modulating T cell responses and preventing dendritic cell maturation.

    PubMed

    Junginger, Johannes; Raue, Katharina; Wolf, Karola; Janecek, Elisabeth; Stein, Veronika M; Tipold, Andrea; Günzel-Apel, Anne-Rose; Strube, Christina; Hewicker-Trautwein, Marion

    2017-09-04

    Parasite co-evolution alongside the mammalian immune system gave rise to several modulatory strategies by which they prevent exaggerated pathology and facilitate a longer worm survival. As little is known about the immunoregulatory potential of the zoonotic canine parasites Ancylostoma caninum and Toxocara canis in the natural host, the present study aimed to investigate whether their larval excretory-secretory (ES) products can modulate the canine immune system. We demonstrated TcES to increase the frequency of CD4+ Foxp3(high) T cells, while both AcES and TcES were associated with elevated Helios expression in Foxp3(high) lymphocytes. ES products were further capable of inducing IL-10 production by lymphocytes, which was mainly attributed to CD8+ T cells. ES treatment of PBMCs prior to mitogen stimulation inhibited polyclonal proliferation of CD4+ and CD8+ T cells. Moreover, monocyte-derived ES-pulsed dendritic cells reduced upregulation of MHC-II and CD80 in response to lipopolysaccharide. The data showed that regulation of the canine immune system by A. caninum and T. canis larvae comprises the modification of antigen-specific and polyclonal T cell responses and dendritic cell maturation.

  11. Photoreceptor phagosome processing defects and disturbed autophagy in retinal pigment epithelium of Cln3Δex1-6 mice modelling juvenile neuronal ceroid lipofuscinosis (Batten disease)

    PubMed Central

    Wavre-Shapton, Silène T.; Calvi, Alessandra A.; Turmaine, Mark; Seabra, Miguel C.; Cutler, Daniel F.; Futter, Clare E.; Mitchison, Hannah M.

    2015-01-01

    Retinal degeneration and visual impairment are the first signs of juvenile neuronal ceroid lipofuscinosis caused by CLN3 mutations, followed by inevitable progression to blindness. We investigated retinal degeneration in Cln3Δex1-6 null mice, revealing classic ‘fingerprint’ lysosomal storage in the retinal pigment epithelium (RPE), replicating the human disease. The lysosomes contain mitochondrial F0-ATP synthase subunit c along with undigested membranes, indicating a reduced degradative capacity. Mature autophagosomes and basal phagolysosomes, the terminal degradative compartments of autophagy and phagocytosis, are also increased in Cln3Δex1-6 RPE, reflecting disruption to these key pathways that underpin the daily phagocytic turnover of photoreceptor outer segments (POS) required for maintenance of vision. The accumulated autophagosomes have post-lysosome fusion morphology, with undigested internal contents visible, while accumulated phagosomes are frequently docked to cathepsin D-positive lysosomes, without mixing of phagosomal and lysosomal contents. This suggests lysosome-processing defects affect both autophagy and phagocytosis, supported by evidence that phagosomes induced in Cln3Δex1-6-derived mouse embryonic fibroblasts have visibly disorganized membranes, unprocessed internal vesicles and membrane contents, in addition to reduced LAMP1 membrane recruitment. We propose that defective lysosomes in Cln3Δex1-6 RPE have a reduced degradative capacity that impairs the final steps of the intimately connected autophagic and phagocytic pathways that are responsible for degradation of POS. A build-up of degradative organellar by-products and decreased recycling of cellular materials is likely to disrupt processes vital to maintenance of vision by the RPE. PMID:26450516

  12. Modulation of K+ conductances by Ca2+ and human chorionic gonadotrophin in Leydig cells from mature rat testis.

    PubMed Central

    Desaphy, J F; Rogier, C; Joffre, M

    1996-01-01

    1. Although the control of steroidogenic activity of the Leydig cell by the peptides luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) is clearly mediated by cAMP, the extent to which Ca2+ controls the Leydig cell function is less well defined. In the present study, the whole-cell configuration of the patch-clamp technique was used to investigate the modulation of potassium conductances by calcium and hCG, in the Leydig cells from mature rat testis. 2. In symmetrical glutamate solutions, depolarizations elicited outwardly rectifying currents, which were mainly carried by potassium and were blocked by tetraethylammonium and 4-aminopyridine. For values of [Ca2+]i below 10(-8) M, transient currents of low amplitudes, insensitive to charybdotoxin (CTX) and iberiotoxin (IBTX), were activated above -40 mV. For [Ca2+]i values of 10(-7) M and above, noisy currents with slow activation kinetics were activated above 0 mV. These currents were sustained and were sensitive to CTX and IBTX. 3. Both current types were modulated by intracellular calcium. Ionomycin and a [Ca2+]i elevation in the range from 10(-9) to 10(-7) M, both inhibited the CTX-insensitive currents, whereas a rise in the calcium concentration above 10(-7) M increased the amplitude and shifted the threshold of activation of the CTX-sensitive currents to less positive levels. 4. hCG (1-50 i.u. ml-1), in conditions where the chloride currents were strongly inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), induced a partial inhibition of the CTX-insensitive currents but was unable to increase the CTX-sensitive currents. 5. No voltage-sensitive calcium current was recorded in control or hCG-stimulated cells. 6. The results indicate that hCG inhibits one kind of Ca(2+)-modulated channel, perhaps as a result of a moderate [Ca2+]i rise, but is unable to increase the intracellular Ca2+ concentration to the range in which large conductance Ca(2+)-dependent channels are

  13. Modulation of K+ conductances by Ca2+ and human chorionic gonadotrophin in Leydig cells from mature rat testis.

    PubMed

    Desaphy, J F; Rogier, C; Joffre, M

    1996-08-15

    1. Although the control of steroidogenic activity of the Leydig cell by the peptides luteinizing hormone (LH) and human chorionic gonadotrophin (hCG) is clearly mediated by cAMP, the extent to which Ca2+ controls the Leydig cell function is less well defined. In the present study, the whole-cell configuration of the patch-clamp technique was used to investigate the modulation of potassium conductances by calcium and hCG, in the Leydig cells from mature rat testis. 2. In symmetrical glutamate solutions, depolarizations elicited outwardly rectifying currents, which were mainly carried by potassium and were blocked by tetraethylammonium and 4-aminopyridine. For values of [Ca2+]i below 10(-8) M, transient currents of low amplitudes, insensitive to charybdotoxin (CTX) and iberiotoxin (IBTX), were activated above -40 mV. For [Ca2+]i values of 10(-7) M and above, noisy currents with slow activation kinetics were activated above 0 mV. These currents were sustained and were sensitive to CTX and IBTX. 3. Both current types were modulated by intracellular calcium. Ionomycin and a [Ca2+]i elevation in the range from 10(-9) to 10(-7) M, both inhibited the CTX-insensitive currents, whereas a rise in the calcium concentration above 10(-7) M increased the amplitude and shifted the threshold of activation of the CTX-sensitive currents to less positive levels. 4. hCG (1-50 i.u. ml-1), in conditions where the chloride currents were strongly inhibited by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS), induced a partial inhibition of the CTX-insensitive currents but was unable to increase the CTX-sensitive currents. 5. No voltage-sensitive calcium current was recorded in control or hCG-stimulated cells. 6. The results indicate that hCG inhibits one kind of Ca(2+)-modulated channel, perhaps as a result of a moderate [Ca2+]i rise, but is unable to increase the intracellular Ca2+ concentration to the range in which large conductance Ca(2+)-dependent channels are

  14. Differential modulation of resistance biomarkers in skin of juvenile and mature pink salmon, Oncorhynchus gorbuscha by the salmon louse, Lepeophtheirus salmonis.

    PubMed

    Braden, Laura M; Barker, Duane E; Koop, Ben F; Jones, Simon R M

    2015-11-01

    Juvenile pink salmon larger than 0.7 g reject the sea louse, Lepeophtheirus salmonis, and are considered resistant to the infection. Robust innate defense responses in the skin contribute to the observed resistance. In contrast adult pink salmon captured at sea or shortly before spawning carry large numbers of the parasite, suggesting inability to control the infection. The purpose of this research is to better understand these apparently contradictory conclusions by comparing a suite of genetic and cellular markers of resistance to L. salmonis in the skin of juvenile and mature pink salmon. The expression of major histocompatibility factor II, C-reactive protein, interleukin-1β, interleukin-8 and cyclooxygenase-2 was down-regulated in mature but not juvenile pink salmon. Similarly, skin at the site of parasite attachment in juvenile salmon was highly populated with MHIIβ(+) and IL-1β(+) cells that were either absent, or at reduced levels at similar sites in mature salmon. In addition, mucocyte density was relatively low in the skin of mature salmon, irrespective of louse infection. In juveniles, the higher mucocyte density decreased following louse attachment. We show that in mature pink salmon, genetic and histological responses in skin are depressed and speculate that salmonid defense against L. salmonis is modulated by maturation.

  15. Mycobacterial escape from macrophage phagosomes to the cytoplasm represents an alternate adaptation mechanism

    PubMed Central

    Jamwal, Shilpa V.; Mehrotra, Parul; Singh, Archana; Siddiqui, Zaved; Basu, Atanu; Rao, Kanury V.S.

    2016-01-01

    Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage. PMID:26980157

  16. Mycobacterial escape from macrophage phagosomes to the cytoplasm represents an alternate adaptation mechanism.

    PubMed

    Jamwal, Shilpa V; Mehrotra, Parul; Singh, Archana; Siddiqui, Zaved; Basu, Atanu; Rao, Kanury V S

    2016-03-16

    Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.

  17. Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis.

    PubMed

    Bernardo, John; Long, Heidi J; Simons, Elizabeth R

    2010-03-01

    Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pH(p)) and oxidative burst, on a cell-by-cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis (Se). The availability of a new, highly sensitive pH probe, pHrodo, permits observation of increasing pH(p). Simultaneous labeling of the organism, applicable to any phagocyte target, with a probe insensitive to pH and oxidative species, such as AlexaFluor350, permits distinction between binding and functional responses to it by ratioing fluorescences. Addition of an extracellular-specific quencher (Trypan blue) permits distinction between bound and phagosome-enclosed targets, so that conditions within the closed phagosome can be studied. We found that opsonization is required for functional activation of PMN by Se, that the organism causes early alkalinization of the phagosome (in contrast to Cf which hyperacidifies it), and that extracellular Ca(2+) is not required for cytoplasmic Ca(2+) signaling but contributes markedly to binding of Se to PMN and to ensuant bactericidal functions. These findings lead to a new approach to the study of select organisms, like Cf and Mtb, which evade killing by manipulating the phagosomal environment.

  18. Cytosolic Access of Mycobacterium tuberculosis: Critical Impact of Phagosomal Acidification Control and Demonstration of Occurrence In Vivo

    PubMed Central

    Simeone, Roxane; Sayes, Fadel; Song, Okryul; Gröschel, Matthias I.; Brodin, Priscille; Brosch, Roland; Majlessi, Laleh

    2015-01-01

    Mycobacterium tuberculosis (Mtb) uses efficient strategies to evade the eradication by professional phagocytes, involving—as recently confirmed—escape from phagosomal confinement. While Mtb determinants, such as the ESX-1 type VII secretion system, that contribute to this phenomenon are known, the host cell factors governing this important biological process are yet unexplored. Using a newly developed flow-cytometric approach for Mtb, we show that macrophages expressing the phagosomal bivalent cation transporter Nramp-1, are much less susceptible to phagosomal rupture. Together with results from the use of the phagosome acidification inhibitor bafilomycin, we demonstrate that restriction of phagosomal acidification is a prerequisite for mycobacterial phagosomal rupture and cytosolic contact. Using different in vivo approaches including an enrichment and screen for tracking rare infected phagocytes carrying the CD45.1 hematopoietic allelic marker, we here provide first and unique evidence of M. tuberculosis-mediated phagosomal rupture in mouse spleen and lungs and in numerous phagocyte types. Our results, linking the ability of restriction of phagosome acidification to cytosolic access, provide an important conceptual advance for our knowledge on host processes targeted by Mtb evasion strategies. PMID:25658322

  19. Role of the Brucella suis Lipopolysaccharide O Antigen in Phagosomal Genesis and in Inhibition of Phagosome-Lysosome Fusion in Murine Macrophages

    PubMed Central

    Porte, Françoise; Naroeni, Aroem; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre

    2003-01-01

    Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types. However, the molecular mechanisms and the microbial factors involved are poorly understood. Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear. In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages. In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes. In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains. Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages. PMID:12595466

  20. CCL-34, a synthetic toll-like receptor 4 activator, modulates differentiation and maturation of myeloid dendritic cells.

    PubMed

    Fu, Shu-Ling; Lin, Chun-Cheng; Hsu, Ming-Ling; Liu, Sheng-Hung; Huang, Yu-Chuen; Chen, Yu-Jen

    2016-03-08

    CCL-34, a synthetic α-galactosylceramide analog, has been reported as an activator of toll-like receptor 4 (TLR4) in macrophages. TLR4 is highly expressed in dendritic cell (DC) and several TLR4 agonists are known to trigger DC maturation. We herein evaluated the effect of CCL-34 on DC maturation. Human CD14+ monocyte-derived immature DC were treated with CCL-34, its inactive structural analog CCL-44, or LPS to assess the DC maturation. CCL-34 induced DC maturation according to their characteristically dendrite-forming morphology, CD83 expression and IL-12p70 production. The allostimulatory activity of DC on proliferation of naive CD4+CD45+RA+ T cells and their secretion of interferon-γ was increased by CCL-34. Phagocytosis, an important function of immature DC, was reduced after CCL-34 treatment. All these effects related to DC maturation were evidently induced by positive control LPS but not by CCL-44 treatment. TLR4 neutralization impaired human DC maturation triggered by CCL-34. The induction of IL-12, a hallmark of DC maturation, by CCL-34 and LPS was only evident in TLR4-competent C3H/HeN, but not in TLR4-defective C3H/HeJ mice. CCL-34 could further elicit the antigen presentation capability in mice inoculated with doxorubicin-treated colorectal cancer cells. In summary, CCL-34 triggers DC maturation via a TLR4-dependent manner, which supports its potential application as an immunostimulator.

  1. Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission

    PubMed Central

    Parra-Lobato, Maria C.; Gomez-Jimenez, Maria C.

    2011-01-01

    After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ–AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ–AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling. PMID:21633085

  2. Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission.

    PubMed

    Parra-Lobato, Maria C; Gomez-Jimenez, Maria C

    2011-08-01

    After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ-AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ-AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling. © 2011 The Author(s).

  3. Class I and class III phosphoinositide 3-kinases are required for actin polymerization that propels phagosomes

    PubMed Central

    Bohdanowicz, Michal; Cosío, Gabriela; Backer, Jonathan M.

    2010-01-01

    Actin polymerization drives the extension of pseudopods that trap and engulf phagocytic targets. The polymerized actin subsequently dissociates as the phagocytic vacuole seals and detaches from the plasma membrane. We found that phagosomes formed by engagement of integrins that serve as complement receptors (CR3) undergo secondary waves of actin polymerization, leading to the formation of “comet tails” that propel the vacuoles inside the cells. Actin tail formation was accompanied by and required de novo formation of PI(3,4)P2 and PI(3,4,5)P3 on the phagosomal membrane by class I phosphoinositide 3-kinases (PI3Ks). Although the phosphatidylinositide phosphatase Inpp5B was recruited to nascent phagosomes, it rapidly detached from the membrane after phagosomes sealed. Detachment of Inpp5B required the formation of PI(3)P. Thus, class III PI3K activity was also required for the accumulation of PI(4,5)P2 and PI(3,4,5)P3 and for actin tail formation. These experiments reveal a new PI(3)P-sensitive pathway leading to PI(3,4)P2 and PI(3,4,5)P3 formation and signaling in endomembranes. PMID:21115805

  4. Retrieval of the Vacuolar H+-ATPase from Phagosomes Revealed by Live Cell Imaging

    PubMed Central

    Clarke, Margaret; Maddera, Lucinda; Engel, Ulrike; Gerisch, Günther

    2010-01-01

    Background The vacuolar H+-ATPase, or V-ATPase, is a highly-conserved multi-subunit enzyme that transports protons across membranes at the expense of ATP. The resulting proton gradient serves many essential functions, among them energizing transport of small molecules such as neurotransmitters, and acidifying organelles such as endosomes. The enzyme is not present in the plasma membrane from which a phagosome is formed, but is rapidly delivered by fusion with endosomes that already bear the V-ATPase in their membranes. Similarly, the enzyme is thought to be retrieved from phagosome membranes prior to exocytosis of indigestible material, although that process has not been directly visualized. Methodology To monitor trafficking of the V-ATPase in the phagocytic pathway of Dictyostelium discoideum, we fed the cells yeast, large particles that maintain their shape during trafficking. To track pH changes, we conjugated the yeast with fluorescein isothiocyanate. Cells were labeled with VatM-GFP, a fluorescently-tagged transmembrane subunit of the V-ATPase, in parallel with stage-specific endosomal markers or in combination with mRFP-tagged cytoskeletal proteins. Principal Findings We find that the V-ATPase is commonly retrieved from the phagosome membrane by vesiculation shortly before exocytosis. However, if the cells are kept in confined spaces, a bulky phagosome may be exocytosed prematurely. In this event, a large V-ATPase-rich vacuole coated with actin typically separates from the acidic phagosome shortly before exocytosis. This vacuole is propelled by an actin tail and soon acquires the properties of an early endosome, revealing an unexpected mechanism for rapid recycling of the V-ATPase. Any V-ATPase that reaches the plasma membrane is also promptly retrieved. Conclusions/Signficance Thus, live cell microscopy has revealed both a usual route and alternative means of recycling the V-ATPase in the endocytic pathway. PMID:20052281

  5. CCL-34, a synthetic toll-like receptor 4 activator, modulates differentiation and maturation of myeloid dendritic cells

    PubMed Central

    Fu, Shu-Ling; Lin, Chun-Cheng; Hsu, Ming-Ling; Liu, Sheng-Hung; Huang, Yu-Chuen; Chen, Yu-Jen

    2016-01-01

    CCL-34, a synthetic α-galactosylceramide analog, has been reported as an activator of toll-like receptor 4 (TLR4) in macrophages. TLR4 is highly expressed in dendritic cell (DC) and several TLR4 agonists are known to trigger DC maturation. We herein evaluated the effect of CCL-34 on DC maturation. Human CD14+ monocyte-derived immature DC were treated with CCL-34, its inactive structural analog CCL-44, or LPS to assess the DC maturation. CCL-34 induced DC maturation according to their characteristically dendrite-forming morphology, CD83 expression and IL-12p70 production. The allostimulatory activity of DC on proliferation of naive CD4+CD45+RA+ T cells and their secretion of interferon-γ was increased by CCL-34. Phagocytosis, an important function of immature DC, was reduced after CCL-34 treatment. All these effects related to DC maturation were evidently induced by positive control LPS but not by CCL-44 treatment. TLR4 neutralization impaired human DC maturation triggered by CCL-34. The induction of IL-12, a hallmark of DC maturation, by CCL-34 and LPS was only evident in TLR4-competent C3H/HeN, but not in TLR4-defective C3H/HeJ mice. CCL-34 could further elicit the antigen presentation capability in mice inoculated with doxorubicin-treated colorectal cancer cells. In summary, CCL-34 triggers DC maturation via a TLR4-dependent manner, which supports its potential application as an immunostimulator. PMID:26883191

  6. Statins reduce amyloid β-peptide production by modulating amyloid precursor protein maturation and phosphorylation through a cholesterol-independent mechanism in cultured neurons.

    PubMed

    Hosaka, Ai; Araki, Wataru; Oda, Akiko; Tomidokoro, Yasushi; Tamaoka, Akira

    2013-03-01

    Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been reported to attenuate amyloid-β peptide (Aβ) production in various cellular models. However, the mechanisms by which statins affect neuronal Aβ production have not yet been clarified. Here, we investigated this issue in rat primary cortical neurons using two statins, pitavastatin (PV) and atorvastatin (AV). Treatment of neurons with 0.2-2.5 μM PV or AV for 4 days induced a concentration- and time-dependent reduction in the secretion of both Aβ40 and Aβ42. Moreover, Western blot analyses of cell lysates showed that treatment with PV or AV significantly reduced expression levels of the mature form of amyloid precursor protein (APP) and Thr668-phosphorylated APP (P-APP), but not immature form of APP; the decreases in P-APP levels were more notable than those of mature APP levels. The statin treatment did not alter expression of BACE1 (β-site APP-cleaving enzyme 1) or γ-secretase complex proteins (presenilin 1, nicastrin, APH-1, and PEN-2). In neurons overexpressing APP via recombinant adenoviruses, PV or AV similarly reduced Aβ secretion and the levels of mature APP and P-APP. Statins also markedly reduced cellular cholesterol content in neurons in a concentration-dependent manner. Co-treatment with mevalonate reversed the statin-induced decreases in Aβ secretion and mature APP and P-APP levels, whereas co-treatment with cholesterol did not, despite recovery of cellular cholesterol levels. Finally, cell-surface biotinylation experiments revealed that both statins significantly reduced the levels of cell-surface P-APP without changing those of cell surface mature APP. These results suggest that statins reduce Aβ production by selectively modulating APP maturation and phosphorylation through a mechanism independent of cholesterol reduction in cultured neurons.

  7. The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum.

    PubMed Central

    Abu Kwaik, Y

    1996-01-01

    Legionella pneumophila is an intracellular parasite of protozoa and human phagocytes. To examine adaptation of this bacterium to parasitize protozoa, the sequence of events of the intracellular infection of the amoeba Hartmannella vermiformis was examined. The previously described uptake phenomenon of coiling phagocytosis by human monocytes was not detected. A 1 h postinfection with wild-type strain AA100, mitochondria were observed within the vicinity of the phagosome. At 2.5 h postinfection, numerous vesicles surrounded the phagosomes and mitochondria were in close proximity to the phagosome. At 5 h postinfection, the bacterium was surrounded by a ribosome-studded multilayer membrane. Bacterial multiplication was evident by 8 h postinfection, and the phagosome was surrounded by a ribosome-studded multilayer membrane until 15 h postinfection. The recruitment of organelles and formation of the ribosome-studded phagosome was defective in an isogenic attenuated mutant of L. pneumophila (strain AA101A) that failed to replicate within amoebae. At 20 h postinfection with wild-type strain AA100, numerous bacteria were present in the phagosome and ribosome were not detected around the phagosome. These data showed that, at the ultrastructural level, the intracellular infection of protozoa by L. pneumophila is highly similar to that of infection of macrophages. Immunocytochemical studies provided evidence that at 5 h postinfection the phagosome containing L. pneumophila acquired an abundant amount of the endoplasmic reticulum-specific protein (BiP). Similar to phagosomes containing heat-killed wild-type L. pneumophila, the BiP protein was not detectable in phagosomes containing the mutant strain AA101A. In addition to the absence of ribosomes and mitochondria, the BiP protein was not detected in the phagosomes at 20 h postinfection with wild-type L. pneumophila. The data indicated that the ability of L. pneumophila to establish the intracellular infection of amoebae is

  8. A novel role for nuclear factor-erythroid 2 in erythroid maturation by modulation of mitochondrial autophagy

    PubMed Central

    Gothwal, Monika; Wehrle, Julius; Aumann, Konrad; Zimmermann, Vanessa; Gründer, Albert; Pahl, Heike L.

    2016-01-01

    We have recently demonstrated that the transcription factor nuclear factor-erythroid 2, which is critical for erythroid maturation and globin gene expression, plays an important role in the pathophysiology of myeloproliferative neoplasms. Myeloproliferative neoplasm patients display elevated levels of nuclear factor-erythroid 2 and transgenic mice overexpressing the transcription factor develop myeloproliferative neoplasm, albeit, surprisingly without erythrocytosis. Nuclear factor-erythroid 2 transgenic mice show both a reticulocytosis and a concomitant increase in iron deposits in the spleen, suggesting both enhanced erythrocyte production and increased red blood cell destruction. We therefore hypothesized that elevated nuclear factor-erythroid 2 levels may lead to increased erythrocyte destruction by interfering with organelle clearance during erythroid maturation. We have previously shown that nuclear factor-erythroid 2 overexpression delays erythroid maturation of human hematopoietic stem cells. Here we report that increased nuclear factor-erythroid 2 levels also impede murine maturation by retarding mitochondrial depolarization and delaying mitochondrial elimination. In addition, ribosome autophagy is delayed in transgenics. We demonstrate that the autophagy genes NIX and ULK1 are direct novel nuclear factor-erythroid 2 target genes, as these loci are bound by nuclear factor-erythroid 2 in chromatin immunoprecipitation assays. Moreover, Nix and Ulk1 expression is increased in transgenic mice and in granulocytes from polycythemia vera patients. This is the first report implying a role for nuclear factor-erythroid 2 in erythroid maturation by affecting autophagy. PMID:27479815

  9. MKP1 dependent PTH modulation of bone matrix mineralization in female mice is osteoblast maturation stage specific and involves P-ERK, P-p38 MAPKs

    PubMed Central

    Mahalingam, Chandrika D; Sampathi, Bharat Reddy; Sharma, Sonali; Datta, Tanuka; Das, Varsha; Abou-Samra, Abdul B; Datta, Nabanita S

    2013-01-01

    Limited information is available on the role of MAPK phosphatase1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation and skeletal responsiveness to parathyroid hormone (PTH). Since MKP1 regulates the phosphorylation status of MAPKs we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knock out (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9–14 week old wild type (WT) and MKP1 KO male and female mice were examined. Western blot analysis revealed down-regulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts, and by U in mature KO cells. Changes in matrix gla protein (MGP) expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1 dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs. PMID:23197743

  10. MKP1-dependent PTH modulation of bone matrix mineralization in female mice is osteoblast maturation stage specific and involves P-ERK and P-p38 MAPKs.

    PubMed

    Mahalingam, Chandrika D; Sampathi, Bharat Reddy; Sharma, Sonali; Datta, Tanuka; Das, Varsha; Abou-Samra, Abdul B; Datta, Nabanita S

    2013-03-01

    Limited information is available on the role of MAPK phosphatase 1 (MKP1) signaling in osteoblasts. We have recently reported distinct roles for MKP1 during osteoblast proliferation, differentiation, and skeletal responsiveness to parathyroid hormone (PTH). As MKP1 regulates the phosphorylation status of MAPKs, we investigated the involvement of P-ERK and P-p38 MAPKs in MKP1 knockout (KO) early and mature osteoblasts with respect to mineralization and PTH response. Calvarial osteoblasts from 9-14-week-old WT and MKP1 KO male and female mice were examined. Western blot analysis revealed downregulation and sustained expressions of P-ERK and P-p38 with PTH treatment in differentiated osteoblasts derived from KO males and females respectively. Exposure of early osteoblasts to p38 inhibitor, SB203580 (S), markedly inhibited mineralization in WT and KO osteoblasts from both genders as determined by von Kossa assay. In osteoblasts from males, ERK inhibitor U0126 (U), not p38 inhibitor (S), prevented the inhibitory effects of PTH on mineralization in early or mature osteoblasts. In osteoblasts from KO females, PTH sustained mineralization in early osteoblasts and decreased mineralization in mature cells. This effect of PTH was attenuated by S in early osteoblasts and by U in mature KO cells. Changes in matrix Gla protein expression with PTH in KO osteoblasts did not correlate with mineralization, indicative of MKP1-dependent additional mechanisms essential for PTH action on osteoblast mineralization. We conclude that PTH regulation of osteoblast mineralization in female mice is maturation stage specific and involves MKP1 modulation of P-ERK and P-p38 MAPKs.

  11. Tfap2a promotes specification and maturation of neurons in the inner ear through modulation of Bmp, Fgf and notch signaling.

    PubMed

    Kantarci, Husniye; Edlund, Renee K; Groves, Andrew K; Riley, Bruce B

    2015-03-01

    Neurons of the statoacoustic ganglion (SAG) transmit auditory and vestibular information from the inner ear to the hindbrain. SAG neuroblasts originate in the floor of the otic vesicle. New neuroblasts soon delaminate and migrate towards the hindbrain while continuing to proliferate, a phase known as transit amplification. SAG cells eventually come to rest between the ear and hindbrain before terminally differentiating. Regulation of these events is only partially understood. Fgf initiates neuroblast specification within the ear. Subsequently, Fgf secreted by mature SAG neurons exceeds a maximum threshold, serving to terminate specification and delay maturation of transit-amplifying cells. Notch signaling also limits SAG development, but how it is coordinated with Fgf is unknown. Here we show that transcription factor Tfap2a coordinates multiple signaling pathways to promote neurogenesis in the zebrafish inner ear. In both zebrafish and chick, Tfap2a is expressed in a ventrolateral domain of the otic vesicle that includes neurogenic precursors. Functional studies were conducted in zebrafish. Loss of Tfap2a elevated Fgf and Notch signaling, thereby inhibiting SAG specification and slowing maturation of transit-amplifying cells. Conversely, overexpression of Tfap2a inhibited Fgf and Notch signaling, leading to excess and accelerated SAG production. However, most SAG neurons produced by Tfap2a overexpression died soon after maturation. Directly blocking either Fgf or Notch caused less dramatic acceleration of SAG development without neuronal death, whereas blocking both pathways mimicked all observed effects of Tfap2a overexpression, including apoptosis of mature neurons. Analysis of genetic mosaics showed that Tfap2a acts non-autonomously to inhibit Fgf. This led to the discovery that Tfap2a activates expression of Bmp7a, which in turn inhibits both Fgf and Notch signaling. Blocking Bmp signaling reversed the effects of overexpressing Tfap2a. Together, these data

  12. [Modulating the survival and maturation system of B lymphocytes: Current and future new therapeutic strategies in systemic lupus erythematosus].

    PubMed

    Valor, Lara; López-Longo, Francisco Javier

    2015-09-07

    Systemic lupus erythematosus is an autoimmune disease associated with an aberrant production of autoantibodies by self-reactive B lymphocytes. The study of the phenotypic characteristics of B lymphocytes and the identification of their surface receptors such as BAFF-R, TACI and BCMA, which are responsible of their survival and maturation, have contributed to the development of new therapeutic strategies in recent years.

  13. Fetal lipopolysaccharide exposure modulates diet-dependent gut maturation and sensitivity to necrotising enterocolitis in pre-term pigs

    USDA-ARS?s Scientific Manuscript database

    Uterine infections during pregnancy predispose to pre-term birth and postnatal morbidity, but it is unknown how prenatal bacterial exposure affects maturation of the immature gut. We hypothesised that a prenatal exposure to gram-negative lipopolysaccharide (LPS) has immunomodulatory effects that imp...

  14. PtdIns(4,5)P2 and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance

    PubMed Central

    Cheng, Shiya; Wang, Kun; Zou, Wei; Miao, Rui; Huang, Yaling; Wang, Haibin

    2015-01-01

    Phagocytosis requires phosphoinositides (PIs) as both signaling molecules and localization cues. How PIs coordinate to control phagosomal sealing and the accompanying switch of organelle identity is unclear. In this study, we followed dynamic changes in PIs during apoptotic cell clearance in Caenorhabditis elegans. We found that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) and phosphatidylinositol-3-phosphate (PtdIns3P), which accumulate transiently on unsealed and fully sealed phagosomes, respectively, are both involved in phagosome closure. We identified PtdIns3P phosphatase MTM-1 as an effector of PtdIns(4,5)P2 to promote phagosomal sealing. MTM-1 coordinates with the class II PI3 kinase PIKI-1 to control PtdIns3P levels on unsealed phagosomes. The SNX9 family protein LST-4 is required for sealing, and its association with unsealed phagosomes is regulated by PtdIns(4,5)P2, PIKI-1, and MTM-1. Loss of LST-4 or its retention on phagosomes disrupts sealing and suppresses PtdIns3P accumulation, indicating close coupling of the two events. Our findings support a coincidence detection mechanism by which phagosomal sealing is regulated and coupled with conversion from PtdIns(4,5)P2 enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes. PMID:26240185

  15. Novel transmembrane receptor involved in phagosome transport of lysozymes and β-hexosaminidase in the enteric protozoan Entamoeba histolytica.

    PubMed

    Furukawa, Atsushi; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2012-02-01

    Lysozymes and hexosaminidases are ubiquitous hydrolases in bacteria and eukaryotes. In phagocytic lower eukaryotes and professional phagocytes from higher eukaryotes, they are involved in the degradation of ingested bacteria in phagosomes. In Entamoeba histolytica, which is the intestinal protozoan parasite that causes amoebiasis, phagocytosis plays a pivotal role in the nutrient acquisition and the evasion from the host defense systems. While the content of phagosomes and biochemical and physiological roles of the major phagosomal proteins have been established in E. histolytica, the mechanisms of trafficking of these phagosomal proteins, in general, remain largely unknown. In this study, we identified and characterized for the first time the putative receptor/carrier involved in the transport of the above-mentioned hydrolases to phagosomes. We have shown that the receptor, designated as cysteine protease binding protein family 8 (CPBF8), is localized in lysosomes and mediates transport of lysozymes and β-hexosaminidase α-subunit to phagosomes when the amoeba ingests mammalian cells or Gram-positive bacillus Clostridium perfringens. We have also shown that the binding of CPBF8 to the cargos is mediated by the serine-rich domain, more specifically three serine residues of the domain, which likely contains trifluoroacetic acid-sensitive O-phosphodiester-linked glycan modifications, of CPBF8. We further showed that the repression of CPBF8 by gene silencing reduced the lysozyme and β-hexosaminidase activity in phagosomes and delayed the degradation of C. perfringens. Repression of CPBF8 also resulted in decrease in the cytopathy against the mammalian cells, suggesting that CPBF8 may also be involved in, besides the degradation of ingested bacteria, the pathogenesis against the mammalian hosts. This work represents the first case of the identification of a transport receptor of hydrolytic enzymes responsible for the degradation of microorganisms in phagosomes.

  16. Cysteine protease-binding protein family 6 mediates the trafficking of amylases to phagosomes in the enteric protozoan Entamoeba histolytica.

    PubMed

    Furukawa, Atsushi; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-05-01

    Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or β-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins.

  17. Cysteine Protease-Binding Protein Family 6 Mediates the Trafficking of Amylases to Phagosomes in the Enteric Protozoan Entamoeba histolytica

    PubMed Central

    Furukawa, Atsushi; Nakada-Tsukui, Kumiko

    2013-01-01

    Phagocytosis plays a pivotal role in nutrient acquisition and evasion from the host defense systems in Entamoeba histolytica, the intestinal protozoan parasite that causes amoebiasis. We previously reported that E. histolytica possesses a unique class of a hydrolase receptor family, designated the cysteine protease-binding protein family (CPBF), that is involved in trafficking of hydrolases to lysosomes and phagosomes, and we have also reported that CPBF1 and CPBF8 bind to cysteine proteases or β-hexosaminidase α-subunit and lysozymes, respectively. In this study, we showed by immunoprecipitation that CPBF6, one of the most highly expressed CPBF proteins, specifically binds to α-amylase and γ-amylase. We also found that CPBF6 is localized in lysosomes, based on immunofluorescence imaging. Immunoblot and proteome analyses of the isolated phagosomes showed that CPBF6 mediates transport of amylases to phagosomes. We also demonstrated that the carboxyl-terminal cytosolic region of CPBF6 is engaged in the regulation of the trafficking of CPBF6 to phagosomes. Our proteome analysis of phagosomes also revealed new potential phagosomal proteins. PMID:23509141

  18. Repeated Cycles of Rapid Actin Assembly and Disassembly on Epithelial Cell PhagosomesV⃞

    PubMed Central

    Yam, Patricia T.; Theriot, Julie A.

    2004-01-01

    We have found that early in infection of the intracellular pathogen Listeria monocytogenes in Madin-Darby canine kidney epithelial cells expressing actin conjugated to green fluorescent protein, F-actin rapidly assembles (∼25 s) and disassembles (∼30 s) around the bacteria, a phenomenon we call flashing. L. monocytogenes strains unable to perform actin-based motility or unable to escape the phagosome were capable of flashing, suggesting that the actin assembly occurs on the phagosome membrane. Cycles of actin assembly and disassembly could occur repeatedly on the same phagosome. Indirect immunofluorescence showed that most bacteria were fully internalized when flashing occurred, suggesting that actin flashing does not represent phagocytosis. Escherichia coli expressing invA, a gene product from Yersinia pseudotuberculosis that mediates cellular invasion, also induced flashing. Furthermore, polystyrene beads coated with E-cadherin or transferrin also induced flashing after internalization. This suggests that flashing occurs downstream of several distinct molecular entry mechanisms and may be a general consequence of internalization of large objects by epithelial cells. PMID:15456901

  19. Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment.

    PubMed

    Schnappinger, Dirk; Ehrt, Sabine; Voskuil, Martin I; Liu, Yang; Mangan, Joseph A; Monahan, Irene M; Dolganov, Gregory; Efron, Brad; Butcher, Philip D; Nathan, Carl; Schoolnik, Gary K

    2003-09-01

    Little is known about the biochemical environment in phagosomes harboring an infectious agent. To assess the state of this organelle we captured the transcriptional responses of Mycobacterium tuberculosis (MTB) in macrophages from wild-type and nitric oxide (NO) synthase 2-deficient mice before and after immunologic activation. The intraphagosomal transcriptome was compared with the transcriptome of MTB in standard broth culture and during growth in diverse conditions designed to simulate features of the phagosomal environment. Genes expressed differentially as a consequence of intraphagosomal residence included an interferon gamma- and NO-induced response that intensifies an iron-scavenging program, converts the microbe from aerobic to anaerobic respiration, and induces a dormancy regulon. Induction of genes involved in the activation and beta-oxidation of fatty acids indicated that fatty acids furnish carbon and energy. Induction of sigmaE-dependent, sodium dodecyl sulfate-regulated genes and genes involved in mycolic acid modification pointed to damage and repair of the cell envelope. Sentinel genes within the intraphagosomal transcriptome were induced similarly by MTB in the lungs of mice. The microbial transcriptome thus served as a bioprobe of the MTB phagosomal environment, showing it to be nitrosative, oxidative, functionally hypoxic, carbohydrate poor, and capable of perturbing the pathogen's cell envelope.

  20. Glutamate Utilization Couples Oxidative Stress Defense and the Tricarboxylic Acid Cycle in Francisella Phagosomal Escape

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Rigard, Mélanie; Dairou, Julien; Dupuis, Marion; Dubail, Iharilalao; Meibom, Karin; Henry, Thomas; Barel, Monique; Charbit, Alain

    2014-01-01

    Intracellular bacterial pathogens have developed a variety of strategies to avoid degradation by the host innate immune defense mechanisms triggered upon phagocytocis. Upon infection of mammalian host cells, the intracellular pathogen Francisella replicates exclusively in the cytosolic compartment. Hence, its ability to escape rapidly from the phagosomal compartment is critical for its pathogenicity. Here, we show for the first time that a glutamate transporter of Francisella (here designated GadC) is critical for oxidative stress defense in the phagosome, thus impairing intra-macrophage multiplication and virulence in the mouse model. The gadC mutant failed to efficiently neutralize the production of reactive oxygen species. Remarkably, virulence of the gadC mutant was partially restored in mice defective in NADPH oxidase activity. The data presented highlight links between glutamate uptake, oxidative stress defense, the tricarboxylic acid cycle and phagosomal escape. This is the first report establishing the role of an amino acid transporter in the early stage of the Francisella intracellular lifecycle. PMID:24453979

  1. Modulation of in vitro activity of zymogenic and mature recombinant human β-secretase by dietary plants.

    PubMed

    Sheean, Paul; Rout, Manoj K; Head, Richard J; Bennett, Louise E

    2012-04-01

    The in vitro activity of human recombinant β-secretase (BACE1) was studied using a fluorogenic substrate based on the cleavage site for the enzyme in the Swedish mutation of amyloid precursor protein. The enzyme was inhibited by a control peptide inhibitor with good repeatability. The enzyme preparation comprised a mixture of pro-enzyme or zymogen and mature enzyme whereby the pro-enzyme sequence forms a 'flap' that can obstruct the binding site. 'Open flap' forms of the zymogen and mature enzyme are active, but the 'closed flap' form of the zymogen is inactive. This mixture of enzyme populations permitted apparent stimulation of enzyme activity under particular conditions, presumably due to facilitating flap-opening of the zymogen. As reported for heparin, enzyme activation was stimulated in the presence of low concentrations of Tween 20 and dimethylsulfoxide before becoming inhibited at higher concentrations. Dietary plant extracts either consistently inhibited (e.g. clove, tea, cinammon) or consistently stimulated (e.g. mushroom, parsley, asparagus) BACE1. Common structural features identified by Fourier transform infrared spectroscopy revealed that BACE1 activity could be explained by differential interactions of either small molecule or polymeric species with mature versus zymogen forms of the enzyme, respectively. Further, enzyme activity could be reversed by mixtures of high and low mass species. These results may have implications for the regulation of β-secretase activity in vivo by either endogenous or possibly dietary factors and for a potential role of BACE1 in stimulation of the production of amyloid beta peptide in sporadic Alzheimer's disease. © 2012 CSIRO. Journal compilation © 2012 FEBS.

  2. Modulation of phenotypic and functional maturation of dendritic cells by intestinal bacteria and gliadin: relevance for celiac disease.

    PubMed

    De Palma, G; Kamanova, J; Cinova, J; Olivares, M; Drasarova, H; Tuckova, L; Sanz, Y

    2012-11-01

    DC maturation and functions are influenced by microbial and environmental stimuli, which could contribute to immune dysfunction. Here, we have investigated the role of enterobacteria (Escherichia coli CBL2 and Shigella CBD8) isolated from CD patients, bifidobacteria (Bifidobacterium longum CECT 7347 and Bifidobacterium bifidum CECT 7365), and gliadins on phenotypic and functional features of MDDCs and in coculture with Caco-2 cells. The ultimate goal of our study is to understand the roles played by specific components of the gut microbiota in CD. Enterobacteria induced marked alterations in MDDC morphology, inducing podosome dissolution and dendrites, and activated MDDC adhesion and spreading. Enterobacteria also induced inflammatory cytokine production (IFN-γ, TNF-α, and IL-12), partially resembling the gliadin-induced Th1-type cytokine profile. B. longum CECT 7347 and B. bifidum CECT 7365 induced minor MDDC morphological changes and activated adhesion and spreading and inflammatory cytokine production to a lesser extent compared with enterobacteria. B. longum CECT 7347 also induced lower CD86 and CD40 expression on MDDCs than the two enterobacteria. The aforementioned bifidobacterial strain also reduced gliadin-induced IFN-γ production and increased IL-10 secretion when both stimuli were combined. Similar trends were detected for MDDCs cocultured with Caco-2 cells. B. longum CECT 7347 reversed the gliadin-reduced ZO-1 expression in Caco-2 cells. Thus, our results suggest that specific components of the gut microbiota may influence phenotypic and functional maturation of DCs differently and their interactions with epithelial cells. This could ultimately define the role of DCs in CD progression.

  3. TLR Signals Induce Phagosomal MHC-I Delivery from the Endosomal Recycling Compartment to Allow Cross-Presentation

    PubMed Central

    Nair-Gupta, Priyanka; Baccarini, Alessia; Tung, Navpreet; Seyffer, Fabian; Florey, Oliver; Huang, Yunjie; Huang, Meenakshi; Overholtzer, Michael; Roche, Paul A.; Tampé, Robert; Brown, Brian D.; Amsen, Derk; Whiteheart, Sidney W.; Blander, J. Magarian

    2014-01-01

    SUMMARY Adaptation of the endoplasmic reticulum (ER) pathway for MHC class I (MHC-I) presentation in dendritic cells enables cross-presentation of peptides derived from phagocytosed microbes, infected cells, or tumor cells to CD8 T cells. How these peptides intersect with MHC-I molecules remains poorly understood. Here, we show that MHC-I selectively accumulate within phagosomes carrying microbial components, which engage Toll-like receptor (TLR) signaling. Although cross-presentation requires Sec22b-mediated phagosomal recruitment of the peptide loading complex from the ER-Golgi intermediate compartment (ERGIC), this step is independent of TLR signaling and does not deliver MHC-I. Instead, MHC-I are recruited from an endosomal recycling compartment (ERC), which is marked by Rab11a, VAMP3/cellubrevin, and VAMP8/endobrevin and holds large reserves of MHC-I. While Rab11a activity stocks ERC stores with MHC-I, MyD88-dependent TLR signals drive IκB-kinase (IKK)2-mediated phosphorylation of phagosome-associated SNAP23. Phospho-SNAP23 stabilizes SNARE complexes orchestrating ERC-phagosome fusion, enrichment of phagosomes with ERC-derived MHC-I, and subsequent cross-presentation during infection. PMID:25083866

  4. Mapping-by-Sequencing Identifies HvPHYTOCHROME C as a Candidate Gene for the early maturity 5 Locus Modulating the Circadian Clock and Photoperiodic Flowering in Barley

    PubMed Central

    Pankin, Artem; Campoli, Chiara; Dong, Xue; Kilian, Benjamin; Sharma, Rajiv; Himmelbach, Axel; Saini, Reena; Davis, Seth J; Stein, Nils; Schneeberger, Korbinian; von Korff, Maria

    2014-01-01

    Phytochromes play an important role in light signaling and photoperiodic control of flowering time in plants. Here we propose that the red/far-red light photoreceptor HvPHYTOCHROME C (HvPHYC), carrying a mutation in a conserved region of the GAF domain, is a candidate underlying the early maturity 5 locus in barley (Hordeum vulgare L.). We fine mapped the gene using a mapping-by-sequencing approach applied on the whole-exome capture data from bulked early flowering segregants derived from a backcross of the Bowman(eam5) introgression line. We demonstrate that eam5 disrupts circadian expression of clock genes. Moreover, it interacts with the major photoperiod response gene Ppd-H1 to accelerate flowering under noninductive short days. Our results suggest that HvPHYC participates in transmission of light signals to the circadian clock and thus modulates light-dependent processes such as photoperiodic regulation of flowering. PMID:24996910

  5. Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

    PubMed Central

    Defacque, Hélène; Bos, Evelyne; Garvalov, Boyan; Barret, Cécile; Roy, Christian; Mangeat, Paul; Shin, Hye-Won; Rybin, Vladimir; Griffiths, Gareth

    2002-01-01

    Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process (Defacque et al., 2000b). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane. PMID:11950931

  6. Phosphoinositides regulate membrane-dependent actin assembly by latex bead phagosomes.

    PubMed

    Defacque, Hélène; Bos, Evelyne; Garvalov, Boyan; Barret, Cécile; Roy, Christian; Mangeat, Paul; Shin, Hye-Won; Rybin, Vladimir; Griffiths, Gareth

    2002-04-01

    Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P(2)-binding proteins ezrin and/or moesin were essential for this process (). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P(2), and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P(2) antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P(2) into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P(2)-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

  7. The phagosomal environment protects virulent Mycobacterium avium from killing and destruction by clarithromycin.

    PubMed Central

    Fréhel, C; Offredo, C; de Chastellier, C

    1997-01-01

    Murine bone marrow-derived macrophages (Mphis) infected with virulent strains of Mycobacterium avium (TMC 724 and a human clinical isolate) or with an avirulent opaque variant that spontaneously dissociates from the virulent human clinical isolate were subjected to a prolonged and continuous treatment with clarithromycin added at the MIC. The efficiency of this antibiotic in terms of inhibition of bacterial growth and bacterial degradation was evaluated during a 21-day treatment period. Growth was assessed by determination of CFU of intracellular bacteria and by a quantitative ultrastructural analysis which allowed us also to determine the extent of bacterial degradation. A similar treatment was applied to the same strains growing in liquid medium. Our data show that in liquid medium, clarithromycin caused a 90% decrease in CFU within 7 days of treatment. When applied to Mphis infected with virulent M. avium, clarithromycin immediately arrested bacterial growth but was unable to fully kill and degrade intracellularly growing virulent bacteria. After 21 days of treatment, 25% of intracellular bacteria were still morphologically intact. These bacteria resumed growth upon removal of the antibiotic, with a normal replication rate. These bacteria had not become more resistant to the drug, since the MIC was unchanged as compared to the one determined for the initial stock used to infect Mphis. Our data therefore suggest that the intraphagosomal environment protects bacteria from degradation. We propose that the inability of the drug to completely destroy bacteria is the result of a limited accessibility of the drug due to prevention of fusions between the immature phagosomes in which virulent bacteria reside and lysosomes in which clarithromycin accumulates. In accord with our proposal, we show that the avirulent opaque variant, which does not prevent phagosome-lysosome fusions (unpublished data), is finally destroyed by clarithromycin even within the phagosomal

  8. Warming modulates the effects of the endocrine disruptor progestin levonorgestrel on the zebrafish fitness, ovary maturation kinetics and reproduction success.

    PubMed

    Cardoso, P G; Rodrigues, D; Madureira, T V; Oliveira, N; Rocha, M J; Rocha, E

    2017-10-01

    Interactive effects between multiple stressors, namely climate drivers (e.g., temperature) and chemical pollution (e.g., endocrine disruptors) are poorly studied. Here, it was for the first time evaluated the combinatory effects of temperature and a synthetic progestin, levonorgestrel (LNG), on the fitness and reproductive-related endpoints of zebrafish (Danio rerio). A multi-factorial design was implemented by manipulating both temperature [setting as baseline an ambient temperature of 27 °C, against warming (+3 °C)] and LNG levels (10 ngL(-1) and 1000 ngL(-1)). Groups of males and females were exposed sub-acutely, for 21-days. Increased temperature caused an overall decrease in the females' gonadosomatic index (GSI), during the pre-reproduction phase, LNG did not affect GSI. In addition, fecundity (number of ovulated eggs) was negatively affected by both temperature and LNG, being the effect of the latter more intense. Fish exposed to the highest LNG concentration (at both temperatures) did not reproduce, but also in those exposed to the lowest dose of progestin at a higher temperature, a complete reproductive failure occurred. These results reflect what was observed in the stereological analysis of the ovary maturation stages prior to reproduction. Accordingly, the higher the LNG concentration, the lower the degree of maturation of the ovary. This was exacerbated by the higher temperature. As to embryonated eggs, they hatched significantly faster at higher temperatures, but exposure to 10 ngL(-1) of LNG (at 27 °C) reduced significantly the hatching rate, comparing to control. Further, the recrudescence of the ovary 48 h after spawning seems to be not affected by both stressors. Our data suggest that in a future scenario of global warming and synthetic hormones exposure, the reproduction of fish species, such as the zebrafish, can be endangered, which can put at risk their success, and consequently affect the structure and functioning of associated

  9. Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties.

    PubMed

    Costa, Federica; Vescovini, Rosanna; Bolzoni, Marina; Marchica, Valentina; Storti, Paola; Toscani, Denise; Accardi, Fabrizio; Notarfranchi, Laura; Dalla Palma, Benedetta; Manferdini, Cristina; Manni, Sabrina; Todaro, Giannalisa; Lisignoli, Gina; Piazza, Francesco; Aversa, Franco; Giuliani, Nicola

    2017-08-08

    The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14(+) cells obtained from patients with multiple myeloma or from a human monocytic cell line. LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3(+) cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes. In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity.

  10. Oleoresinosis in Grand Fir (Abies grandis) Saplings and Mature Trees (Modulation of this Wound Response by Light and Water Stresses).

    PubMed Central

    Lewinsohn, E.; Gijzen, M.; Muzika, R. M.; Barton, K.; Croteau, R.

    1993-01-01

    The stem content of diterpene resin acids (rosin) increases dramatically following wounding of grand fir (Abies grandis) saplings, but the level of monoterpene olefins (turpentine) in the stem decreases following injury, in spite of a significant increase in monoterpene cyclase (synthase) activity. However, this observation was explained when rapid evaporative losses of the volatile monoterpenes from the wound site was demonstrated by trapping experiments, a finding consistent with a role of turpentine as a solvent for the mobilization and deposition of rosin to seal the injury. Mature forest trees responded to stem wounding by the enhancement of monoterpene cyclization capacity in a manner similar to 2-year-old grand fir saplings raised in the greenhouse. Light and water stresses greatly reduced the constitutive level of monoterpene cyclase activity and abolished the wound-induced response. The diminution in monoterpene biosynthetic capacity was correlated with a dramatic decrease in cyclase protein as demonstrated by immunoblotting. Relief of stress conditions resulted in the restoration of cyclase activity (both constitutive and wound induced) to control levels. The results of these experiments indicate that grand fir saplings are a suitable model for studies of the regulation of defensive oleoresinosis in conifers. PMID:12231755

  11. 5-Aza-2'-deoxycytidine acts as a modulator of chondrocyte hypertrophy and maturation in chick caudal region chondrocytes in culture

    PubMed Central

    2016-01-01

    This study was carried out to explore the effect of DNA hypomethylation on chondrocytes phenotype, in particular the effect on chondrocyte hypertrophy, maturation, and apoptosis. Chondrocytes derived from caudal region of day 17 embryonic chick sterna were pretreated with hypomethylating drug 5-aza-2'-deoxycytidine for 48 hours and then maintained in the normal culture medium for up to 14 days. Histological studies showed distinct morphological changes occurred in the pretreated cultures when compared to the control cultures. The pretreated chondrocytes after 7 days in culture became bigger in size and acquired more flattened fibroblastic phenotype as well as a loss of cartilage specific extracellular matrix. Scanning electron microscopy at day 7 showed chondrocytes to have increased in cell volume and at day 14 in culture the extracellular matrix of the pretreated cultures showed regular fibrillar structure heavily embedded with matrix vesicles, which is the characteristic feature of chondrocyte hypertrophy. Transmission electron microscopic studies indicated the terminal fate of the hypertrophic cells in culture. The pretreated chondrocytes grown for 14 days in culture showed two types of cells: dark cells which had condense chromatin in dark patches and dark cytoplasm. The other light chondrocytes appeared to be heavily loaded with endoplasmic reticulum indicative of very active protein and secretory activity; their cytoplasm had large vacuoles and disintegrating cytoplasm. The biosynthetic profile showed that the pretreated cultures were actively synthesizing and secreting type X collagen and alkaline phosphatase as a major biosynthetic product. PMID:27382512

  12. Role of inhibin and activin in the modulation of gonadotropin- and steroid-induced oocyte maturation in the teleost Fundulus heteroclitus

    PubMed Central

    Petrino, Teresa R; Toussaint, Gesulla; Lin, Yu-Wai P

    2007-01-01

    demonstrate the presence of activin/inhibin subunits in the ovary of F. heteroclitus, these in vitro findings indicate that inhibin and activin are local regulators in the teleost ovary and have opposing effects in modulating oocyte maturation. PMID:17550604

  13. Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties

    PubMed Central

    Costa, Federica; Vescovini, Rosanna; Bolzoni, Marina; Marchica, Valentina; Storti, Paola; Toscani, Denise; Accardi, Fabrizio; Notarfranchi, Laura; Dalla Palma, Benedetta; Manferdini, Cristina; Manni, Sabrina; Todaro, Giannalisa; Lisignoli, Gina; Piazza, Francesco; Aversa, Franco; Giuliani, Nicola

    2017-01-01

    The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14+ cells obtained from patients with multiple myeloma or from a human monocytic cell line. LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes. In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity. PMID:28881793

  14. TLR3-Induced Maturation of Murine Dendritic Cells Regulates CTL Responses by Modulating PD-L1 Trafficking

    PubMed Central

    Varthaman, Aditi; Moreau, Hélène D.; Maurin, Mathieu; Benaroch, Philippe

    2016-01-01

    Targeting TLR3 through formulations of polyI:C is widely studied as an adjuvant in cancer immunotherapy. The efficacy of such targeting has been shown to increase in combination with anti-PD-L1 treatment. Nevertheless, the mechanistic details of the effect of polyI:C on DC maturation and the impact on T-DC interactions upon PD-L1 blockade is largely unknown. Here we found that although DC treatment with polyI:C induced a potent inflammatory response including the production of type I interferon, polyI:C treatment of DCs impaired activation of peptide specific CD8+ T cells mainly due to PD-L1. Interestingly, we found that PD-L1 trafficking to the cell surface is regulated in two waves in polyI:C-treated DCs. One induced upon overnight treatment and a second more rapid one, specific to polyI:C treatment, was induced upon CD40 signaling leading to a further increase in surface PD-L1 in DCs. The polyI:C-induced cell surface PD-L1 reduced the times of contact between DCs and T cells, potentially accounting for limited T cell activation. Our results reveal a novel CD40-dependent regulation of PD-L1 trafficking induced upon TLR3 signaling that dictates its inhibitory activity. These results provide a mechanistic framework to understand the efficacy of anti-PD-L1 cancer immunotherapy combined with TLR agonists. PMID:27911948

  15. NK cell killer Ig-like receptor repertoire acquisition and maturation are strongly modulated by HLA class I molecules.

    PubMed

    Sleiman, Marwan; Brons, Nicolaas H C; Kaoma, Tony; Dogu, Figen; Villa-Forte, Alexandra; Lenoble, Patrick; Hentges, François; Kotsch, Katja; Gadola, Stephan D; Vilches, Carlos; Zimmer, Jacques

    2014-03-15

    The interaction between clonally distributed inhibitory receptors and their activating counterparts on NK cells and HLA class I molecules defines NK cell functions, but the role of HLA class I ligands in the acquisition of their receptors during NK development is still unclear. Although some studies demonstrated that HLA-C affects the expression of killer Ig-like receptors (KIR), other studies showed that NK cells acquire their KIR repertoire in a stochastic manner. Only when infected with human CMV is an expansion of self-specific KIR(+) NKG2C(+) NK cells detected. To gain more insight into this question, we compared the coexpression of different KIR molecules, NKG2A, CD8, and CD57, on NK cells in healthy donors and seven patients with deficient HLA class I expression due to mutations in one of the TAP genes. Our results show a correlation between the presence/absence of HLA class I molecules and the coexpression of their receptors. In an HLA class I low-expression context, an increase in KIR molecules' coexpression is detected on the NKG2A(+) CD8(+) subset. In functional assays, hyporesponsiveness was observed for TAP-deficient NK cells derived from four patients. In contrast, NK cells from patient five were functional, whereas CD107a(+) and IFN-γ(+) CD56(dim) NK cells presented a different pattern of HLA class I receptors compared with healthy donors. Taken together, our results provide strong evidence for the role of HLA class I molecules in NK cell maturation and KIR repertoire acquisition.

  16. Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

    PubMed Central

    Ramond, Elodie; Gesbert, Gael; Guerrera, Ida Chiara; Chhuon, Cerina; Dupuis, Marion; Rigard, Mélanie; Henry, Thomas; Barel, Monique; Charbit, Alain

    2015-01-01

    Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584). PMID:25616868

  17. Targeted Protein Degradation by Salmonella under Phagosome-Mimicking Culture Conditions Investigated Using Comparative Peptidomics

    SciTech Connect

    Manes, Nathan P.; Gustin, Jean K.; Rue, Joanne; Mottaz, Heather M.; Purvine, Samuel O.; Norbeck, Angela D.; Monroe, Matthew E.; Zimmer, Jennifer S.; Metz, Thomas O.; Adkins, Joshua N.; Smith, Richard D.; Heffron, Fred

    2007-04-01

    The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Due to the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g., contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study Salmonella enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal compartment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified 5,163 distinct peptides originating from 682 proteins and the data clearly indicated that compared to cells cultured in the rich medium, Salmonella cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed by using bottom-up proteomics, and when the peptidomic and proteomic data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

  18. The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages.

    PubMed

    Sauer, John-Demian; Bachman, Michael A; Swanson, Michele S

    2005-07-12

    Differentiation in response to environmental cues is integral to the success of many intracellular pathogens. By characterizing a Legionella pneumophila mutant defective for differentiation in broth and replication in macrophages, we identified a subfamily of major facilitator superfamily transporters, here named Pht (phagosomal transporter), that also is conserved in two other vacuolar pathogens, Coxiella burnetii and Francisella tularensis. Biolog phenotype microarray analysis indicated that PhtA transports threonine, an essential amino acid. Either excess threonine or threonine peptides bypass phtA function. In minimal medium, phtA mutants do not replicate; in rich broth, the bacteria prematurely differentiate to the transmissive phase, as judged by the kinetics of flaA-gfp expression, heat resistance, and sodium sensitivity. PhtA is dispensable for transmissive L. pneumophila to establish and persist within a replication vacuole but is essential for their differentiation to the replicative phase, based on phenotypic and RT-PCR analysis. Accordingly, we propose that the Pht transporter family equips transmissive L. pneumophila, C. burnetii, and F. tularensis to assess their phagosomal nutrient supply before committing to reenter the cell cycle.

  19. Phagosomal degradation increases TLR access to bacterial ligands and enhances macrophage sensitivity to bacteria

    PubMed Central

    Wolf, Andrea J.; Arruda, Andrea; Reyes, Christopher N.; Kaplan, Amber T.; Shimada, Takahiro; Shimada, Kenichi; Arditi, Moshe; Liu, George; Underhill, David M.

    2011-01-01

    Signaling by innate immune receptors initiates and orchestrates the overall immune responses to infection. Macrophage receptors recognizing pathogens can be broadly grouped into surface receptors and receptors restricted to intracellular compartments, such as phagosomes and the cytoplasm. There is an expectation that ingestion and degradation of microorganisms by phagocytes contributes to activation of intracellular innate receptors, although direct demonstrations of this are rare and many model ligands are studied in soluble form, outside of their microbial context. By comparing a wild-type strain of Staphylococcus aureus and a lysozyme-sensitive mutant, we have been able to directly address the role of degradation of live bacteria by mouse macrophages in determining the overall innate cellular inflammatory response. Our investigations revealed a biphasic response to S. aureus that consisted of an initial signal resulting from the engagement of surface TLR2, followed by a later, second wave on inflammatory gene induction. This second wave of inflammatory signaling was dependent on and correlated with the timing of bacterial degradation in phagosomes. We found that TLR2 signaling followed by TLR2/TLR9 signaling enhanced sensitivity to small numbers of bacteria. We further found that treating wild-type bacteria with the peptidoglycan synthesis-inhibiting antibiotic vancomycin made S. aureus more susceptible to degradation and resulted in increased inflammatory responses, similar to those observed for mutant degradation-sensitive bacteria. PMID:22031762

  20. Assessment of training effects on autonomic modulation of the cardiovascular system in mature rats using power spectral analysis of heart rate variability.

    PubMed

    Kumae, Takashi

    2012-09-01

    To clarify the effects of forced or voluntary exercise on autonomic modulation of the cardiovascular system, we monitored changes in autonomic nervous activity in a mature rat by spectral analysis of the heart rate (HR) during a 10-week training period. Male Wistar rats implanted with a radio-telemetry system were divided into three groups at 18 weeks of age: (1) Control group (n = 8); (2) Voluntary group (n = 6), which were housed separately in a cage with a running wheel; (3) Forced group (n = 6), which were exercised on a treadmill (35 m/min, 15 min/day, 5 days/week). The electrocardiogram was analyzed by the maximum entropy method into two main oscillations, low-frequency (LF) and high-frequency (HF) oscillations, respectively. LF and HF are considered to be markers of both sympathetic and parasympathetic modulations and parasympathetic modulation, respectively. Average running distances of the Voluntary group were more than twofold higher than those of the Forced group. HR levels in the Forced group were lower than those in the Control group. LF and HF levels in the Control and the Forced groups were almost the same during the experiment, and those in the Voluntary group showed a tendency to decrease. The results in the Voluntary and the Forced groups suggest that cardiovascular adjustments are not simply caused by the quantity of exercise. In the Voluntary group, both sympathetic and parasympathetic activity may decrease with a predominance of sympathetic activity. Conversely, in the Forced group, the baroreflex may be hyper-activated by the undesired treadmill running and handling stress.

  1. Sexual maturation modulates expression of nuclear receptor types in laser-captured single cells of the cichlid (Oreochromis niloticus) pituitary.

    PubMed

    Kitahashi, Takashi; Ogawa, Satoshi; Soga, Tomoko; Sakuma, Yasuo; Parhar, Ishwar

    2007-12-01

    The role of steroid/thyroid hormones in the regulation of endocrine cells at the level of the pituitary has remained unclear. Therefore, using single-cell quantitative real-time PCR, we examined absolute amounts of transcripts for nuclear receptors [estrogen receptors (ERs) alpha, beta, and gamma; androgen receptors (ARs) a and b; glucocorticoid receptors (GRs) 1, 2a, and 2b; and thyroid hormone receptors (TRs) alpha1, alpha2, and beta] in pituitary cells of immature (IM) and mature (M) male tilapia, Oreochromis niloticus. In the two reproductive stages, ACTH cells expressed only ERbeta, whereas all other pituitary cell types expressed ERalpha + beta, and a subpopulation coexpressed ARa, ARb, GR1, GR2b, and TRbeta but lacked ERgamma, GR2a, TRalpha1, and TRalpha2. IM males had high percentages of LH cells (IM 46.0% vs. M 10.0%), GH cells (IM 23.3% vs. M 7.9%), and prolactin cells (IM 68.8% vs. M 6.0%) with ERbeta, and TSH cells (IM 19.2% vs. M 0.0%) and MSH cells (IM 25.6% vs. M 0.0%) with ERalpha + TRbeta. A high percentage of FSH cells in IM males expressed ERbeta (IM 46.9% vs. M 18.8%), and FSH cells in M males showed significantly high GR1 transcripts (IM 76.0 +/- 5.0 vs. M 195.0 +/- 10.7 copies per cell; P < 0.05), suggesting that FSH cells are regulated differently in the two reproductive stages. Coexpression of ERalpha + beta in high percentages of cells of the GH family (GH, IM 43.8% vs. M 14.3%; prolactin, IM 8.3% vs. M 59.7%; somatolactin, IM 22.2% vs. M 42.2%) suggests that the expression of both ERs is important for functionality. Thus, differential coexpression of genes for nuclear receptors in subpopulations of pituitary cell types suggests multiple steroid/thyroid hormone regulatory pathways at the level of the pituitary during the two reproductive stages.

  2. LuxS promotes biofilm maturation and persistence of nontypeable haemophilus influenzae in vivo via modulation of lipooligosaccharides on the bacterial surface.

    PubMed

    Armbruster, Chelsie E; Hong, Wenzhou; Pang, Bing; Dew, Kristin E; Juneau, Richard A; Byrd, Matthew S; Love, Cheraton F; Kock, Nancy D; Swords, W Edward

    2009-09-01

    Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.

  3. LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface▿ †

    PubMed Central

    Armbruster, Chelsie E.; Hong, Wenzhou; Pang, Bing; Dew, Kristin E.; Juneau, Richard A.; Byrd, Matthew S.; Love, Cheraton F.; Kock, Nancy D.; Swords, W. Edward

    2009-01-01

    Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo. PMID:19564381

  4. A small molecule deubiquitinase inhibitor increases localization of inducible nitric oxide synthase to the macrophage phagosome and enhances bacterial killing.

    PubMed

    Burkholder, Kristin M; Perry, Jeffrey W; Wobus, Christiane E; Donato, Nicholas J; Showalter, Hollis D; Kapuria, Vaibhav; O'Riordan, Mary X D

    2011-12-01

    Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

  5. The ability of Listeria monocytogenes PI-PLC to facilitate escape from the macrophage phagosome is dependent on host PKCbeta.

    PubMed

    Poussin, Mathilde A; Leitges, Michael; Goldfine, Howard

    2009-01-01

    Listeria monocytogenes are facultative intracellular pathogenic bacteria that can infect macrophages as well as non-professional phagocytes. After entry in the host cell, the bacteria escape from the phagosome into the cytoplasm. In murine macrophages and in cell lines derived from these cells, escape of L. monocytogenes from the phagosome is absolutely dependent on listeriolysin O (LLO) and facilitated by a secreted phosphatidylinositol-specific phospholipase C (PI-PLC). Work in this laboratory has previously demonstrated a LLO and PI-PLC-dependent translocation of host PKCbeta isoforms. Pharmacological inhibition of PKCbeta resulted in a significant reduction in permeabilization of the phagosome, and in the number of bacteria reaching the cytosol. These findings led to the prediction that the bacterial PI-PLC promotes escape through the production of diacylglycerol leading to the activation of host PKCbeta. To test this hypothesis, bone marrow-derived macrophages (BMMf) obtained from PKCbeta knockout (PKCbetaKO) or C57Bl/6 mice were infected with L. monocytogenes. We observed that wild-type L. monocytogenes escapes from the phagosome of PKCbetaKO BMMf as well as from C57Bl/6 BMMf. However, in PKCbetaKO BMMf, L. monocytogenes uses a PI-PLC-independent, but phosphatidylcholine-preferring PLC (PC-PLC)-dependent pathway to facilitate escape. These findings strongly support the hypothesis that PI-PLC promotes escape through mobilization of host PKCbeta.

  6. Effects of zinc oxide nanoparticles on Kupffer cell phagosomal motility, bacterial clearance, and liver function

    PubMed Central

    Watson, Christa Y; Molina, Ramon M; Louzada, Andressa; Murdaugh, Kimberly M; Donaghey, Thomas C; Brain, Joseph D

    2015-01-01

    Background Zinc oxide engineered nanoparticles (ZnO ENPs) have potential as nanomedicines due to their inherent properties. Studies have described their pulmonary impact, but less is known about the consequences of ZnO ENP interactions with the liver. This study was designed to describe the effects of ZnO ENPs on the liver and Kupffer cells after intravenous (IV) administration. Materials and methods First, pharmacokinetic studies were conducted to determine the tissue distribution of neutron-activated 65ZnO ENPs post-IV injection in Wistar Han rats. Then, a noninvasive in vivo method to assess Kupffer cell phagosomal motility was employed using ferromagnetic iron particles and magnetometry. We also examined whether prior IV injection of ZnO ENPs altered Kupffer cell bactericidal activity on circulating Pseudomonas aeruginosa. Serum and liver tissues were collected to assess liver-injury biomarkers and histological changes, respectively. Results We found that the liver was the major site of initial uptake of 65ZnO ENPs. There was a time-dependent decrease in tissue levels of 65Zn in all organs examined, refecting particle dissolution. In vivo magnetometry showed a time-dependent and transient reduction in Kupffer cell phagosomal motility. Animals challenged with P. aeruginosa 24 hours post-ZnO ENP injection showed an initial (30 minutes) delay in vascular bacterial clearance. However, by 4 hours, IV-injected bacteria were cleared from the blood, liver, spleen, lungs, and kidneys. Seven days post-ZnO ENP injection, creatine phosphokinase and aspartate aminotransferase levels in serum were significantly increased. Histological evidence of hepatocyte damage and marginated neutrophils were observed in the liver. Conclusion Administration of ZnO ENPs transiently inhibited Kupffer cell phagosomal motility and later induced hepatocyte injury, but did not alter bacterial clearance from the blood or killing in the liver, spleen, lungs, or kidneys. Our data show that

  7. The unique trafficking pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry.

    PubMed Central

    Rathman, M; Barker, L P; Falkow, S

    1997-01-01

    Although it has been known for some time that Salmonella typhimurium is able to survive and even replicate in the normally bactericidal environment of the macrophage phagosome, the mechanisms by which this organism accomplishes this feat remain obscure. In this study, a murine macrophage cell line and confocal immunofluorescence microscopy were used to more thoroughly define the specific nature of phagosomes containing latex beads or wild-type S. typhimurium (viable or heat-killed organisms). Live S. typhimurium organisms were observed to reside in phagosomes that diverge from the degradative pathway of the macrophage. These compartments contain lysosomal glycoproteins and lysosomal acid phosphatase, endocytic markers delivered to vacuoles by mannose 6-phosphate receptor-independent mechanisms, but are devoid of the mannose 6-phosphate receptor and cathepsin L. In contrast, phagosomes containing latex beads or heat-killed organisms appeared to be processed along the degradative pathway of the host cell; these compartments colocalized not only with lysosomal glycoproteins and lysosomal acid phosphatases but also with mannose 6-phosphate receptors and cathepsin L. The uniqueness of the phagosome containing viable S. typhimurium was confirmed by the observation that these compartments, in comparison to phagosomes containing latex beads, do not readily interact with incoming endocytic traffic. Finally, we show that an isogenic, noninvasive mutant of S. typhimurium, BJ66, ends up in an intracellular compartment identical to the wild-type S. typhimurium-containing phagosome. Thus, modifications of the Salmonella-containing compartment occur independently of the mechanism of bacterial entry. PMID:9119490

  8. Silica crystals and aluminum salts mediate NALP-3 inflammasome activation via phagosomal destabilization

    PubMed Central

    Hornung, Veit; Bauernfeind, Franz; Halle, Annett; Samstad, Eivind O.; Kono, Hajime; Rock, Kenneth L.; Fitzgerald, Katherine A.; Latz, Eicke

    2010-01-01

    Inhalation of silica crystals causes inflammation in the alveolar space. Prolonged silica exposure can lead to the development of silicosis, an irreversible, fibrotic pulmonary disease. The mechanisms by which silica and other crystals activate immune cells are not well understood. Here, we demonstrate that silica and aluminum salt crystals activate the NALP3 inflammasome. NALP3 activation requires crystal phagocytosis and crystal uptake leads to lysosomal damage and rupture. Sterile lysosomal damage is also sufficient to induce NALP3 activation and inhibition of phagosomal acidification or cathepsin B impairs NALP3 activation. These results indicate that the NALP3 inflammasome can sense lysosomal damage induced by various means as an endogenous danger signal. PMID:18604214

  9. Salmonella transcriptional signature in Tetrahymena phagosomes and role of acid tolerance in passage through the protist

    PubMed Central

    Rehfuss, Marc Yi Ming; Parker, Craig Thomas; Brandl, Maria Theresa

    2011-01-01

    Salmonella enterica Typhimurium remains undigested in the food vacuoles of the common protist, Tetrahymena. Contrary to its interaction with Acanthamoeba spp., S. Typhimurium is not cytotoxic to Tetrahymena and is egested as viable cells in its fecal pellets. Through microarray gene expression profiling we investigated the factors in S. Typhimurium that are involved in its resistance to digestion by Tetrahymena. The transcriptome of S. Typhimurium in Tetrahymena phagosomes showed that 989 and 1282 genes were altered in expression compared with that in water and in LB culture medium, respectively. A great proportion of the upregulated genes have a role in anaerobic metabolism and the use of alternate electron acceptors. Many genes required for survival and replication within macrophages and human epithelial cells also had increased expression in Tetrahymena, including mgtC, one of the most highly induced genes in all three cells types. A ΔmgtC mutant of S. Typhimurium did not show decreased viability in Tetrahymena, but paradoxically, was egested at a higher cell density than the wild type. The expression of adiA and adiY, which are involved in arginine-dependent acid resistance, also was increased in the protozoan phagosome. A ΔadiAY mutant had lower viability after passage through Tetrahymena, and a higher proportion of S. Typhimurium wild-type cells within pellets remained viable after exposure to pH 3.4 as compared with uningested cells. Our results provide evidence that acid resistance has a role in the resistance of Salmonella to digestion by Tetrahymena and that passage through the protist confers physiological advantages relevant to its contamination cycle. PMID:20686510

  10. Salmonella transcriptional signature in Tetrahymena phagosomes and role of acid tolerance in passage through the protist.

    PubMed

    Rehfuss, Marc Yi Ming; Parker, Craig Thomas; Brandl, Maria Theresa

    2011-02-01

    Salmonella enterica Typhimurium remains undigested in the food vacuoles of the common protist, Tetrahymena. Contrary to its interaction with Acanthamoeba spp., S. Typhimurium is not cytotoxic to Tetrahymena and is egested as viable cells in its fecal pellets. Through microarray gene expression profiling we investigated the factors in S. Typhimurium that are involved in its resistance to digestion by Tetrahymena. The transcriptome of S. Typhimurium in Tetrahymena phagosomes showed that 989 and 1282 genes were altered in expression compared with that in water and in LB culture medium, respectively. A great proportion of the upregulated genes have a role in anaerobic metabolism and the use of alternate electron acceptors. Many genes required for survival and replication within macrophages and human epithelial cells also had increased expression in Tetrahymena, including mgtC, one of the most highly induced genes in all three cells types. A ΔmgtC mutant of S. Typhimurium did not show decreased viability in Tetrahymena, but paradoxically, was egested at a higher cell density than the wild type. The expression of adiA and adiY, which are involved in arginine-dependent acid resistance, also was increased in the protozoan phagosome. A ΔadiAY mutant had lower viability after passage through Tetrahymena, and a higher proportion of S. Typhimurium wild-type cells within pellets remained viable after exposure to pH 3.4 as compared with uningested cells. Our results provide evidence that acid resistance has a role in the resistance of Salmonella to digestion by Tetrahymena and that passage through the protist confers physiological advantages relevant to its contamination cycle.

  11. Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist Entamoeba histolytica.

    PubMed

    Picazarri, Karina; Nakada-Tsukui, Kumiko; Tsuboi, Kumiko; Miyamoto, Eri; Watanabe, Naoko; Kawakami, Eiryo; Nozaki, Tomoyoshi

    2015-10-01

    Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan Entamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy-related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E. histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory-maintained and recently established clinical isolates and appeared to be lipid-modified in logarithmic growth phase, suggesting a role of Atg8 in non-stress and proliferative conditions. These findings are in contrast to those for Entamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En. histolytica by antisense small RNA-mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote.

  12. Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist E ntamoeba histolytica

    PubMed Central

    Picazarri, Karina; Nakada‐Tsukui, Kumiko; Tsuboi, Kumiko; Miyamoto, Eri; Watanabe, Naoko; Kawakami, Eiryo

    2015-01-01

    Summary Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan E ntamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy‐related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E . histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory‐maintained and recently established clinical isolates and appeared to be lipid‐modified in logarithmic growth phase, suggesting a role of Atg8 in non‐stress and proliferative conditions. These findings are in contrast to those for E ntamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En . histolytica by antisense small RNA‐mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote. PMID:25923949

  13. Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis

    PubMed Central

    1982-01-01

    Certain species of Chlorella live within the digestive cells of the fresh water cnidarian Hydra viridis. When introduced into the hydra gut, these symbiotic algae are phagocytized by digestive cells but avoid host digestion and persist at relatively constant numbers within host cells. In contrast, heat-killed symbionts are rapidly degraded after phagocytosis. Live symbionts appear to persist because host lysosomes fail to fuse with phagosomes containing live symbionts. Neither acid phosphatase nor ferritin was delivered via lysosomes into phagosomes containing live symbionts, whereas these lysosomal markers were found in 50% of the vacuoles containing heat-killed symbionts 1 h after phagocytosis. Treatment of symbiotic algae before phagocytosis with polycationic polypeptides abolishes algal persistence and perturbs the ability of these algae to control the release of photosynthate in vitro. Similarly, inhibition of photosynthesis and hence of the release of photosynthetic products as a result of prolonged darkness and 3-(3,4- dichlorophenyl)-1,1-dimethyl urea (DCMU) treatment also abolishes persistence. Symbiotic algae are not only protected from host digestive attack but are also selectively transported within host cells, moving from the apical site of phagocytosis to a basal position of permanent residence. This process too is disrupted by polycationic polypeptides, DCMU and darkness. Both algal persistence and transport may, therefore, be a function of the release of products from living, photosynthesizing symbionts. Vinblastine treatment of host animals blocked the movement of algae within host cells but did not perturb algal persistence: algal persistence and the transport of algae may be initiated by the same signal, but they are not interdependent processes. PMID:7119017

  14. Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae.

    PubMed

    Chen, Ming-Chyuan; Cheng, Ying-Min; Hong, Min-Chang; Fang, Lee-Shing

    2004-11-19

    The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts.

  15. Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae.

    PubMed

    Chen, Ming-Chyuan; Cheng, Ying-Min; Sung, Ping-Jyun; Kuo, Cham-En; Fang, Lee-Shing

    2003-08-29

    The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.

  16. Coronin-1a inhibits autophagosome formation around Mycobacterium tuberculosis-containing phagosomes and assists mycobacterial survival in macrophages.

    PubMed

    Seto, Shintaro; Tsujimura, Kunio; Koide, Yukio

    2012-05-01

    Mycobacterium tuberculosis is an intracellular bacterium that can survive within macrophages. Such survival is potentially associated with Coronin-1a (Coro1a). We investigated the mechanism by which Coro1a promotes the survival of M. tuberculosis in macrophages and found that autophagy was involved in the inhibition of mycobacterial survival in Coro1a knock-down (KD) macrophages. Fluorescence microscopy and immunoblot analyses revealed that LC3, a representative autophagic protein, was recruited to M. tuberculosis-containing phagosomes in Coro1a KD macrophages. Thin-section electron microscopy demonstrated that bacilli were surrounded by the multiple membrane structures in Coro1a KD macrophages. The proportion of LC3-positive mycobacterial phagosomes colocalized with p62/SQSTM1, ubiquitin or LAMP1 increased in Coro1a KD macrophages during infection. These results demonstrate the formation of autophagosomes around M. tuberculosis in Coro1a KD macrophages. Phosphorylation of p38 mitogen-activated protein kinase (MAPK) was induced in response to M. tuberculosis infection in Coro1a KD macrophages, suggesting that Coro1a blocks the activation of the p38 MAPK pathway involved in autophagosome formation. LC3 recruitment to M. tuberculosis-containing phagosomes was also observed in Coro1a KD alveolar or bone marrow-derived macrophages. These results suggest that Coro1a inhibits autophagosome formation in alveolar macrophages, thereby facilitating M. tuberculosis survival within the lung. © 2012 Blackwell Publishing Ltd.

  17. Yersinia pestis survival and replication within human neutrophil phagosomes and uptake of infected neutrophils by macrophages.

    PubMed

    Spinner, Justin L; Winfree, Seth; Starr, Tregei; Shannon, Jeffrey G; Nair, Vinod; Steele-Mortimer, Olivia; Hinnebusch, B Joseph

    2014-03-01

    Yersinia pestis, the bacterial agent of plague, is transmitted by fleas. The bite of an infected flea deposits Y. pestis into the dermis and triggers recruitment of innate immune cells, including phagocytic PMNs. Y. pestis can subvert this PMN response and survive at the flea-bite site, disseminate, and persist in the host. Although its genome encodes a number of antiphagocytic virulence factors, phagocytosis of Y. pestis by PMNs has been observed. This study tests the hypotheses that Y. pestis, grown at the ambient temperature of the flea vector (21°C), where the major antiphagocytic virulence factors are not produced, can survive and replicate within human PMNs and can use PMNs as a route to infect macrophages subsequently. We show that Y. pestis is localized within PMN phagosomes, predominately as individual bacteria, and that intracellular bacteria can survive and replicate. Within 12 h of infection, ~70% of infected PMNs had PS on their surface and were plausibly competent for efferocytosis. With the use of live cell confocal imaging, we show that autologous HMDMs recognize and internalize infected PMNs and that Y. pestis survives and replicates within these HMDMs following efferocytosis. Addition of HMDMs to infected PMNs resulted in decreased secretion of inflammatory cytokines (compared with HMDMs incubated directly with pCD1(-) Y. pestis) and increased secretion of the anti-inflammatory cytokine IL-1ra. Thus, Y. pestis can survive and replicate within PMNs, and infected PMNs may be a route for noninflammatory infection of macrophages.

  18. Fatty acid composition of porcine cumulus oocyte complexes (COC) during maturation: effect of the lipid modulators trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) and forskolin.

    PubMed

    Prates, E G; Alves, S P; Marques, C C; Baptista, M C; Horta, A E M; Bessa, R J B; Pereira, R M

    2013-05-01

    The effect of maturation and of two lipid modulators supplementation along in vitro maturation (IVM) on fatty acid (FA) and dimethylacetal (DMA) composition of porcine cumulus oocyte complexes (COC) were studied. Abattoir-derived immature COC were analyzed for FA and DMA or submitted to IVM as follows: control group; t10,c12 CLA group, t10,c12 CLA supplementation for 44 h; Forskolin group, forskolin supplementation during the initial 2 h; t10,c12 CLA + forskolin group, t10,c12 CLA for 44 h and forskolin for just 2h. Each experimental group had five replicates. FA analysis of oocytes, cumulus cells (CC), follicular fluid, and culture media were performed by gas-liquid chromatography. Oocytes and their CC had different FA composition. Oocytes were richer in saturated FA (SFA) preferentially maintaining their FA profile during maturation. Mature CC had the highest polyunsaturated FA (PUFA) content. Five individual and total SFA, and monounsaturated FA (MUFA), notably oleic acid (c9-18:1), percentages were lower (P ≤ 0.023) in mature than in immature CC. t10,c12 CLA was accumulated by COC from t10,c12 CLA and t10,c12 CLA + forskolin groups, mostly in CC where MUFA and an eicosatrienoic isomer decreased (P ≤ 0.043). Nevertheless, PUFA or FA and DMA total content were not affected. Arachidonic acid was reduced in t10,c12 CLA + forskolin CC and hexadecanal-DMA-16:0 in t10,c12 CLA CC. Forskolin alone increased (P ≤ 0.043) c9-18:1 in oocytes. In conclusion, maturation process clearly changed porcine COC FA and DMA profiles, mostly of CC, also more susceptible to modifications induced by t10,c12 CLA. This possibility of manipulating COC lipid composition during IVM could be used to improve oocyte quality/cryopreservation efficiency.

  19. Brain maturation and epilepsy.

    PubMed

    Dulac, Olivier; Milh, Mathieu; Holmes, Gregory L

    2013-01-01

    At full term, both glutamate and gamma-amino-butyric acid (GABA) are excitatory; cortical synapses are beginning to appear, there is little myelin in the cerebral hemispheres, and long tracts hardly start to develop. Neonatal myoclonic encephalopathy can result from premature activation of N-methyl-D-aspartate (NMDA) transmission. Benign neonatal seizures and migrating partial seizures in infancy could involve excessive or premature excitability of deep cortical layers. Benign rolandic epilepsy and continuous spike waves in slow sleep are consistent with an excess of both excitatory and inhibitory cortical synapses. West and Lennox-Gastaut syndromes express age-related diffuse cortical hyperexcitability, the pattern depending on the age of occurrence; synchronization of spikes is becoming possible with maturation of the myelin. Idiopathic generalized epilepsy is itself modulated by maturation that causes frontal hyperexcitability generating myoclonic-astatic seizures, between the ages of infantile and juvenile myoclonic epilepsies. Physiological delay of hippocampo-neocortical pathways maturation could account for the delayed occurrence of mesial temporal epilepsy following infantile damage, whereas premature maturation could contribute to fronto-temporal damage characteristic of fever-induced epileptic encephalopathy in school-age children, a dramatic school-age epileptic encephalopathy.

  20. Virus maturation.

    PubMed

    Delgui, Laura R; Rodríguez, José F

    2013-01-01

    The formation of infectious virus particles is a highly complex process involving a series of sophisticated molecular events. In most cases, the assembly of virus structural elements results in the formation of immature virus particles unable to initiate a productive infection. Accordingly, for most viruses the final stage of the assembly pathway entails a set of structural transitions and/or biochemical modifications that transform inert precursor particles into fully infectious agents. In this chapter, we review the most relevant maturation mechanisms involved in the generation of infectious virions for a wide variety of viruses.

  1. Phagosome-lysosome fusions in macrophages infected by Mycobacterium avium: role of mycosides-C and other cells surface components.

    PubMed

    Fréhel, C; Rastogi, N

    1989-01-01

    The phagosome-lysosome fusions (PLE) were assessed in case of bone-marrow macrophages infected by the opportunistic species Mycobacterium avium, employing the acid-phosphatase (AcPase) electron-cytochemistry. The role of surface components was evaluated by coating the bacteria prior to phagocytosis by specific M. avium antiserum or the anti-mycosides-C serum raised in rabbit. PLF was evaluated under the electron microscope during (2, 4 hours), or after (24 hours) phagocytosis. The preliminary results suggest that although M. avium surface components intervene in PLF inhibition, the role of mycosides-C among these surface components (effectively intervening in PLF inhibition) is questionable.

  2. The Microvesicle Component of HIV-1 Inocula Modulates Dendritic Cell Infection and Maturation and Enhances Adhesion to and Activation of T Lymphocytes

    PubMed Central

    Mercier, Sarah K.; Donaghy, Heather; Botting, Rachel A.; Turville, Stuart G.; Harman, Andrew N.; Nasr, Najla; Ji, Hong; Kusebauch, Ulrike; Mendoza, Luis; Shteynberg, David; Sandgren, Kerrie; Simpson, Richard J.; Moritz, Robert L.; Cunningham, Anthony L.

    2013-01-01

    HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45+ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45+ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24+) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects

  3. Genetic manipulation of RPS5 gene expression modulates the initiation of commitment of MEL cells to erythroid maturation: Implications in understanding ribosomopathies.

    PubMed

    Vizirianakis, Ioannis S; Papachristou, Eleni T; Andreadis, Panagiotis; Zopounidou, Elena; Matragkou, Christina N; Tsiftsoglou, Asterios S

    2015-07-01

    Impairment of ribosome biogenesis contributes to the molecular pathophysiology of ribosomopathies by deregulating cell-lineage specific proliferation, differentiation and apoptosis decisions of haematopoietic progenitor cells. Here, using pro-erythroblast-like murine erythroleukemia (MEL) cells, a model system of erythroid maturation, we aimed to investigate whether genetic manipulation of RPS5 expression affects the capacity of cells to grow and differentiate in culture. Parental MEL cells stably transfected with full length RPS5 cDNA in sense (MEL-C14 culture) or antisense (MEL-antisenseRPS5 culture) orientation, as well as MEL cells transiently transfected with siRNAs specific for RPS5 gene silencing (MEL-RPS5siRNA culture) were assessed for their ability to fully execute their erythroid maturation program in culture. The data obtained thus far indicate that: a) MEL-antisenseRPS5 exhibit a pronounced delay in the initiation of differentiation, as well as an impairment of commitment, since the continuous presence of the inducer in culture is required for the cells to fully execute their erythroid maturation program. b) RNAi-mediating silencing of RPS5 gene expression resulted in the inability of MEL cells to differentiate; however, when these cells were allowed to recapitulate normal RPS5 gene expression levels they regained their differentiation capacity by accumulating high proportion of erythroid mature cells. c) Interestingly the latter, is accompanied by morphological changes of cells and an impairment of their proliferation and apoptosis potential. Such data for the first time correlate the RPS5 gene expression levels with the differentiation capacity of MEL cells in vitro, a fact that might also have implications in understanding ribosomopathies.

  4. Cytosolic Delivery of Liposomal Vaccines by Means of the Concomitant Photosensitization of Phagosomes.

    PubMed

    Hjálmsdóttir, Ásdís; Bühler, Céline; Vonwil, Vera; Roveri, Maurizio; Håkerud, Monika; Wäckerle-Men, Ying; Gander, Bruno; Johansen, Pål

    2016-02-01

    One of the greatest pharmaceutical challenges in vaccinology is the delivery of antigens to the cytosol of antigen-presenting cells (APCs) in order to allow for the stimulation of major histocompatibility complex (MHC) class I-restricted CD8(+) T-cell responses, which may act on intracellular infections or cancer. Recently, we described a novel method for cytotoxic T-lymphocyte (CTL) vaccination by combining antigens with a photosensitizer and light for cytosolic antigen delivery. The goal of the current project was to test this immunization method with particle-based formulations. Liposomes were prepared from dipalmitoylphosphatidylcholine and cholesterol, and the antigen ovalbumin (OVA) or the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) was separately encapsulated. C57BL/6 mice were immunized intradermally with OVA liposomes or a combination of OVA and TPCS2a liposomes, and light was applied the next day for activation of the photosensitizer resulting in cytosolic release of antigen from phagosomes. Immune responses were tested both after a prime only regime and after a prime-boost scheme with a repeat immunization 2 weeks post priming. Antigen-specific CD8(+) T-cell responses and antibody responses were analyzed ex vivo by flow cytometry and ELISA methods. The physicochemical stability of liposomes upon storage and light exposure was analyzed in vitro. Immunization with both TPCS2a- and OVA-containing liposomes greatly improved CD8(+) T-cell responses as compared to immunization without TPCS2a and as measured by proliferation in vivo and cytokine secretion ex vivo. In contrast, OVA-specific antibody responses (IgG1 and IgG2c) were reduced after immunization with TPCS2a-containing liposomes. The liposomal formulation protected the photosensitizer from light-induced inactivation during storage. In conclusion, the photosensitizer TPCS2a was successfully formulated in liposomes and enabled a shift from MHC class II to MHC class I antigen processing and

  5. LIMP-2 Links Late Phagosomal Trafficking with the Onset of the Innate Immune Response to Listeria monocytogenes

    PubMed Central

    Carrasco-Marín, Eugenio; Fernández-Prieto, Lorena; Rodriguez-Del Rio, Estela; Madrazo-Toca, Fidel; Reinheckel, Thomas; Saftig, Paul; Alvarez-Dominguez, Carmen

    2011-01-01

    The innate immune response to Listeria monocytogenes depends on phagosomal bacterial degradation by macrophages. Here, we describe the role of LIMP-2, a lysosomal type III transmembrane glycoprotein and scavenger-like protein, in Listeria phagocytosis. LIMP-2-deficient mice display a macrophage-related defect in Listeria innate immunity. They produce less acute phase pro-inflammatory cytokines/chemokines, MCP-1, TNF-α, and IL-6 but normal levels of IL-12, IL-10, and IFN-γ and a 25-fold increase in susceptibility to Listeria infection. This macrophage defect results in a low listericidal potential, poor response to TNF-α activation signals, impaired phago-lysosome transformation into antigen-processing compartments, and uncontrolled LM cytosolic growth that fails to induce normal levels of acute phase pro-inflammatory cytokines. LIMP-2 transfection of CHO cells confirmed that LIMP-2 participates in the degradation of Listeria within phagosomes, controls the late endosomal/lysosomal fusion machinery, and is linked to the activation of Rab5a. Therefore, the role of LIMP-2 appears to be connected to the TNF-α-dependent and early activation of Listeria macrophages through internal signals linking the regulation of late trafficking events with the onset of the innate Listeria immune response. PMID:21123180

  6. Dictyostelium Nramp1, which is structurally and functionally similar to mammalian DMT1 transporter, mediates phagosomal iron efflux.

    PubMed

    Buracco, Simona; Peracino, Barbara; Cinquetti, Raffaella; Signoretto, Elena; Vollero, Alessandra; Imperiali, Francesca; Castagna, Michela; Bossi, Elena; Bozzaro, Salvatore

    2015-09-01

    The Nramp (Slc11) protein family is widespread in bacteria and eukaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum has two Nramp proteins. Nramp1, like its mammalian ortholog (SLC11A1), is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and the transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, and electrophysiological and radiochemical assays, we show that Nramp1, similar to rat DMT1 (also known as SLC11A2), transports Fe(2+) and manganese, not Fe(3+) or copper. Metal ion transport is electrogenic and proton dependent. By contrast, Nramp2 transports only Fe(2+) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.

  7. Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

    PubMed Central

    Brodmann, Maj; Dreier, Roland F.; Broz, Petr; Basler, Marek

    2017-01-01

    Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture. PMID:28621333

  8. Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

    PubMed Central

    Wells, M Y; Rikihisa, Y

    1988-01-01

    Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed in E. risticii-containing vacuoles in untreated cells, while significantly greater P-L fusion with ehrlichia-containing vacuoles was observed after oxytetracycline treatment. When latex beads were introduced into uninfected cell cultures, P-L fusion was observed with vacuoles containing latex. Fusion of lysosomes with latex-containing vacuoles in cells was significantly greater than fusion of lysosomes with ehrlichia-containing vacuoles in the same infected cells. These findings indicate that E. risticii is able to inhibit P-L fusion, whereas oxytetracycline deprives organisms of this ability. Images PMID:3182078

  9. Modulation of cell-cycle dynamics is required to regulate the number of cerebellar GABAergic interneurons and their rhythm of maturation.

    PubMed

    Leto, Ketty; Bartolini, Alice; Di Gregorio, Alessandra; Imperiale, Daniele; De Luca, Annarita; Parmigiani, Elena; Filipkowski, Robert K; Kaczmarek, Leszek; Rossi, Ferdinando

    2011-08-01

    The progenitors of cerebellar GABAergic interneurons proliferate up to postnatal development in the prospective white matter, where they give rise to different neuronal subtypes, in defined quantities and according to precise spatiotemporal sequences. To investigate the mechanisms that regulate the specification of distinct interneuron phenotypes, we examined mice lacking the G1 phase-active cyclin D2. It has been reported that these mice show severe reduction of stellate cells, the last generated interneuron subtype. We found that loss of cyclin D2 actually impairs the whole process of interneuron genesis. In the mutant cerebella, progenitors of the prospective white matter show reduced proliferation rates and enhanced tendency to leave the cycle, whereas young postmitotic interneurons undergo severe delay of their maturation and migration. As a consequence, the progenitor pool is precociously exhausted and the number of interneurons is significantly reduced, although molecular layer interneurons are more affected than those of granular layer or deep nuclei. The characteristic inside-out sequence of interneuron placement in the cortical layers is also reversed, so that later born cells occupy deeper positions than earlier generated ones. Transplantation experiments show that the abnormalities of cyclin D2(-/-) interneurons are largely caused by cell-autonomous mechanisms. Therefore, cyclin D2 is not required for the specification of particular interneuron subtypes. Loss of this protein, however, disrupts regulatory mechanisms of cell cycle dynamics that are required to determine the numbers of interneurons of different types and impairs their rhythm of maturation and integration in the cerebellar circuitry.

  10. Dental pulp in mature replanted human teeth: morphological alterations and metalloproteineses-2 and -9, Annexin-5, BCL-2 and iNOS modulation.

    PubMed

    Leone, A; Angelova Volponi, A; Uzzo, M L; Spatola, G F; Jurjus, A; Vandevska-Radunovic, V

    2015-01-01

    Tooth replantation, as a treatment concept, has been subject to controversies regarding the mechanism as well as the various parameters underlying this process. This work aimed to study time-related changes in the pulp of replanted mature human premolars through the changes in the levels of certain factors involved in the underlying mechanisms of pulpal tissue healing after replantation. Eleven experimental mature teeth were extracted, immediately replanted in the original socket and left without any other intervention for 1, 2, 3 and 12 weeks before re-extraction. Three premolars served as control. All specimens were subject to histological analysis and the levels of MMP-2, MMP-9, Annexin V, iNOS and BCL-2 (anti-apoptotic family) were analyzed employing immunohistochemistry. The results showed degradation of the extracellular matrix (ECM), inflammatory cell infiltrate, loss in pulpo-dentine interface and loss of odontoblasts in the dental pulp tissue. This was accompanied by increase over time of MMP-9, Annexin V, iNOS and a decrease of BCL-2 and MMP-2, suggesting that apoptosis increased throughout the experimental period.

  11. Vacuolar protein sorting protein 13A, TtVPS13A, localizes to the tetrahymena thermophila phagosome membrane and is required for efficient phagocytosis.

    PubMed

    Samaranayake, Haresha S; Cowan, Ann E; Klobutcher, Lawrence A

    2011-09-01

    Vacuolar protein sorting 13 (VPS13) proteins have been studied in a number of organisms, and mutations in VPS13 genes have been implicated in two human genetic disorders, but the function of these proteins is poorly understood. The TtVPS13A protein was previously identified in a mass spectrometry analysis of the Tetrahymena thermophila phagosome proteome (M. E. Jacobs et al., Eukaryot. Cell 5:1990-2000, 2006), suggesting that it is involved in phagocytosis. In this study, we analyzed the structure of the macronuclear TtVPS13A gene, which was found to be composed of 17 exons spanning 12.5 kb and was predicted to encode a protein of 3,475 amino acids (aa). A strain expressing a TtVPS13A-green fluorescent protein (GFP) fusion protein was constructed, and the protein was found to associate with the phagosome membrane during the entire cycle of phagocytosis. In addition, Tetrahymena cells with a TtVPS13A knockout mutation displayed impaired phagocytosis. Specifically, they grew slowly under conditions where phagocytosis is essential, they formed few phagosomes, and the digestion of phagosomal contents was delayed compared to wild-type cells. Overall, these results provide evidence that the TtVPS13A protein is required for efficient phagocytosis.

  12. The Role of Brain-Derived Neurotrophic Factor Receptors in the Mature Hippocampus: Modulation of Long-Term Potentiation through a Presynaptic Mechanism involving TrkB

    PubMed Central

    Xu, Baoji; Gottschalk, Wolfram; Chow, Ana; Wilson, Rachel I.; Schnell, Eric; Zang, Keling; Wang, Denan; Nicoll, Roger A.; Lu, Bai; Reichardt, Louis F.

    2009-01-01

    The neurotrophin BDNF has been shown to modulate long-term potentiation (LTP) at Schaffer collateral-CA1 hippocampal synapses. Mutants in the BDNF receptor gene trkB and antibodies to its second receptor p75NTR have been used to determine the receptors and cells involved in this response. Inhibition of p75NTR does not detectably reduce LTP or affect presynaptic function, but analyses of newly generated trkB mutants implicate TrkB. One mutant has reduced expression in a normal pattern of TrkB throughout the brain. The second mutant was created by cre-loxP-mediated removal of TrkB in CA1 pyramidal neurons of this mouse. Neither mutant detectably impacts survival or morphology of hippocampal neurons. TrkB reduction, however, affects presynaptic function and reduces the ability of tetanic stimulation to induce LTP. Postsynaptic glutamate receptors are not affected by TrkB reduction, indicating that BDNF does not modulate plasticity through postsynaptic TrkB. Consistent with this, elimination of TrkB in postsynaptic neurons does not affect LTP. Moreover, normal LTP is generated in the mutant with reduced TrkB by a depolarization–low-frequency stimulation pairing protocol that puts minimal demands on presynaptic terminal function. Thus, BDNF appears to act through TrkB presynaptically, but not postsynaptically, to modulate LTP. PMID:10995833

  13. Co-culture with pig membrana granulosa cells modulates the activity of cdc2 and MAP kinase in maturing cattle oocytes.

    PubMed

    Motlík, J; Sutovský, P; Kalous, J; Kubelka, M; Moos, J; Schultz, R M

    1996-08-01

    membrana granulosa cells can cause a prompt decrease in histone H1 and MAP kinase activities, and metaphase I oocytes. While these events are fully reversible in late diakinesis oocytes, metaphase I oocytes did not complete maturation after release from co-culture.

  14. An essential role of discoidin domain receptor 2 (DDR2) in osteoblast differentiation and chondrocyte maturation via modulation of Runx2 activation.

    PubMed

    Zhang, Yan; Su, Jin; Yu, Jiangtian; Bu, Xin; Ren, Tingting; Liu, Xinping; Yao, Libo

    2011-03-01

    Discoidin domain receptor 2 (DDR2) belongs to receptor tyrosine kinase (RTK) family and is activated by collagen binding. Although the bone defects in Ddr2 null mice have been reported for a decade, the molecular mechanism remains unclear. This study sought to investigate the function and detailed mechanism of DDR2 in osteogenic and chondrogenic differentiation. Herein we found that in preosteoblastic cells, DDR2 activation was enhanced by osteogenic induction but was not paralleled with the alteration of DDR2 expression. Under differentiated condition, downregulation of endogenous DDR2 through specific shRNA dramatically repressed osteoblastic marker gene expression and osteogenic differentiation. Enforced expression of constitutively activated DDR2 increased the expression of bone markers in both undifferentiated and differentiated osteoblasts. Importantly, molecular evidence showed that DDR2 regulated the transactivity of Runx2, a master transcription factor involved in skeletal development, by modulating its phosphorylation. Analysis of candidate protein kinases indicated that extracellular signal-regulated kinase (ERK) activation is responsive to DDR2 signaling and involved in DDR2 regulation of Runx2 phosphorylation and transcriptional activity. Notably, a gain-of-function mutant of Runx2 with enhanced ERK-independent phosphorylation rescued the impaired osteogenic phenotypes observed in Ddr2-silenced cells, whereas a Runx2 mutant devoid of phosphorylation regulation by ERK inhibited DDR2 induction of osteogenesis. In addition, DDR2 facilitated Runx2 transactivation and type X collagen expression in hypertrophic chondrocytes. Thus this study reveals for the first time that DDR2 plays an essential role in osteoblast and chondrocyte differentiation. The mechanism disclosure may provide therapeutic targets for human genetic disorders caused by DDR2 deficiency.

  15. Early Induction of Interleukin-10 Limits Antigen-Specific CD4+ T Cell Expansion, Function, and Secondary Recall Responses during Persistent Phagosomal Infection

    PubMed Central

    Singh, Abinav Kumar

    2014-01-01

    Diverse pathogens have evolved to survive and replicate in the endosomes or phagosomes of the host cells and establish persistent infection. Ehrlichiae are Gram-negative, intracellular bacteria that are transmitted by ticks. Ehrlichiae reside in the endosomes of the host phagocytic or endothelial cells and establish persistent infection in their vertebrate reservoir hosts. CD4+ T cells play a critical role in protection against phagosomal infections. In the present study, we investigated the expansion, maintenance, and functional status of antigen-specific CD4+ T cells during persistent Ehrlichia muris infection in wild-type and interleukin-10 (IL-10)-deficient mice. Our study indicated that early induction of IL-10 led to reduced inflammatory responses and impaired bacterial clearance during persistent Ehrlichia infection. Notably, we demonstrated that the functional production of gamma interferon (IFN-γ) by antigen-specific CD4+ T cells maintained during a persistent phagosomal infection progressively deteriorates. The functional loss of IFN-γ production by antigen-specific CD4+ T cells was reversed in the absence of IL-10. Furthermore, we demonstrated that transient blockade of IL-10 receptor during the T cell priming phase early in infection was sufficient to enhance the magnitude and the functional capacity of antigen-specific effector and memory CD4+ T cells, which translated into an enhanced recall response. Our findings provide new insights into the functional status of antigen-specific CD4+ T cells maintained during persistent phagosomal infection. The study supports the concept that a better understanding of the factors that influence the priming and differentiation of CD4+ T cells may provide a basis to induce a protective immune response against persistent infections. PMID:25024370

  16. Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga.

    PubMed

    Molmeret, Maëlle; Bitar, Dina M; Han, Lihui; Kwaik, Yousef Abu

    2004-07-01

    Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion

  17. Virulent Mycobacterium fortuitum Restricts NO Production by a Gamma Interferon-Activated J774 Cell Line and Phagosome-Lysosome Fusion

    PubMed Central

    da Silva, TÂnia Regina Marques; de Freitas, Juliana Ribeiro; Silva, Queilan Chagas; Figueira, Cláudio Pereira; Roxo, Eliana; Leão, Sylvia Cardoso; de Freitas, Luiz Antônio Rodrigues; Veras, Patrícia Sampaio Tavares

    2002-01-01

    The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mφ) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-γ) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-γ-stimulated Mφ. Indeed, the amount of IFN-γ-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 μM) compared to SmTr infection (3.9 to 4.8 μM) (P = 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-γ-induced NO production by Mφ was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-dl-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-γ-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes. PMID:12228291

  18. Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2.

    PubMed

    Nigorikawa, Kiyomi; Hazeki, Kaoru; Sasaki, Junko; Omori, Yumio; Miyake, Mikiko; Morioka, Shin; Guo, Ying; Sasaki, Takehiko; Hazeki, Osamu

    2015-01-01

    Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

  19. Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2

    PubMed Central

    Nigorikawa, Kiyomi; Hazeki, Kaoru; Sasaki, Junko; Omori, Yumio; Miyake, Mikiko; Morioka, Shin; Guo, Ying; Sasaki, Takehiko; Hazeki, Osamu

    2015-01-01

    Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P. PMID:26535897

  20. The transcriptomic response of the coral Acropora digitifera to a competent Symbiodinium strain: the symbiosome as an arrested early phagosome.

    PubMed

    Mohamed, A R; Cumbo, V; Harii, S; Shinzato, C; Chan, C X; Ragan, M A; Bourne, D G; Willis, B L; Ball, E E; Satoh, N; Miller, D J

    2016-07-01

    Despite the ecological significance of the relationship between reef-building corals and intracellular photosynthetic dinoflagellates of the genus Symbiodinium, very little is known about the molecular mechanisms involved in its establishment. Indeed, microarray-based analyses point to the conclusion that host gene expression is largely or completely unresponsive during the establishment of symbiosis with a competent strain of Symbiodinium. In this study, the use of Illumina RNA-Seq technology allowed detection of a transient period of differential expression involving a small number of genes (1073 transcripts; <3% of the transcriptome) 4 h after the exposure of Acropora digitifera planulae to a competent strain of Symbiodinium (a clade B strain). This phenomenon has not previously been detected as a consequence of both the lower sensitivity of the microarray approaches used and the sampling times used. The results indicate that complex changes occur, including transient suppression of mitochondrial metabolism and protein synthesis, but are also consistent with the hypothesis that the symbiosome is a phagosome that has undergone early arrest, raising the possibility of common mechanisms in the symbiotic interactions of corals and symbiotic sea anemones with their endosymbionts. © 2016 John Wiley & Sons Ltd.

  1. Human oocyte maturation in vitro.

    PubMed

    Coticchio, Giovanni; Dal-Canto, Mariabeatrice; Guglielmo, Maria-Cristina; Mignini-Renzini, Mario; Fadini, Rubens

    2012-01-01

    Oocytes from medium-sized antral follicles have already completed their growth phase and, if released from the follicular environment and cultured in vitro, are able to resume the meiotic process and mature. However, in vitro maturation (IVM) does not entirely support all the nuclear and cytoplasmic changes that occur physiologically as an effect of the ovulatory stimulus. Regardless, oocyte IVM is widely applied for the breeding of agriculturally important species. In assisted reproduction technology, IVM has been proposed as an alternative treatment to circumvent the drawbacks of standard ovarian stimulation regimens. Initially introduced to eliminate the risks of ovarian hyperstimulation syndrome afflicting women presenting with polycystic ovaries, subsequently IVM has been suggested to represent an additional approach suitable also for normovulatory patients. So far, in children born from IVM cycles, no doubts of an increased incidence of congenital abnormalities have been raised. Many more births would be achieved if novel IVM systems, currently dominated by empiricism, could be conceived according to more physiological criteria. Recent findings shedding new light on the control of meiotic progression, the support of cumulus cells to the oocyte cellular reorganization occurring during maturation, and the modulation of the stimulus that promotes oocyte maturation downstream the mid-cycle gonadotropin signal are likely to provide crucial hints for the development of more efficient IVM systems.

  2. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  3. Mature Teachers Matter

    ERIC Educational Resources Information Center

    Berl, Patricia Scallan

    2005-01-01

    In this article, the author discusses the consequences of losing mature teachers due to voluntary separation or retirement and the mindset of a mature teacher that is different from younger teachers in a number of ways. Mature teachers are colleagues over 45 years of age possessing significant experience in the field. Future trends in teacher…

  4. Ankyrin-repeat proteins from sponge symbionts modulate amoebal phagocytosis.

    PubMed

    Nguyen, Mary T H D; Liu, Michael; Thomas, Torsten

    2014-03-01

    Bacteria-eukaryote symbiosis occurs in all stages of evolution, from simple amoebae to mammals, and from facultative to obligate associations. Sponges are ancient metazoans that form intimate symbiotic interactions with complex communities of bacteria. The basic nutritional requirements of the sponge are in part satisfied by the phagocytosis of bacterial food particles from the surrounding water. How bacterial symbionts, which are permanently associated with the sponge, survive in the presence of phagocytic cells is largely unknown. Here, we present the discovery of a genomic fragment from an uncultured gamma-proteobacterial sponge symbiont that encodes for four proteins, whose closest known relatives are found in a sponge genome. Through recombinant approaches, we show that these four eukaryotic-like, ankyrin-repeat proteins (ARP) when expressed in Eschericha coli can modulate phagocytosis of amoebal cells and lead to accumulation of bacteria in the phagosome. Mechanistically, two ARPs appear to interfere with phagosome development in a similar way to reduced vacuole acidification, by blocking the fusion of the early phagosome with the lysosome and its digestive enzymes. Our results show that ARP from sponge symbionts can function to interfere with phagocytosis, and we postulate that this might be one mechanism by which symbionts can escape digestion in a sponge host.

  5. The magnesium inhibition and arrested phagosome hypotheses: new perspectives on the evolution and ecology of Symbiodinium symbioses.

    PubMed

    Malcolm, Hill; April, Hill

    2012-11-01

    Zooxanthella symbioses are arguably the most important ecological interaction on coral reefs because they energetically subsidize the entire community, and enhance the calcification process that provides structure for all other organisms. While we have developed a detailed understanding of the diversity among and within the Symbiodinium clades, we currently lack a mechanistic explanation for which factors favoured zooxanthella invasion of the intracellular habitat in heterotrophic hosts, and for what molecular mechanisms permit residence within the cell. We propose two hypotheses that explain important evolutionary and ecological features of zooxanthella symbioses. The magnesium inhibition hypothesis (MIH) states that increases in the Mg/Ca ratio in sea water that occurred over the last 100 million years created a situation where Mg(2+) inhibited Ca(2+) transport to zooxanthellae. The MIH predicts, among other things, that the intracellular niche was invaded as a response to this abiotic stressor. The arrested phagosome hypothesis (APH) states that Symbiodinium spp. mimic host cell endosomal digestive machinery via the symbiosome to appear like digesting prey through perpetual release of zooxanthella-derived compounds. The APH represents a subtle but important distinction from previous hypotheses regarding interactions between symbiont and host at the cellular level. The APH predicts that symbionts tune rates of material release to match expectations of host cellular machinery. An outcome of the APH is that intra-host residence time becomes a vital parameter to consider. Both hypotheses shift control of the symbiosis away from the host, and instead focus attention on the niche requirements of Symbiodinium spp. © 2012 The Authors. Biological Reviews © 2012 Cambridge Philosophical Society.

  6. Phagosomal pH and glass fiber dissolution in cultured nasal epithelial cells and alveolar macrophages: a preliminary study.

    PubMed Central

    Johnson, N F

    1994-01-01

    The dissolution rate of glass fibers has been shown to be pH sensitive using in vitro lung fluid simulant models. The current study investigated whether there is a difference in phagosomal pH (ppH) between rat alveolar macrophages (AM) and rat nasal epithelial cells (RNEC) and whether such a difference would influence the dissolution of glass fibers. The ppH was measured in cultured AM and RNEC using flow cytometric, fluorescence-emission rationing techniques with fluorescein-labeled, amorphous silica particles. Glass fiber dissolution was determined in AM and RNEC cultured for 3 weeks with fast dissolving glass fibers (GF-A) or slow dissolving ones (GF-B). The mean diameters of GF-A were 2.7 microns and of GF-B, 2.6 microns, the average length of both fibers was approximately 22 to 25 microns. Dissolution was monitored by measuring the length and diameter of intracellular fibers and estimating the volume, assuming a cylindrical morphology. The ppH of AM was 5.2 to 5.8, and the ppH of RNEC was 7.0 to 7.5. The GF-A dissolved more slowly in RNEC than in AM, and no dissolution was evident in either cell type with GF-B. The volume loss with GF-A after a 3-week culture with AM was 66% compared to 45% for cultured RNEC. These results are different from those obtained using in vitro lung fluid-simulant models where dissolution is faster at higher pH. This difference suggests that dissolution rates of glass fibers in AM should not be applied to the dissolution of fibers in epithelial cells. Images Figure 1. a Figure 1. b Figure 2. a Figure 2. b Figure 3. a Figure 3. b PMID:7882965

  7. Phagosomal Acidification Prevents Macrophage Inflammatory Cytokine Production to Malaria, and Dendritic Cells Are the Major Source at the Early Stages of Infection

    PubMed Central

    Wu, Xianzhu; Gowda, Nagaraj M.; Gowda, D. Channe

    2015-01-01

    Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H+-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors. PMID:26240140

  8. Myo1 localizes to phagosomes, some of which traffic to the nucleus in a Myo1-dependent manner in Tetrahymena thermophila.

    PubMed

    Hosein, Roland E; Gavin, R H

    2007-12-01

    Myo1 is one of 13 myosins in Tetrahymena thermophila. Initially, twelve of the myosins in Tetrahymena were assigned to Class XX in the myosin superfamily but recently re-assigned to a subclass within Class XIV. In a previous study, we reported that genomic knockout of MYO1 affected phagocytosis and macronuclear amitosis. These two phenotypes have appeared disparate because a possible mechanism linking phagocytosis and amitosis was unknown. In the present study, Myo1 localization was investigated in order to further link machinery for phagocytosis and amitosis. Antibodies directed against the Myo1 motor domain detected an immunospecific polypeptide at 175-180 kDa on immunoblots of wild-type proteins. The 175-180 kDa polypeptide was not detected on immunoblots of proteins from the knockout strain. For immunofluorescence microscopy, cells were allowed to internalize fluorescent beads as markers for phagosomes. In wild-type cells, anti-Myo1 and anti-actin antibodies co-localized to the periphery of phagosomes and the macronucleus. In the MYO1-knockout strain only background fluorescence was observed with anti-Myo1 antibody. Confocal x-z series through macronuclei revealed fluorescent beads within the nucleoplasm. Statistical analysis showed a significant difference between the mean distributions of fluorescent beads in the nucleoplasm of wild-type and MYO1-knockout cells. A fluorescent dye was used to label plasma membrane in living cells. Dye-labeled vacuoles trafficked to the macronucleus. Trafficking of phagosomes to the macronucleus in a myosin-dependent manner is a novel finding and a possible mechanism for targeting myosin and actin to the nucleus.

  9. Phagosomal Acidification Prevents Macrophage Inflammatory Cytokine Production to Malaria, and Dendritic Cells Are the Major Source at the Early Stages of Infection: IMPLICATION FOR MALARIA PROTECTIVE IMMUNITY DEVELOPMENT.

    PubMed

    Wu, Xianzhu; Gowda, Nagaraj M; Gowda, D Channe

    2015-09-18

    Inflammatory cytokines produced at the early stages of malaria infection contribute to shaping protective immunity and pathophysiology. To gain mechanistic insight into these processes, it is important to understand the cellular origin of cytokines because both cytokine input and cytokine-producing cells play key roles. Here, we determined cytokine responses by monocytes, macrophages, and dendritic cells (DCs) to purified Plasmodium falciparum and Plasmodium berghei ANKA, and by spleen macrophages and DCs from Plasmodium yoelii 17NXL-infected and P. berghei ANKA-infected mice. The results demonstrate that monocytes and macrophages do not produce inflammatory cytokines to malaria parasites and that DCs are the primary source early in infection, and DC subsets differentially produce cytokines. Importantly, blocking of phagosomal acidification by inhibiting vacuolar-type H(+)-ATPase enabled macrophages to elicit cytokine responses. Because cytokine responses to malaria parasites are mediated primarily through endosomal Toll-like receptors, our data indicate that the inability of macrophages to produce cytokines is due to the phagosomal acidification that disrupts endosomal ligand-receptor engagement. Macrophages efficiently produced cytokines to LPS upon simultaneously internalizing parasites and to heat-killed Escherichia coli, demonstrating that phagosomal acidification affects endosomal receptor-mediated, but not cell surface receptor-mediated, recognition of Toll-like receptor agonists. Enabling monocytes/macrophages to elicit immune responses to parasites by blocking endosomal acidification can be a novel strategy for the effective development of protective immunity to malaria. The results have important implications for enhancing the efficacy of a whole parasite-based malaria vaccine and for designing strategies for the development of protective immunity to pathogens that induce immune responses primarily through endosomal receptors.

  10. The Type I NADH Dehydrogenase of Mycobacterium tuberculosis Counters Phagosomal NOX2 Activity to Inhibit TNF-α-Mediated Host Cell Apoptosis

    PubMed Central

    Miller, Jessica L.; Velmurugan, Kamalakannan; Cowan, Mark J.; Briken, Volker

    2010-01-01

    The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persisting, intracellular pathogens such as the human pathogen Mycobacterium tuberculosis (Mtb) to inhibit infection-induced apoptosis of macrophages is important for virulence. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase, NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis, but the molecular mechanism of this host pathogen interaction remains elusive. Here we show that the apoptogenic phenotype of MtbΔnuoG was significantly reduced in human macrophages treated with caspase-3 and -8 inhibitors, TNF-α-neutralizing antibodies, and also after infection of murine TNF−/− macrophages. Interestingly, incubation of macrophages with inhibitors of reactive oxygen species (ROS) reduced not only the apoptosis induced by the nuoG mutant, but also its capacity to increase macrophage TNF-α secretion. The MtbΔnuoG phagosomes showed increased ROS levels compared to Mtb phagosomes in primary murine and human alveolar macrophages. The increase in MtbΔnuoG induced ROS and apoptosis was abolished in NOX-2 deficient (gp91−/−) macrophages. These results suggest that Mtb, via a NuoG-dependent mechanism, can neutralize NOX2-derived ROS in order to inhibit TNF-α-mediated host cell apoptosis. Consistently, an Mtb mutant deficient in secreted catalase induced increases in phagosomal ROS and host cell apoptosis, both of which were dependent upon macrophage NOX-2 activity. In conclusion, these results serendipitously reveal a novel connection between NOX2 activity, phagosomal ROS, and TNF-α signaling during infection-induced apoptosis in macrophages. Furthermore, our study reveals a novel function of NOX2 activity in innate immunity beyond the initial respiratory burst, which is the sensing of persistent intracellular pathogens and subsequent induction of host

  11. [Adolescent brain maturation].

    PubMed

    Holzer, L; Halfon, O; Thoua, V

    2011-05-01

    Recent progress in neuroscience has yielded major findings regarding brain maturation during adolescence. Unlike the body, which reaches adult size and morphology during this period, the adolescent brain is still maturing. The prefrontal cortex appears to be an important locus of maturational change subserving executive functions that may regulate emotional and motivational issues. The recent expansion of the adolescent period has increased the lag between the onset of emotional and motivational changes activated by puberty and the completion of cognitive development-the maturation of self-regulatory capacities and skills that are continuing to develop long after puberty has occurred. This "disconnect" predicts risk for a broad set of behavioral and emotional problems. Adolescence is a critical period for high-level cognitive functions such as socialization that rely on maturation of the prefrontal cortex. Intervention during the period of adolescent brain development provides opportunities and requires an interdisciplinary approach.

  12. Transcriptomic analysis of the head kidney of Topmouth culter (Culter alburnus) infected with Flavobacterium columnare with an emphasis on phagosome pathway.

    PubMed

    Zhao, Lijuan; Tu, Jiagang; Zhang, Yulei; Wang, Jinfu; Yang, Ling; Wang, Weimin; Wu, Zaohe; Meng, Qinglei; Lin, Li

    2016-10-01

    Flavobacterium columnare (FC) has caused worldwide fish columnaris disease with high mortality and great economic losses in cultured fish, including Topmouth culter (Culter alburnus). However, the knowledge about the host factors involved in FC infection is little known. In this study, the transcriptomic profiles of the head kidney from Topmouth culter with or without FC infection were obtained using HiSeq™ 2500 (Illumina). Totally 79,641 unigenes with high quality were obtained. Among them, 4037 differently expressed genes, including 1217 up-regulated and 2820 down-regulated genes, were identified and enriched using databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The differently expressed genes were mainly associated with pathways such as immune response, carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Since phagocytosis is a central mechanism of innate immune response by host cells to defense against infectious agents, genes related to the phagosome pathway were scrutinized and 9 differently expressed phagosome-related genes were identified including 3 up-regulated and 6 down-regulated genes. Five of them were further validated by quantitative real-time polymerase chain reaction (qRT-PCR). This transcriptomic analysis of host genes in response to FC infection provides data towards understanding the infection mechanisms and will shed a new light on the prevention of columnaris.

  13. Posttesticular sperm maturation, infertility, and hypercholesterolemia

    PubMed Central

    Whitfield, Marjorie; Pollet-Villard, Xavier; Levy, Rachel; Drevet, Joël R; Saez, Fabrice

    2015-01-01

    Cholesterol is a key molecule in the mammalian physiology of especial particular importance for the reproductive system as it is the common precursor for steroid hormone synthesis. Cholesterol is also a recognized modulator of sperm functions, not only at the level of gametogenesis. Cholesterol homeostasis regulation is crucial for posttesticular sperm maturation, and imbalanced cholesterol levels may particularly affect these posttesticular events. Metabolic lipid disorders (dyslipidemia) affect male fertility but are most of the time studied from the angle of endocrine/testicular consequences. This review will focus on the deleterious effects of a particular dyslipidemia, i.e., hypercholesterolemia, on posttesticular maturation of mammalian spermatozoa. PMID:26067871

  14. Toward Teacher Maturity.

    ERIC Educational Resources Information Center

    Pickle, Judy

    1985-01-01

    The essence of teacher maturity can be synthesized into personal, professional, and process domains. Although overlapping, these categories add a multidimensional approach to the search for what is good in teaching and provide a model for professional development. (MT)

  15. Oxidative stress-dependent activation of the eIF2α–ATF4 unfolded protein response branch by skin sensitizer 1-fluoro-2,4-dinitrobenzene modulates dendritic-like cell maturation and inflammatory status in a biphasic manner [corrected].

    PubMed

    Luís, Andreia; Martins, João Demétrio; Silva, Ana; Ferreira, Isabel; Cruz, Maria Teresa; Neves, Bruno Miguel

    2014-12-01

    The pathogenesis of allergic contact dermatitis, the most common manifestation of immunotoxicity in humans, is intimately connected to hapten-induced maturation of dendritic cells (DC). The molecular mechanisms driving this maturational program are not completely known; however, initial danger signals such as the generation of reactive oxygen species (ROS) were shown to play a critical role. Recent evidence linking ROS production, endoplasmic reticulum (ER) stress, and the pathogenesis of several inflammatory diseases led us to analyze, in the present work, the ability of the skin sensitizer 1-fluoro-2,4-dinitrobenzene (DNFB) to evoke ER stress in DC-like THP-1 cells and the concomitant consequences to their immunobiology. We found that DNFB triggers a ROS-dependent activation of the PERK-eIFα-ATF4 unfolded protein response (UPR) branch conferring cytoprotection and modulating the maturation/proinflammatory cell status in a biphasic manner. Early DNFB induction of ATF4 positively modulates autophagy-related genes MAP1LC3B and ATG3 and stabilizes the transcription factor Nrf2, causing a strong induction of the HMOX1-detoxifying gene. Moreover, we observed that in a first phase, DNFB-induced ATF4 upregulates IL8 mRNA levels while blocking CD86, IL1B, IL12B, and CXL10 transcription. Later, following ATF4 decay, HMOX1 and IL8 transcription drastically decrease and CD86, IL1B, and Il12B are upregulated. Overall, our results evidence a connection between sensitizer-induced redox imbalance and the establishment of ER stress in DC-like cells and provide new insights into the role of UPR effectors such as ATF4 to the complex DC maturational program.

  16. Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

    PubMed Central

    Monteith, Andrew J.; Kang, SunAh; Scott, Eric; Hillman, Kai; Rajfur, Zenon; Jacobson, Ken; Costello, M. Joseph; Vilen, Barbara J.

    2016-01-01

    Defects in clearing apoptotic debris disrupt tissue and immunological homeostasis, leading to autoimmune and inflammatory diseases. Herein, we report that macrophages from lupus-prone MRL/lpr mice have impaired lysosomal maturation, resulting in heightened ROS production and attenuated lysosomal acidification. Impaired lysosomal maturation diminishes the ability of lysosomes to degrade apoptotic debris contained within IgG–immune complexes (IgG-ICs) and promotes recycling and the accumulation of nuclear self-antigens at the membrane 72 h after internalization. Diminished degradation of IgG-ICs prolongs the intracellular residency of nucleic acids, leading to the activation of Toll-like receptors. It also promotes phagosomal membrane permeabilization, allowing dsDNA and IgG to leak into the cytosol and activate AIM2 and TRIM21. Collectively, these events promote the accumulation of nuclear antigens and activate innate sensors that drive IFNα production and heightened cell death. These data identify a previously unidentified defect in lysosomal maturation that provides a mechanism for the chronic activation of intracellular innate sensors in systemic lupus erythematosus. PMID:27035940

  17. Maturation in Larch 1

    PubMed Central

    Greenwood, Michael S.; Hopper, Catherine A.; Hutchison, Keith W.

    1989-01-01

    The time course of maturation in eastern larch (Larix laricina [Du Roi] K. Koch) was examined by grafting scions from trees of different ages onto 2-year-old root stock and following scion development for several years. Height, diameter, foliar chlorophyll content, and rooting ability of scion-derived cuttings all varied linearly as a function of log10 age. Chlorophyll content (milligrams per gram of dry weight) increased while height, diameter, and ability to root decreased with age (P < 0.01). The tendency toward orthotropic growth and branch formation per centimeter of main stem decreased abruptly between age 1 and 5 years (P < 0.01). Total chlorophyll content of both long and short shoot foliage increased by 30 to 50% with increasing age, but the chlorophyll a/b ratio did not change. Also, juvenile long shoot needles were significantly longer than mature (P < 0.01). Surprisingly, the juvenile scions produced more total strobili over two successive years, but the mature scions produced a significantly higher proportion of male strobili (P < 0.001 year 1; P < 0.02 year 2). The age-related changes in foliar traits were not associated with changes in DNA methylation between juvenile and mature scions. Using HPLC, we found that 20% of foliar DNA cytosine residues were methylated in both scion types. Images Figure 1 PMID:16666785

  18. Jealousy and Moral Maturity.

    ERIC Educational Resources Information Center

    Mathes, Eugene W.; Deuger, Donna J.

    Jealousy may be perceived as either good or bad depending upon the moral maturity of the individual. To investigate this conclusion, a study was conducted testing two hypothesis: a positive relationship exists between conventional moral reasoning (reference to norms and laws) and the endorsement and level of jealousy; and a negative relationship…

  19. Mature Students Studying Mathematics.

    ERIC Educational Resources Information Center

    Hirst, Keith

    1999-01-01

    Discusses mature students in the single subject area of mathematics in a single institution and makes comparisons with traditional universities. Reviews some features of the age distribution, entry qualifications, degree-class distribution, non-completion rates and gender distribution. (Author/ASK)

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

    PubMed

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

    2016-02-01

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

  1. Delayed visual maturation.

    PubMed Central

    Cole, G F; Hungerford, J; Jones, R B

    1984-01-01

    Sixteen blind babies who were considered to be showing the characteristics of delayed visual maturation were studied prospectively. The diagnosis was made on clinical grounds, and the criteria for this are discussed. All of these infants developed visual responses between 4 and 6 months of age and had normal or near normal visual acuities by 1 year of age. Long term follow up, however, has shown neurological abnormalities in some of these children. PMID:6200080

  2. Meristem maturation and inflorescence architecture--lessons from the Solanaceae.

    PubMed

    Park, Soon Ju; Eshed, Yuval; Lippman, Zachary B

    2014-02-01

    Plant apical meristems (AMs) grow continuously by delicately balancing cells leaving at the periphery to form lateral organs with slowly dividing central domain cells that replenish reservoirs of pluripotent cells. This balance can be modified by signals originating from within and outside the meristem, and their integration results in a gradual maturation process that often culminates with the meristem differentiating into a flower. Accompanying this 'meristem maturation' are changes in spacing and size of lateral organs and in rates at which lateral meristems are released from apical dominance. Modulation of distinct meristem maturation parameters through environmental and genetic changes underlies the remarkable diversity of shoot architectures. Here, we discuss recent studies relating the dynamics of meristem maturation with organization of floral branching systems--inflorescences--in the nightshades. From this context, we suggest general principles on how factors coordinating meristem maturation impact shoot organization more broadly. Published by Elsevier Ltd.

  3. Vocational Maturity and Self Concepts.

    ERIC Educational Resources Information Center

    Helbing, Hans

    The relationship between separate dimensions of vocational maturity and different self-concept and identity variables were examined. Subjects were Dutch students, age 14-18 years. The vocational maturity dimensions were measured by Dutch adaptations of American vocational maturity scales. Instruments for self-concept and identity measurement were…

  4. Capability Maturity Model for Software,

    DTIC Science & Technology

    1991-08-01

    This paper provides a technical overview of the Capability Maturity Model for Software and reflects the most current version. Specifically, this...paper, in combination with the Key Practices of the Capability Maturity Model , is intended to help software organizations use the CMM as a guide to improve the maturity of their software process.

  5. Dissociation of motor maturation.

    PubMed

    DiMario, Francis J

    2003-06-01

    We prospectively acquired clinical data regarding the presentation, evaluation, and developmental progress of all patients identified with dissociated motor maturation to define their clinical outcomes. Children (N = 8) referred for evaluation of suspected cerebral palsy because of delayed sitting or walking and identified to have dissociated motor maturation were followed with serial clinical examination. All displayed the characteristic "sitting on air" posture while held in vertical suspension and had otherwise normal developmental assessments. This posture is composed of the hips held in flexion and abduction with the knees extended and feet plantar or dorsiflexed. Three children were initially evaluated at 10 months of age owing to absence of sitting and five other children were evaluated at a mean of 14 months (range 12-19 months) owing to inability to stand. Follow-up evaluations were conducted over a mean of 10.5 months (range 5-34 months). Five children were born prematurely at 34 to 36 weeks gestation. Denver Developmental Screening Test and general and neurologic examinations were normal except to note hypotonia in six children and the "sitting on air" posture in all of the children. Four children have older siblings or parents who "walked late" (after 15 months). On average, the children attained sitting by 8 months (range 7-10 months). One child did not crawl prior to independent walking, two children scooted rather than crawled, and five children crawled at an average of 13.5 months (range 10-16 months). All children cruised by a mean of 18 months (range 16-21.5 months) and attained independent walking by 20.1 months (range 18-25 months). Neuroimaging and serum creatine kinase enzyme testing were normal in two children who were tested. These eight children conform to the syndrome of dissociated motor maturation. The "sitting on air" posture serves as a diagnostic sign and anticipated excellent prognosis, but follow-up is required to ensure a normal

  6. Maturation in Larch 1

    PubMed Central

    Hutchison, Keith W.; Sherman, Christopher D.; Weber, Jill; Smith, Sandra Schiller; Singer, Patricia B.; Greenwood, Michael S.

    1990-01-01

    The effect of maturation on the morphological and photosynthetic characteristics, as well as the expression of two genes involved in photosynthesis in the developing, current year foliage of Eastern larch (Larix laricina [Du Roi]) is described. These effects were observed on foliage during the third growing season after grafting of scions from trees of different ages onto 2 year old rootstock. Specific leaf weight (gram dry weight per square meter), leaf cross-sectional area (per square millimeter), and chlorophyll content (milligram per gram dry weight) all increase with increasing age in long shoot foliage from both indoor- and outdoor-grown trees. Net photosynthesis (NPS) (mole of CO2 per square millimeter per second) increases with age on indoor- but not outdoor-grown trees. NPS also increases with increased chlorophyll content, but outdoor-grown scions of all ages had higher chlorophyll content, and chlorophyll does not appear to be limiting for NPS outdoors. To extend these studies of maturation-related differences in foliar morphology and physiology to the molecular genetic level, sequences were cloned from the cab and rbsS gene families of larch. Both cab and rbcS gene families are expressed in foliage but not in roots, and they are expressed in light-grown seedlings of larch but only at very low levels in dark-grown seedlings (~2% of light-grown seedlings). Steady-state cab mRNA levels are relatively higher (~40%) in newly expanding short shoot foliage from juvenile plants compared to mature plants. Unlike cab, the expression of the rbcS gene family did not seem to vary with age. These data show that the maturation-related changes in morphological and physiological phenotypes are associated with changes in gene expression. No causal relationship has been established, however. Indeed, we conclude that the faster growth of juvenile scions reported previously (MS Greenwood, CA Hopper, KW Hutchison [1989] Plant Physiol 90: 406-412) is not due to increased NPS

  7. The src-family protein-tyrosine kinase p59hck is located on the secretory granules in human neutrophils and translocates towards the phagosome during cell activation.

    PubMed Central

    Möhn, H; Le Cabec, V; Fischer, S; Maridonneau-Parini, I

    1995-01-01

    The src-family protein-tyrosine kinase p59hck is mainly expressed in neutrophils; however, its functional role in these cells is unknown. Several other src-family members are localized on secretory vesicles and have been proposed to regulate intracellular traffic. We have established here the subcellular localization of p59hck in human neutrophils. Immunoblotting of subcellular fractions showed that approx. 60% of the p59hck per cell is localized on the secretory granules; the other 40% is distributed equally between non-granular membranes and the cytosol. Immunofluorescence of neutrophils and HL60 cells suggests that the p59hck-positive granules are azurophil granules. Granular p59hck is highly susceptible to degradation by an azurophil-granule proteinase. Different forms of p59hck occur in the three subcellular compartments: a 61 kDa form is mainly found in the granules, a 59 kDa form is predominant in the non-granular membranes, whereas cytosolic p59hck migrates as a doublet at 63 kDa. During the process of phagocytosis-linked degranulation, induced by serum-opsonized zymosan in neutrophils or HL60 cells, granular p59hck translocates towards the phagosome. The subcellular localization of p59hck suggests that the enzyme could be involved in the regulation of the degranulation process. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7626033

  8. Correlation between dental maturity and cervical vertebral maturity.

    PubMed

    Chen, Jianwei; Hu, Haikun; Guo, Jing; Liu, Zeping; Liu, Renkai; Li, Fan; Zou, Shujuan

    2010-12-01

    The aim of this study was to investigate the association between dental and skeletal maturity. Digital panoramic radiographs and lateral skull cephalograms of 302 patients (134 boys and 168 girls, ranging from 8 to 16 years of age) were examined. Dental maturity was assessed by calcification stages of the mandibular canines, first and second premolars, and second molars, whereas skeletal maturity was estimated by the cervical vertebral maturation (CVM) stages. The Spearman rank-order correlation coefficient was used to measure the association between CVM stage and dental calcification stage of individual teeth. The mean chronologic age of girls was significantly lower than that of boys in each CVM stage. The Spearman rank-order correlation coefficients between dental maturity and cervical vertebral maturity ranged from 0.391 to 0.582 for girls and from 0.464 to 0.496 for boys (P < 0.05). In girls, the mandibular second molar had the highest and the canine the lowest correlation. In boys, the canine had the highest and the first premolar the lowest correlation. Tooth calcification stage was significantly correlated with cervical vertebral maturation stage. The development of the mandibular second molar in females and that of the mandibular canine in males had the strongest correlations with cervical vertebral maturity. Therefore, it is practical to consider the relationship between dental and skeletal maturity when planning orthodontic treatment. Copyright © 2010 Mosby, Inc. All rights reserved.

  9. CFD - Mature Technology?

    NASA Technical Reports Server (NTRS)

    Kwak, Dochan

    2005-01-01

    Over the past 30 years, numerical methods and simulation tools for fluid dynamic problems have advanced as a new discipline, namely, computational fluid dynamics (CFD). Although a wide spectrum of flow regimes are encountered in many areas of science and engineering, simulation of compressible flow has been the major driver for developing computational algorithms and tools. This is probably due to a large demand for predicting the aerodynamic performance characteristics of flight vehicles, such as commercial, military, and space vehicles. As flow analysis is required to be more accurate and computationally efficient for both commercial and mission-oriented applications (such as those encountered in meteorology, aerospace vehicle development, general fluid engineering and biofluid analysis) CFD tools for engineering become increasingly important for predicting safety, performance and cost. This paper presents the author's perspective on the maturity of CFD, especially from an aerospace engineering point of view.

  10. In vitro maturation of oocytes.

    PubMed

    Smith, G D

    2001-10-01

    In vitro maturation (IVM) of human oocytes is an emerging assisted reproductive technology with great promise. To be successful, this process must entail both nuclear and cytoplasmic maturation. Endogenous regulation of oocyte maturation is a complex sequence of events regulated by endocrine parameters, oocyte/follicular cross-talk, and intra-oocyte kinase/phosphatase interactions. Although nuclear maturation during human oocyte IVM progresses normally, cytoplasmic maturation is significantly lacking, as exemplified by poor embryonic developmental competence and pregnancy rates. Advances made in immature oocyte isolation and oocyte and embryo culture conditions have increased the clinical feasibility of IVM. However, in order to achieve acceptable birth rates, future studies should focus on characterization and regulation of oocyte cytoplasmic maturation, and how oocyte-derived factors influence zygotic genome activation and embryonic developmental competence.

  11. Mechanisms of Hierarchical Cortical Maturation

    PubMed Central

    Chomiak, Taylor; Hu, Bin

    2017-01-01

    Cortical information processing is structurally and functionally organized into hierarchical pathways, with primary sensory cortical regions providing modality specific information and associative cortical regions playing a more integrative role. Historically, there has been debate as to whether primary cortical regions mature earlier than associative cortical regions, or whether both primary and associative cortical regions mature simultaneously. Identifying whether primary and associative cortical regions mature hierarchically or simultaneously will not only deepen our understanding of the mechanisms that regulate brain maturation, but it will also provide fundamental insight into aspects of adolescent behavior, learning, neurodevelopmental disorders and computational models of neural processing. This mini-review article summarizes the current evidence supporting the sequential and hierarchical nature of cortical maturation, and then proposes a new cellular model underlying this process. Finally, unresolved issues associated with hierarchical cortical maturation are also addressed. PMID:28959187

  12. Localization of mature neprilysin in lipid rafts.

    PubMed

    Sato, Kimihiko; Tanabe, Chiaki; Yonemura, Yoji; Watahiki, Haruhiko; Zhao, Yimeng; Yagishita, Sosuke; Ebina, Maiko; Suo, Satoshi; Futai, Eugene; Murata, Masayuki; Ishiura, Shoichi

    2012-04-01

    Alzheimer's disease (AD) is characterized by senile plaques caused by amyloid-β peptide (Aβ) accumulation. It has been reported that Aβ generation and accumulation occur in membrane microdomains, called lipid rafts, which are enriched in cholesterol and glycosphingolipids. Moreover, the ablation of cholesterol metabolism has been implicated in AD. Neprilysin (NEP), a neutral endopeptidase, is one of the major Aβ-degrading enzymes in the brain. Activation of NEP is a possible therapeutic target. However, it remains unknown whether the activity of NEP is regulated by its association with lipid rafts. Here we show that only the mature form of NEP, which has been glycosylated in the Golgi, exists in lipid rafts, where it is directly associated with phosphatidylserine. Moreover, the localization of NEP in lipid rafts is enhanced by its dimerization, as shown using the NEP E403C homodimerization mutant. However, the protease activities of the mature form of NEP, as assessed by in vitro peptide hydrolysis, did not differ between lipid rafts and nonlipid rafts. We conclude that cholesterol and other lipids regulate the localization of mature NEP to lipid rafts, where the substrate Aβ accumulates but does not modulate the protease activity of NEP.

  13. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  14. Maturational and Non-Maturational Factors in Heritage Language Acquisition

    ERIC Educational Resources Information Center

    Moon, Ji Hye

    2012-01-01

    This dissertation aims to understand the maturational and non-maturational aspects of early bilingualism and language attrition in heritage speakers who have acquired their L1 incompletely in childhood. The study highlights the influential role of age and input dynamics in early L1 development, where the timing of reduction in L1 input and the…

  15. Mycobacterium tuberculosis Chaperonin 10 Is Secreted in the Macrophage Phagosome: Is Secretion Due to Dissociation and Adoption of a Partially Helical Structure at the Membrane?

    PubMed Central

    Fossati, Gianluca; Izzo, Gaetano; Rizzi, Emanuele; Gancia, Emanuela; Modena, Daniela; Moras, Maria Luisa; Niccolai, Neri; Giannozzi, Elena; Spiga, Ottavia; Bono, Letizia; Marone, Piero; Leone, Eugenio; Mangili, Francesca; Harding, Stephen; Errington, Neil; Walters, Christopher; Henderson, Brian; Roberts, Michael M.; Coates, Anthony R. M.; Casetta, Bruno; Mascagni, Paolo

    2003-01-01

    To confirm that Mycobacterium tuberculosis chaperonin 10 (Cpn10) is secreted outside the live bacillus, infected macrophages were examined by electron microscopy. This revealed that the mycobacterial protein accumulates both in the wall of the bacterium and in the matrix of the phagosomes in which ingested mycobacteria survive within infected macrophages. To understand the structural implications underlying this secretion, a structural study of M. tuberculosis Cpn10 was performed under conditions that are generally believed to mimic the membrane environment. It was found that in buffer-organic solvent mixtures, the mycobacterial protein forms two main species, namely, a partially helical monomer that prevails in dilute solutions at room temperature and a dimer that folds into a β-sheet-dominated structure and prevails in either concentrated protein solutions at room temperature or in dilute solutions at low temperature. A partially helical monomer was also found and was completely associated with negatively charged detergents in a micelle-bound state. Remarkably, zwitterionic lipids had no effect on the protein structure. By using N- and C-truncated forms of the protein, the C- and N-terminal sequences were identified as possessing an amphiphilic helical character and as selectively associating with acidic detergent micelles. When the study was extended to other chaperonins, it was found that human Cpn10 is also monomeric and partially helical in dilute organic solvent-buffer mixtures. In contrast, Escherichia coli Cpn10 is mostly dimeric and predominately β-sheet in both dilute and concentrated solutions. Interestingly, human Cpn10 also crosses biological membranes, whereas the E. coli homologue is strictly cytosolic. These results suggest that dissociation to partially helical monomers and interaction with acidic lipids may be two important steps in the mechanism of secretion of M. tuberculosis Cpn10 to the external environment. PMID:12837802

  16. The Mature Athlete

    PubMed Central

    McCarthy, Moira M.; Hannafin, Jo A.

    2014-01-01

    Context: Aging changes the biology, healing capacity, and biomechanical function of tendons and ligaments and results in common clinical pathologies that present to orthopedic surgeons, primary care physicians, physical therapists, and athletic trainers. A better understanding of the age-related changes in these connective tissues will allow better patient care. Evidence Acquisition: The PubMed database was searched in December 2012 for English-language articles pertaining to age-related changes in tendons and ligaments. Level of Evidence: Level 5. Results: The mature athlete faces challenges associated with age-dependent changes in the rotator cuff, Achilles tendon, lateral humeral epicondylar tendons, quadriceps tendon, and patellar tendon. The anterior cruciate ligament and the medial collateral ligament are the most studied intra-articular and extra-articular ligaments, and both are associated with age-dependent changes. Conclusion: Tendons and ligaments are highly arranged connective tissue structures that maintain joint motion and joint stability. These structures are subject to vascular and compositional changes with increasing age that alter their mechanotransduction, biology, healing capacity, and biomechanical function. Emerging research into the etiology of age-dependent changes will provide further information to help combat the age-related clinical complications associated with the injuries that occur to tendons and ligaments. PMID:24427441

  17. Career Maturity of Welfare Recipients.

    ERIC Educational Resources Information Center

    Beckman, Carol M.

    To investigate the career maturity of welfare recipients, this thesis examines six independent variables: (1) race; (2) sex; (3) age; (4) level of formal education; (5) general intelligence; and (6) locus of control. Scales taken from the Career Maturity Inventory served as the dependent variables. The sample consisted of 83 welfare recipients who…

  18. Treating mature stands for wildlife

    Treesearch

    William H. Healy; Gary F. Houf

    1989-01-01

    Stands older than 60 years or that are medium to large sawtimber size generally provide good wildlife habitat. Mature trees usually produce abundant mast and provide den sites (see fig. 1 in Note 9.04 Treating Immature Stands). The undergrowth in these stands produces moderate amounts of browse and herbage. Mature stands also provide opportunities for management...

  19. Financial maturity of yellow birch

    Treesearch

    William B. Leak

    1969-01-01

    The methods used to compute financial maturity of yellow birch sawtimber are similar to those used for paper birch sawtimber, except for minor differences in detail. The procedure followed for yellow-birch veneer-log trees was also similar, except that local veneer grades and local veneer-log prices were used as the basis for the financial maturity computations.

  20. Career Education and Career Maturity.

    ERIC Educational Resources Information Center

    Trebilco, Geoffrey R.

    1984-01-01

    Investigated the relationships between career maturity and career curriculum in 38 Melbourne metropolitan secondary schools (N=2280 students) using an Australian adaption of the Career Development Inventory. Results confirmed that schools with career education programs achieved higher gains in student career maturity. (JAC)

  1. Predictive Capability Maturity Model (PCMM).

    SciTech Connect

    Swiler, Laura Painton; Knupp, Patrick Michael; Urbina, Angel

    2010-10-01

    Predictive Capability Maturity Model (PCMM) is a communication tool that must include a dicussion of the supporting evidence. PCMM is a tool for managing risk in the use of modeling and simulation. PCMM is in the service of organizing evidence to help tell the modeling and simulation (M&S) story. PCMM table describes what activities within each element are undertaken at each of the levels of maturity. Target levels of maturity can be established based on the intended application. The assessment is to inform what level has been achieved compared to the desired level, to help prioritize the VU activities & to allocate resources.

  2. Maturation of sugar maple seed

    Treesearch

    Clayton M., Jr. Carl; Albert G., Jr. Snow; Albert G. Snow

    1971-01-01

    The seeds of a sugar maple tree (Acer saccharum Marsh.) do not mature at the same time every year. And different trees mature their seeds at different times. So time of year is not a reliable measure of when seeds are ripe. Better criteria are needed. In recent studies we have found that moisture content and color are the best criteria for judging when sugar maple...

  3. Naturally Engineered Maturation of Cardiomyocytes

    PubMed Central

    Scuderi, Gaetano J.; Butcher, Jonathan

    2017-01-01

    Ischemic heart disease remains one of the most prominent causes of mortalities worldwide with heart transplantation being the gold-standard treatment option. However, due to the major limitations associated with heart transplants, such as an inadequate supply and heart rejection, there remains a significant clinical need for a viable cardiac regenerative therapy to restore native myocardial function. Over the course of the previous several decades, researchers have made prominent advances in the field of cardiac regeneration with the creation of in vitro human pluripotent stem cell-derived cardiomyocyte tissue engineered constructs. However, these engineered constructs exhibit a functionally immature, disorganized, fetal-like phenotype that is not equivalent physiologically to native adult cardiac tissue. Due to this major limitation, many recent studies have investigated approaches to improve pluripotent stem cell-derived cardiomyocyte maturation to close this large functionality gap between engineered and native cardiac tissue. This review integrates the natural developmental mechanisms of cardiomyocyte structural and functional maturation. The variety of ways researchers have attempted to improve cardiomyocyte maturation in vitro by mimicking natural development, known as natural engineering, is readily discussed. The main focus of this review involves the synergistic role of electrical and mechanical stimulation, extracellular matrix interactions, and non-cardiomyocyte interactions in facilitating cardiomyocyte maturation. Overall, even with these current natural engineering approaches, pluripotent stem cell-derived cardiomyocytes within three-dimensional engineered heart tissue still remain mostly within the early to late fetal stages of cardiomyocyte maturity. Therefore, although the end goal is to achieve adult phenotypic maturity, more emphasis must be placed on elucidating how the in vivo fetal microenvironment drives cardiomyocyte maturation. This

  4. A proliferation-inducing ligand sustains the proliferation of human naïve (CD27⁻) B cells and mediates their differentiation into long-lived plasma cells in vitro via transmembrane activator and calcium modulator and cyclophilin ligand interactor and B-cell mature antigen.

    PubMed

    Matsuda, Yoshiko; Haneda, Masataka; Kadomatsu, Kenji; Kobayashi, Takaaki

    2015-06-01

    Long-lived plasma cells (PCs) contribute to humoral immunity through an undefined mechanism. Memory B cells, but not human naïve B cells, can be induced to differentiate into long-lived PCs in vitro. Because evidence links a proliferation-inducing ligand (APRIL), a tumor necrosis factor family member, to the ability of bone marrow to mediate long-term PC survival, we reasoned that APRIL influences the proliferation and differentiation of naïve B cells. We describe here the development of a simple cell culture system that allowed us to show that APRIL sustained the proliferation of naïve human B cells and induced them to differentiate into long-lived PCs. Blocking the transmembrane activator and calcium modulator and cyclophilin ligand interactor or B-cell mature antigen shows they were required for the differentiation of naïve B cells into long-lived PCs in vitro. Our in vitro culture system will reveal new insights into the biology of long-lived PCs.

  5. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the normal...

  6. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the normal...

  7. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure the proper completion of the normal...

  8. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure...

  9. 7 CFR 51.3746 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Honey Dew and Honey Ball Type Melons Definitions § 51.3746 Mature. Mature means that the melon has reached the stage of maturity which will insure...

  10. Toward the Decision Tree for Inferring Requirements Maturation Types

    NASA Astrophysics Data System (ADS)

    Nakatani, Takako; Kondo, Narihito; Shirogane, Junko; Kaiya, Haruhiko; Hori, Shozo; Katamine, Keiichi

    Requirements are elicited step by step during the requirements engineering (RE) process. However, some types of requirements are elicited completely after the scheduled requirements elicitation process is finished. Such a situation is regarded as problematic situation. In our study, the difficulties of eliciting various kinds of requirements is observed by components. We refer to the components as observation targets (OTs) and introduce the word “Requirements maturation.” It means when and how requirements are elicited completely in the project. The requirements maturation is discussed on physical and logical OTs. OTs Viewed from a logical viewpoint are called logical OTs, e.g. quality requirements. The requirements of physical OTs, e.g., modules, components, subsystems, etc., includes functional and non-functional requirements. They are influenced by their requesters' environmental changes, as well as developers' technical changes. In order to infer the requirements maturation period of each OT, we need to know how much these factors influence the OTs' requirements maturation. According to the observation of actual past projects, we defined the PRINCE (Pre Requirements Intelligence Net Consideration and Evaluation) model. It aims to guide developers in their observation of the requirements maturation of OTs. We quantitatively analyzed the actual cases with their requirements elicitation process and extracted essential factors that influence the requirements maturation. The results of interviews of project managers are analyzed by WEKA, a data mining system, from which the decision tree was derived. This paper introduces the PRINCE model and the category of logical OTs to be observed. The decision tree that helps developers infer the maturation type of an OT is also described. We evaluate the tree through real projects and discuss its ability to infer the requirements maturation types.

  11. Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS.

    PubMed

    Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui

    2017-02-20

    Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

  12. Loop nucleotides control primary and mature miRNA function in target recognition and repression

    PubMed Central

    Yue, Si-Biao; Deis Trujillo, Robin; Tang, Yujie; O'Gorman, William E

    2011-01-01

    MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5′ end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation. PMID:22142974

  13. Space Station Photovoltaic power modules

    NASA Technical Reports Server (NTRS)

    Tatro, Charles A.

    1988-01-01

    Silicon cell Photovoltaic (PV) power modules are key components of the Space Station Electrical Power System (EPS) scheduled to begin deployment in 1994. Four PV power modules, providing 75 KWe of user ac power, form the cornerstone of the EPS; which is comprised of Photovoltaic (PV) power modules, Solar Dynamic (SD) power modules, and the Power Management and Distribution (PMAD) system. The PV modules are located on rotating outboard sections of the Space Station (SS) structure and each module incorporates its own nickel-hydrogen energy storage batteries, its own thermal control system, and some autonomous control features. The PV modules are a cost-effective and technologically mature approach for providing reliable SS electrical power and are a solid base for EPS growth, which is expected to reach 300 KWe by the end of the Space Station's 30-year design lifetime.

  14. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 10 2012-01-01 2012-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  15. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  16. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2013-07-01 2013-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  17. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2014-07-01 2014-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  18. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2012-07-01 2012-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  19. 38 CFR 7.7 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 1 2011-07-01 2011-07-01 false Maturity. 7.7 Section 7.7... Soldiers' and Sailors' Civil Relief Act Amendments of 1942 § 7.7 Maturity. (a) The phrase maturity of a policy as a death claim or otherwise (SSCRA, as amended) will not include a termination or maturity of a...

  20. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 10 2014-01-01 2014-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  1. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 10 2013-01-01 2013-01-01 false Maturity dates. 1421.101 Section 1421.101 Agriculture... Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the... filed and disbursed except, for transferred marketing assistance loan collateral. The maturity date for...

  2. Career Maturity and Physically Disabled College Students.

    ERIC Educational Resources Information Center

    Burkhead, E. Jane; Cope, Corrine S.

    1984-01-01

    Examined the relationships between career maturity, sex, physical disability, and grades in 40 disabled and 46 nondisabled college students. Results showed disabled students were more vocationally mature than nondisabled students and female students were more vocationally mature than males. Type of disability was not related to career maturity.…

  3. 7 CFR 51.2841 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.2841 Section 51.2841 Agriculture... Creole Types) Definitions § 51.2841 Mature. Mature means well cured. Midseason onions which are not customarily held in storage shall be considered mature when harvested in accordance with good commercial...

  4. 7 CFR 51.2841 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.2841 Section 51.2841 Agriculture... Mature. Mature means well cured. Midseason onions which are not customarily held in storage shall be considered mature when harvested in accordance with good commercial practice at a stage which will not result...

  5. 7 CFR 51.484 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.484 Section 51.484 Agriculture Regulations..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Cantaloups 1 Definitions § 51.484 Mature. Mature means that the cantaloup has reached the stage of maturity which will insure the proper completion...

  6. 7 CFR 51.484 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.484 Section 51.484 Agriculture Regulations..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Cantaloups 1 Definitions § 51.484 Mature. Mature means that the cantaloup has reached the stage of maturity which will insure the proper completion...

  7. 7 CFR 51.2841 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.2841 Section 51.2841 Agriculture... Creole Types) Definitions § 51.2841 Mature. Mature means well cured. Midseason onions which are not customarily held in storage shall be considered mature when harvested in accordance with good commercial...

  8. Ribosome maturation in E. coli.

    PubMed

    Silengo, L; Altruda, F; Dotto, G P; Lacquaniti, F; Perlo, C; Turco, E; Mangiarotti, G

    1977-01-01

    In vivo and in vitro experiments have shown that processing of ribosomal RNA is a late event in ribosome biogenesis. The precursor form of RNA is probably necessary to speed up the assembly of ribomal proteins. Newly formed ribosomal particles which have already entered polyribosomes differ from mature ribosomes not only in their RNA content but also in their susceptibility to unfolding in low Mg concentration and to RNase attack. Final maturation of new ribosomes is probably dependent on their functioning in protein synthesis. Thus only those ribosomes which have proven to be functional may be converted into stable cellular structures.

  9. Enticing Mature Females into College.

    ERIC Educational Resources Information Center

    Loseth, Lexie; Moreau, Linda

    Following a review of the literature on mature female students, this paper examines enrollment trends in a selection of colleges in Alberta (Canada) and presents the findings of a survey of returning women students at Red Deer College. The literature review highlights factors related to the personal and professional development of women graduates…

  10. Psychosocial Maturity or Social Desirability?

    ERIC Educational Resources Information Center

    Greenberger, Ellen

    The psychosocial maturity scale (PSM) described in several earlier papers is a self-report questionnaire. It is vulnerable, as are other questionnaires of this type, to respondents' wishes to present themselves in a socially desirable light. In this study, scores on two social desirability scales are examined in relation to PSM. Correlations…

  11. Vineland Social Maturity Scale Profile.

    ERIC Educational Resources Information Center

    Pedrini, D. T.; Pedrini, Bonnie C.

    The Vineland Social Maturity Scale (VSMS), despite its limitations, is an excellent clinical technique and includes psychometric and questionnaire characteristics. It is a good single measure of adaptive behavior. The VSMS Profile in this paper uses content categories different from the original Scale, but based upon the same items. It lends…

  12. The People Capability Maturity Model

    ERIC Educational Resources Information Center

    Wademan, Mark R.; Spuches, Charles M.; Doughty, Philip L.

    2007-01-01

    The People Capability Maturity Model[R] (People CMM[R]) advocates a staged approach to organizational change. Developed by the Carnegie Mellon University Software Engineering Institute, this model seeks to bring discipline to the people side of management by promoting a structured, repeatable, and predictable approach for improving an…

  13. Enticing Mature Females into College.

    ERIC Educational Resources Information Center

    Loseth, Lexie; Moreau, Linda

    Following a review of the literature on mature female students, this paper examines enrollment trends in a selection of colleges in Alberta (Canada) and presents the findings of a survey of returning women students at Red Deer College. The literature review highlights factors related to the personal and professional development of women graduates…

  14. Adolescent Maturation in Transitioning Cultures.

    ERIC Educational Resources Information Center

    Mulroy, Kevin; Palacios, Anna; Reid, Robert E.

    This is a theoretical study of adolescent maturation within a cultural context. Personality development and disintegration due to the pressure of a dominant culture on a minority culture is considered. An attempt is made to understand how teachers might assist students to work out their psychological growth by story telling. The need for cultural…

  15. Financial maturity of paper birch

    Treesearch

    Joseph J. Mendel

    1969-01-01

    One objective in forestry is to earn the greatest possible return on the capital invested in growing timber. To do this, the forester not only must know which silvicultural methods to use, but also ought to know the methods of economic analysis that will help him make the decisions that will lead to the greatest return. The financial maturity concept provides a method...

  16. Motivational Maturity and Helping Behavior

    ERIC Educational Resources Information Center

    Haymes, Michael; Green, Logan

    1977-01-01

    Maturity in conative development (type of motivation included in Maslow's needs hierarchy) was found to be predictive of helping behavior in middle class white male college students. The effects of safety and esteem needs were compared, and the acceptance of responsibility was also investigated. (GDC)

  17. Rate of meristem maturation determines inflorescence architecture in tomato

    PubMed Central

    Park, Soon Ju; Jiang, Ke; Schatz, Michael C.; Lippman, Zachary B.

    2012-01-01

    Flower production and crop yields are highly influenced by the architectures of inflorescences. In the compound inflorescences of tomato and related nightshades (Solanaceae), new lateral inflorescence branches develop on the flanks of older branches that have terminated in flowers through a program of plant growth known as “sympodial.” Variability in the number and organization of sympodial branches produces a remarkable array of inflorescence architectures, but little is known about the mechanisms underlying sympodial growth and branching diversity. One hypothesis is that the rate of termination modulates branching. By performing deep sequencing of transcriptomes, we have captured gene expression dynamics from individual shoot meristems in tomato as they gradually transition from a vegetative state to a terminal flower. Surprisingly, we find thousands of age-dependent expression changes, even when there is little change in meristem morphology. From these data, we reveal that meristem maturation is an extremely gradual process defined molecularly by a “meristem maturation clock.” Using hundreds of stage-enriched marker genes that compose this clock, we show that extreme branching, conditioned by loss of expression of the COMPOUND INFLORESCENCE gene, is driven by delaying the maturation of both apical and lateral meristems. In contrast, we find that wild tomato species display a delayed maturation only in apical meristems, which leads to modest branching. Our systems genetics approach reveals that the program for inflorescence branching is initiated surprisingly early during meristem maturation and that evolutionary diversity in inflorescence architecture is modulated by heterochronic shifts in the acquisition of floral fate. PMID:22203998

  18. Plastic downregulation of the transcriptional repressor BCL6 during maturation of human dendritic cells

    SciTech Connect

    Pantano, Serafino . E-mail: serafino.pantano@unil.ch; Jarrossay, David; Saccani, Simona; Bosisio, Daniela; Natoli, Gioacchino

    2006-05-01

    Dendritic cell (DC) maturation links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. Here, we describe an as yet unrecognized modulator of human DC maturation, the transcriptional repressor BCL6. We found that both myeloid and plasmacytoid DCs constitutively express BCL6, which is rapidly downregulated following maturation triggered by selected stimuli. Both in unstimulated and maturing DCs, control of BCL6 protein levels reflects the convergence of several mechanisms regulating BCL6 stability, mRNA transcription and nuclear export. By regulating the induction of several genes implicated in the immune response, including inflammatory cytokines, chemokines and survival genes, BCL6 may represent a pivotal modulator of the afferent branch of the immune response.

  19. Maturation of Oocytes in Vitro.

    PubMed

    Lonergan, Patrick; Fair, Trudee

    2016-01-01

    Only a fraction of oocytes present in the ovaries at birth are ever ovulated during the lifetime of a female mammal. In vitro maturation (IVM) offers the possibility to exploit what is a largely untapped biological resource. Although IVM is used routinely for the in vitro production of embryos in domestic species, especially cattle, its clinical use in human-assisted reproduction is still evolving. The successful recapitulation in vitro of the events associated with successful oocyte maturation is not always achieved, with the majority of immature oocytes typically failing to develop to the blastocyst stage. Evidence suggests that although culture conditions throughout in vitro embryo production may have a modest influence on the developmental potential of the early embryo, the quality of the oocyte at the start of the process is the key factor determining the proportion of oocytes developing to the blastocyst stage.

  20. Maturation of the adolescent brain

    PubMed Central

    Arain, Mariam; Haque, Maliha; Johal, Lina; Mathur, Puja; Nel, Wynand; Rais, Afsha; Sandhu, Ranbir; Sharma, Sushil

    2013-01-01

    Adolescence is the developmental epoch during which children become adults – intellectually, physically, hormonally, and socially. Adolescence is a tumultuous time, full of changes and transformations. The pubertal transition to adulthood involves both gonadal and behavioral maturation. Magnetic resonance imaging studies have discovered that myelinogenesis, required for proper insulation and efficient neurocybernetics, continues from childhood and the brain’s region-specific neurocircuitry remains structurally and functionally vulnerable to impulsive sex, food, and sleep habits. The maturation of the adolescent brain is also influenced by heredity, environment, and sex hormones (estrogen, progesterone, and testosterone), which play a crucial role in myelination. Furthermore, glutamatergic neurotransmission predominates, whereas gamma-aminobutyric acid neurotransmission remains under construction, and this might be responsible for immature and impulsive behavior and neurobehavioral excitement during adolescent life. The adolescent population is highly vulnerable to driving under the influence of alcohol and social maladjustments due to an immature limbic system and prefrontal cortex. Synaptic plasticity and the release of neurotransmitters may also be influenced by environmental neurotoxins and drugs of abuse including cigarettes, caffeine, and alcohol during adolescence. Adolescents may become involved with offensive crimes, irresponsible behavior, unprotected sex, juvenile courts, or even prison. According to a report by the Centers for Disease Control and Prevention, the major cause of death among the teenage population is due to injury and violence related to sex and substance abuse. Prenatal neglect, cigarette smoking, and alcohol consumption may also significantly impact maturation of the adolescent brain. Pharmacological interventions to regulate adolescent behavior have been attempted with limited success. Since several factors, including age, sex

  1. Maturation of the adolescent brain.

    PubMed

    Arain, Mariam; Haque, Maliha; Johal, Lina; Mathur, Puja; Nel, Wynand; Rais, Afsha; Sandhu, Ranbir; Sharma, Sushil

    2013-01-01

    Adolescence is the developmental epoch during which children become adults - intellectually, physically, hormonally, and socially. Adolescence is a tumultuous time, full of changes and transformations. The pubertal transition to adulthood involves both gonadal and behavioral maturation. Magnetic resonance imaging studies have discovered that myelinogenesis, required for proper insulation and efficient neurocybernetics, continues from childhood and the brain's region-specific neurocircuitry remains structurally and functionally vulnerable to impulsive sex, food, and sleep habits. The maturation of the adolescent brain is also influenced by heredity, environment, and sex hormones (estrogen, progesterone, and testosterone), which play a crucial role in myelination. Furthermore, glutamatergic neurotransmission predominates, whereas gamma-aminobutyric acid neurotransmission remains under construction, and this might be responsible for immature and impulsive behavior and neurobehavioral excitement during adolescent life. The adolescent population is highly vulnerable to driving under the influence of alcohol and social maladjustments due to an immature limbic system and prefrontal cortex. Synaptic plasticity and the release of neurotransmitters may also be influenced by environmental neurotoxins and drugs of abuse including cigarettes, caffeine, and alcohol during adolescence. Adolescents may become involved with offensive crimes, irresponsible behavior, unprotected sex, juvenile courts, or even prison. According to a report by the Centers for Disease Control and Prevention, the major cause of death among the teenage population is due to injury and violence related to sex and substance abuse. Prenatal neglect, cigarette smoking, and alcohol consumption may also significantly impact maturation of the adolescent brain. Pharmacological interventions to regulate adolescent behavior have been attempted with limited success. Since several factors, including age, sex, disease

  2. Maturity model for enterprise interoperability

    NASA Astrophysics Data System (ADS)

    Guédria, Wided; Naudet, Yannick; Chen, David

    2015-01-01

    Historically, progress occurs when entities communicate, share information and together create something that no one individually could do alone. Moving beyond people to machines and systems, interoperability is becoming a key factor of success in all domains. In particular, interoperability has become a challenge for enterprises, to exploit market opportunities, to meet their own objectives of cooperation or simply to survive in a growing competitive world where the networked enterprise is becoming a standard. Within this context, many research works have been conducted over the past few years and enterprise interoperability has become an important area of research, ensuring the competitiveness and growth of European enterprises. Among others, enterprises have to control their interoperability strategy and enhance their ability to interoperate. This is the purpose of the interoperability assessment. Assessing interoperability maturity allows a company to know its strengths and weaknesses in terms of interoperability with its current and potential partners, and to prioritise actions for improvement. The objective of this paper is to define a maturity model for enterprise interoperability that takes into account existing maturity models while extending the coverage of the interoperability domain. The assessment methodology is also presented. Both are demonstrated with a real case study.

  3. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a...

  4. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a...

  5. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a...

  6. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  7. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND THE EGG PRODUCTS INSPECTION ACT FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION... Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  8. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... AND THE EGG PRODUCTS INSPECTION ACT FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1 2 (INSPECTION... Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  9. 7 CFR 51.2651 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades for Sweet Cherries 1 Definitions § 51.2651 Mature. Mature means that the cherries have reached the stage of growth which will insure the...

  10. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a stage of growth which will insure a proper completion of...

  11. Career Maturity: The Construct and its Measurement.

    ERIC Educational Resources Information Center

    Savickas, Mark L.

    1984-01-01

    Describes vocational maturity and assists counselors in identifying what the various career maturity instruments measure. Discusses task variable measures, intervening variable measures, response variable measures, and methods of choosing an instrument. (JAC)

  12. Career Maturity: The Construct and its Measurement.

    ERIC Educational Resources Information Center

    Savickas, Mark L.

    1984-01-01

    Describes vocational maturity and assists counselors in identifying what the various career maturity instruments measure. Discusses task variable measures, intervening variable measures, response variable measures, and methods of choosing an instrument. (JAC)

  13. Maturation of widely distributed brain function subserves cognitive development.

    PubMed

    Luna, B; Thulborn, K R; Munoz, D P; Merriam, E P; Garver, K E; Minshew, N J; Keshavan, M S; Genovese, C R; Eddy, W F; Sweeney, J A

    2001-05-01

    Cognitive and brain maturational changes continue throughout late childhood and adolescence. During this time, increasing cognitive control over behavior enhances the voluntary suppression of reflexive/impulsive response tendencies. Recently, with the advent of functional MRI, it has become possible to characterize changes in brain activity during cognitive development. In order to investigate the cognitive and brain maturation subserving the ability to voluntarily suppress context-inappropriate behavior, we tested 8-30 year olds in an oculomotor response-suppression task. Behavioral results indicated that adult-like ability to inhibit prepotent responses matured gradually through childhood and adolescence. Functional MRI results indicated that brain activation in frontal, parietal, striatal, and thalamic regions increased progressively from childhood to adulthood. Prefrontal cortex was more active in adolescents than in children or adults; adults demonstrated greater activation in the lateral cerebellum than younger subjects. These results suggest that efficient top-down modulation of reflexive acts may not be fully developed until adulthood and provide evidence that maturation of function across widely distributed brain regions lays the groundwork for enhanced voluntary control of behavior during cognitive development.

  14. Differing roles of pyruvate dehydrogenase kinases during mouse oocyte maturation

    PubMed Central

    Hou, Xiaojing; Zhang, Liang; Han, Longsen; Ge, Juan; Ma, Rujun; Zhang, Xuesen; Moley, Kelle; Schedl, Tim; Wang, Qiang

    2015-01-01

    ABSTRACT Pyruvate dehydrogenase kinases (PDKs) modulate energy homeostasis in multiple tissues and cell types, under various nutrient conditions, through phosphorylation of the α subunit (PDHE1α, also known as PDHA1) of the pyruvate dehydrogenase (PDH) complex. However, the roles of PDKs in meiotic maturation are currently unknown. Here, by undertaking knockdown and overexpression analysis of PDK paralogs (PDK1–PDK4) in mouse oocytes, we established the site-specificity of PDKs towards the phosphorylation of three serine residues (Ser232, Ser293 and Ser300) on PDHE1α. We found that PDK3-mediated phosphorylation of Ser293-PDHE1α results in disruption of meiotic spindle morphology and chromosome alignment and decreased total ATP levels, probably through inhibition of PDH activity. Unexpectedly, we discovered that PDK1 and PDK2 promote meiotic maturation, as their knockdown disturbs the assembly of the meiotic apparatus, without significantly altering ATP content. Moreover, phosphorylation of Ser232-PDHE1α was demonstrated to mediate PDK1 and PDK2 action in meiotic maturation, possibly through a mechanism that is distinct from PDH inactivation. These findings reveal that there are divergent roles of PDKs during oocyte maturation and indicate a new mechanism controlling meiotic structure. PMID:25991547

  15. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1554 Section 51.1554 Agriculture...-tenth of the skin missing or “feathered.” ...

  16. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature means that the outer skin (epidermis... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

  17. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature means that the outer skin (epidermis... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...

  18. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.1554 Section 51.1554 Agriculture...-tenth of the skin missing or “feathered.” ...

  19. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  20. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  1. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  2. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  3. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  4. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  5. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Loan maturities. 201.11 Section 201... PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities. (a... the original loan to the final maturity of the refinanced loan shall not exceed: (i) In the case of a...

  6. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  7. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  8. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  9. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  10. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  11. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  12. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  13. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  14. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  15. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  16. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  17. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  18. 7 CFR 1710.115 - Final maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 11 2010-01-01 2010-01-01 false Final maturity. 1710.115 Section 1710.115 Agriculture... Basic Policies § 1710.115 Final maturity. (a) RUS is authorized to make loans and loan guarantees with a final maturity of up to 35 years. The borrower may elect a repayment period for a loan not longer than...

  19. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  20. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 13 Business Credit and Assistance 1 2012-01-01 2012-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  1. 24 CFR 200.82 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Maturity. 200.82 Section 200.82 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements for Existing Projects Mortgage Provisions § 200.82 Maturity. The mortgage shall have a maturity...

  2. 7 CFR 29.3039 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.3039 Section 29.3039 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. Tobacco is mature when it reaches its prime state of development. The...

  3. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  4. 7 CFR 906.11 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Maturity. 906.11 Section 906.11 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS... RIO GRANDE VALLEY IN TEXAS Order Regulating Handling Definitions § 906.11 Maturity. Maturity means...

  5. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  6. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 13 Business Credit and Assistance 1 2014-01-01 2014-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  7. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  8. 25 CFR 101.14 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Maturity. 101.14 Section 101.14 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR FINANCIAL ACTIVITIES LOANS TO INDIANS FROM THE REVOLVING LOAN FUND § 101.14 Maturity. The maturity of any United States direct loan shall not exceed thirty years. Loans...

  9. 24 CFR 242.27 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Maturity. 242.27 Section 242.27 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... FOR HOSPITALS Mortgage Requirements § 242.27 Maturity. The mortgage shall have a maturity not to...

  10. 13 CFR 120.933 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 13 Business Credit and Assistance 1 2013-01-01 2013-01-01 false Maturity. 120.933 Section 120.933... Program (504) 504 Loans and Debentures § 120.933 Maturity. From time to time, SBA will publish in the Federal Register the available maturities for a 504 loan and the Debenture that funds it. Such available...

  11. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These orange maturity requirements are contained in the Florida Citrus Code, Chapter...

  12. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These tangerine maturity requirements are contained in the Florida Citrus Code, Chapter...

  13. 7 CFR 51.1158 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... § 51.1158 Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These orange maturity requirements are contained in the Florida Citrus Code, Chapter...

  14. 7 CFR 51.1823 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These tangerine maturity requirements are contained in the Florida Citrus Code, Chapter...

  15. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These grapefruit maturity requirements are contained in the Florida Citrus Code, Chapter...

  16. 7 CFR 51.767 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Mature. Mature shall have the same meaning assigned the term in the Florida Citrus Code, Chapter 601, 1995 Edition, and the Official Rules Affecting the Florida Citrus Industry, in effect as of February 12, 1995. These grapefruit maturity requirements are contained in the Florida Citrus Code, Chapter...

  17. Career Maturity in High School Age Females.

    ERIC Educational Resources Information Center

    Pedro, Joan Daniels

    1982-01-01

    Examined career maturity in high school females by using a set of general career-maturity and gender-specific, career-related measures, and an alternate career-maturity criterion measure, career-planning involvement. Results indicated significant relationships between achievement orientation and occupational information and knowledge of women's…

  18. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Pears for Canning Definitions § 51.1351 Mature. Mature means that the pear has reached the...

  19. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.1238 Mature. Mature means that the shells are firm and well developed. ...

  20. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.1238 Mature. Mature means that the shells are firm and well developed. ...

  1. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1554 Section 51.1554 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes are generally firmly set and not more than 5 percent of the...

  2. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Standards for Pears for Canning Definitions § 51.1351 Mature. Mature...

  3. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Cleaned Virginia Type Peanuts in the Shell Definitions § 51.1238 Mature. Mature means that the...

  4. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Standards for Pears for Canning Definitions § 51.1351 Mature. Mature...

  5. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1554 Section 51.1554 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes...

  6. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature...

  7. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.3203 Mature. Mature means that the onion is fairly well cured, and at least fairly firm. ...

  8. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Bermuda-Granex-Grano Type Onions Definitions § 51.3203 Mature. Mature means that the...

  9. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature means that the outer skin (epidermis...

  10. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.3203 Mature. Mature means that the onion is fairly well cured, and at least fairly firm. ...

  11. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.693 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  12. 7 CFR 51.1907 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1907 Section 51.1907 Agriculture..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Fresh Tomatoes Definitions § 51.1907 Mature. Mature means that the tomato has reached the stage of development which will insure a proper completion...

  13. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.631 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  14. 7 CFR 51.1351 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1351 Section 51.1351 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Pears for Canning Definitions § 51.1351 Mature. Mature means that the pear has reached the...

  15. 7 CFR 51.1585 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1585 Section 51.1585 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Potatoes Definitions § 51.1585 Mature. Mature...

  16. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.2930 Section 51.2930 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of development which will insure a proper completion of the...

  17. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Bermuda-Granex-Grano Type Onions Definitions § 51.3203 Mature. Mature means that the...

  18. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Florida, California, and Arizona) Definitions § 51.631 Mature. Mature shall have the same meaning...

  19. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.2930 Section 51.2930 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of development which will insure a proper completion of the...

  20. 7 CFR 51.1554 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.1554 Section 51.1554 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Potatoes 1 Definitions § 51.1554 Mature. Mature means that the skins of the potatoes are generally firmly set and not more than 5 percent of the...

  1. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.2930 Section 51.2930 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of...

  2. 7 CFR 51.2930 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.2930 Section 51.2930 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Apricots Definitions § 51.2930 Mature. Mature means having reached the stage of...

  3. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.693 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  4. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.631 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  5. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., California, and Arizona) Definitions § 51.693 Mature. Mature shall have the same meaning currently assigned...

  6. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1238 Section 51.1238 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Cleaned Virginia Type Peanuts in the Shell Definitions § 51.1238 Mature. Mature means that the...

  7. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Florida, California, and Arizona) Definitions § 51.631 Mature. Mature shall have the same meaning...

  8. 7 CFR 51.631 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.631 Section 51.631 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...) Definitions § 51.631 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  9. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... § 51.693 Mature. Mature shall have the same meaning currently assigned that term in the laws and...

  10. 7 CFR 51.1907 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.1907 Section 51.1907 Agriculture..., CERTIFICATION, AND STANDARDS) United States Consumer Standards for Fresh Tomatoes Definitions § 51.1907 Mature. Mature means that the tomato has reached the stage of development which will insure a proper completion...

  11. 7 CFR 51.3203 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.3203 Section 51.3203 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Bermuda-Granex-Grano Type Onions Definitions § 51.3203 Mature. Mature means that the...

  12. 7 CFR 51.693 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.693 Section 51.693 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing..., California, and Arizona) Definitions § 51.693 Mature. Mature shall have the same meaning currently assigned...

  13. Micropropagation of juvenile and mature american beech

    Treesearch

    Melanie J. Barker; Paula M. Pijut; Michael E. Ostry; David R. Houston

    1997-01-01

    The purpose of this study was to micropropagate juvenile and mature American beech (Fagus grandifolia Ehrh.) resistant to beech bark disease. Shoot tips (from juvenile seedlings and root sprouts of mature trees) and buds from branches of mature trees, were cultured and multiplied on aspen culture medium supplemented with 0.89 ?M 6-benzyladenine, 0.27 ?M a-...

  14. Regulatory Mechanisms Controlling Maturation of Serotonin Neuron Identity and Function.

    PubMed

    Spencer, William C; Deneris, Evan S

    2017-01-01

    The brain serotonin (5-hydroxytryptamine; 5-HT) system has been extensively studied for its role in normal physiology and behavior, as well as, neuropsychiatric disorders. The broad influence of 5-HT on brain function, is in part due to the vast connectivity pattern of 5-HT-producing neurons throughout the CNS. 5-HT neurons are born and terminally specified midway through embryogenesis, then enter a protracted period of maturation, where they functionally integrate into CNS circuitry and then are maintained throughout life. The transcriptional regulatory networks controlling progenitor cell generation and terminal specification of 5-HT neurons are relatively well-understood, yet the factors controlling 5-HT neuron maturation are only recently coming to light. In this review, we first provide an update on the regulatory network controlling 5-HT neuron development, then delve deeper into the properties and regulatory strategies governing 5-HT neuron maturation. In particular, we discuss the role of the 5-HT neuron terminal selector transcription factor (TF) Pet-1 as a key regulator of 5-HT neuron maturation. Pet-1 was originally shown to positively regulate genes needed for 5-HT synthesis, reuptake and vesicular transport, hence 5-HT neuron-type transmitter identity. It has now been shown to regulate, both positively and negatively, many other categories of genes in 5-HT neurons including ion channels, GPCRs, transporters, neuropeptides, and other transcription factors. Its function as a terminal selector results in the maturation of 5-HT neuron excitability, firing characteristics, and synaptic modulation by several neurotransmitters. Furthermore, there is a temporal requirement for Pet-1 in the control of postmitotic gene expression trajectories thus indicating a direct role in 5-HT neuron maturation. Proper regulation of the maturation of cellular identity is critical for normal neuronal functioning and perturbations in the gene regulatory networks controlling

  15. Sterols in spermatogenesis and sperm maturation

    PubMed Central

    Keber, Rok; Rozman, Damjana; Horvat, Simon

    2013-01-01

    Mammalian spermatogenesis is a complex developmental program in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. One intriguing aspect of sperm production is the dynamic change in membrane lipid composition that occurs throughout spermatogenesis. Cholesterol content, as well as its intermediates, differs vastly between the male reproductive system and nongonadal tissues. Accumulation of cholesterol precursors such as testis meiosis-activating sterol and desmosterol is observed in testes and spermatozoa from several mammalian species. Moreover, cholesterogenic genes, especially meiosis-activating sterol-producing enzyme cytochrome P450 lanosterol 14α-demethylase, display stage-specific expression patterns during spermatogenesis. Discrepancies in gene expression patterns suggest a complex temporal and cell-type specific regulation of sterol compounds during spermatogenesis, which also involves dynamic interactions between germ and Sertoli cells. The functional importance of sterol compounds in sperm production is further supported by the modulation of sterol composition in spermatozoal membranes during epididymal transit and in the female reproductive tract, which is a prerequisite for successful fertilization. However, the exact role of sterols in male reproduction is unknown. This review discusses sterol dynamics in sperm maturation and describes recent methodological advances that will help to illuminate the complexity of sperm formation and function. PMID:23093550

  16. [Arrest of maturation in spermatogenesis].

    PubMed

    Francavilla, S; Bellocci, M; Martini, M; Bruno, B; Moscardelli, S; Fabbrini, A; Properzi, G

    1982-07-30

    The ultrastructural aspects of the germinal epithelium of 10 infertile men affected by maturative arrest of spermatogenesis were studied. We noted an increased number of malformed germinal cells. Marginal nuclear vescicles were present in spermatogonia of patients affected by spermatogonial arrest. The few spermatid present in the germinal epithelium of the patients affected by a spermatidic arrest presented changes of the nuclear condensation, the acrosome, and the tail. The Sertoli cells presented an immature aspect of the nucleus and changes of the "mantle". A possible correlation between the Sertoli cells changes and the altered spermatogenesis was proposed.

  17. Phenotype of normal hairline maturation.

    PubMed

    Rassman, William R; Pak, Jae P; Kim, Jino

    2013-08-01

    Hairlines change shape with age, starting at birth. A good head of hair is frequently present some time after ages 3 to 5 years. The look of childhood has its corresponding hairline, and, as the child grows and develops into adulthood, facial morphology migrate changes from a childlike look to a more mature look. This article discusses the dynamics of hairline evolution and the phenotypic variations of the front and side hairlines in men and women. A modeling system is introduced that provides a common language to define the various anatomic points of the full range of hairlines.

  18. Academic Achievement of High School Students in Relation to Their Anxiety, Emotional Maturity and Social Maturity

    ERIC Educational Resources Information Center

    Puar, Surjit Singh

    2013-01-01

    The present study has been designed to investigate the non-cognitive variables like anxiety, emotional maturity and social maturity and their relationship with academic achievement and also to see the locale-wise differences on the basis of their anxiety, emotional maturity and social maturity. The study was conducted over a sample of 400 (200…

  19. Validation of endoscopy for determination of maturity in small salmonids and sex of mature individuals

    Treesearch

    Erica A. Swenson; Amanda E. Rosenberger; Philip J. Howell

    2007-01-01

    Fish maturity status, sex ratio, and age and size at first maturity are important parameters in population assessments and life history studies. In most empirical studies of these variables, fish are sacrificed and dissected to obtain data. However, maturity status and the sex of mature individuals can be determined by inserting an endoscope through a small incision in...

  20. The Relationship Between Cognitive Career Maturity and Self-Reported Career Maturity of High School Students.

    ERIC Educational Resources Information Center

    Westbrook, Bert W.; And Others

    1987-01-01

    Investigated relationship between scores on measures of cognitive career maturity and self-reported career maturity in high school sophomores (N=391) and juniors (N=283). Results suggest that there is no relationship between measured career maturity competencies and self-reported career maturity competencies of high school students. (Author/NB)

  1. A Maturity Model for Enterprise Interoperability

    NASA Astrophysics Data System (ADS)

    Guédria, Wided; Chen, David; Naudet, Yannick

    Existing interoperability maturity models are fragmented and only cover some interoperability aspects. This paper tentatively proposes a maturity model for enterprise interoperability which is elaborated on the basis of existing ones. It is also consistent to the Enterprise Interoperability Framework currently under the standardization process. After a brief introduction, the paper reviews existing maturity models for interoperability and recalls the basic concepts of the Enterprise Interoperability Framework. Then the proposed maturity model for enterprise interoperability is discussed in details. Metrics for determining maturity levels are presented as well. Finally the last part of the paper gives the conclusions and perspectives for future work.

  2. Sexual maturation of female Saguinus oedipus oedipus

    SciTech Connect

    Tardif, S.D.

    1982-01-01

    This study is an examination of the process of female sexual maturation in the cotton-top tamarin, Saguinus oedipus oedipus, a South-American primate of the family, Callitrichidae. Two types of questions are addressed. The first question is whether the type of social grouping in which a young female lives affects the rate of her sexual maturation. Specifically, is there a difference between the maturation rate of a female housed with a strange adult male and a female housed with her natal group (i.e., her parents and various siblings). Second, the effect of sexual maturation on various social interactions is examined. Specifically are male-female interactions in mated pairs and mother-daughter interactions in natal groups changed by the sexual maturation of the young females. The mother's presence was not related to the daughter's maturation age. However, whether the natal group, as a whole, inhibited maturation, or unrelated males accelerated maturation, or both, remains unknown. Most of the behavioral interactions involving maturing females were unchanged by maturation. There was some indication that certain behaviors were affected by maturation, but only if a strange unrelated male was present.

  3. Neural networks predict tomato maturity stage

    NASA Astrophysics Data System (ADS)

    Hahn, Federico

    1999-03-01

    Almost 40% of the total horticultural produce exported from Mexico the USA is tomato, and quality is fundamental for maintaining the market. Many fruits packed at the green-mature stage do not mature towards a red color as they were harvested before achieving its physiological maturity. Tomato gassed for advancing maturation does not respond on those fruits, and repacking is necessary at terminal markets, causing losses to the producer. Tomato spectral signatures are different on each maturity stage and tomato size was poorly correlated against peak wavelengths. A back-propagation neural network was used to predict tomato maturity using reflectance ratios as inputs. Higher success rates were achieved on tomato maturity stage recognition with neural networks than with discriminant analysis.

  4. Physiological thermoregulation of mature alligators.

    PubMed

    Smith, E N; Standora, E A; Robertson, S L

    1984-01-01

    A 67.1 kg alligator (Alligator mississippiensis), tested in air, heated twice as fast as it cooled. The cooling thermal time constant was 425 min while alive. Warming and cooling thermal time constants were 421 min after death. The thermal time constant was not appropriate in describing warming in air of mature alligators. Surface and subdermal heat flow measurements of the 67.1 kg animal indicate greater blood flow in the skin during warming compared to cooling. Two mature alligators, 49.9 and 103 kg, were heated and cooled in water. Warming time constants were 67 and 110 min respectively. Cooling time constants were 180 and 246 min. Data from this study were combined with previously published thermal time constants for alligators providing regression equations for alligators ranging from 37 g to 103 kg. Regression equations for alligators tested in water are: tau w = 8.81 M050 tau c = 12.6 M0.62. Time constants (tau) are in minutes (w = warming, c = cooling) with all measurements in stirred water; mass, M, is in kg. Thermal conductance and metabolism data are combined to provide an estimate of the amount the body temperature of theoretical alligators ranging from 50 g to 1000 kg would be elevated by metabolism. A body temperature of 34.2 degrees C is predicted for a 1000 kg theoretical alligator in 30 degrees C water.

  5. Membrane remodeling during reticulocyte maturation

    PubMed Central

    Liu, Jing; Guo, Xinhua; Mohandas, Narla; Chasis, Joel A.

    2010-01-01

    The transition of reticulocytes into erythrocytes is accompanied by extensive changes in the structure and properties of the plasma membrane. These changes include an increase in shear resistance, loss of surface area, and acquisition of a biconcave shape. The processes by which these changes are effected have remained largely undefined. Here we examine how the expression of 30 distinct membrane proteins and their interactions change during murine reticulocyte maturation. We show that tubulin and cytosolic actin are lost, whereas the membrane content of myosin, tropomyosin, intercellular adhesion molecule-4, glucose transporter-4, Na-K-ATPase, sodium/hydrogen exchanger 1, glycophorin A, CD47, Duffy, and Kell is reduced. The degradation of tubulin and actin is, at least in part, through the ubiquitin-proteasome degradation pathway. In regard to the protein-protein interactions, the formation of membrane-associated spectrin tetramers from dimers is unperturbed, whereas the interactions responsible for the formation of the membrane-skeletal junctions are weaker in reticulocytes, as is the attachment of transmembrane proteins to these structures. This weakness, in part, results from the elevated phosphorylation of 4.1R in reticulocytes, which leads to a decrease in shear resistance by reducing its interaction with spectrin and actin. These observations begin to unravel the mechanistic basis of crucial changes accompanying reticulocyte maturation. PMID:20038785

  6. Modulation techniques

    NASA Technical Reports Server (NTRS)

    Schilling, D. L.

    1982-01-01

    Bandwidth efficient digital modulation techniques, proposed for use on and/or applied to satellite channels, are reviewed. In a survey of recent works on digital modulation techniques, the performance of several schemes operating in various environments are compared. Topics covered include: (1) quadrature phase shift keying; (2) offset - QPSK and MSK; (3) combined modulation and coding; and (4) spectrally efficient modulation techniques.

  7. Sexual maturation in kokanee Oncorhynchus nerka

    USGS Publications Warehouse

    Patterson, S.D.; Scarnecchia, D.L.; Congleton, J.L.

    2008-01-01

    We used observational and experimental approaches to obtain information on factors affecting the timing of maturation of kokanee Oncorhynchus nerka, a semelparous, landlocked salmon. Gonadal staging criteria were developed and applied to three kokanee populations in Idaho lakes and reservoirs. Testes were classified into three stages: immature (stage one, S1), maturing (S2), and mature (S3). Ovaries were classified into eight stages: immature (S1-S3), transitional (stage S4), maturing (S5-S7), and mature (S8). Males entered the maturing stage (S2) in February through April of the spawning year. Females entered maturing stage (S5) as early as July of the year before the spawning year, and as late as March of the spawning year. Three hatchery experiments demonstrated that attainment of a larger body size 10 to 16 months before spawning increased the likelihood of initiation of maturation in both sexes. No gonads in a state of regression were observed. A gonadosomatic index above 0.1 by early July was a good indicator of a maturing male, and a gonadosomatic index above 1.0 by early July was a good indicator of a maturing female. Instantaneous growth rates were not good predictors of maturation, but attaining a size threshold of 18 to 19 cm in the fall was a good predictor of maturation the following year. This improved knowledge of kokanee maturation will permit more effectively management of the species for age, growth and size at maturity as well as for contributions to fisheries. ?? 2008 by the Northwest Scientific Association. All rights reserved.

  8. Mechanism of the Intracellular Killing and Modulation of Antibiotic Susceptibility of Listeria monocytogenes in THP-1 Macrophages Activated by Gamma Interferon

    PubMed Central

    Ouadrhiri, Youssef; Scorneaux, Bernard; Sibille, Yves; Tulkens, Paul M.

    1999-01-01

    Listeria monocytogenes, a facultative intracellular pathogen, readily enters cells and multiplies in the cytosol after escaping from phagosomal vacuoles. Macrophages exposed to gamma interferon, one of the main cellular host defenses against Listeria, become nonpermissive for bacterial growth while containing Listeria in the phagosomes. Using the human myelomonocytic cell line THP-1, we show that the combination of l-monomethyl arginine and catalase restores bacterial growth without affecting the phagosomal containment of Listeria. A previous report (B. Scorneaux, Y. Ouadrhiri, G. Anzalone, and P. M. Tulkens, Antimicrob. Agents Chemother. 40:1225–1230, 1996) showed that intracellular Listeria was almost equally sensitive to ampicillin, azithromycin, and sparfloxacin in control cells but became insensitive to ampicillin and more sensitive to azithromycin and sparfloxacin in gamma interferon-treated cells. We show here that these modulations of antibiotic activity are largely counteracted by l-monomethyl arginine and catalase. In parallel, we show that gamma interferon enhances the cellular accumulation of azithromycin and sparfloxacin, an effect which is not reversed by addition of l-monomethyl arginine and catalase and which therefore cannot account for the increased activity of these antibiotics in gamma interferon-treated cells. We conclude that (i) the control exerted by gamma interferon on intracellular multiplication of Listeria in THP-1 macrophages is dependent on the production of nitric oxide and hydrogen peroxide; (ii) intracellular Listeria may become insensitive to ampicillin in macrophages exposed to gamma interferon because the increase in reactive oxygen and nitrogen intermediates already controls bacterial growth; and (iii) azithromycin and still more sparfloxacin cooperate efficiently with gamma interferon, one of the main cellular host defenses in Listeria infection. PMID:10223943

  9. Maturity group classification and maturity locus genotyping of early-maturing soybean varieties from high-latitude cold regions.

    PubMed

    Jia, Hongchang; Jiang, Bingjun; Wu, Cunxiang; Lu, Wencheng; Hou, Wensheng; Sun, Shi; Yan, Hongrui; Han, Tianfu

    2014-01-01

    With the migration of human beings, advances of agricultural sciences, evolution of planting patterns and global warming, soybeans have expanded to both tropical and high-latitude cold regions (HCRs). Unlike other regions, HCRs have much more significant and diverse photoperiods and temperature conditions over seasons or across latitudes, and HCR soybeans released there show rich diversity in maturity traits. However, HCR soybeans have not been as well classified into maturity groups (MGs) as other places. Therefore, it is necessary to identify MGs in HCRs and to genotype the maturity loci. Local varieties were collected from the northern part of Northeast China and the far-eastern region of Russia. Maturity group reference (MGR) soybeans of MGs MG000, MG00, and MG0 were used as references during field experiments. Both local varieties and MGR soybeans were planted for two years (2010-2011) in Heihe (N 50°15', E 127°27', H 168.5 m), China. The days to VE (emergence), R1 (beginning bloom) and R7 (beginning maturity) were recorded and statistically analyzed. Furthermore, some varieties were further genotyped at four molecularly-identified maturity loci E1, E2, E3 and E4. The HCR varieties were classified into MG0 or even more early-maturing. In Heihe, some varieties matured much earlier than MG000, which is the most early-maturing known MG, and clustered into a separate group. We designated the group as MG0000, following the convention of MGs. HCR soybeans had relatively stable days to beginning bloom from emergence. The HCR varieties diversified into genotypes of E1, E2, E3 and E4. These loci had different effects on maturity. HCRs diversify early-maturing MGs of soybean. MG0000, a new MG that matures much earlier than known MGs, was developed. HCR soybean breeding should focus more on shortening post-flowering reproductive growth. E1, E2, E3, and E4 function differentially.

  10. Postradiation atrophy of mature bone

    SciTech Connect

    Ergun, H.; Howland, W.J.

    1980-01-01

    The primary event of radiation damage to bone is atrophy and true necrosis of bone is uncommon. The postradiation atrophic changes of bone are the result of combined cellular and vascular damage, the former being more important. The damage to the osteoblast resulting in decreased matrix production is apparently the primary histopathologic event. Radiation damaged bone is susceptible to superimposed complications of fracture, infection, necrosis, and sarcoma. The primary radiographic evidence of atrophy, localized osteopenia, is late in appearing. Contrary to former views, the mature bone is quite radiosensitive and reacts quickly to even small doses of radiation. The differentiation of postirradiation atrophy and metastasis may be difficult. Biopsy should be the last resort because of the possibility of causing true necrosis in atrophic bone by trauma and infection.

  11. Maturation of the MOUTh Intervention

    PubMed Central

    Jablonski-Jaudon, Rita A.; Kolanowski, Ann M.; Winstead, Vicki; Jones-Townsend, Corteza; Azuero, Andres

    2016-01-01

    The purpose of the current article is to describe a personalized practice originally conceived as a way to prevent and minimize care-resistant behavior to provide mouth care to older adult with dementia. The original intervention, Managing Oral Hygiene Using Threat Reduction Strategies (MOUTh), matured during the clinical trial study into a relationship-centered intervention with emphasis on developing strategies that support residents behavioral health and staff involved in care. Relationships that were initially pragmatic (i.e., focused on the task of completing mouth care) developed into more personal and responsive relationships that involved deeper engagement between mouth care providers and nursing home (NH) residents. Mouth care was accomplished and completed in a manner enjoyable to NH residents and mouth care providers. The MOUTh intervention may also concurrently affirm the dignity and personhood of the care recipient because of its emphasis on connecting with older adults. PMID:26934969

  12. Optical maturation of asteroid surfaces

    NASA Astrophysics Data System (ADS)

    Shestopalov, D. I.; Golubeva, L. F.; Cloutis, E. A.

    2013-07-01

    Analysis of laboratory experiments simulating space weathering optical effects on atmosphereless planetary bodies reveals that the time needed to alter the spectrum of an ordinary chondrite meteorite to resemble the overall spectral shape and slope of an S-type asteroid is about ˜105 yr. The time required to reduce the visible albedo of samples to ˜0.05 is ˜106 yr. Since both these timescales are much less than the average collisional lifetime of asteroids larger than several kilometers in size, numerous low-albedo asteroids having reddish spectra with subdued absorption bands should be observed instead of an S-type dominated population. This is not the case because asteroid surfaces cannot be considered as undisturbed, unlike laboratory samples. We have estimated the number of collisions occurring in the time of ˜105 yr between asteroids and projectiles of various sizes and show that impact-activated motions of regolith particles counteract the progress of optical maturation of asteroid surfaces. We suppose that the maturation level of asteroid surfaces may be a compromise resulting from a competition between impact resurfacing and solar wind darkening, and that after reaching some steady state after a relatively short time (˜7 × 105 yr), thereafter depends only slightly on time. Spectroscopic analysis, using relatively invariant spectral parameters, such as band centers and band area ratios, can determine whether the surface of an S asteroid has chondritic composition or not. In this sense, the bodies with the ordinary chondrite composition cannot be masked among S asteroids. Differences in the environment of the main asteroid belt versus that at 1 AU, and the physical difference between the Moon and main belt asteroids (i.e., size) can account for the lack of lunar-type weathering on main belt asteroids.

  13. Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63

    PubMed Central

    Matte, Christine; Casgrain, Pierre-André; Séguin, Olivier; Moradin, Neda; Hong, Wan Jin; Descoteaux, Albert

    2016-01-01

    The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery. PMID:27280768

  14. Optimizing IV and V for Mature Organizations

    NASA Technical Reports Server (NTRS)

    Fuhman, Christopher

    2003-01-01

    NASA is intending for its future software development agencies to have at least a Level 3 rating in the Carnegie Mellon University Capability Maturity Model (CMM). The CMM has built-in Verification and Validation (V&V) processes that support higher software quality. Independent Verification and Validation (IV&V) of software developed by mature agencies can be therefore more effective than for software developed by less mature organizations. How is Independent V&V different with respect to the maturity of an organization? Knowing a priori the maturity of an organization's processes, how can IV&V planners better identify areas of need choose IV&V activities, etc? The objective of this research is to provide a complementary set of guidelines and criteria to assist the planning of IV&V activities on a project using a priori knowledge of the measurable levels of maturity of the organization developing the software.

  15. Optimizing IV and V for Mature Organizations

    NASA Technical Reports Server (NTRS)

    Fuhman, Christopher

    2003-01-01

    NASA is intending for its future software development agencies to have at least a Level 3 rating in the Carnegie Mellon University Capability Maturity Model (CMM). The CMM has built-in Verification and Validation (V&V) processes that support higher software quality. Independent Verification and Validation (IV&V) of software developed by mature agencies can be therefore more effective than for software developed by less mature organizations. How is Independent V&V different with respect to the maturity of an organization? Knowing a priori the maturity of an organization's processes, how can IV&V planners better identify areas of need choose IV&V activities, etc? The objective of this research is to provide a complementary set of guidelines and criteria to assist the planning of IV&V activities on a project using a priori knowledge of the measurable levels of maturity of the organization developing the software.

  16. Skeletal maturation determined by cervical vertebrae development.

    PubMed

    San Román, Paloma; Palma, Juan Carlos; Oteo, M Dolores; Nevado, Esther

    2002-06-01

    The aim of this study was to determine the validity of cervical vertebrae radiographic assessment to predict skeletal maturation. Left hand-wrist and lateral cephalometric radiographs of 958 Spanish children from 5 to 18 years of age were measured. On the left hand-wrist radiographs the classification of Grave and Brown was used to assess skeletal maturation. Cervical vertebrae maturation was evaluated with lateral cephalometric radiographs using the stages described by Lamparski and by Hassel and Farman. A new method to evaluate the cervical maturation by studying the changes in the concavity of the lower border, height, and shape of the vertebral body was created. Correlation coefficients were calculated to establish the relationship between skeletal maturation values obtained by the three classifications of vertebral and skeletal maturation measured at the wrist. All correlation values obtained were statistically significant (P < 0.001). The results suggest that this new method to determine skeletal maturation is very reliable. A simple method based on morphological characteristics of the cervical vertebral bodies to evaluate the maturation stage has been designed. In the population investigated, this method is as accurate as the Hassel and Farman classification and superior to the Lamparski classification. The morphological vertebral parameter best able to estimate the maturation is the concavity of the lower border of the body.

  17. Ghrelin promotion of oocyte maturation via ERK1/2 pathway in ovis aries.

    PubMed

    Bai, R; Zhao, P; Cao, G; Wen, S; Li, Q; Meng, Q

    2012-12-05

    Ghrelin has recently garnered increasing attention in biomolecular studies. Ghrelin's growth hormone secretagogue receptor type 1a (GHS--R) is a pleiotropic modulator of diverse biological functions, including energy homeostasis and reproduction. This study sought to understand the ways in which ghrelin impacts ERK1/2 and p90rsk during the ovis aries oocyte maturation process. We applied different concentrations of ghrelin and of ghrelin receptor inhibitor (D--Lys3--GHRP--6) to ovis aries oocytes and observed the effects on the ERK1/2 and p90rsk pathway. The ERK1/2 and p90rsk pathway plays an essential role in the in vitro maturation of ovis aries oocytes. This study discovered that ERK1/2 and p90 rsk pathway, during the ovis aries oocyte maturation, was associated with maturation of ovis aries oocyte in vitro.

  18. Structural dynamics control the MicroRNA maturation pathway

    PubMed Central

    Dallaire, Paul; Tan, Huiping; Szulwach, Keith; Ma, Christopher; Jin, Peng; Major, François

    2016-01-01

    MicroRNAs (miRNAs) are crucial gene expression regulators and first-order suspects in the development and progression of many diseases. Comparative analysis of cancer cell expression data highlights many deregulated miRNAs. Low expression of miR-125a was related to poor breast cancer prognosis. Interestingly, a single nucleotide polymorphism (SNP) in miR-125a was located within a minor allele expressed by breast cancer patients. The SNP is not predicted to affect the ground state structure of the primary transcript or precursor, but neither the precursor nor mature product is detected by RT-qPCR. How this SNP modulates the maturation of miR-125a is poorly understood. Here, building upon a model of RNA dynamics derived from nuclear magnetic resonance studies, we developed a quantitative model enabling the visualization and comparison of networks of transient structures. We observed a high correlation between the distances between networks of variants with that of their respective wild types and their relative degrees of maturation to the latter, suggesting an important role of transient structures in miRNA homeostasis. We classified the human miRNAs according to pairwise distances between their networks of transient structures. PMID:27651454

  19. Symbiotic commensal bacteria direct maturation of the host immune system.

    PubMed

    Edelman, Sanna M; Kasper, Dennis L

    2008-11-01

    Although commensal bacteria are known to play an important role in the proper maturation of the immune system of their mammalian hosts, the molecular mechanisms underlying this immunomodulation are poorly characterized. The present review summarizes recent findings in the field and describes new knowledge on the interplay of the innate and adaptive arms of the immune response induced by symbiotic bacterial carbohydrate antigens. Commensal bacteria in the intestine not only interact directly with dendritic cells but also engage in cross-talk with epithelial cells. These interactions lead to the induction of tolerogenic antigen-presenting cells in the lamina propria and ultimately to the regulation of functional maturation of effector T cells. Upon recognition of capsular polysaccharide antigens of commensal bacteria by dendritic cells (through toll-like receptor 2), innate immune responses facilitate and act in conjunction with adaptive responses to promote optimal Th1 polarization. In contrast, adaptive immunoglobulin A responses to symbiotic bacteria regulate the magnitude of oxidative innate immune responses in the mucosa as well as bacterial epitope expression in the lumen. Accumulating evidence is elucidating surface carbohydrate structures of symbiotic bacteria that drive the modulation of the intestinal immune system, resulting in mature, balanced immune responses and oral tolerance.

  20. Advanced Stirling Convertor (ASC) Technology Maturation

    NASA Technical Reports Server (NTRS)

    Wong, Wayne A.; Wilson, Scott; Collins, Josh; Wilson, Kyle

    2015-01-01

    The Advanced Stirling Convertor (ASC) development effort was initiated by NASA Glenn Research Center (GRC) with contractor Sunpower Inc. to develop high efficiency thermal-to-electric power conversion technology for NASA Radioisotope Power Systems. Early successful performance demonstrations led to the expansion of the project as well as adoption of the technology by the Department of Energy (DOE) and system integration contractor Lockheed Martin Space Systems Company as part of the Advanced Stirling Radioisotope Generator (ASRG) flight project. The ASRG integrates a pair of ASCs to convert the heat from a pair of General Purpose Heat Source (GPHS) modules into electrical power. The expanded NASA ASC effort included development of several generations of ASC prototypes or Engineering Units to help prepare the ASC technology and Sunpower for flight implementation. Sunpower later had two parallel contracts allowing the last of the NASA Engineering Units called ASC-E3 to serve as pathfinders for the ASC-F flight convertors being built for DOE. The ASC-E3 convertors utilized the ASC-F flight specifications and were built using the ASC-F design and process documentation. Shortly after the first ASC-F Pair achieved initial operation, due to budget constraints, the DOE ASRG flight development contract was terminated. NASA continues to invest in the development of Stirling RPS technology including continued production of the ASC-E3 convertors, seven of which have been delivered with one additional unit in production. Starting in FY2015, Stirling Convertor Technology Maturation has been reorganized as an element of the RPS Stirling Cycle Technology Development (SCTD) Project and long-term plans for continued Stirling technology advancement are in reformulation. This paper provides a status on the ASC project, an overview of advancements made in the design and production of the ASC at Sunpower, and a summary of acceptance tests, reliability tests, and tactical tests at NASA

  1. Advanced Stirling Convertor (ASC) Technology Maturation

    NASA Technical Reports Server (NTRS)

    Wong, Wayne A.; Wilson, Scott; Collins, Josh; Wilson, Kyle

    2016-01-01

    The Advanced Stirling Convertor (ASC) development effort was initiated by NASA Glenn Research Center with contractor Sunpower, Inc., to develop high-efficiency thermal-to-electric power conversion technology for NASA Radioisotope Power Systems (RPSs). Early successful performance demonstrations led to the expansion of the project as well as adoption of the technology by the Department of Energy (DOE) and system integration contractor Lockheed Martin Space Systems Company as part of the Advanced Stirling Radioisotope Generator (ASRG) flight project. The ASRG integrates a pair of ASCs to convert the heat from a pair of General Purpose Heat Source (GPHS) modules into electrical power. The expanded NASA ASC effort included development of several generations of ASC prototypes or engineering units to help prepare the ASC technology and Sunpower for flight implementation. Sunpower later had two parallel contracts allowing the last of the NASA engineering units called ASC-E3 to serve as pathfinders for the ASC-F flight convertors being built for DOE. The ASC-E3 convertors utilized the ASC-F flight specifications and were built using the ASC-F design and process documentation. Shortly after the first ASC-F pair achieved initial operation, due to budget constraints, the DOE ASRG flight development contract was terminated. NASA continues to invest in the development of Stirling RPS technology including continued production of the ASC-E3 convertors, seven of which have been delivered with one additional unit in production. Starting in fiscal year 2015, Stirling Convertor Technology Maturation has been reorganized as an element of the RPS Stirling Cycle Technology Development (SCTD) Project and long-term plans for continued Stirling technology advancement are in reformulation. This paper provides a status on the ASC project, an overview of advancements made in the design and production of the ASC at Sunpower, and a summary of acceptance tests, reliability tests, and tactical

  2. Multichip module study

    NASA Astrophysics Data System (ADS)

    Sferrino, V. J.

    1992-03-01

    Multichip module (MCM) technology addresses the large gap that exists between the speed and circuit densities achieved in monolithic integrated circuits versus those achieved at the board and subsystem level using conventional through-hole and surface mount package technology. Multichip modules promise not only to improve board-level circuit densities but also to support dramatic increases in clock rate and reductions in overall power dissipation. This new technology is driven by the realization that current printed circuit board technologies are inadequate to achieve the speed and system throughput capabilities inherent in the chips that are now becoming available. Multichip module technology centers on the high-density interconnection of bare die on a suitable substrate, resulting in a module with up to 95 percent of the substrate area devoted to active circuits. The technology features substrates that are generally made of silicon or ceramic with insulating layers of polyimide. Various other materials are employed by a host of vendors, and the technology, which is available now, is continuing to mature at a rapid rate. MCM technology is supplanting printed circuit board technology for most high-performance applications and will provide a vehicle for leading-edge digital systems in the 1990s.

  3. Evolutionary and Developmental Modules

    PubMed Central

    Lacquaniti, Francesco; Ivanenko, Yuri P.; d’Avella, Andrea; Zelik, Karl E.; Zago, Myrka

    2013-01-01

    The identification of biological modules at the systems level often follows top-down decomposition of a task goal, or bottom-up decomposition of multidimensional data arrays into basic elements or patterns representing shared features. These approaches traditionally have been applied to mature, fully developed systems. Here we review some results from two other perspectives on modularity, namely the developmental and evolutionary perspective. There is growing evidence that modular units of development were highly preserved and recombined during evolution. We first consider a few examples of modules well identifiable from morphology. Next we consider the more difficult issue of identifying functional developmental modules. We dwell especially on modular control of locomotion to argue that the building blocks used to construct different locomotor behaviors are similar across several animal species, presumably related to ancestral neural networks of command. A recurrent theme from comparative studies is that the developmental addition of new premotor modules underlies the postnatal acquisition and refinement of several different motor behaviors in vertebrates. PMID:23730285

  4. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes

    PubMed Central

    Isidor, Marie S.; Winther, Sally; Basse, Astrid L.; Petersen, M. Christine H.; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B.

    2016-01-01

    ABSTRACT Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo “browning.” In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  5. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    PubMed

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells.

  6. Effect of maturity and hybrid on ruminal and intestinal digestion of corn silage in dry cows.

    PubMed

    Peyrat, J; Baumont, R; Le Morvan, A; Nozière, P

    2016-01-01

    The aim of this study was to evaluate the effect of stage of maturity at harvest on extent of starch, neutral detergent fiber (NDF), and protein digestion, and rumen fermentation in dry cows fed whole-plant corn silage from different hybrids. Four nonlactating Holstein cows cannulated at the rumen and proximal duodenum were fed 4 corn silages differing in hybrid (flint vs. flint-dent) and maturity stage (early vs. late) in a 4 × 4 Latin square design. From early to late maturity, starch content increased (from 234.5 to 348.5 g/kg), whereas total-tract (99.7 to 94.5%) and ruminal starch digestibility (91.3 to 86.5%) decreased significantly. The decrease in ruminal starch digestibility with increasing maturity was similar between hybrids. No effects were found of maturity, hybrid, or maturity × hybrid interaction on total-tract NDF digestibility, ruminal NDF digestibility, true digestibility of N and organic matter in the rumen, or microbial synthesis. Harvesting at later maturity led to increased ruminal ammonia, total volatile fatty acid concentrations, and acetate/propionate ratio but not pH. This study concludes that delaying date of harvest modifies the proportions of digestible starch and NDF supplied to cattle. Adjusting date of corn harvest to modulate amount of rumen-digested starch could be used as a strategy to control nutrient delivery to ruminants.

  7. Pet-1 Switches Transcriptional Targets Postnatally to Regulate Maturation of Serotonin Neuron Excitability

    PubMed Central

    Wyler, Steven C.; Spencer, W. Clay; Green, Noah H.; Rood, Benjamin D.; Crawford, LaTasha; Craige, Caryne; Gresch, Paul; McMahon, Douglas G.; Beck, Sheryl G.

    2016-01-01

    Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1−/− neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. 5-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. SIGNIFICANCE STATEMENT The

  8. Postradiation atrophy of mature bone

    SciTech Connect

    Erguen, H.; Howland, W.J.

    1980-01-01

    The growing number of oncological patients subjected to radiotherapy require the diagnostic radiologist to be aware of expected bone changes following irradiation and the differentiation of this entity from metastasis. The primary event of radiation damage to bone is atrophy and true necrosis of bone is uncommon. The postradiation atrophic changes of bone are the result of combined cellular and vascular damage, the former being more important. The damage to the osteoblast resulting in decreased matrix production is apparently the primary histopathologic event. Radiation damaged bone is susceptible to superimposed complications of fracture, infection, necrosis, and sarcoma. The primary radiographic evidence of atrophy, localized osteopenia, is late in appearing, mainly because of the relative insensitivity of radiographs in detecting demineralization. Contrary to former views, the mature bone is quite radiosensitive and reacts quickly to even small doses of radiation. In vivo midrodensitometric analysis and radionuclide bone and bone marrow scans can reveal early changes following irradiation. The differentiation of postirradiation atrophy and metastasis may be difficult. Biopsy should be the last resort because of the possibility of causing true necrosis in atrophic bone by trauma and infection.

  9. Postradiation atrophy of mature bone

    SciTech Connect

    Ergun, H.; Howland, W.J.

    1980-01-01

    The growing number of oncological patients subjected to radiotherapy require the diagnostic radiologist to be aware of expected bone changes following irradiation and the differentiation of this entity from metastasis. The primary event of radiation damage to bone is atrophy and true necrosis of bone is uncommon. The postradiation atrophic changes of bone are the result of combined cellular and vascular damage, the former being more important. The damage to the osteoblast resulting in decreased matrix production is apparently the primary histopathologic event. Radiation damaged bone is susceptible to superimposed complications of fracture, infection, necrosis, and sarcoma. The primary radiographic evidence of atrophy, localized osteopenia, is late in appearing, mainly because of the relative insensitivity of radiographs in detecing demineralization. Contrary to former views, the mature bone is quite radiosensitive and reacts quickly to even small doses of radiation. In vivo midrodensitometric analysis and radionuclide bone and bone marrow scans can reveal early changes following irradiation. The differentiation of postirradiation atrophy and metastasis may be difficult. Biopsy should be the last resort because of the possibility of causing true necrosis in atrophic bone by trauma and infection.

  10. Smart Grid Interoperability Maturity Model

    SciTech Connect

    Widergren, Steven E.; Levinson, Alex; Mater, J.; Drummond, R.

    2010-04-28

    The integration of automation associated with electricity resources (including transmission and distribution automation and demand-side resources operated by end-users) is key to supporting greater efficiencies and incorporating variable renewable resources and electric vehicles into the power system. The integration problems faced by this community are analogous to those faced in the health industry, emergency services, and other complex communities with many stakeholders. To highlight this issue and encourage communication and the development of a smart grid interoperability community, the GridWise Architecture Council (GWAC) created an Interoperability Context-Setting Framework. This "conceptual model" has been helpful to explain the importance of organizational alignment in addition to technical and informational interface specifications for "smart grid" devices and systems. As a next step to building a community sensitive to interoperability, the GWAC is investigating an interoperability maturity model (IMM) based on work done by others to address similar circumstances. The objective is to create a tool or set of tools that encourages a culture of interoperability in this emerging community. The tools would measure status and progress, analyze gaps, and prioritize efforts to improve the situation.

  11. Motivational maturity and helping behavior.

    PubMed

    Haymes, M; Green, L

    1977-12-01

    This study was undertaken to examine the independent influences of conative development (the Maslow needs hierarchy) upon behavioral aspects of prosocial orientations. It provides a behavioral demonstration of conative effects in a helping paradigm, among college-age men. A comparison of the conative data across the ages of 15-22 provided a cross-sectional view of conative development itself. Conative maturity was found to be predictive of greater helping among college-age men. Situational demands were demonstrated which tended to mask, but not override, these predispositional influences on helping. The cross-sectional data on conative development point to probable movement to early esteem concerns among high school men who have reached the conative level of love and belonging. On the other hand, the stability across the years of 15-22 of proportion of safety concerns suggests fixation of such concerns in those exhibiting them in high school. Results are discussed in terms of conative growth for development of prosocial orientations.

  12. Prolonged maturation of auditory perception and learning in gerbils

    PubMed Central

    Sarro, Emma C.; Sanes, Dan H.

    2011-01-01

    In humans, auditory perception reaches maturity over a broad age range, extending through adolescence. Despite this slow maturation, children are considered to be outstanding learners, suggesting that immature perceptual skills might actually be advantageous to improvement on an acoustic task as a result of training (perceptual learning). Previous non-human studies have not employed an identical task when comparing perceptual performance of young and mature subjects, making it difficult to assess learning. Here, we used an identical procedure on juvenile and adult gerbils to examine the perception of amplitude modulation (AM), a stimulus feature that is an important component of most natural sounds. On average, Adult animals could detect smaller fluctuations in amplitude (i.e. smaller modulation depths) than Juveniles, indicating immature perceptual skills in Juveniles. However, the population variance was much greater for Juveniles, a few animals displaying adult-like AM detection. To determine whether immature perceptual skills facilitated learning, we compared naïve performance on the AM detection task with the amount of improvement following additional training. The amount of improvement in Adults correlated with naïve performance: those with the poorest naïve performance improved the most. In contrast, the naïve performance of Juveniles did not predict the amount of learning. Those Juveniles with immature AM detection thresholds did not display greater learning than Adults. Furthermore, for several of the Juveniles with adult-like thresholds, AM detection deteriorated with repeated testing. Thus, immature perceptual skills in young animals were not associated with greater learning. PMID:20506133

  13. Probiotics can induce follicle maturational competence: the Danio rerio case.

    PubMed

    Gioacchini, Giorgia; Giorgini, Elisabetta; Merrifield, Daniel L; Hardiman, Gary; Borini, Andrea; Vaccari, Lisa; Carnevali, Oliana

    2012-03-01

    In the present study, the effects of the probiotic Lactobacillus rhamnosus IMC 501 on the acquisition of oocyte maturational competence was examined in zebrafish (Danio rerio). L. rhamnosus administration induced the responsiveness of incompetent follicles (stage IIIa) to 17,20-dihydroxy-4-pregnen-3-one and their in vitro maturation. Acquisition of competence by the stage IIIa follicles was further validated by changes of lhr, mprb, inhbaa (activin betaA1), tgfb1, and gdf9 gene expression, which have recently emerged as key regulators of oocyte acquisition of maturational competence, and pou5f1 gene expression, which in other models has been shown to govern the establishment of developmental competence of oocytes. In addition, a DNA microarray experiment was conducted using the same follicles, and with relative gene ontology (GO) data analysis, the molecular effects of probiotic administration emerged. Molecular analysis using PCR-DGGE (denaturing gradient gel electrophoresis) approach, providing information about only the most abundant bacterial members of the microbial community, revealed that the probiotic was able to populate the gastrointestinal tract and modulate the microbial communities, causing a clear shift in them and specifically enhancing the presence of the lactic acid bacteria Streptococcus thermophilus. At the same time, PCR-DGGE analysis revealed that the probiotic was not directly associated with the ovaries. Finally, the effects of probiotic treatment on zebrafish follicle development were also analyzed by FPA (focal plane array) Fourier transform-infrared (FT-IR) imaging, a technique that provides the overall biochemical composition of samples. Changes were found above all in stage IIIa follicles from probiotic-exposed females; the modifications, observed in protein secondary structures as well as in hydration and in bands related to phosphate moieties, allowed us to hypothesize that probiotics act at this follicle stage, affecting the

  14. 7 CFR 51.3058 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... AND THE EGG PRODUCTS INSPECTION ACT FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Florida Avocados Definitions § 51.3058 Mature. Mature means that the avocado has reached a stage of growth which will insure a proper completion of the...

  15. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  16. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  17. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  18. 24 CFR 201.11 - Loan maturities.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Loan maturities. 201.11 Section 201... DEVELOPMENT MORTGAGE AND LOAN INSURANCE PROGRAMS UNDER NATIONAL HOUSING ACT AND OTHER AUTHORITIES TITLE I PROPERTY IMPROVEMENT AND MANUFACTURED HOME LOANS Loan and Note Provisions § 201.11 Loan maturities....

  19. 7 CFR 51.1238 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing Practices), DEPARTMENT OF AGRICULTURE REGULATIONS AND STANDARDS UNDER THE AGRICULTURAL MARKETING ACT OF 1946... Standards for Cleaned Virginia Type Peanuts in the Shell Definitions § 51.1238 Mature. Mature means that...

  20. Motivation and Maturity Patterns in Marital Success.

    ERIC Educational Resources Information Center

    McClelland, David C.; And Others

    1978-01-01

    Married couples rated their marital satisfaction and played interpersonal competitive games which revealed the success with which they interacted. Younger husbands who scored more maturely on the Stewart measure of psychosocial maturity belonged to more successful marriages, as did college-educated wives who showed less immaturity and more phallic…

  1. Thinned Mature Deciduous Forest Silvopastures for Appalachia

    USDA-ARS?s Scientific Manuscript database

    Little information is available on effective management and utilization of silvopastures developed from the ubiquitous mature woodlots which comprise 40-50% of small Appalachian farm acreage. We thinned a white oak dominated mature second growth forested area establishing two 0.5 ha, eight-paddock,...

  2. 7 CFR 29.1034 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.1034 Section 29.1034 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 92) § 29.1034 Maturity. The degree of ripeness. (See Elements of Quality Chart.) ...

  3. 24 CFR 241.1060 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 24 Housing and Urban Development 2 2013-04-01 2013-04-01 false Maturity. 241.1060 Section 241.1060 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements § 241.1060 Maturity. (a) Equity loans shall have a term not to exceed 40 years; and (b...

  4. 7 CFR 29.3531 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.3531 Section 29.3531 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 95) § 29.3531 Maturity. The degree of ripeness. (See Elements of Quality, § 29.3586, and Rule 16...

  5. 7 CFR 29.3531 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.3531 Section 29.3531 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 95) § 29.3531 Maturity. The degree of ripeness. (See Elements of Quality, § 29.3586, and Rule 16...

  6. 7 CFR 29.2281 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.2281 Section 29.2281 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. (See chart, § 29.2351.) ...

  7. 24 CFR 241.1060 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Maturity. 241.1060 Section 241.1060 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements § 241.1060 Maturity. (a) Equity loans shall have a term not to exceed 40 years; and (b...

  8. 24 CFR 241.1060 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 24 Housing and Urban Development 2 2012-04-01 2012-04-01 false Maturity. 241.1060 Section 241.1060 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements § 241.1060 Maturity. (a) Equity loans shall have a term not to exceed 40 years; and (b...

  9. 7 CFR 29.2281 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.2281 Section 29.2281 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. (See chart, § 29.2351.) ...

  10. 7 CFR 29.2533 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.2533 Section 29.2533 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2533 Maturity. The degree of ripeness. (See...

  11. 24 CFR 241.1060 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 24 Housing and Urban Development 2 2014-04-01 2014-04-01 false Maturity. 241.1060 Section 241.1060 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements § 241.1060 Maturity. (a) Equity loans shall have a term not to exceed 40 years; and (b...

  12. 7 CFR 29.1034 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.1034 Section 29.1034 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 92) § 29.1034 Maturity. The degree of ripeness. (See Elements of Quality Chart.) [42 FR 21092, Apr...

  13. 7 CFR 29.2281 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.2281 Section 29.2281 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. (See chart, § 29.2351.) ...

  14. 7 CFR 29.6026 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.6026 Section 29.6026 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6026 Maturity. The degree of ripeness. (See chart.) ...

  15. 7 CFR 29.1034 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.1034 Section 29.1034 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 92) § 29.1034 Maturity. The degree of ripeness. (See Elements of Quality Chart.) [42 FR 21092, Apr...

  16. 7 CFR 29.2281 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.2281 Section 29.2281 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. (See chart, § 29.2351.) ...

  17. 7 CFR 29.6026 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.6026 Section 29.6026 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6026 Maturity. The degree of ripeness. (See chart.) ...

  18. 7 CFR 29.3531 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.3531 Section 29.3531 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 95) § 29.3531 Maturity. The degree of ripeness. (See Elements of Quality, § 29.3586, and Rule 16...

  19. 7 CFR 29.1034 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.1034 Section 29.1034 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 92) § 29.1034 Maturity. The degree of ripeness. (See Elements of Quality Chart.) [42 FR 21092, Apr...

  20. 7 CFR 29.6026 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.6026 Section 29.6026 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6026 Maturity. The degree of ripeness. (See chart.) ...

  1. 7 CFR 29.2281 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.2281 Section 29.2281 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Maturity. The degree of ripeness. (See chart, § 29.2351.) ...

  2. 24 CFR 241.1060 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Maturity. 241.1060 Section 241.1060 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF... Requirements § 241.1060 Maturity. (a) Equity loans shall have a term not to exceed 40 years; and (b...

  3. 7 CFR 29.3531 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.3531 Section 29.3531 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 95) § 29.3531 Maturity. The degree of ripeness. (See Elements of Quality, § 29.3586, and Rule 16...

  4. 7 CFR 29.6026 - Maturity.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Maturity. 29.6026 Section 29.6026 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6026 Maturity. The degree of ripeness. (See chart.) ...

  5. 7 CFR 29.1034 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.1034 Section 29.1034 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 92) § 29.1034 Maturity. The degree of ripeness. (See Elements of Quality Chart.) ...

  6. 7 CFR 29.2533 - Maturity.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Maturity. 29.2533 Section 29.2533 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2533 Maturity. The degree of ripeness. (See...

  7. 7 CFR 29.2533 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.2533 Section 29.2533 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2533 Maturity. The degree of ripeness. (See...

  8. 7 CFR 29.2533 - Maturity.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Maturity. 29.2533 Section 29.2533 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2533 Maturity. The degree of ripeness. (See...

  9. 7 CFR 29.2533 - Maturity.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Maturity. 29.2533 Section 29.2533 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing...-Cured Tobacco (u.s. Types 22, 23, and Foreign Type 96) § 29.2533 Maturity. The degree of ripeness. (See...

  10. 7 CFR 29.6026 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.6026 Section 29.6026 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... INSPECTION Standards Definitions § 29.6026 Maturity. The degree of ripeness. (See chart.) ...

  11. 7 CFR 29.3531 - Maturity.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Maturity. 29.3531 Section 29.3531 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Type 95) § 29.3531 Maturity. The degree of ripeness. (See Elements of Quality, § 29.3586, and Rule 16...

  12. Relationship of Vocational Maturity to Work Values

    ERIC Educational Resources Information Center

    Miller, Michael F.

    1974-01-01

    Two hypotheses were tested: (1) Vocational maturity is positively related to differentiation of work values within subjects. (2) Vocational maturity is positively associated with intrinsic work values and negatively associated with extrinsic work values. Data analyses supported hypothesis 1 for females, but not for males, and partially supported…

  13. Cone and Seed Maturation of Southern Pines

    Treesearch

    James P. Barnett

    1976-01-01

    If slightly reduced yields and viability are acceptable, loblolly and slash cone collections can begin 2 to 3 weeks before maturity if the cones are stored before processing. Longleaf(P. palestris Mill.) pine cones should be collected only when mature, as storage decreased germination of seeds from immature cones. Biochemical analyses to determine reducing sugar...

  14. Canopy temperature and maturity in cotton

    USDA-ARS?s Scientific Manuscript database

    Heat units are a widely used indicator of maturity in cotton. It is generally assumed that it takes approximately 2200°F (1222°C) heat units for a cotton plant on the South High Plains of Texas to mature. This value is based on a typical planting date of May 15 with ample irrigation. As water for c...

  15. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Fresh Plums and Prunes Definitions § 51.1530 Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  16. 7 CFR 51.1313 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States... stage of maturity which will insure the proper completion of the ripening process. (b) Before a mature pear becomes overripe it will show varying degrees of firmness depending upon the stage of the ripening...

  17. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Fresh Plums and Prunes Definitions § 51.1530 Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  18. 7 CFR 51.1530 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States Standards for Grades of Fresh Plums and Prunes Definitions § 51.1530 Mature. “Mature” means that the fruit has reached the stage of maturity which will insure a proper completion of the ripening process. ...

  19. 7 CFR 51.1313 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... FRESH FRUITS, VEGETABLES AND OTHER PRODUCTS 1,2 (INSPECTION, CERTIFICATION, AND STANDARDS) United States... stage of maturity which will insure the proper completion of the ripening process. (b) Before a mature pear becomes overripe it will show varying degrees of firmness depending upon the stage of the ripening...

  20. Toward the Measurement of Psychosocial Maturity.

    ERIC Educational Resources Information Center

    Greenberger, Ellen; And Others

    The concept of psychosocial maturity is reviewed in preparation for the exploration of the feasibility of constructing a scale that measures maturity. Investigation produced a preliminary 54-item scale with high reliability and moderate validity, which is appended. A factor analysis of the scale supports the a priori structure by the theoretical…

  1. Egg maturation dynamics of Homalodisca vitripennis

    USDA-ARS?s Scientific Manuscript database

    The egg maturation dynamics of holometabolous insects are particularly well studied compared to those of hemimetabolous insects. The wealth of knowledge produced from studies on holometabolous insects has allowed researchers to test for correlations between egg maturation schedule and specific life...

  2. Technology Evaluation Cycles and Maturity Assessment

    DTIC Science & Technology

    2006-05-01

    reserved. tManager: Technology Selection Requirements View Concept Refinement View Maturity View Us er Ne ed s eBusiness ServicesRisk Analysis C ustom er...Technology Selection Requirements View Concept Refinement View Maturity View Us er Ne ed s eBusiness ServicesRisk Analysis C ustom er D eveloper

  3. Maturation of Sweetgum and American Sycamore Seeds

    Treesearch

    F. T. Bonner

    1972-01-01

    Over three consecutive years in central Mississippi, sweetgum (Liquidambar styraciflua L.) and sycamore (Platanus occidentalis L.) fruits had nearly reached full-size by late June. Sweetgum seeds were physiologically mature by mid-August, but dry weight increased until late September. As sweetgum seeds matured, the crude fat level rose to 27 percent of seed dry weight...

  4. A Spectropolarimetric Maturity Index of Lunar Soil

    NASA Astrophysics Data System (ADS)

    Shevchenko, V. V.; Skobeleva, T. P.; Kvaratskhelia, O. I.

    2003-05-01

    The lunar soil maturity is the most important parameter of the Moon's surface material. The degree of regolith processing should be taken into account in remote determinations of the chemical and mineralogical surface compositions. However, the possibilities for directly determining the lunar regolith maturity are limited to laboratory studies of the fine fraction and microparticles of samples returned to Earth. In these conditions, the urgency of developing methods for remotely determining the lunar soil maturity increases sharply. The suggested method of using spectropolarimetric data to quantitatively estimate the maturity of the surface material has an advantage that the derived maturity index is determined only by structural parameters of the reflecting layer and is completely free from the effects of chemical and mineralogical surface rock compositions. The reference catalog of spectropolarimetric indices contains values for 92 objects on the Moon's visible hemisphere and includes a wide range of structures with various degrees of maturity of the surface material. We obtained correlations with other maturity indices determined by laboratory and remote sensing techniques and the time scale that represents the correspondence between the spectropolarimetric maturity index and the soil exposure age.

  5. The FMI: Dimensions of Follower Maturity

    ERIC Educational Resources Information Center

    Moore, Loren I.

    1976-01-01

    The Follower Maturity Index (FMI) is an instrument derived from leadership theory and based on observations of verbal and nonverbal behavior of followers in task groups. Dimensions of follower maturity--achievement, responsibility, experience, activity, dependence, variety, interests, perspective, position, and awareness--are discussed. For…

  6. Motivation and Maturity Patterns in Marital Success.

    ERIC Educational Resources Information Center

    McClelland, David C.; And Others

    1978-01-01

    Married couples rated their marital satisfaction and played interpersonal competitive games which revealed the success with which they interacted. Younger husbands who scored more maturely on the Stewart measure of psychosocial maturity belonged to more successful marriages, as did college-educated wives who showed less immaturity and more phallic…

  7. 7 CFR 51.3153 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 2 2013-01-01 2013-01-01 false Mature. 51.3153 Section 51.3153 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Nectarines Definitions § 51.3153 Mature. “Mature” means that the nectarine has reached the stage of growth which will insure a proper completion of...

  8. 7 CFR 51.1218 - Mature.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Mature. 51.1218 Section 51.1218 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Peaches Definitions § 51.1218 Mature. “Mature” means that the peach has reached the...

  9. 7 CFR 51.3153 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 2 2014-01-01 2014-01-01 false Mature. 51.3153 Section 51.3153 Agriculture..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Nectarines Definitions § 51.3153 Mature. “Mature” means that the nectarine has reached the stage of growth which will insure a proper completion of...

  10. 7 CFR 51.1218 - Mature.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 2 2012-01-01 2012-01-01 false Mature. 51.1218 Section 51.1218 Agriculture Regulations of the Department of Agriculture AGRICULTURAL MARKETING SERVICE (Standards, Inspections, Marketing... Standards for Grades of Peaches Definitions § 51.1218 Mature. “Mature” means that the peach has reached the...

  11. Quantifying Semantic Linguistic Maturity in Children

    ERIC Educational Resources Information Center

    Hansson, Kristina; Bååth, Rasmus; Löhndorf, Simone; Sahlén, Birgitta; Sikström, Sverker

    2016-01-01

    We propose a method to quantify "semantic linguistic maturity" (SELMA) based on a high dimensional semantic representation of words created from the co-occurrence of words in a large text corpus. The method was applied to oral narratives from 108 children aged 4;0-12;10. By comparing the SELMA measure with maturity ratings made by human…

  12. New definitions for cotton fiber maturity ratio

    USDA-ARS?s Scientific Manuscript database

    Cotton fiber maturity affects fiber physical, mechanical, and chemical properties, as well as the processability and qualities of yarn and fabrics. New definitions of cotton fiber maturity ratio are introduced. The influences of sampling, sample preparation, measurement method, and correlations am...

  13. The Mature Woman and the Community College

    ERIC Educational Resources Information Center

    Elliot, Jeffrey M.; Mantz, Concetta M.

    1976-01-01

    The factors and motivations contributing to the presence of increasing numbers of mature women in college are examined, and seven proposals are offered, representing an attempt to develop a total community college program which will meet the needs of mature women students. (NHM)

  14. Quantifying Semantic Linguistic Maturity in Children

    ERIC Educational Resources Information Center

    Hansson, Kristina; Bååth, Rasmus; Löhndorf, Simone; Sahlén, Birgitta; Sikström, Sverker

    2016-01-01

    We propose a method to quantify "semantic linguistic maturity" (SELMA) based on a high dimensional semantic representation of words created from the co-occurrence of words in a large text corpus. The method was applied to oral narratives from 108 children aged 4;0-12;10. By comparing the SELMA measure with maturity ratings made by human…

  15. Overview of the People Capability Maturity Model.

    DTIC Science & Technology

    1995-09-01

    This document provides an overview and an introduction to the People Capability Maturity Model (P-CMM) (Curtis95). Specifically, this document...capability. The document is intended to provide an overview of the concepts of the P-CMM, while the People Capability Maturity Model (Curtis95) describes the key practices for each level of the P-CMM.

  16. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of firmness...

  17. 7 CFR 51.312 - Mature.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ..., CERTIFICATION, AND STANDARDS) United States Standards for Grades of Apples Definitions § 51.312 Mature. “Mature” means that the apples have reached the stage of development which will insure the proper completion of the ripening process. Before a mature apple becomes overripe it will show varying degrees of firmness...

  18. 7 CFR 1421.101 - Maturity dates.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... AGRICULTURE LOANS, PURCHASES, AND OTHER OPERATIONS GRAINS AND SIMILARLY HANDLED COMMODITIES-MARKETING ASSISTANCE LOANS AND LOAN DEFICIENCY PAYMENTS FOR 2008 THROUGH 2012 Marketing Assistance Loans § 1421.101 Maturity dates. (a)(1) All marketing assistance loans shall mature on demand by CCC and no later than the...

  19. Module Configuration

    DOEpatents

    Oweis, Salah; D'Ussel, Louis; Chagnon, Guy; Zuhowski, Michael; Sack, Tim; Laucournet, Gaullume; Jackson, Edward J.

    2002-06-04

    A stand alone battery module including: (a) a mechanical configuration; (b) a thermal management configuration; (c) an electrical connection configuration; and (d) an electronics configuration. Such a module is fully interchangeable in a battery pack assembly, mechanically, from the thermal management point of view, and electrically. With the same hardware, the module can accommodate different cell sizes and, therefore, can easily have different capacities. The module structure is designed to accommodate the electronics monitoring, protection, and printed wiring assembly boards (PWAs), as well as to allow airflow through the module. A plurality of modules may easily be connected together to form a battery pack. The parts of the module are designed to facilitate their manufacture and assembly.

  20. ANTIGENIC MODULATION

    PubMed Central

    Old, Lloyd J.; Stockert, Elisabeth; Boyse, Edward A.; Kim, Jae Ho

    1968-01-01

    Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro. An ascites leukemia, phenotype TL.1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon. Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr. The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test. The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C. Thus modulation apparently is an active cellular process. Antigens TL. 1,2, and 3 are all modulated by anti-TL.1,3 serum and by anti-TL.3 serum. This modulation affects all three TL components together, even when antibody to one or two of them is lacking. aAnti-TL.2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies. The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro. Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region. Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions. PMID:5636556