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Sample records for active mature form

  1. Active Site Inhibitors Protect Protein Kinase C from Dephosphorylation and Stabilize Its Mature Form*

    PubMed Central

    Gould, Christine M.; Antal, Corina E.; Reyes, Gloria; Kunkel, Maya T.; Adams, Ryan A.; Ziyar, Ahdad; Riveros, Tania; Newton, Alexandra C.

    2011-01-01

    Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C. PMID:21715334

  2. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    PubMed Central

    Lum, Michelle A.; Balaburski, Gregor M.; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Although alterations in stimulus-induced degradation of PKC have been implicated in disease, mechanistic understanding of this process remains limited. Evidence supports the existence of both proteasomal and lysosomal mechanisms of PKC processing. An established pathway involves rate-limiting priming site dephosphorylation of the activated enzyme and proteasomal clearance of the dephosphorylated protein. However, here we show that agonists promote down-regulation of endogenous PKCα with minimal accumulation of a nonphosphorylated species in multiple cell types. Furthermore, proteasome and lysosome inhibitors predominantly protect fully phosphorylated PKCα, pointing to this form as a substrate for degradation. Failure to detect substantive dephosphorylation of activated PKCα was not due to rephosphorylation because inhibition of Hsp70/Hsc70, which is required for re-priming, had only a minor effect on agonist-induced accumulation of nonphosphorylated protein. Thus, PKC degradation can occur in the absence of dephosphorylation. Further analysis revealed novel functions for Hsp70/Hsc70 and Hsp90 in the control of agonist-induced PKCα processing. These chaperones help to maintain phosphorylation of activated PKCα but have opposing effects on degradation of the phosphorylated protein; Hsp90 is protective, whereas Hsp70/Hsc70 activity is required for proteasomal processing of this species. Notably, down-regulation of nonphosphorylated PKCα shows little Hsp70/Hsc70 dependence, arguing that phosphorylated and nonphosphorylated species are differentially targeted for proteasomal degradation. Finally, lysosomal processing of activated PKCα is not regulated by phosphorylation or Hsps. Collectively, these data demonstrate that phosphorylated PKCα is a direct target for agonist-induced proteasomal degradation via an Hsp-regulated mechanism, and highlight the existence of a novel pathway of PKC desensitization in cells. PMID:23900841

  3. Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

    PubMed Central

    Sudiman, Jaqueline; Sutton-McDowall, Melanie L.; Ritter, Lesley J.; White, Melissa A.; Mottershead, David G.; Thompson, Jeremy G.; Gilchrist, Robert B.

    2014-01-01

    Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies. PMID:25058588

  4. Select forms of tumor cell apoptosis induce dendritic cell maturation.

    PubMed

    Demaria, Sandra; Santori, Fabio R; Ng, Bruce; Liebes, Leonard; Formenti, Silvia C; Vukmanovic, Stanislav

    2005-03-01

    Dendritic cells (DC) play a crucial role in initiating immune responses to tumors. DC can efficiently present antigens from apoptotic tumor cells, but apoptotic cells are thought to lack the inflammatory signals required to induce DC maturation. Here, we show that apoptosis of 67NR mouse carcinoma cells via the Fas (CD95) pathway or induced by the anticancer drug bortezomib (PS-341) but not by ultraviolet irradiation is associated with the production of maturation signals for DC. These data have important implications for the effects of chemotherapy on antitumor immunity in solid and hematologic malignancies. PMID:15569694

  5. Revision of the Career Maturity Inventory: The Adaptability Form

    ERIC Educational Resources Information Center

    Savickas, Mark L.; Porfeli, Erik J.

    2011-01-01

    Initially administered in 1961, the Career Maturity Inventory (CMI) was the first paper-and-pencil measure of vocational development. The present research revised the CMI to reestablish its usefulness as a succinct, reliable, and valid measure of career choice readiness, with a few theoretically relevant and practically useful content scales for…

  6. Reactive oxygen scavenging activity of matured whiskey and its active polyphenols.

    PubMed

    Koga, K; Taguchi, A; Koshimizu, S; Suwa, Y; Yamada, Y; Shirasaka, N; Yoshizumi, H

    2007-04-01

    The quality of whiskey is known to improve remarkably by its storage over many years. This process is commonly termed "maturing." In this process, polyphenols derived from lignin and tannin of the barrel have an important role in not only forming the matured flavor and taste but also contributing to the advance of clustering ethanol and water in whiskey. It is also likely that polyphenols generally possess reactive oxygen (RO) scavenging activity. The present study evaluated the RO scavenging activity (free-radical scavenging activity, H(2)O(2) reduction activity under peroxidase coculture, and H(2)O(2)scavenging activity) of 24 single malt whiskeys with a maturation age of 10 to 30 y produced in Japanese, Scotch (Islay), or Scotch (Speyside and Highland) regions. Single malt whiskey not only showed RO scavenging activity but there was also a positive correlation between this activity and the maturation age of whiskey exceeding the difference resulting from the manufacturing region. A nonvolatile fraction derived from the barrel was responsible for RO scavenging activity. In particular, the contents of ellagic and gallic acids and lyoniresinol, the main polyphenolic compounds in whiskey, increased with maturation age. For the free-radical scavenging activity per molecule, each compound was 1.68 to 3.14 times that of trolox (a water-soluble vitamin E). The activities of ellagic acid, gallic acid, and lyoniresinol in the whiskey (Yamazaki 18) were equivalent to that of 80.3, 31.2, and 11.1 ppm trolox, respectively. Accordingly, the total activity of these 3 compounds accounted for about 20% of the activity of the whiskey (630.7 ppm trolox). PMID:17995817

  7. Technology maturation project on optimization of sheet metal forming of aluminum for use in transportation systems

    NASA Astrophysics Data System (ADS)

    Johnson, Ken I.; Smith, Mark T.; Lavender, Curt A.; Khalell, Mohammad A.

    1994-10-01

    Using aluminum instead of steel in transportation systems could dramatically reduce the weight of vehicles, an effective way of decreasing energy consumption and emissions. The current cost of sheet metal formed (SMF) aluminum alloys (about $4 per pound) and the relatively long forming times of current materials are serious drawbacks to the widespread use of SMF in industry. The interdependence of materials testing and model development is critical to optimizing SMF since the current process is conducted in a heated, pressurized die where direct measurement of critical SMF parameters is extremely difficult. Numerical models provide a means of tracking the forming process, allowing the applied gas pressure to be adjusted to maintain the optimum SMF behavior throughout the forming process. Thus, models can help produce the optimum SMF component in the least amount of time. The Pacific Northwest Laboratory is integrating SMF model development with research in improved aluminum alloys for SMF. The objectives of this research are: develop and characterize competitively priced aluminum alloys for SMF applications in industry; improve numerical models to accurately predict the optimum forming cycle for reduced forming time and improved quality; and verify alloy performance and model accuracy with forming tests conducted in PNL's Superplastic Forming User Facility. The activities performed in this technology maturation project represent a critical first step in achieving these objectives through cooperative research among industry, PNL, and universities.

  8. Monogenic Forms of Diabetes: Neonatal Diabetes Mellitus and Maturity-Onset Diabetes of the Young

    MedlinePlus

    ... For More Information American Diabetes Association JDRF MedlinePlus Diabetes Disease Organizations Many organizations provide support to patients ... 293 KB). Alternate Language URL Monogenic Forms of Diabetes: Neonatal Diabetes Mellitus and Maturity-onset Diabetes of ...

  9. Physical activity and biological maturation: a systematic review

    PubMed Central

    Bacil, Eliane Denise Araújo; Mazzardo, Oldemar; Rech, Cassiano Ricardo; Legnani, Rosimeide Francisco dos Santos; de Campos, Wagner

    2015-01-01

    OBJECTIVE: To analyze the association between physical activity (PA) and biological maturation in children and adolescents. DATA SOURCE: We performed a systematic review in April 2013 in the electronic databases of PubMed/MEDLINE, SportDiscus, Web of Science and LILACS without time restrictions. A total of 628 potentially relevant articles were identified and 10 met the inclusion criteria for this review: cross-sectional or longitudinal studies, published in Portuguese, English or Spanish, with schoolchildren aged 9-15 years old of both genders. DATA SYNTHESIS: Despite the heterogeneity of the studies, there was an inverse association between PA and biological maturation. PA decreases with increased biological and chronological age in both genders. Boys tend to be more physically active than girls; however, when controlling for biological age, the gender differences disappear. The association between PA and timing of maturation varies between the genders. Variation in the timing of biological maturation affects the tracking of PA in early adolescent girls. This review suggests that mediators (BMI, depression, low self-esteem, and concerns about body weight) can explain the association between PA and biological maturation. CONCLUSIONS: There is an association between PA and biological maturation. PA decreases with increasing biological age with no differences between genders. As for the timing of biological maturation, this association varies between genders. PMID:25583624

  10. Tissue factor activity. A marker of alveolar macrophage maturation in rabbits. Effects of granulomatous pneumonitis.

    PubMed Central

    Rothberger, H; McGee, M P; Lee, T K

    1984-01-01

    amounts of tissue factor activity due to the presence of large numbers of mature alveolar macrophage forms that had high levels of the procoagulant. Thus, tissue factor activity in alveolar macrophages is a marker of cellular maturation in vivo and in vitro. Increased amounts of this initiator of the extrinsic clotting pathway, as found in alveolar macrophage populations from animals with granulomatous pneumonitis induced by BCG hypersensitivity, suggest that alveolar macrophage tissue factor may contribute to the pathology of immune lung diseases. PMID:6373826

  11. Neuronal activity is not required for the initial formation and maturation of visual selectivity.

    PubMed

    Hagihara, Kenta M; Murakami, Tomonari; Yoshida, Takashi; Tagawa, Yoshiaki; Ohki, Kenichi

    2015-12-01

    Neuronal activity is important for the functional refinement of neuronal circuits in the early visual system. At the level of the cerebral cortex, however, it is still unknown whether the formation of fundamental functions such as orientation selectivity depends on neuronal activity, as it has been difficult to suppress activity throughout development. Using genetic silencing of cortical activity starting before the formation of orientation selectivity, we found that the orientation selectivity of neurons in the mouse visual cortex formed and matured normally despite a strong suppression of both spontaneous and visually evoked activity throughout development. After the orientation selectivity formed, the distribution of the preferred orientations of neurons was reorganized. We found that this process required spontaneous activity, but not visually evoked activity. Thus, the initial formation and maturation of orientation selectivity is largely independent of neuronal activity, and the initial selectivity is subsequently modified depending on neuronal activity. PMID:26523644

  12. Organizational Learning from the Perspective of Knowledge Maturing Activities

    ERIC Educational Resources Information Center

    Kaschig, A.; Maier, R.; Sandow, A.; Lazoi, M.; Schmidt, A.; Barnes, S.-A.; Bimrose, J.; Brown, A.; Bradley, C.; Kunzmann, C.; Mazarakis, A.

    2013-01-01

    The level of similarity of knowledge work across occupations and industries allows for the design of supportive information and communication technology (ICT) that can be widely used. In a previous ethnographically informed study, we identified activities that can be supported to increase knowledge maturing, conceptualized as goal-oriented…

  13. The structure of procaspase 6 is similar to that of active mature caspase 6.

    PubMed Central

    Kang, Byoung Heon; Ko, Eunsil; Kwon, Oh-Keun; Choi, Kwan Yong

    2002-01-01

    To investigate the structural characteristics and activation mechanism of the precursor caspase, genes encoding the inactive pro-form and the active mature form of caspase 6 were expressed in Escherichia coli and the proteins of both forms were purified to homogeneity. The structure of each protein was characterized by chemical cross-linking, size-exclusion chromatography, CD and fluorescence spectroscopies. The pro-form caspase 6 exhibits a dimeric structure and its overall secondary structure was found to be similar to that of the mature caspase 6. Upon the maturation of procaspase 6, the maximum fluorescence wavelength lambda(max) was red-shifted from 330 to 337 nm and the fluorescence intensity of lambda(max) was increased. This fluorescence spectral change indicates that the environment of a tryptophan residue in the substrate-binding site can be changed to a more polar one when the procaspase 6 is processed. Taken together, our results strongly demonstrate that precursor caspase 6 exists as a dimer and its overall structure is similar to that of the active caspase 6. Our results also suggest that the local conformational change at the substrate-binding site, with no drastic change in the overall structure, seems to enable precursor caspase 6 to become the active mature enzyme. PMID:12049625

  14. Evolution of robusta green coffee redox enzymatic activities with maturation.

    PubMed

    Montavon, Philippe; Bortlik, Karlheinz

    2004-06-01

    Oxidation reactions in coffee involve redox-sensitive polyphenols and appear to control the fragmentation of coffee storage proteins both in solution and during roasting. Coffee-specific nitrogenous flavor precursors may derive from this process. Accordingly, data converge to suggest that the redox status of the green bean before roasting might control the development of subsequent redox reactions during roasting. Consequently, we decided to identify biological events that may trigger or prevent oxidation during maturation of the coffee cherry and set the final redox status of the green bean. In a previous study, we observed that the sensitivity of green coffee to oxidative processes decreased along maturation. By using the very same samples originating from open-pollinated Robusta clones, we followed the activity of three essential redox enzymes: catalase (CAT), peroxidase (POD) and polyphenoloxidase (PPO). While CAT and POD activities increased with maturation, PPO activities decreased. Thanks to the identification of an atypical immature subclass, it appeared that CAT might be an essential factor in setting the final redox status of the green bean before the roasting event. PMID:15161235

  15. Cathepsin L maturation and activity is impaired in macrophages harboring M. avium and M. tuberculosis.

    PubMed

    Nepal, Rajeev M; Mampe, Stephanie; Shaffer, Brian; Erickson, Ann H; Bryant, Paula

    2006-06-01

    Mycobacterium tuberculosis-infected macrophages demonstrate diminished capacity to present antigens via class II MHC molecules. Since successful class II MHC-restricted antigen presentation relies on the actions of endocytic proteases, we asked whether the activities of cathepsins (Cat) B, S and L-three major lysosomal cysteine proteases-are modulated in macrophages infected with pathogenic Mycobacterium spp. Infection of murine bone marrow-derived macrophages with either Mycobacterium avium or M. tuberculosis had no obvious effect on Cat B or Cat S activity. In contrast, the activity of Cat L was altered in infected cells. Specifically, whereas the 24-kDa two-chain mature form of active Cat L predominated in uninfected cells, we observed an increase in the steady-state activity of the precursor single-chain (30 kDa) and 25-kDa two-chain forms of the enzyme in cells infected with either M. avium or M. tuberculosis. Pulse-chase analyses revealed that maturation of nascent, single-chain Cat L into the 25-kDa two-chain form was impaired in infected macrophages, and that maturation into the 24-kDa two-chain form did not occur. Consistent with these data, M. avium infection inhibited the IFNgamma-induced secretion of active two-chain Cat L by macrophages. Viable bacilli were not required to disrupt Cat L maturation, suggesting that a constitutively expressed mycobacterial component was responsible. The absence of the major active form of lysosomal Cat L in M. avium- and M. tuberculosis-infected macrophages may influence the types of T cell epitopes generated in these antigen-presenting cells, and/or the rate of class II MHC peptide loading. PMID:16636015

  16. The Sandia MEMS passive shock sensor : FY07 maturation activities.

    SciTech Connect

    Houston, Jack E.; Blecke, Jill; Mitchell, John Anthony; Wittwer, Jonathan W.; Crowson, Douglas A.; Clemens, Rebecca C.; Walraven, Jeremy Allen; Epp, David S.; Baker, Michael Sean

    2008-08-01

    This report describes activities conducted in FY07 to mature the MEMS passive shock sensor. The first chapter of the report provides motivation and background on activities that are described in detail in later chapters. The second chapter discusses concepts that are important for integrating the MEMS passive shock sensor into a system. Following these two introductory chapters, the report details modeling and design efforts, packaging, failure analysis and testing and validation. At the end of FY07, the MEMS passive shock sensor was at TRL 4.

  17. Recombinant expression, purification and crystallographic studies of the mature form of human mitochondrial aspartate aminotransferase.

    PubMed

    Jiang, Xiuping; Wang, Jia; Chang, Haiyang; Zhou, Yong

    2016-02-01

    Mitochondrial aspartate aminotransferase (mAspAT) was recognized as a moonlighting enzyme because it has not only aminotransferase activity but also a high-affinity long-chain fatty acids (LCFA) binding site. This enzyme plays a key role in amino acid metabolism, biosynthesis of kynurenic acid and transport of the LCFA. Therefore, it is important to study the structure-function relationships of human mAspAT protein. In this work, the mature form of human mAspAT was expressed to a high level in Escherichia coli periplasmic space using pET-22b vector, purified by a combination of immobilized metal-affinity chromatography and cation exchange chromatography. Optimal activity of the enzyme occurred at a temperature of 47.5ºC and a pH of 8.5. Crystals of human mAspAT were grown using the hanging-drop vapour diffusion method at 277K with 0.1 M HEPES pH 6.8 and 25%(v/v) Jeffamine(®) ED-2001 pH 6.8. The crystals diffracted to 2.99 Å and belonged to the space group P1 with the unit-cell parameters a =56.7, b = 76.1, c = 94.2 Å, α =78.0, β =85.6, γ = 78.4º. Elucidation of mAspAT structure can provide a molecular basis towards understanding catalysis mechanism and substrate binding site of enzyme. PMID:26902786

  18. Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form.

    PubMed

    Schröder, Heinz C; Wang, Xiaohong; Manfrin, Alberto; Yu, Shu-Hong; Grebenjuk, Vlad A; Korzhev, Michael; Wiens, Matthias; Schlossmacher, Ute; Müller, Werner E G

    2012-06-22

    Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties. PMID:22544742

  19. Cassava root membrane proteome reveals activities during storage root maturation.

    PubMed

    Naconsie, Maliwan; Lertpanyasampatha, Manassawe; Viboonjun, Unchera; Netrphan, Supatcharee; Kuwano, Masayoshi; Ogasawara, Naotake; Narangajavana, Jarunya

    2016-01-01

    Cassava (Manihot esculenta Crantz) is one of the most important crops of Thailand. Its storage roots are used as food, feed, starch production, and be the important source for biofuel and biodegradable plastic production. Despite the importance of cassava storage roots, little is known about the mechanisms involved in their formation. This present study has focused on comparison of the expression profiles of cassava root proteome at various developmental stages using two-dimensional gel electrophoresis and LC-MS/MS. Based on an anatomical study using Toluidine Blue, the secondary growth was confirmed to be essential during the development of cassava storage root. To investigate biochemical processes occurring during storage root maturation, soluble and membrane proteins were isolated from storage roots harvested from 3-, 6-, 9-, and 12-month-old cassava plants. The proteins with differential expression pattern were analysed and identified to be associated with 8 functional groups: protein folding and degradation, energy, metabolism, secondary metabolism, stress response, transport facilitation, cytoskeleton, and unclassified function. The expression profiling of membrane proteins revealed the proteins involved in protein folding and degradation, energy, and cell structure were highly expressed during early stages of development. Integration of these data along with the information available in genome and transcriptome databases is critical to expand knowledge obtained solely from the field of proteomics. Possible role of identified proteins were discussed in relation with the activities during storage root maturation in cassava. PMID:26547558

  20. Technology maturation project on optimization of sheet metal forming of aluminum for use in transportation systems: Final project report

    SciTech Connect

    Johnson, K.I.; Smith, M.T.; Lavender, C.A.; Khalell, M.A.

    1994-10-01

    Using aluminum instead of steel in transportation systems could dramatically reduce the weight of vehicles--an effective way of decreasing energy consumption and emissions. The current cost of SMF aluminum alloys (about $4 per pound) and the relatively long forming times of current materials are serious drawbacks to the widespread use of SMF in industry. The interdependence of materials testing and model development is critical to optimizing SMF since the current process is conducted in a heated, pressurized die where direct measurement of critical SMF parameters is extremely difficult. Numerical models provide a means of tracking the forming process, allowing the applied gas pressure to be adjusted to maintain the optimum SMF behavior throughout the forming process. Thus, models can help produce the optimum SMF component in the least amount of time. The Pacific Northwest Laboratory is integrating SMF model development with research in improved aluminum alloys for SMF. The objectives of this research are: develop and characterize competitively priced aluminum alloys for SMF applications in industry; improve numerical models to accurately predict the optimum forming cycle for reduced forming time and improved quality; verify alloy performance and model accuracy with forming tests conducted in PNL`s Superplastic Forming User Facility. The activities performed in this technology maturation project represent a critical first step in achieving these objectives through cooperative research among industry, PNL, and universities.

  1. An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+.

    PubMed

    Sinsereekul, Nitat; Foophow, Tita; Yamanouchi, Mai; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

    2011-06-01

    Pro-Tk-SP from Thermococcus kodakaraensis consists of the four domains: N-propeptide, subtilisin (EC 3.4.21.62) domain, β-jelly roll domain and C-propeptide. To analyze the maturation process of this protein, the Pro-Tk-SP derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*) were constructed. Pro-Tk-S359C was purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC-Tk-S359C, instead of the ProN:ProC-Tk-S359C complex. The C-propeptide was autoprocessed and degraded when ProC-Tk-S359C was incubated at 80 °C in the absence of Ca(2+). However, it was not autoprocessed in the presence of Ca(2+). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca(2+) suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca(2+). We propose that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) represents a mature form of Pro-Tk-SP in a natural environment. The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the T(m) value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9 °C in the absence of Ca(2+) and 7.5 °C in the presence of Ca(2+), indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C. PMID:21443525

  2. Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

    PubMed

    Hegele-Hartung, C; Kuhnke, J; Lessl, M; Grøndahl, C; Ottesen, J; Beier, H M; Eisner, S; Eichenlaub-Ritter, U

    1999-11-01

    Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization. PMID:10529286

  3. Maturation of neuronal form and function in a mouse thalamo-cortical circuit.

    PubMed

    Warren, R A; Jones, E G

    1997-01-01

    Postnatal development of physiological properties underlying slow intrathalamic oscillations was studied by whole-cell recording from synaptically coupled neurons of the reticular nucleus (RTN) and ventral posterior nucleus (VPN) of mouse brain slices in vitro and compared with the morphological development of dye-injected cells. Between postnatal days 3 and 11 (P3-P11), progressive changes in RTN and VPN neurons included shortening of the membrane time constant, decreasing input resistance, and lowering of the resting membrane potential (RMP). Low-threshold Ca2+ spikes (LTS) were present from P3, but their capacity to sustain multispike bursts was limited before P11. Synaptic responses were evoked in RTN and VPN neurons by electrical stimulation of the internal capsule from P3. Younger RTN neurons responded with a single spike, but their capacity to fire bursts gradually improved as the RMP reached levels below the LTS activation potential. Concomitantly, as the reversal potential of the inhibitory postsynaptic potential in VPN neurons became more negative, its capacity to deinactivate the LTS increased, and rebound bursts that could maintain oscillations were produced; sustained oscillations became the typical response to internal capsule stimulation at P12. The functional maturation of the intrathalamic circuitry, particularly between P10 and P14, occurs in parallel with the morphological maturation (size, dendritic growth, and dendritic field structure) of individual RTN and VPN neurons, as studied by confocal microscopy. Maturation of RTN cells led that of VPN cells by 2-3 d. The appearance of intrathalamic oscillations is probably correlated with the appearance of slow-wave sleep in postnatal animals. PMID:8987755

  4. Notch signaling promotes osteoclast maturation and resorptive activity.

    PubMed

    Ashley, Jason W; Ahn, Jaimo; Hankenson, Kurt D

    2015-11-01

    The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta-like1 or by suppression with γ-secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context-specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors. PMID:25914241

  5. Effect of different activators on development of activated in vitro matured caprine oocytes

    PubMed Central

    Sharma, J. R.; Agarwal, S.; Kharche, S. D.; Goel, A. K.; Jindal, S. K.; Agarwal, S. K.

    2015-01-01

    This study was designed to compare the effectiveness of different activation treatments for activation of in vitro matured oocytes and their developmental potency in mCR2aa medium so as to obtain maximum number of embryos. A total of 1090 cumulus oocyte complexes (COC’s) were collected from 480 ovaries. In vitro matured oocytes were randomly divided into four groups. Group 1 in vitro matured oocytes (n=226) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Group 2 in vitro matured oocytes (n=294) were exposed to 7% ethanol for 5 min followed by treatment with 10 µg/ml CHX for 4 h in mCR2aa medium. Group 3 in vitro matured oocytes (n=325) were exposed to 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP and 10 µg/ml CHX for 4 h in mCR2aa medium. Group 4 in vitro matured oocytes (n=108) were cultured for 4 h without any chemical treatment in mCR2aa medium (control). The cleavage rate in groups 1, 2, 3 and 4 was 54.42%, 44.55%, 51.69% and 0.00%, respectively. The percentage of morula and blastocyst production in group 1, group 2 and group 3 was 26.01%, 29.77% and 29.76% and 2.43%, 1.52% and 1.78%, respectively. These results suggest that the activation of in vitro matured oocytes by 7% ethanol for 5 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa is most favorable for parthenogenetic caprine embryos production. PMID:27175149

  6. The Rho GTPase Cdc42 Is Essential for the Activation and Function of Mature B Cells

    PubMed Central

    Gerasimcik, Natalija; Dahlberg, Carin I. M.; Baptista, Marisa A. P.; Massaad, Michel J.; Geha, Raif S.; Westerberg, Lisa S.

    2015-01-01

    The Rho GTPase Cdc42 coordinates regulation of the actin and the microtubule cytoskeleton by binding and activating the Wiskott–Aldrich syndrome protein. We sought to define the role of intrinsic expression of Cdc42 by mature B cells in their activation and function. Mice with inducible deletion of Cdc42 in mature B cells formed smaller germinal centers and had a reduced Ab response, mostly of low affinity to T cell–dependent Ag, compared with wild-type (WT) controls. Spreading formation of long protrusions that contain F-actin, microtubules, and Cdc42-interacting protein 4, and assumption of a dendritic cell morphology in response to anti-CD40 plus IL-4 were impaired in Cdc42-deficient B cells compared with WT B cells. Cdc42-deficient B cells had an intact migratory response to chemokine in vitro, but their homing to the B cell follicles in the spleen in vivo was significantly impaired. Cdc42-deficient B cells induced a skewed cytokine response in CD4+ T cells, compared with WT B cells. Our results demonstrate a critical role for Cdc42 in the motility of mature B cells, their cognate interaction with T cells, and their differentiation into Ab-producing cells. PMID:25870239

  7. 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

    2016-03-01

    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

  8. 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

  9. Antioxidant and antiproliferative activities in different maturation stages of broccoli (Brassica oleracea Italica) biofortified with selenium.

    PubMed

    Bachiega, Patricia; Salgado, Jocelem Mastrodi; de Carvalho, João Ernesto; Ruiz, Ana Lúcia T G; Schwarz, Kélin; Tezotto, Tiago; Morzelle, Maressa Caldeira

    2016-01-01

    In this work, three different broccoli maturity stages subjected to biofortification with selenium were evaluated for antioxidant and antiproliferative activities. Antioxidant trials have shown that the maturation stages biofortified with selenium had significantly higher amounts of phenolic compounds and antioxidant activity, especially seedlings. Although non-polar extracts of all samples show antiproliferative activity, the extract of broccoli seedlings biofortified with selenium stood out, presenting cytocidal activity for a glioma line (U251, GI50 28.5 mg L(-1)). PMID:26213037

  10. Loss of H2 histamine receptor activity in rabbit aorta after maturity

    SciTech Connect

    Holl, J.E.; Mokler, C.M.

    1982-01-01

    The activity of H1 and H2 histaminergic receptors was studied in aortic strips taken from young (6 weeks) and mature (7-8 months) rabbits. H1-mediated contractile activity was similar in both age groups, but H2 receptor relaxant activity was greatly diminished in mature rabbits. H2 receptor activity was evaluated with two experimental approaches: its modifying effect on blockade of H1 receptors by diphenhydramine (DPN), and its capacity to directly relax precontracted strips. Schild plot evaluation of DPN blockade revealed that the slope of the plot was below 1.0 in strips from young rabbits (0.77), but not in strips from mature rabbits (0.94). Propranolol pretreatment did not increase the Schild plot slope (0.66) of young aortic strips, but metiamide pretreatment did (0.92). Metiamide treatment did not further in crease slope of mature strips (0.95). When the slopes of the plots were near 1.0, as in the mature and metiamide-treated mature and young strips, the pA2 of DPN was very close (7.51, 7.70, and 7.65). Lost H2 receptor activity in maturity was confirmed by relaxing norepinephrine-precontracted strips with histamine or dimaprit. Histamine-induced relaxation of precontracted strips was slightly diminished in mature strips, while the relaxing activity of dimaprit was greatly reduced in mature tissue. The same strips relaxed vigorously when exposed to nitroglycerin or NaNO2, demonstrating that the strips were capable of relaxing. Therefore, it appears that H2 receptor activity in rabbit aorta is greatly diminished as the animal matures.

  11. The Main Sequence of Explosive Solar Active Regions: Comparison of Emerging and Mature Active Regions

    NASA Technical Reports Server (NTRS)

    Falconer, David; Moore, Ron

    2011-01-01

    For mature active regions, an active region s magnetic flux content determines the maximum free energy the active region can have. Most Large flares and CMEs occur in active regions that are near their free-energy limit. Active-region flare power radiated in the GOES 1-8 band increases steeply as the free-energy limit is approached. We infer that the free-energy limit is set by the rate of release of an active region s free magnetic energy by flares, CMEs and coronal heating balancing the maximum rate the Sun can put free energy into the active region s magnetic field. This balance of maximum power results in explosive active regions residing in a "mainsequence" in active-region (flux content, free energy content) phase space, which sequence is analogous to the main sequence of hydrogen-burning stars in (mass, luminosity) phase space.

  12. The Native Form and Maturation Process of Hepatitis C Virus Core Protein

    PubMed Central

    Yasui, Kohichiroh; Wakita, Takaji; Tsukiyama-Kohara, Kyoko; Funahashi, Shin-Ichi; Ichikawa, Masumi; Kajita, Tadahiro; Moradpour, Darius; Wands, Jack R.; Kohara, Michinori

    1998-01-01

    The maturation and subcellular localization of hepatitis C virus (HCV) core protein were investigated with both a vaccinia virus expression system and CHO cell lines stably transformed with HCV cDNA. Two HCV core proteins, with molecular sizes of 21 kDa (p21) and 23 kDa (p23), were identified. The C-terminal end of p23 is amino acid 191 of the HCV polyprotein, and p21 is produced as a result of processing between amino acids 174 and 191. The subcellular localization of the HCV core protein was examined by confocal laser scanning microscopy. Although HCV core protein resided predominantly in the cytoplasm, it was also found in the nucleus and had the same molecular size as p21 in both locations, as determined by subcellular fractionation. The HCV core proteins had different immunoreactivities to a panel of monoclonal antibodies. Antibody 5E3 stained core protein in both the cytoplasm and the nucleus, C7-50 stained core protein only in the cytoplasm, and 499S stained core protein only in the nucleus. These results clearly indicate that the p23 form of HCV core protein is processed to p21 in the cytoplasm and that the core protein in the nucleus has a higher-order structure different from that of p21 in the cytoplasm. HCV core protein in sera of patients with HCV infection was analyzed in order to determine the molecular size of genuinely processed HCV core protein. HCV core protein in sera was found to have exactly the same molecular weight as the p21 protein. These results suggest that p21 core protein is a component of native viral particles. PMID:9621068

  13. Monogenic Forms of Diabetes: Neonatal Diabetes Mellitus and Maturity-Onset Diabetes of the Young

    MedlinePlus

    ... Neonatal Diabetes Mellitus and MODY Monogenic Forms of Diabetes: Neonatal Diabetes Mellitus and MODY The most common forms of ... is inherited from each parent. Monogenic Forms of Diabetes Some rare forms of diabetes result from mutations ...

  14. Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

    SciTech Connect

    Moore, Michael D.; Fu, William; Soheilian, Ferri; Nagashima, Kunio; Ptak, Roger G.; Pathak, Vinay K.; Hu, Wei-Shau

    2008-09-15

    Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC{sub 90}), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation.

  15. Gas6 Downregulation Impaired Cytoplasmic Maturation and Pronuclear Formation Independent to the MPF Activity

    PubMed Central

    Kim, Kyeoung-Hwa; Kim, Eun-Young; Kim, Yuna; Kim, Eunju; Lee, Hyun-Seo; Yoon, Sook-Young; Lee, Kyung-Ah

    2011-01-01

    Previously, we found that the growth arrest-specific gene 6 (Gas6) is more highly expressed in germinal vesicle (GV) oocytes than in metaphase II (MII) oocytes using annealing control primer (ACP)-PCR technology. The current study was undertaken to investigate the role of Gas6 in oocyte maturation and fertilization using RNA interference (RNAi). Interestingly, despite the specific and marked decrease in Gas6 mRNA and protein expression in GVs after Gas6 RNAi, nuclear maturation including spindle structures and chromosome segregation was not affected. The only discernible effect induced by Gas6 RNAi was a change in maturation promoting factor (MPF) activity. After parthenogenetic activation, Gas6 RNAi-treated oocytes at the MII stage had not developed further and arrested at MII (90.0%). After stimulation with Sr2+, Gas6-silenced MII oocytes had markedly reduced Ca2+ oscillation and exhibited no exocytosis of cortical granules. In these oocytes, sperm penetration occurred during fertilization but not pronucleus (PN) formation. By roscovitine and colcemid treatment, we found that the Gas6 knockdown affected cytoplasmic maturation directly, independent to the changed MPF activity. These results strongly suggest that 1) the Gas6 signaling itself is important to the cytoplasmic maturation, but not nuclear maturation, and 2) the decreased Gas6 expression and decreased MPF activity separately or mutually influence sperm head decondensation and PN formation. PMID:21850267

  16. Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract

    PubMed Central

    Sami, Amtul Jamil; Sami, Amtul Naseer; Kanwal, Noreen

    2007-01-01

    This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at −20 °C. The total protein extract was isolated from 5 samples in each case (mature and hyper mature cataract) and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 °C. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. β mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates. PMID:17657864

  17. Comparison in effect of different metal ions, pH and reducing agent on the protease activity in human hyper mature and mature cataract.

    PubMed

    Sami, Amtul Jamil; Sami, Amtul Naseer; Kanwal, Noreen

    2007-08-01

    This study was undertaken to isolate and characterize the protease activity of human eye lens sample of mature and hyper mature cataract. Samples were collected just after surgery of the cataract lens and were stored at -20 degrees C. The total protein extract was isolated from 5 samples in each case (mature and hyper mature cataract) and clear supernatant obtained after centrifugation was used as an enzyme source. The optimum pH for the proteases of mature cataract was 7.5 while the proteases of hyper mature cataract were recorded for maximum activity at pH 5.5 and 7.5. The optimum temperature for both enzyme sources was 50 degrees C. Effect of different metal ions such as potassium, lead, silver, zinc and borate was studied. In each case protease activity was increased. Reducing agent e.g. beta mercaptoethanol also caused an increase in activity indicating the involvement of sulfhydryl groups. Protease activity was also located on agar plates. PMID:17657864

  18. Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation.

    PubMed Central

    Aoufouchi, S; Shall, S

    1997-01-01

    Poly(ADP-ribose) polymerase (PARP) is an abundant nuclear enzyme that is dependent on DNA breaks and nicks for its enzyme activity. These DNA nicks and breaks function as allosteric effectors of the enzyme activity. This reaction is important for efficient DNA base excision repair, although it is not a component of the elementary repair pathway itself. The physiological relevance of this reaction might be to ensure correct and efficient DNA repair. We have examined the enzyme activity of PARP in oocytes and eggs of Xenopus laevis. Although both oocytes and eggs contain approximately the same amounts of enzyme protein, there is no detectable enzyme activity in the oocytes, whereas in the eggs the enzyme is active. Enzyme activity appears during oocyte maturation, approx. 4 h after induction by progesterone. This enzyme activation coincides with the appearance of active maturation-promoting factor. Enzyme activation is accompanied by a shift in the electrophoretic mobility of the polypeptide, from an apparent molecular mass of 116 kDa to 125 kDa. Treatment with either bacterial or potato phosphatase reverses the mobility shift and abolishes enzyme activity. Incubation of maturing X. laevis eggs with radioactive inorganic phosphate and subsequent immunoprecipitation demonstrate that the PARP protein is phosphorylated in vivo. We show that maturation-promoting factor (Cyclin B/cdc2) cannot itself be responsible for the phosphorylation and activation of PARP in maturing X. laevis eggs. Together, these results demonstrate that the enzyme activity of PARP in X. laevis oocytes and eggs is regulated by post-translational, covalent phosphorylation. PMID:9230139

  19. The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture

    PubMed Central

    MORITA, Yasuhiro; TANIGUCHI, Masayasu; TANIHARA, Fuminori; ITO, Aya; NAMULA, Zhao; DO, Lanh Thi Kim; TAKAGI, Mitsuhiro; TAKEMOTO, Tatsuya; OTOI, Takeshige

    2016-01-01

    The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts. PMID:26947170

  20. Investigations on iodothyronine deiodinase activity in the maturing rat brain.

    PubMed

    Ködding, R; Fuhrmann, H; von zur Mühlen, A

    1986-04-01

    5-Monodeiodination of T4 and T3 and 5'-monodeiodination of T4 and rT3 were studied in brain homogenates of male Sprague-Dawley rats, aged 1-60 days. Portions of the homogenates were incubated with the substrates at 37 C for 30 min. The reaction products were estimated by specific RIAs. All of the four reactions were dependent upon time, temperature, pH, and upon the concentrations of substrate, thiol, and tissue protein. Maximal reactions were obtained between 40 and 160 mM dithioerythritol. T4 5'-deiodination proceeded optimally at pH 7.4 and 0.4 microM substrate, the other reactions at pH 8.5 and 10 microM substrate. The four reactions were inactivated by heat (56 C, 30 min) and inhibited by 10(-5) M iopanoic acid. Only rT3 5'-deiodination was inhibited by 3 X 10(-4) M propylthiouracil (greater than 95%). In cerebellum, basal ganglia, brainstem, and hypothalamus both T4 and T3 5-deiodinase activity were very high in perinatal rats [up to 5.56 pmol/(min X mg protein) in hypothalamus], and decreased rapidly with age. In cortex and olfactory bulb these enzyme activities were low after birth, followed by an increase during the growth spurt [up to 632 fmol/(min X mg protein) in olfactory bulb]. T4 and rT3 5'-deiodinase activity in all brain regions studied were at their lowest in perinatal rats. During and after the growth spurt an increase was observed [up to 457 fmol/(min X mg protein) in cerebellum]. The reciprocal course of 5- and 5'-deiodination between birth and growth spurt in most of the brain regions studied might lead to a reduced intracellular thyromimetic activity during the perinatal period. PMID:3948784

  1. Reactive oxygen species production and antioxidant enzyme activity during epididymal sperm maturation in Corynorhinus mexicanus bats.

    PubMed

    Arenas-Ríos, Edith; Rosado García, Adolfo; Cortés-Barberena, Edith; Königsberg, Mina; Arteaga-Silva, Marcela; Rodríguez-Tobón, Ahiezer; Fuentes-Mascorro, Gisela; León-Galván, Miguel Angel

    2016-03-01

    Prolonged sperm storage in the epididymis of Corynorhinus mexicanus bats after testicular regression has been associated with epididymal sperm maturation in the caudal region, although the precise factors linked with this phenomenon are unknown. The aim of this work is to determine the role of reactive oxygen species (ROS) and changes in antioxidant enzymatic activity occurring in the spermatozoa and epididymal fluid over time, in sperm maturation and storage in the caput, corpus and cauda of the bat epididymis. Our data showed that an increment in ROS production coincided with an increase in superoxide dismutase (SOD) activity in epididymal fluid and with a decrease in glutathione peroxidase (GPX) activity in the spermatozoa in at different time points and epididymal regions. The increase in ROS production was not associated with oxidative damage measured by lipid peroxidation. The results of the current study suggest the existence of a shift in the redox balance, which might be associated with sperm maturation and storage. PMID:26952757

  2. Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation.

    PubMed

    Waiz, Syma Ashraf; Raies-Ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T Sridhar; Chauhan, M S; Upadhyay, R C

    2016-09-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS. PMID:26781547

  3. Heat stress and antioxidant enzyme activity in bubaline (Bubalus bubalis) oocytes during in vitro maturation

    NASA Astrophysics Data System (ADS)

    Waiz, Syma Ashraf; Raies-ul-Haq, Mohammad; Dhanda, Suman; Kumar, Anil; Goud, T. Sridhar; Chauhan, M. S.; Upadhyay, R. C.

    2016-01-01

    In vitro environments like heat stress usually increase the production of reactive oxygen species in bubaline oocytes which have been implicated as one of the major causes for reduced developmental competence. Oocytes during meiotic maturation are sensitive to oxidative stress, and heat stress accelerates cellular metabolism, resulting in the higher production of free radicals. Therefore, the aim of present work was to assess the impact of heat stress during meiotic maturation on bubaline cumulus-oocyte complexes (COC), denuded oocytes (DO), and cumulus cell mass in terms of their oxidative status. Accordingly, for control group, COC were matured at 38.5 °C for complete 24 h of meiotic maturation and heat stress of 40.5 and 41.5 °C was applied to COC during the first 12 h of maturation and then moved to 38.5 °C for rest of the 12 h. In another group, COC after maturation were denuded from the surrounding cumulus cells by manual pipetting. Results indicated that the production of reactive oxygen species (ROS), lipid peroxides, and nitric oxide (NO) was significantly (P < 0.05) higher in the oocytes subjected to heat stress (40.5 and 41.5 °C) during meiotic maturation compared to the oocytes matured under standard in vitro culture conditions (38.5 °C). Also, the antioxidant enzymatic activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase were significantly (P < 0.05) increased in all the treatment groups compared to the control group. Therefore, the present study clearly establishes that heat stress ensues oxidative stress in bubaline oocytes which triggers the induction of antioxidant enzymatic defense system for scavenging the ROS.

  4. ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed.

    PubMed

    Gonzalez-Jorge, Sabrina; Mehrshahi, Payam; Magallanes-Lundback, Maria; Lipka, Alexander E; Angelovici, Ruthie; Gore, Michael A; DellaPenna, Dean

    2016-07-01

    Elucidation of the carotenoid biosynthetic pathway has enabled altering the composition and content of carotenoids in various plants, but to achieve desired nutritional impacts, the genetic components regulating carotenoid homeostasis in seed, the plant organ consumed in greatest abundance, must be elucidated. We used a combination of linkage mapping, genome-wide association studies (GWAS), and pathway-level analysis to identify nine loci that impact the natural variation of seed carotenoids in Arabidopsis (Arabidopsis thaliana). ZEAXANTHIN EPOXIDASE (ZEP) was the major contributor to carotenoid composition, with mutants lacking ZEP activity showing a remarkable 6-fold increase in total seed carotenoids relative to the wild type. Natural variation in ZEP gene expression during seed development was identified as the underlying mechanism for fine-tuning carotenoid composition, stability, and ultimately content in Arabidopsis seed. We previously showed that two CAROTENOID CLEAVAGE DIOXYGENASE enzymes, CCD1 and CCD4, are the primary mediators of seed carotenoid degradation, and here we demonstrate that ZEP acts as an upstream control point of carotenoid homeostasis, with ZEP-mediated epoxidation targeting carotenoids for degradation by CCD enzymes. Finally, four of the nine loci/enzymatic activities identified as underlying natural variation in Arabidopsis seed carotenoids also were identified in a recent GWAS of maize (Zea mays) kernel carotenoid variation. This first comparison of the natural variation in seed carotenoids in monocots and dicots suggests a surprising overlap in the genetic architecture of these traits between the two lineages and provides a list of likely candidates to target for selecting seed carotenoid variation in other species. PMID:27208224

  5. A comparative study of parthenogenetic activation and in vitro fertilization of in vitro matured caprine oocytes

    PubMed Central

    Kouamo, J; Kharche, S. D.

    2015-01-01

    The aim of the study was to compare the parthenogenetic activation and in vitro fertilization (IVF) of in vitro matured caprine oocytes. A total of 881 cumulus-oocyte complexes (COC’s) were collected from 243 ovaries. Oocytes were matured in TCM-199 medium containing eCG (20 IU/ml), hCG (20 IUµg/ml), oestradiol-17β (1 µg/ml), BSA embryo tested (3 mg/ml) supplemented with 10% fetal bovine serum at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. Based on cumulus expansion, the maturation rate was 86.86%. Morphological matured oocytes (n=749) were selected, denuded and randomly divided into two groups. Group 1 (n=223) in vitro matured oocytes activated with 5 µm calcium ionophore for 5 min and cultured in mCR2aa medium containing 5 mM DMAP for 4 h. After 4 h of DMAP treatment, the presumptive zygotes were washed and cultured in the embryo culture medium. Group 2 (n=526) in vitro matured oocytes processed for IVF in mTALP using fresh semen of a fertile pure bred adult Sirohi buck and in vitro culture in mCR2aa medium. Development of putative zygotes was observed every 24 h till day 9 post activation or fertilization under inverted phase contrast microscope. The cleavage rate, morula and blastocyst percentage in groups 1 and 2 were 67.36%, 23.07% and 9.23%, and 30.99%, 19.63% and 9.82%, respectively. The results indicated that the cleavage rate was comparatively higher following parthenogenetic activation with ionomycin/6-DMAP than IVF. PMID:27175145

  6. A comparative study of parthenogenetic activation and in vitro fertilization of in vitro matured caprine oocytes.

    PubMed

    Kouamo, J; Kharche, S D

    2015-01-01

    The aim of the study was to compare the parthenogenetic activation and in vitro fertilization (IVF) of in vitro matured caprine oocytes. A total of 881 cumulus-oocyte complexes (COC's) were collected from 243 ovaries. Oocytes were matured in TCM-199 medium containing eCG (20 IU/ml), hCG (20 IUµg/ml), oestradiol-17β (1 µg/ml), BSA embryo tested (3 mg/ml) supplemented with 10% fetal bovine serum at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. Based on cumulus expansion, the maturation rate was 86.86%. Morphological matured oocytes (n=749) were selected, denuded and randomly divided into two groups. Group 1 (n=223) in vitro matured oocytes activated with 5 µm calcium ionophore for 5 min and cultured in mCR2aa medium containing 5 mM DMAP for 4 h. After 4 h of DMAP treatment, the presumptive zygotes were washed and cultured in the embryo culture medium. Group 2 (n=526) in vitro matured oocytes processed for IVF in mTALP using fresh semen of a fertile pure bred adult Sirohi buck and in vitro culture in mCR2aa medium. Development of putative zygotes was observed every 24 h till day 9 post activation or fertilization under inverted phase contrast microscope. The cleavage rate, morula and blastocyst percentage in groups 1 and 2 were 67.36%, 23.07% and 9.23%, and 30.99%, 19.63% and 9.82%, respectively. The results indicated that the cleavage rate was comparatively higher following parthenogenetic activation with ionomycin/6-DMAP than IVF. PMID:27175145

  7. Subcellular localization of calcium and Ca-ATPase activity during nuclear maturation in Bufo arenarum oocytes.

    PubMed

    Ramos, Inés; Cisint, Susana B; Crespo, Claudia A; Medina, Marcela F; Fernández, Silvia N

    2009-08-01

    The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process. PMID:19397840

  8. Relationship Between Kernel Moisture Content and Water Activity in Different Maturity Stages of Peanut

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The water activity (aw) and moisture content (KMC) of individual peanut kernels representing five different maturity stages were measured during a period of late-season drought stress leading up to normal harvest time. Curves were generated describing the relationship between aw and KMC for yellow 1...

  9. Effects of sex pheromones and sexual maturation on locomotor activity in female sea lamprey (Petromyzon marinus).

    PubMed

    Walaszczyk, Erin J; Johnson, Nicholas S; Steibel, Juan Pedro; Li, Weiming

    2013-06-01

    Synchronization of male and female locomotor rhythmicity can play a vital role in ensuring reproductive success. Several physiological and environmental factors alter these locomotor rhythms. As sea lamprey, Petromyzon marinus, progress through their life cycle, their locomotor activity rhythm changes multiple times. The goal of this study was to elucidate the activity patterns of adult female sea lamprey during the sexual maturation process and discern the interactions of these patterns with exposure to male pheromones. During these stages, preovulated and ovulated adult females are exposed to sex pheromone compounds, which are released by spermiated males and attract ovulated females to the nest for spawning. The locomotor behavior of adult females was monitored in a natural stream with a passive integrated tag responder system as they matured, and they were exposed to a sex pheromone treatment (spermiated male washings) or a control (prespermiated male washings). Results showed that, dependent on the hour of day, male sex pheromone compounds reduce total activity (p < 0.05) and cause increases in activity during several daytime hours in preovulated and ovulated females. These results are one of the first examples of how sex pheromones modulate a locomotor rhythm in a vertebrate, and they suggest that the interaction between maturity stage and sex pheromone exposure contributes to the differential locomotor rhythms found in adult female sea lamprey. This phenomenon may contribute to the reproductive synchrony of mature adults, thus increasing reproductive success in this species. PMID:23735501

  10. Effects of sex pheromones and sexual maturation on locomotor activity in female sea lamprey (Petromyzon marinus)

    USGS Publications Warehouse

    Walaszczyk, Erin J.; Johnson, Nicholas S.; Steibel, Juan Pedro; Li, Weiming

    2013-01-01

    Synchronization of male and female locomotor rhythmicity can play a vital role in ensuring reproductive success. Several physiological and environmental factors alter these locomotor rhythms. As sea lamprey, Petromyzon marinus, progress through their life cycle, their locomotor activity rhythm changes multiple times. The goal of this study was to elucidate the activity patterns of adult female sea lamprey during the sexual maturation process and discern the interactions of these patterns with exposure to male pheromones. During these stages, preovulated and ovulated adult females are exposed to sex pheromone compounds, which are released by spermiated males and attract ovulated females to the nest for spawning. The locomotor behavior of adult females was monitored in a natural stream with a passive integrated tag responder system as they matured, and they were exposed to a sex pheromone treatment (spermiated male washings) or a control (prespermiated male washings). Results showed that, dependent on the hour of day, male sex pheromone compounds reduce total activity (p < 0.05) and cause increases in activity during several daytime hours in preovulated and ovulated females. These results are one of the first examples of how sex pheromones modulate a locomotor rhythm in a vertebrate, and they suggest that the interaction between maturity stage and sex pheromone exposure contributes to the differential locomotor rhythms found in adult female sea lamprey. This phenomenon may contribute to the reproductive synchrony of mature adults, thus increasing reproductive success in this species.

  11. The Absence of a Mature Cell Wall Sacculus in Stable Listeria monocytogenes L-Form Cells Is Independent of Peptidoglycan Synthesis

    PubMed Central

    Studer, Patrick; Borisova, Marina; Schneider, Alexander; Ayala, Juan A.; Mayer, Christoph; Schuppler, Markus; Loessner, Martin J.

    2016-01-01

    L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan. PMID:27149671

  12. The Absence of a Mature Cell Wall Sacculus in Stable Listeria monocytogenes L-Form Cells Is Independent of Peptidoglycan Synthesis.

    PubMed

    Studer, Patrick; Borisova, Marina; Schneider, Alexander; Ayala, Juan A; Mayer, Christoph; Schuppler, Markus; Loessner, Martin J; Briers, Yves

    2016-01-01

    L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan. PMID:27149671

  13. Inhibitory activity of thymol on native and mature Gardnerella vaginalis biofilms: in vitro study.

    PubMed

    Braga, Pier Carlo; Dal Sasso, Monica; Culici, Maria; Spallino, Alessandra

    2010-01-01

    Bacterial vaginosis (BV) is the most frequent diagnosis made in women with lower genital tract symptoms. It has recently been observed that 90 % of subjects with BV show the growth of bacteria in the form of biofilms as against only 10% without BV, and that Gardnerella vaginalis was the predominant species. The propensity of G. vaginalis to form biofilm is clinically relevant because this form of growth allows it to tolerate higher concentrations of certain antibiotics, thus increasing the possibilty of recurrent BV even after apparently curative therapy. The aim of this study was to investigate whether thymol (CAS 89-83-8), a molecule present in thyme essential oil, that is credited with having a series of pharmacological properties including antimicrobial and antifungal effects, can interfere with newly formed and mature G. vaginalis biofilms. The ability of G. vaginalis ATCC 49145 and two G. vaginalis strains isolated from human BV to form biofilm in flat-bottomed 96-well microtitre plates was verified, and the effects of thymol concentrations ranging from 1 to 1/16 MIC (minimum inhibitory concentration) on preformed and mature biofilms was investigated by means of spectrophotometric analysis, Nomarski interference contrast microscopy, and fluorescence microscopy with live-dead cell visualisation (SYTO 9 and propidium iodide). Native biofilm was inhibited by concentrations ranging from 1 MIC to 1/8 MIC (32.77% +/- 2.37 to 11.39% +/- 1.46), and mature biofilm was inhibited by concentrations ranging from 1 MIC to 1/4 MIC (26.18% +/- 1.36 to 13.20% +/- 1.44). Nomarski interference contrast and fluorescence microscopy visually confirmed these findings. As biofilm is a multi-factorial phenomenon, the multiple mechanisms of thymol may act on different steps in the evolution of mature biofilm. PMID:21175040

  14. Mature vs. Active Deep-Seated Landslides: A Comparison Through Two Case Histories in the Alps

    NASA Astrophysics Data System (ADS)

    Delle Piane, Luca; Perello, Paolo; Baietto, Alessandro; Giorza, Alessandra; Musso, Alessia; Gabriele, Piercarlo; Baster, Ira

    2016-06-01

    Two case histories are presented, concerning the still poorly known alpine deep-seated gravitational slope deformations (DSD) located nearby Lanzada (central Italian Alps), and Sarre (north-western Italian Alps). The Lanzada DSD is a constantly monitored, juvenile, and active phenomenon, partly affecting an existing hydropower plant. Its well-developed landforms allow a precise field characterization of the instability-affected area. The Sarre DSD is a mature, strongly remodeled phenomenon, where the only hazard factor is represented by secondary instability processes at the base of the slope. In this case, the remodeling imposed the adoption of complementary analytical techniques to support the field work. The two presented studies had to be adapted to external factors, namely (a) available information, (b) geological and geomorphological setting, and (c) final scope of the work. The Lanzada case essentially relied upon accurate field work; the Sarre case was mostly based on digital image and DTM processing. In both cases a sound field structural analysis formed the necessary background to understand the mechanisms leading to instability. A back-analysis of the differences between the study methods adopted in the two cases is finally presented, leading to suggestions for further investigations and design.

  15. Stationary phase and mature infectious forms of Legionella pneumophila produce distinct viable but non-culturable cells.

    PubMed

    Al-Bana, Badii H; Haddad, Moreen T; Garduño, Rafael A

    2014-02-01

    Legionella pneumophila is an intracellular bacterial parasite of freshwater protozoa and an accidental waterborne human pathogen. L. pneumophila is highly pleomorphic showing several forms that differentiate within its developmental cycle. In water, L. pneumophila produces viable but non-culturable cells (VBNCCs), which remain largely uncharacterized. We produced VBNCCs from two developmental forms of L. pneumophila [stationary phase forms (SPFs) and mature infectious forms (MIFs)] in two water microcosms [double-deionized (dd) and tap water] at 45°C. In contrast with SPFs, MIFs upheld a robust ultrastructure and high viability in the two water microcosms. In dd-water, MIFs and SPFs lost their culturability faster than in tap water and did not consume their poly-β-hydroxybutyrate inclusions. Resuscitation in Acanthamoeba castellani was only possible for VBNCCs produced from SPFs in tap water. Addition of salts to dd-water prolonged L. pneumophila culturability to tap water levels, suggesting that L. pneumophila requires ions to maintain its readiness to resume growth. VBNCCs resisted detergent lysis and digestion in the ciliate Tetrahymena, except for VBNCCs produced from SPFs in dd-water. L. pneumophila VBNCCs thus show distinct traits according to its originating developmental form and the surrounding water microcosm. PMID:23968544

  16. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

    PubMed

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

  17. Maternal immune activation differentially impacts mature and adult-born hippocampal neurons in male mice.

    PubMed

    Zhang, Zhi; van Praag, Henriette

    2015-03-01

    Schizophrenia is associated with deficits in the hippocampus, a brain area important for learning and memory. The dentate gyrus (DG) of the hippocampus develops both before and after birth. To study the relative contribution of mature and adult-born DG granule cells to disease etiology, we compared both cell populations in a mouse model of psychiatric illness resulting from maternal immune activation. Polyriboinosinic-polyribocytidilic acid (PolyIC, 5mg/kg) or saline was given on gestation day 15 to pregnant female C57Bl/6 mice. Male offspring (n=105), was administered systemic bromodeoxyuridine (BrdU, 50mg/kg) (n=52) or intracerebral retroviral injection into the DG (n=53), to label dividing cells at one month of age. Two months later behavioral tests were performed to evaluate disease phenotype. Immunohistochemistry and whole-cell patch clamping were used to assess morphological and physiological characteristics of DG cells. Three-month-old PolyIC exposed male offspring exhibited deficient pre-pulse inhibition, spatial maze performance and motor coordination, as well as increased depression-like behavior. Histological analysis showed reduced DG volume and parvalbumin positive interneuron number. Both mature and new hippocampal neurons showed modifications in intrinsic properties such as increased input resistance and lower current threshold, and decreased action potential number. Reduced GABAergic inhibitory transmission was observed only in mature DG neurons. Differential impairments in mature DG cells and adult-born new neurons may have implications for behavioral deficits associated with maternal immune activation. PMID:25449671

  18. Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system

    PubMed Central

    Desguin, Benoît; Goffin, Philippe; Viaene, Eric; Kleerebezem, Michiel; Martin-Diaconescu, Vlad; Maroney, Michael J; Declercq, Jean-Paul; Soumillion, Patrice; Hols, Pascal

    2014-01-01

    Racemases catalyze the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel α/β fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes. PMID:24710389

  19. The Mediating Role of Physical Self-Concept on Relations between Biological Maturity Status and Physical Activity in Adolescent Females

    ERIC Educational Resources Information Center

    Cumming, Sean P.; Standage, Martyn; Loney, Tom; Gammon, Catherine; Neville, Helen; Sherar, Lauren B.; Malina, Robert M.

    2011-01-01

    The current study examined the mediating role of physical self-concept on relations between biological maturity status and self-reported physical activity in adolescent British females. Biological maturity status, physical self-concept and physical activity were assessed in 407 female British year 7-9 pupils (M age = 13.2 years, SD = 1.0).…

  20. 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

    2016-01-01

    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. PMID:26928472

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

    PubMed Central

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

    2016-01-01

    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. PMID:26928472

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

    PubMed

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

    2016-04-12

    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

  3. Active superconducting devices formed of thin films

    DOEpatents

    Martens, Jon S.; Beyer, James B.; Nordman, James E.; Hohenwarter, Gert K. G.

    1991-05-28

    Active superconducting devices are formed of thin films of superconductor which include a main conduction channel which has an active weak link region. The weak link region is composed of an array of links of thin film superconductor spaced from one another by voids and selected in size and thickness such that magnetic flux can propagate across the weak link region when it is superconducting. Magnetic flux applied to the weak link region will propagate across the array of links causing localized loss of superconductivity in the links and changing the effective resistance across the links. The magnetic flux can be applied from a control line formed of a superconducting film deposited coplanar with the main conduction channel and weak link region on a substrate. The devices can be formed of any type to superconductor but are particularly well suited to the high temperature superconductors since the devices can be entirely formed from coplanar films with no overlying regions. The devices can be utilized for a variety of electrical components, including switching circuits, amplifiers, oscillators and modulators, and are well suited to microwave frequency applications.

  4. Impact of the Maturation of Human Primary Bone-Forming Cells on Their Behavior in Acute or Persistent Staphylococcus aureus Infection Models

    PubMed Central

    Josse, Jérôme; Guillaume, Christine; Bour, Camille; Lemaire, Flora; Mongaret, Céline; Draux, Florence; Velard, Frédéric; Gangloff, Sophie C.

    2016-01-01

    Staphylococcus aureus is one of the most frequently involved pathogens in bacterial infections such as skin abscess, pneumonia, endocarditis, osteomyelitis, and implant-associated infection. As for bone homeostasis, it is partly altered during infections by S. aureus by the induction of various responses from osteoblasts, which are the bone-forming cells responsible for extracellular matrix synthesis and its mineralization. Nevertheless, bone-forming cells are a heterogeneous population with different stages of maturation and the impact of the latter on their responses toward bacteria remains unclear. We describe the impact of S. aureus on two populations of human primary bone-forming cells (HPBCs) which have distinct maturation characteristics in both acute and persistent models of interaction. Cell maturation did not influence the internalization and survival of S. aureus inside bone-forming cells or the cell death related to the infection. By studying the expression of chemokines, cytokines, and osteoclastogenic regulators by HPBCs, we observed different profiles of chemokine expression according to the degree of cell maturation. However, there was no statistical difference in the amounts of proteins released by both populations in the presence of S. aureus compared to the non-infected counterparts. Our findings show that cell maturation does not impact the behavior of HPBCs infected with S. aureus and suggest that the role of bone-forming cells may not be pivotal for the inflammatory response in osteomyelitis. PMID:27446812

  5. Impact of the Maturation of Human Primary Bone-Forming Cells on Their Behavior in Acute or Persistent Staphylococcus aureus Infection Models.

    PubMed

    Josse, Jérôme; Guillaume, Christine; Bour, Camille; Lemaire, Flora; Mongaret, Céline; Draux, Florence; Velard, Frédéric; Gangloff, Sophie C

    2016-01-01

    Staphylococcus aureus is one of the most frequently involved pathogens in bacterial infections such as skin abscess, pneumonia, endocarditis, osteomyelitis, and implant-associated infection. As for bone homeostasis, it is partly altered during infections by S. aureus by the induction of various responses from osteoblasts, which are the bone-forming cells responsible for extracellular matrix synthesis and its mineralization. Nevertheless, bone-forming cells are a heterogeneous population with different stages of maturation and the impact of the latter on their responses toward bacteria remains unclear. We describe the impact of S. aureus on two populations of human primary bone-forming cells (HPBCs) which have distinct maturation characteristics in both acute and persistent models of interaction. Cell maturation did not influence the internalization and survival of S. aureus inside bone-forming cells or the cell death related to the infection. By studying the expression of chemokines, cytokines, and osteoclastogenic regulators by HPBCs, we observed different profiles of chemokine expression according to the degree of cell maturation. However, there was no statistical difference in the amounts of proteins released by both populations in the presence of S. aureus compared to the non-infected counterparts. Our findings show that cell maturation does not impact the behavior of HPBCs infected with S. aureus and suggest that the role of bone-forming cells may not be pivotal for the inflammatory response in osteomyelitis. PMID:27446812

  6. Structure-Activity Relationships of the Human Immunodeficiency Virus Type 1 Maturation Inhibitor PF-46396

    PubMed Central

    Murgatroyd, Christopher; Pirrie, Lisa; Tran, Fanny; Smith, Terry K.

    2016-01-01

    ABSTRACT HIV-1 maturation inhibitors are a novel class of antiretroviral compounds that consist of two structurally distinct chemical classes: betulinic acid derivatives and the pyridone-based compound PF-46396. It is currently believed that both classes act by similar modes of action to generate aberrant noninfectious particles via inhibition of CA-SP1 cleavage during Gag proteolytic processing. In this study, we utilized a series of novel analogues with decreasing similarity to PF-46396 to determine the chemical groups within PF-46396 that contribute to antiviral activity, Gag binding, and the relationship between these essential properties. A spectrum of antiviral activity (active, intermediate, and inactive) was observed across the analogue series with respect to CA-SP1 cleavage and HIV-1 (NL4-3) replication kinetics in Jurkat T cells. We demonstrate that selected inactive analogues are incorporated into wild-type (WT) immature particles and that one inactive analogue is capable of interfering with PF-46396 inhibition of CA-SP1 cleavage. Mutations that confer PF-46396 resistance can impose a defective phenotype on HIV-1 that can be rescued in a compound-dependent manner. Some inactive analogues retained the capacity to rescue PF-46396-dependent mutants (SP1-A3V, SP1-A3T, and CA-P157S), implying that they can also interact with mutant Gag. The structure-activity relationships observed in this study demonstrate that (i) the tert-butyl group is essential for antiviral activity but is not an absolute requirement for Gag binding, (ii) the trifluoromethyl group is optimal but not essential for antiviral activity, and (iii) the 2-aminoindan group is important for antiviral activity and Gag binding but is not essential, as its replacement is tolerated. IMPORTANCE Combinations of antiretroviral drugs successfully treat HIV/AIDS patients; however, drug resistance problems make the development of new mechanistic drug classes an ongoing priority. HIV-1 maturation

  7. [The effect of in-situ nerve growth factor from different biological sources on the reinitiation of mouse oocyte meiotic maturation in culture and on parthenogenetic activation].

    PubMed

    Fedorushchenko, A N; Koval', T Iu; Khamidov, D Kh

    1999-01-01

    We studied the capacity of mouse oocytes to complete meiotic maturation in vitro and form the female pronucleus upon parthenogenetic activation by cycloheximide, in response to a single injection into the mouse ovaries in situ of a purified fraction of 2.5 S NGF from mouse submaxillary glands and beta-NGF from bovine sperm. Injection of NGF from both sources at 10 ng/ml with subsequent incubation of the ovaries for 1 h increased the capacity of matured oocytes for parthenogenetic formation of the pronucleus. The frequency of pronucleus formation in both "naked oocyte" and oocytes surrounded by the cumulus cells was four times that in the control. PMID:10624718

  8. Top 10 Research Questions Related to Growth and Maturation of Relevance to Physical Activity, Performance, and Fitness

    ERIC Educational Resources Information Center

    Malina, Robert M.

    2014-01-01

    Growth, maturation, and development dominate the daily lives of children and adolescents for approximately the first 2 decades of life. Growth and maturation are biological processes, while development is largely a behavioral process. The 3 processes occur simultaneously and interact. They can be influenced by physical activity and also can…

  9. IMPORTANCE OF GLUTATHIONE IN THE ACQUISITION AND MAINTENANCE OF SPERM NUCLEAR DECONDENSING ACTIVITY IN MATURING HAMSTER OOCYTES

    EPA Science Inventory

    Sperm nuclear decondensing activity in mammalian oocytes is dependent upon the maturational state of the oocyte. It is maximal in mature, metaphase II oocytes and minimal or absent in immature germinal vesicle (GV) and fertilized pronuclear oocytes. Previous studies suggested tha...

  10. In vitro biosynthesis of iron-molybdenum cofactor and maturation of the nif-encoded apodinitrogenase. Effect of substitution for NifH with site-specifically altered forms of NifH.

    PubMed

    Rangaraj, P; Ryle, M J; Lanzilotta, W N; Ludden, P W; Shah, V K

    1999-07-01

    NifH has three different roles in the nitrogenase enzyme system. Apart from serving as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (apodinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required for the aforementioned processes have been investigated by the use of site-specifically altered forms of the enzyme. The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH to bind and not hydrolyze MgATP is required for it to function in these processes. We also present evidence that the ability of NifH to function in these processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase. Evidence toward the existence of separate, overlapping sites on NifH for each of its functions (substrate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is presented. PMID:10391920

  11. Alkyl Amine Bevirimat Derivatives Are Potent and Broadly Active HIV-1 Maturation Inhibitors

    PubMed Central

    Urano, Emiko; Ablan, Sherimay D.; Mandt, Rebecca; Pauly, Gary T.; Sigano, Dina M.; Schneider, Joel P.; Martin, David E.; Nitz, Theodore J.; Wild, Carl T.

    2015-01-01

    Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1. PMID:26482309

  12. Maturity effects on colony-forming units of Macrophomina phaseolina infection as measured using near-isogenic lines of soybeans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Charcoal rot (Macrophomina phaseolina) causes significant yield losses in soybean [Glycine max( L.) Merr.] production worldwide. The effect of maturity on the severity of charcoal rot is not well understood mainly because maturity is generally confounded with genotypic background. Therefore, the re...

  13. Phosphoinositide 3-kinase-gamma induces Xenopus oocyte maturation via lipid kinase activity.

    PubMed Central

    Hehl, S; Stoyanov, B; Oehrl, W; Schönherr, R; Wetzker, R; Heinemann, S H

    2001-01-01

    Type-I phosphoinositide 3-kinases (PI3Ks) were characterized as a group of intracellular signalling proteins expressing both protein and lipid kinase activities. Recent studies implicate PI3Ks as mediators of oocyte maturation, but the molecular mechanisms are poorly defined. Here we used the Xenopus oocyte expression system as a model to investigate a possible contribution of the gamma-isoform of PI3K (PI3Kgamma) in the different pathways leading to cell-cycle progression by monitoring the time course of germinal vesicle breakdown (GVBD). Expression of a constitutive active PI3Kgamma (PI3Kgamma-CAAX) induced GVBD and increased the levels of phosphorylated Akt/protein kinase B and mitogen-activated protein kinase (MAPK). Furthermore, PI3Kgamma-CAAX accelerated progesterone-induced GVBD, but had no effect on GVBD induced by insulin. The effects of PI3Kgamma-CAAX could be suppressed by pre-incubation of the oocytes with LY294002, PD98059 or roscovitine, inhibitors of PI3K, MEK (MAPK/extracellular-signal-regulated protein kinase kinase) and cdc2/cyclin B kinase, respectively. Mutants of PI3Kgamma-CAAX, in which either lipid kinase or both lipid and protein kinase activities were altered or eliminated, did not induce significant GVBD. Our data demonstrate that expression of PI3Kgamma in Xenopus oocytes accelerates their progesterone-induced maturation and that lipid kinase activity is required to induce this effect. PMID:11736661

  14. The mediating role of physical self-concept on relations between biological maturity status and physical activity in adolescent females.

    PubMed

    Cumming, Sean P; Standage, Martyn; Loney, Tom; Gammon, Catherine; Neville, Helen; Sherar, Lauren B; Malina, Robert M

    2011-06-01

    The current study examined the mediating role of physical self-concept on relations between biological maturity status and self-reported physical activity in adolescent British females. Biological maturity status, physical self-concept and physical activity were assessed in 407 female British year 7-9 pupils (M age = 13.2 years, SD = 1.0). Participants completed the Physical Activity Questionnaire for Adolescents (Kowalski, Crocker, & Donen, 2004) and the Children and Youth Physical Self-Perceptions Profile (Whitehead, 1995). Percentage of predicted adult height attained at measurement was used as an estimate of biological maturity status. Structural equation modelling using maximum likelihood estimation and bootstrapping procedures revealed that perceptions of sports competence, body attractiveness and physical self-worth mediated an inverse relation between maturity status and physical activity. The results provide partial support for Petersen and Taylor's (1980) Mediated Effects Model of Psychological and Behavioural Adaptation to Puberty within the context of physical activity. PMID:20655102

  15. Maturation of convulsogenic activity induced by leptazol in the albino rat

    PubMed Central

    De Casrilevitz, Mira; Engelhardt, E.; Esbérard, C. A.

    1971-01-01

    1. Maturation of the excitatory and inhibitory neuromechanisms at various levels of the central nervous system was demonstrated by the convulsogenic activity induced by leptazol in the developing albino rat. 2. The somatomotor end points considered (myoclonic jerk, myoclonic seizure, tonic seizure and catalepsy) were not observed in all age groups. Tonic seizure was seen at birth, myoclonic jerks at 2 weeks of age, myoclonic seizure and catalepsy at 3 weeks of age. 3. The convulsive sequences described presented three different patterns, defining three age groups: the infant pattern (infant group: newborn-1 week old animals); the transitional pattern (transitional group: 2 week old animals); and the adult pattern (adult group: 3 week old-adult animals). 4. Effective doses were determined for the three types of convulsive sequence: MJ50 for the myoclonic major sequence (maximal end point: myoclonic jerk), MS50 for the myoclonic major sequence (maximal end point: myoclonic seizure) and the TS50 for the myoclonic-tonic-clonic sequence (maximal end point: tonic seizure). 5. The correlation of the convulsive patterns with the dose and latency variations suggests that: (a) the neuromechanisms responsible for the tonic seizure and clonic seizure, located at brainstem and spinal cord levels, function at birth and reach maturity at 3 weeks of age; (b) the neuromechanisms responsible for the myoclonic manifestations and for catalepsy, located at the striato-thalamocortical level, start functioning at 2-3 weeks of age, indicating the later maturation of the more cephalic structures. PMID:5104003

  16. Conformational and thermal stability of mature dimeric human myeloperoxidase and a recombinant monomeric form from CHO cells.

    PubMed

    Banerjee, Srijib; Stampler, Johanna; Furtmüller, Paul G; Obinger, Christian

    2011-02-01

    Myeloperoxidase (MPO) is a lysosomal heme enzyme present in the azurophilic granules of human neutrophils and monocytes. It is a critical element of the human innate immune system by exerting antimicrobial effects. It is a disulfide bridged dimer with each monomer containing a light and a heavy polypeptide and its biosynthesis and intracellular transport includes several posttranslational processing steps. By contrast, MPO recombinantly produced in Chinese hamster ovary cell lines is monomeric, partially unprocessed and contains a N-terminal propeptide (proMPO). It mirrors a second form of MPO constitutively secreted from normal bone marrow myeloid precursors. In order to clarify the impact of posttranslational modifications on the structural integrity and enzymology of these two forms of human myeloperoxidase, we have undertaken an investigation on the conformational and thermal stability of leukocyte MPO and recombinant proMPO by using complementary biophysical techniques including UV-Vis, circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Mature leucocyte MPO exhibits a peculiar high chemical and thermal stability under oxidizing conditions but is significantly destabilized by addition of dithiothreitol. Unfolding of secondary and tertiary structure occurs concomitantly with denaturation of the heme cavity, reflecting the role of three MPO-typical heme to protein linkages and of six intra-chain disulfides for structural integrity by bridging N- and C-terminal regions of the protein. Recombinant monomeric proMPO follows a similar unfolding pattern but has a lower conformational and thermal stability. Spectroscopic and thermodynamic data of unfolding are discussed with respect to the known three-dimensional structure of leukocyte MPO as well as to known physiological roles. PMID:20933108

  17. Nascent Integrin Adhesions Form on All Matrix Rigidities after Integrin Activation.

    PubMed

    Changede, Rishita; Xu, Xiaochun; Margadant, Felix; Sheetz, Michael P

    2015-12-01

    Integrin adhesions assemble and mature in response to ligand binding and mechanical factors, but the molecular-level organization is not known. We report that ∼100-nm clusters of ∼50 β3-activated integrins form very early adhesions under a wide variety of conditions on RGD surfaces. These adhesions form similarly on fluid and rigid substrates, but most adhesions are transient on rigid substrates. Without talin or actin polymerization, few early adhesions form, but expression of either the talin head or rod domain in talin-depleted cells restores early adhesion formation. Mutation of the integrin binding site in the talin rod decreases cluster size. We suggest that the integrin clusters constitute universal early adhesions and that they are the modular units of cell matrix adhesions. They require the association of activated integrins with cytoplasmic proteins, in particular talin and actin, and cytoskeletal contraction on them causes adhesion maturation for cell motility and growth. PMID:26625956

  18. Glutathione S-transferase activity in follicular fluid from women undergoing ovarian stimulation: role in maturation.

    PubMed

    Meijide, Susana; Hernández, M Luisa; Navarro, Rosaura; Larreategui, Zaloa; Ferrando, Marcos; Ruiz-Sanz, José Ignacio; Ruiz-Larrea, M Begoña

    2014-10-01

    Female infertility involves an emotional impact for the woman, often leading to a state of anxiety and low self-esteem. The assisted reproduction techniques (ART) are used to overcome the problem of infertility. In a first step of the in vitro fertilization therapy women are subjected to an ovarian stimulation protocol to obtain mature oocytes, which will result in competent oocytes necessary for fertilization to occur. Ovarian stimulation, however, subjects the women to a high physical and psychological stress, thus being essential to improve ART and to find biomarkers of dysfunction and fertility. GSH is an important antioxidant, and is also used in detoxification reactions, catalysed by glutathione S-transferases (GST). In the present work, we have investigated the involvement of GST in follicular maturation. Patients with fertility problems and oocyte donors were recruited for the study. From each woman follicles at two stages of maturation were extracted at the preovulatory stage. Follicular fluid was separated from the oocyte by centrifugation and used as the enzyme source. GST activity was determined based on its conjugation with 3,4-dichloronitrobenzene and the assay was adapted to a 96-well microplate reader. The absorbance was represented against the incubation time and the curves were adjusted to linearity (R(2)>0.990). Results showed that in both donors and patients GST activity was significantly lower in mature oocytes compared to small ones. These results suggest that GST may play a role in the follicle maturation by detoxifying xenobiotics, thus contributing to the normal development of the oocyte. Supported by FIS/FEDER (PI11/02559), Gobierno Vasco (Dep. Educación, Universiades e Investigación, IT687-13), and UPV/EHU (CLUMBER UFI11/20 and PES13/58). The work was approved by the Ethics Committee of the UPV/EHU (CEISH/96/2011/RUIZLARREA), and performed according to the UPV/EHU and IVI-Bilbao agreement (Ref. 2012/01). PMID:26461371

  19. Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

    PubMed

    Sirohi, Sunil; Walker, Brendan M

    2015-11-01

    Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function. PMID:26257334

  20. Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms

    PubMed Central

    Ferreira, Inês Santos; Bettencourt, Ana F; Gonçalves, Lídia MD; Kasper, Stefanie; Bétrisey, Bertrand; Kikhney, Judith; Moter, Annette; Trampuz, Andrej; Almeida, António J

    2015-01-01

    The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was

  1. Activity of daptomycin- and vancomycin-loaded poly-epsilon-caprolactone microparticles against mature staphylococcal biofilms.

    PubMed

    Ferreira, Inês Santos; Bettencourt, Ana F; Gonçalves, Lídia M D; Kasper, Stefanie; Bétrisey, Bertrand; Kikhney, Judith; Moter, Annette; Trampuz, Andrej; Almeida, António J

    2015-01-01

    The aim of the present study was to develop novel daptomycin-loaded poly-epsilon-caprolactone (PCL) microparticles with enhanced antibiofilm activity against mature biofilms of clinically relevant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) and polysaccharide intercellular adhesin-positive Staphylococcus epidermidis. Daptomycin was encapsulated into PCL microparticles by a double emulsion-solvent evaporation method. For comparison purposes, formulations containing vancomycin were also prepared. Particle morphology, size distribution, encapsulation efficiency, surface charge, thermal behavior, and in vitro release were assessed. All formulations exhibited a spherical morphology, micrometer size, and negative surface charge. From a very early time stage, the released concentrations of daptomycin and vancomycin were higher than the minimal inhibitory concentration and continued so up to 72 hours. Daptomycin presented a sustained release profile with increasing concentrations of the drug being released up to 72 hours, whereas the release of vancomycin stabilized at 24 hours. The antibacterial activity of the microparticles was assessed by isothermal microcalorimetry against planktonic and sessile MRSA and S. epidermidis. Regarding planktonic bacteria, daptomycin-loaded PCL microparticles presented the highest antibacterial activity against both strains. Isothermal microcalorimetry also revealed that lower concentrations of daptomycin-loaded microparticles were required to completely inhibit the recovery of mature MRSA and S. epidermidis biofilms. Further characterization of the effect of daptomycin-loaded PCL microparticles on mature biofilms was performed by fluorescence in situ hybridization. Fluorescence in situ hybridization showed an important reduction in MRSA biofilm, whereas S. epidermidis biofilms, although inhibited, were not eradicated. In addition, an important attachment of the microparticles to MRSA and S. epidermidis biofilms was

  2. A Tec kinase BTK inhibitor ibrutinib promotes maturation and activation of dendritic cells

    PubMed Central

    Natarajan, Gayathri; Oghumu, Steve; Terrazas, Cesar; Varikuti, Sanjay; Byrd, John C.; Satoskar, Abhay R.

    2016-01-01

    ABSTRACT Ibrutinib, a BTK inhibitor, is currently used to treat various hematological malignancies. We evaluated whether ibrutinib treatment during development of murine bone marrow-derived dendritic cells (DCs) modulates their maturation and activation. Ibrutinib treatment increased the proportion of CD11c+ DCs, upregulated the expression of MHC-II and CD80 and downregulated Ly6C expression by DCs. Additionally, ibrutinib treatment led to an increase in MHC-II+, CD80+ and CCR7+ DCs but a decrease in CD86+ DCs upon LPS stimulation. LPS/ibrutinib-treated DCs displayed increased IFNβ and IL-10 synthesis and decreased IL-6, IL-12 and NO production compared to DCs stimulated with LPS alone. Finally, LPS/ibrutinib-treated DCs promoted higher rates of CD4+ T cell proliferation and cytokine production compared to LPS only stimulated DCs. Taken together, our results indicate that ibrutinib enhances the maturation and activation of DCs to promote CD4+ T cell activation which could be exploited for the development of DC-based cancer therapies. PMID:27471620

  3. Late Holocene eolian activity in the mineralogically mature Nebraska Sand Hills

    USGS Publications Warehouse

    Muhs, D.R.; Stafford, Thomas W., Jr.; Swinehart, J.B.; Cowherd, S.D.; Mahan, S.A.; Bush, C.A.; Madole, R.F.; Maat, P.B.

    1997-01-01

    The age of sand dunes in the Nebraska Sand Hills has been controversial, with some investigators suggesting a full-glacial age and others suggesting that they were last active in the late Holocene. New accelerator mass spectrometry radiocarbon ages of unaltered bison bones and organic-rich sediments suggest that eolian sand deposition occurred at least twice in the past 3000 14C yr B.P. in three widely separated localities and as many as three times in the past 800 14C yr at three other localities. These late Holocene episodes of eolian activity are probably the result of droughts more intense than the 1930s "Dust Bowl" period, based on independent Great Plains climate records from lake sediments and tree rings. However, new geochemical data indicate that the Nebraska Sand Hills are mineralogically mature. Eolian sands in Nebraska have lower K-feldspar (and K2O, Rb, and Ba) contents than most possible source sediments and lower K-feldspar contents than dunes of similar age in Colorado. The most likely explanation for mineralogical maturity is reduction of sand-sized K-feldspar to silt-sized particles via ballistic impacts due to strong winds over many cycles of eolian activity. Therefore, dunes of the Nebraska Sand Hills must have had a long history, probably extending over more than one glacial-interglacial cycle, and the potential for reactivation is high, with or without a future greenhouse warming. ?? 1997 University of Washington.

  4. A Tec kinase BTK inhibitor ibrutinib promotes maturation and activation of dendritic cells.

    PubMed

    Natarajan, Gayathri; Oghumu, Steve; Terrazas, Cesar; Varikuti, Sanjay; Byrd, John C; Satoskar, Abhay R

    2016-06-01

    Ibrutinib, a BTK inhibitor, is currently used to treat various hematological malignancies. We evaluated whether ibrutinib treatment during development of murine bone marrow-derived dendritic cells (DCs) modulates their maturation and activation. Ibrutinib treatment increased the proportion of CD11c(+) DCs, upregulated the expression of MHC-II and CD80 and downregulated Ly6C expression by DCs. Additionally, ibrutinib treatment led to an increase in MHC-II(+), CD80(+) and CCR7(+) DCs but a decrease in CD86(+) DCs upon LPS stimulation. LPS/ibrutinib-treated DCs displayed increased IFNβ and IL-10 synthesis and decreased IL-6, IL-12 and NO production compared to DCs stimulated with LPS alone. Finally, LPS/ibrutinib-treated DCs promoted higher rates of CD4(+) T cell proliferation and cytokine production compared to LPS only stimulated DCs. Taken together, our results indicate that ibrutinib enhances the maturation and activation of DCs to promote CD4(+) T cell activation which could be exploited for the development of DC-based cancer therapies. PMID:27471620

  5. The Cytochrome c Maturation Components CcmF, CcmH, and CcmI Form a Membrane-integral Multisubunit Heme Ligation Complex*

    PubMed Central

    Sanders, Carsten; Turkarslan, Serdar; Lee, Dong-Woo; Onder, Ozlem; Kranz, Robert G.; Daldal, Fevzi

    2008-01-01

    Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus. PMID:18753134

  6. Mature adipocytes in bone marrow protect myeloma cells against chemotherapy through autophagy activation.

    PubMed

    Liu, Zhiqiang; Xu, Jingda; He, Jin; Liu, Huan; Lin, Pei; Wan, Xinhai; Navone, Nora M; Tong, Qiang; Kwak, Larry W; Orlowski, Robert Z; Yang, Jing

    2015-10-27

    A major problem in patients with multiple myeloma is chemotherapy resistance, which develops in myeloma cells upon interaction with bone marrow stromal cells. However, few studies have determined the role of bone marrow adipocytes, a major component of stromal cells in the bone marrow, in myeloma chemotherapy resistance. We reveal that mature human adipocytes activate autophagy and upregulate the expression of autophagic proteins, thereby suppressing chemotherapy-induced caspase cleavage and apoptosis in myeloma cells. We found that adipocytes secreted known and novel adipokines, such as leptin and adipsin. The addition of these adipokines enhanced the expression of autophagic proteins and reduced apoptosis in myeloma cells. In vivo studies further demonstrated the importance of bone marrow-derived adipocytes in the reduced response of myeloma cells to chemotherapy. Our findings suggest that adipocytes, adipocyte-secreted adipokines, and adipocyte-activated autophagy are novel targets for combatting chemotherapy resistance and enhancing treatment efficacy in myeloma patients. PMID:26455377

  7. Mature cathepsin L is substantially active in the ionic milieu of the extracellular medium.

    PubMed

    Dehrmann, F M; Coetzer, T H; Pike, R N; Dennison, C

    1995-12-01

    The activity of cathepsin L is affected by ionic strength, resulting in the measured pH optimum being higher in acetate-4-morpholineethane sulfonic acid (MES)-Tris buffers of constant ionic strength than in phosphate buffers of constant molarity (and hence varying ionic strength). In acetate-MES-Tris and phosphate buffers of constant ionic strength across the pH range, the catalytic constant, kcat, generally peaked at ca. pH 6.5 and essentially independently of ionic strength. Km values, of ca. 5 microM, manifested a slight rising trend with increasing ionic strength, with a sharp increase to 20-25 microM, specifically at pH 6.5 and I = 0.4. At physiological ionic strengths, the specific buffer ions present affected the activity of mature cathepsin L, kcat/Km declining above pH 6.5 in phosphate buffer, but only above pH 7 in acetate-MES-Tris buffer. In Hanks' balanced salt solution, a model of the extracellular fluid, measured values at pH 7.2 were kcat, 18.9 s-1; Km, 13.5 microM; and kcat/Km, 1.4 x 10(6) M-1 s-1. The stability of cathepsin L in the physiological pH range was also differentially affected by the specific buffer ions, generally in parallel with the enzyme activity. In Hanks' balanced salt solution, mature cathepsin L was substantially active and stable, having a half-life of 179 s at pH 7.2 and 657 s at pH 6.8 (the peritumor pH). PMID:7503566

  8. Chronic alcohol consumption enhances iNKT cell maturation and activation

    SciTech Connect

    Zhang, Hui Zhang, Faya; Zhu, Zhaohui; Luong, Dung; Meadows, Gary G.

    2015-01-15

    Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1{sup −} iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1{sup +}CD44{sup hi} mature iNKT cells but does not alter the number of NK1.1{sup −} immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1{sup −} iNKT cells, especially the NK1.1{sup −}CD44{sup lo} Stage I iNKT cells. The percentage of NKG2A{sup +} iNKT cells increases in all of the tissues and organs examined; whereas CXCR3{sup +} iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response. - Highlights: • Chronic alcohol consumption increases iNKT cells in the thymus and liver • Chronic alcohol consumption enhances thymic Stage I iNKT cell proliferation • Chronic alcohol consumption enhances iNKT cell maturation in thymus and periphery • Chronic alcohol

  9. Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

    PubMed Central

    Schulz, Anna M.; Stutte, Susanne; Hogl, Sebastian; Luckashenak, Nancy; Dudziak, Diana; Leroy, Céline; Forné, Ignasi; Imhof, Axel; Müller, Stephan A.; Brakebusch, Cord H.; Lichtenthaler, Stefan F.

    2015-01-01

    Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface. PMID:26553928

  10. Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation.

    PubMed

    Di Niro, Roberto; Lee, Seung-Joo; Vander Heiden, Jason A; Elsner, Rebecca A; Trivedi, Nikita; Bannock, Jason M; Gupta, Namita T; Kleinstein, Steven H; Vigneault, Francois; Gilbert, Tamara J; Meffre, Eric; McSorley, Stephen J; Shlomchik, Mark J

    2015-07-21

    The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies. PMID:26187411

  11. Expression and functional activity of PACAP and its receptors on cumulus cells: effects on oocyte maturation.

    PubMed

    Barberi, Marzia; Di Paolo, Virginia; Latini, Stefania; Guglielmo, Maria Cristina; Cecconi, Sandra; Canipari, Rita

    2013-08-15

    Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect several parameters associated with the ovulatory process. We investigated the expression of PACAP and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and efficiently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of gonadotropin and growth factors during ovulation and fertilisation. PMID:23684890

  12. Maturation of suprathreshold auditory nerve activity involves cochlear CGRP-receptor complex formation.

    PubMed

    Dickerson, Ian M; Bussey-Gaborski, Rhiannon; Holt, Joseph C; Jordan, Paivi M; Luebke, Anne E

    2016-07-01

    In adult animals, the neuropeptide calcitonin gene-related peptide (CGRP) is contained in cochlear efferent fibers projecting out to the cochlea, and contributes to increased suprathreshold sound-evoked activity in the adult auditory nerve. Similarly, CGRP applied to the lateral-line organ (hair cell organ) increases afferent nerve activity in adult frogs (post-metamorphic day 30), yet this increase is developmentally delayed from post-metamorphic day 4-30. In this study, we discovered that there was also a developmental delay in increased suprathreshold sound-evoked activity auditory nerve between juvenile and adult mice similar to what had been observed previously in frog. Moreover, juvenile mice with a targeted deletion of the αCGRP gene [CGRP null (-/-)] did not show a similar developmental increase in nerve activity, suggesting CGRP signaling is involved. This developmental delay is not due to a delay in CGRP expression, but instead is due to a delay in receptor formation. We observed that the increase in sound-evoked nerve activity is correlated with increased formation of cochlear CGRP receptors, which require three complexed proteins (CLR, RAMP1, RCP) to be functional. CGRP receptor formation in the cochlea was incomplete at 1 month of age (juvenile), but complete by 3 months (adult), which corresponded to the onset of suprathreshold enhancement of sound-evoked activity in wild-type animals. Taken together, these data support a model for cochlear function that is enhanced by maturation of CGRP receptor complexes. PMID:27440744

  13. MULTIDRUG RESISTANT TRANSPORT ACTIVITY PROTECTS OOCYTES FROM CHEMOTHERAPEUTIC AGENTS AND CHANGES DURING OOCYTE MATURATION

    PubMed Central

    Brayboy, Lynae M.; Oulhen, Nathalie; Witmyer, Jeannine; Robins, Jared; Carson, Sandra; Wessel, Gary M.

    2013-01-01

    Objective To determine the multidrug resistant (MDR) transporter activity in oocytes and their potential role in oocyte susceptibility to chemotherapy. Design Experimental laboratory study Setting University and Academic Center for reproductive medicine. Patients/Animals Women with eggs retrieved for ICSI cycles and adult female FVBN and B6C3F1 mouse strains. Intervention Inhibition of MDR activity in oocytes. Main Outcome measure(s) Efflux activity of MDRs using quantitative fluorescent dye efflux and oocyte cell death when exposed to chemotherapy. Results Oocytes effluxed fluorescent reporters and this activity was significantly reduced in the presence of the MDR inhibitor PSC 833. GV oocytes are more efficient at efflux compared to M2 oocytes. Human oocytes exposed to cyclophosphamide and PSC 833 showed cell death using two different viability assays compared to controls and those exposed to cyclophosphamide alone. Immunoblots detected MDR-1 in all oocytes with the greatest accumulation in the GV stage. Conclusions Oocytes have a vast repertoire of active MDRs. The implications of this study are that these protective mechanisms are important during oogenesis, and these activities change with maturation, increasing susceptibility to toxicants. Future directions may exploit the up regulation of these transporters during gonadotoxic therapy. PMID:23953328

  14. Multiple active forms of thrombin. IV. Relative activities of meizothrombins

    SciTech Connect

    Doyle, M.F.; Mann, K.G. )

    1990-06-25

    The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes.

  15. Melanoma-derived factors alter the maturation and activation of differentiated tissue-resident dendritic cells.

    PubMed

    Hargadon, Kristian M; Bishop, Johnathan D; Brandt, John P; Hand, Zachary C; Ararso, Yonathan T; Forrest, Osric A

    2016-01-01

    Dendritic cells (DCs) are key regulators of host immunity that are capable of inducing either immune tolerance or activation. In addition to their well-characterized role in shaping immune responses to foreign pathogens, DCs are also known to be critical for the induction and maintenance of anti-tumor immune responses. Therefore, it is important to understand how tumors influence the function of DCs and the quality of immune responses they elicit. Although the majority of studies in this field to date have utilized either immortalized DC lines or DC populations that have been generated under artificial conditions from hematopoietic precursors in vitro, we wished to investigate how tumors impact the function of already differentiated, tissue-resident DCs. Therefore, we used both an ex vivo and in vivo model system to assess the influence of melanoma-derived factors on DC maturation and activation. In ex vivo studies with freshly isolated splenic DCs, we demonstrate that the extent to which DC maturation and activation are altered by these factors correlates with melanoma tumorigenicity, and we identify partial roles for tumor-derived transforming growth factor (TGF)β1 and vascular endothelial growth factor (VEGF)-A in the altered functionality of DCs. In vivo studies using a lung metastasis model of melanoma also demonstrate tumorigenicity-dependent alterations to the function of lung-resident DCs, and skewed production of proinflammatory cytokines and chemokines by these tumor-altered cells is associated with recruitment of an immune infiltrate that may ultimately favor tumor immune escape and outgrowth. PMID:26010746

  16. Chronic alcohol consumption enhances iNKT cell maturation and activation.

    PubMed

    Zhang, Hui; Zhang, Faya; Zhu, Zhaohui; Luong, Dung; Meadows, Gary G

    2015-01-15

    Alcohol consumption exhibits diverse effects on different types of immune cells. NKT cells are a unique T cell population and play important immunoregulatory roles in different types of immune responses. The effects of chronic alcohol consumption on NKT cells remain to be elucidated. Using a mouse model of chronic alcohol consumption, we found that alcohol increases the percentage of NKT cells, especially iNKT cells in the thymus and liver, but not in the spleen or blood. Alcohol consumption decreases the percentage of NK1.1(-) iNKT cells in the total iNKT cell population in all of the tissues and organs examined. In the thymus, alcohol consumption increases the number of NK1.1(+)CD44(hi) mature iNKT cells but does not alter the number of NK1.1(-) immature iNKT cells. A BrdU incorporation assay shows that alcohol consumption increases the proliferation of thymic NK1.1(-) iNKT cells, especially the NK1.1(-)CD44(lo) Stage I iNKT cells. The percentage of NKG2A(+) iNKT cells increases in all of the tissues and organs examined; whereas CXCR3(+) iNKT cells only increases in the thymus of alcohol-consuming mice. Chronic alcohol consumption increases the percentage of IFN-γ-producing iNKT cells and increases the blood concentration of IFN-γ and IL-12 after in vivo α-galactosylceramide (αGalCer) stimulation. Consistent with the increased cytokine production, the in vivo activation of iNKT cells also enhances the activation of dendritic cells (DC) and NK, B, and T cells in the alcohol-consuming mice. Taken together the data indicate that chronic alcohol consumption enhances iNKT cell maturation and activation, which favors the Th1 immune response. PMID:25499027

  17. Contractile activity is required for Z-disc sarcomere maturation in vivo

    PubMed Central

    Geach, Timothy J; Hirst, Elizabeth MA; Zimmerman, Lyle B

    2015-01-01

    Sarcomere structure underpins structural integrity, signaling, and force transmission in the muscle. In embryos of the frog Xenopus tropicalis, muscle contraction begins even while sarcomerogenesis is ongoing. To determine whether contractile activity plays a role in sarcomere formation in vivo, chemical tools were used to block acto-myosin contraction in embryos of the frog X. tropicalis, and Z-disc assembly was characterized in the paralyzed dicky ticker mutant. Confocal and ultrastructure analysis of paralyzed embryos showed delayed Z-disc formation and defects in thick filament organization. These results suggest a previously undescribed role for contractility in sarcomere maturation in vivo. genesis 53:299–307, 2015. © 2015 The Authors. Genesis Published by Wiley Periodicals, Inc. PMID:25845369

  18. Phosphatase activity in relation to key litter and soil properties in mature subtropical forests in China.

    PubMed

    Hou, Enqing; Chen, Chengrong; Wen, Dazhi; Liu, Xian

    2015-05-15

    Phosphatase-mediated phosphorus (P) mineralization is one of the critical processes in biogeochemical cycling of P and determines soil P availability in forest ecosystems; however, the regulation of soil phosphatase activity remains elusive. This study investigated the potential extracellular activities of acid phosphomonoesterase (AcPME) and phosphodiesterase (PDE) and how they were related to key edaphic properties in the L horizon (undecomposed litter) and F/H horizon (fermented and humified litter) and the underlying mineral soil at the 0-15cm depth in eight mature subtropical forests in China. AcPME activity decreased significantly in the order of F/H horizon>L horizon>mineral soil horizon, while the order for PDE activity was L horizon=F/H horizon>mineral soil horizon. AcPME (X axis) and PDE (Y axis) activities were positively correlated in all horizons with significantly higher slope in the L and F/H horizons than in the mineral soil horizon. Both AcPME and PDE activities were positively related to microbial biomass C, moisture content and water-holding capacity in the L horizon, and were positively related to soil C:P, N:P and C:N ratios and fine root (diameter≤2mm) biomass in the mineral soil horizon. Both enzyme activities were also interactively affected by forest and horizon, partly due to the interactive effect of forest and horizon on microbial biomass. Our results suggest that modulator(s) of the potential extracellular activity of phosphatases vary with horizon, depending on the relative C, P and water availability of the horizon. PMID:25700362

  19. Neuronal activity controls Bdnf expression via Polycomb de-repression and CREB/CBP/JMJD3 activation in mature neurons

    PubMed Central

    Palomer, Ernest; Carretero, Javier; Benvegnù, Stefano; Dotti, Carlos G.; Martin, Mauricio G.

    2016-01-01

    It has been recently described that in embryonic stem cells, the expression of some important developmentally regulated genes is repressed, but poised for fast activation under the appropriate stimuli. In this work we show that Bdnf promoters are repressed by Polycomb Complex 2 in mature hippocampal neurons, and basal expression is guaranteed by the coexistence with activating histone marks. Neuronal stimulation triggered by N-methyl-D-aspartate application induces the transcription of these promoters by H3K27Me3 demethylation and H3K27Me3 phosphorylation at Serine 28 leading to displacement of EZH2, the catalytic subunit of Polycomb Repressor Complex 2. Our data show that the fast transient expression of Bdnf promoters II and VI after neuronal stimulation is dependent on acetylation of histone H3K27 by CREB-p/CBP. Thus, regulatory mechanisms established during development seem to remain after differentiation controlling genes induced by different stimuli, as would be the case of early memory genes in mature neurons. PMID:27010597

  20. Rab3A, Rab27A, and Rab35 regulate different events during mouse oocyte meiotic maturation and activation.

    PubMed

    Wang, H H; Cui, Q; Zhang, T; Wang, Z B; Ouyang, Y C; Shen, W; Ma, J Y; Schatten, H; Sun, Q Y

    2016-06-01

    Rab family members play important roles in membrane trafficking, cell growth, and differentiation. Almost all components of the cell endomembrane system, the nucleus, and the plasma membrane are closely related to RAB proteins. In this study, we investigated the distribution and functions of three members of the Rab family, Rab3A, Rab27A, and Rab35, in mouse oocyte meiotic maturation and activation. The three Rab family members showed different localization patterns in oocytes. Microinjection of siRNA, antibody injection, or inhibitor treatment showed that (1) Rab3A regulates peripheral spindle and cortical granule (CG) migration, polarity establishment, and asymmetric division; (2) Rab27A regulates CG exocytosis following MII-stage oocyte activation; and (3) Rab35 plays an important role in spindle organization and morphology maintenance, and thus meiotic nuclear maturation. These results show that Rab proteins play important roles in mouse oocyte meiotic maturation and activation and that different members exert different distinct functions. PMID:26791531

  1. The maturation of task-set related activation supports late developmental improvements in inhibitory control

    PubMed Central

    Velanova, Katerina; Wheeler, Mark E.; Luna, Beatriz

    2009-01-01

    The ability to voluntarily inhibit a single response is evident early in development, even as the ability to maintain an inhibitory “task set” continues to improve. To date, functional neuroimaging studies have detailed developmental changes in systems supporting inhibitory control exerted at the single-trial level, but changes underlying the ability to maintain an inhibitory task set remain little understood. Here we present findings from a functional magnetic resonance imaging (fMRI) study that characterizes the development of systems supporting both transient (trial-related) and sustained (task-set-related) activation during performance of the antisaccade (AS) task—an oculomotor test of inhibitory control (Hallett, 1978). Transient activation decreased from childhood to adolescence in regions known to support inhibitory processes and oculomotor control, likely reflecting less effortful response production. In contrast, sustained activation increased to adulthood in regions implicated in control. Our results suggest that development of the ability to maintain a task set is primary to the maturation of inhibitory control, and further, that this ability is still immature in adolescence. PMID:19812330

  2. Optimizing the conditions for in vitro maturation and artificial activation of sika deer (Cervus nippon hortulorum) oocytes.

    PubMed

    Yin, Y; Tang, L; Zhang, P; Kong, D; Wang, Z; Guan, J; Song, G; Tang, B; Li, Z

    2013-02-01

    With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle-stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6-dimethylaminopurine (6-DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique. PMID:22458270

  3. Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases.

    PubMed

    Hamon, Yveline; Legowska, Monika; Hervé, Virginie; Dallet-Choisy, Sandrine; Marchand-Adam, Sylvain; Vanderlynden, Lise; Demonte, Michèle; Williams, Rich; Scott, Christopher J; Si-Tahar, Mustapha; Heuzé-Vourc'h, Nathalie; Lalmanach, Gilles; Jenne, Dieter E; Lesner, Adam; Gauthier, Francis; Korkmaz, Brice

    2016-04-15

    The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked ∼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases. PMID:26884336

  4. Activation of Protein Kinase A in Mature Osteoblasts Promotes a Major Bone Anabolic Response.

    PubMed

    Tascau, Liana; Gardner, Thomas; Anan, Hussein; Yongpravat, Charlie; Cardozo, Christopher P; Bauman, William A; Lee, Francis Y; Oh, Daniel S; Tawfeek, Hesham A

    2016-01-01

    Protein kinase A (PKA) regulates osteoblast cell function in vitro and is activated by important bone mass modulating agents. We determined whether PKA activation in osteoblasts is sufficient to mediate a bone anabolic response. Thus, a mouse model conditionally expressing a constitutively active PKA (CA-PKA) in osteoblasts (CA-PKA-OB mouse) was developed by crossing a 2.3-kb α1 (I)-collagen promoter-Cre mouse with a floxed-CA-PKA mouse. Primary osteoblasts from the CA-PKA-OB mice exhibited higher basal PKA activity than those from control mice. Microcomputed tomographic analysis revealed that CA-PKA-OB female mice had an 8.6-fold increase in femoral but only 1.16-fold increase in lumbar 5 vertebral bone volume/total volume. Femur cortical thickness and volume were also higher in the CA-PKA-OB mice. In contrast, alterations in many femoral microcomputed tomographic parameters in male CA-PKA-OB mice were modest. Interestingly, the 3-dimensional structure model index was substantially lower both in femur and lumbar 5 of male and female CA-PKA-OB mice, reflecting an increase in the plate to rod-like structure ratio. In agreement, femurs from female CA-PKA-OB mice had greater load to failure and were stiffer compared with those of control mice. Furthermore, the CA-PKA-OB mice had higher levels of serum bone turnover markers and increased osteoblast and osteoclast numbers per total tissue area compared with control animals. In summary, constitutive activation of PKA in osteoblasts is sufficient to increase bone mass and favorably modify bone architecture and improve mechanical properties. PKA activation in mature osteoblasts is, therefore, an important target for designing anabolic drugs for treating diseases with bone loss. PMID:26488807

  5. DYNAMICS OF NASCENT AND ACTIVE ZONE ULTRASTRUCTURE AS SYNAPSES ENLARGE DURING LTP IN MATURE HIPPOCAMPUS

    PubMed Central

    Bell, Maria Elizabeth; Bourne, Jennifer N.; Chirillo, Michael A.; Mendenhall, John M.; Kuwajima, Masaaki; Harris, Kristen M.

    2014-01-01

    Nascent zones and active zones are adjacent synaptic regions that share a postsynaptic density, but nascent zones lack the presynaptic vesicles found at active zones. Here dendritic spine synapses were reconstructed through serial section electron microscopy (3DEM) and EM tomography to investigate nascent zone dynamics during long-term potentiation (LTP) in mature rat hippocampus. LTP was induced with theta-burst stimulation and comparisons were made to control stimulation in the same hippocampal slices at 5 minutes, 30 minutes, and 2 hours post-induction and to perfusion-fixed hippocampus in vivo. Nascent zones were present at the edges of ~35% of synapses in perfusion-fixed hippocampus and as many as ~50% of synapses in some hippocampal slice conditions. By 5 minutes, small dense core vesicles known to transport active zone proteins moved into more presynaptic boutons. By 30 minutes, nascent zone area decreased without significant change in synapse area, suggesting that presynaptic vesicles were recruited to pre-existing nascent zones. By 2 hours, both nascent and active zones were enlarged. Immunogold labeling revealed that glutamate receptors can be found in nascent zones; however, average distances from nascent zones to docked presynaptic vesicles ranged from 170±5 nm in perfusion-fixed hippocampus to 251±4 nm at enlarged synapses by 2 hours during LTP. Prior stochastic modeling suggests that falloff in glutamate concentration reduces the probability of glutamate receptor activation from 0.4 at the center of release to 0.1 just 200 nm away. Thus, conversion of nascent zones to functional active zones likely requires the recruitment of presynaptic vesicles during LTP. PMID:25043676

  6. Comparative Studies on Bioactive Constituents in Hawk Tea Infusions with Different Maturity Degree and Their Antioxidant Activities

    PubMed Central

    Yuan, Ming; Jia, Xuejing; Ding, Chunbang; Yuan, Shu; Zhang, Zhongwei; Chen, Yanger

    2014-01-01

    Hawk tea (Litsea coreana var. lanuginose) is a very popular herbal tea in the southwest of China. According to the maturity degree of raw materials, Hawk tea can usually be divided into three types: Hawk bud tea (HB), Hawk primary leaf tea (HP), and Hawk mature leaf tea (HM). In this study, some of the bioactive constituents and antioxidant properties of the three kinds of Hawk tea infusions were comparatively investigated. The results showed that the contents of total flavonoids, vitamin C, and carbohydrates in Hawk bud tea infusion (HBI) were higher than those in Hawk primary leaf tea infusion (HPI) and Hawk mature leaf tea infusion (HMI). HPI had higher contents of total polyphenols and exhibited better DPPH radical scavenging activity and ferric reducing activity power. HBI could provide more effective protection against erythrocyte hemolysis. As age is going from bud to mature leaf, the ability to inhibit the formation of low density lipoprotein (LDL) conjugated diene and the loss of tryptophan fluorescence decreased. The bioactive constituents and antioxidant activities of Hawk tea infusions were significantly affected by the maturity degree of the raw material. PMID:25133263

  7. 76 FR 42129 - Agency Information Collection Activities: Case Submission Form, Case Assistance Form

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-18

    ... FY2010. We are requesting a two year approval for the form anticipating Government Paperwork Elimination... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY Agency Information Collection Activities: Case Submission Form, Case Assistance Form (Form...

  8. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost.

    PubMed

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun

    2016-10-01

    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems. PMID:26895274

  9. Transcriptional regulation of auxin metabolism and ethylene biosynthesis activation during apple (Malus × domestica) fruit maturation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Variation exists among apple genotypes in fruit maturation and ripening patterns that influences at13 harvest fruit firmness and postharvest storability. Based on the results from our previous large-scale 14 transcriptome profiling on apple fruit maturation and well-documented auxin-ethylene crossta...

  10. Neural network with formed dynamics of activity

    SciTech Connect

    Dunin-Barkovskii, V.L.; Osovets, N.B.

    1995-03-01

    The problem of developing a neural network with a given pattern of the state sequence is considered. A neural network structure and an algorithm, of forming its bond matrix which lead to an approximate but robust solution of the problem are proposed and discussed. Limiting characteristics of the serviceability of the proposed structure are studied. Various methods of visualizing dynamic processes in a neural network are compared. Possible applications of the results obtained for interpretation of neurophysiological data and in neuroinformatics systems are discussed.

  11. 75 FR 26782 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of an Existing Information Collection; Comment Request ACTION: 60-Day Notice of Information Collection Under Review; Form I- 864, Affidavit of...

  12. Evidence for Tectonic Activity During the Mature Harappan Civilization, 2600-1800 BCE

    NASA Astrophysics Data System (ADS)

    Grijalva, K. A.; Kovach, R. L.; Nur, A. M.

    2006-12-01

    The mature Harappan civilization located in Pakistan and India dates from 2600 to 1800 BCE. By combining seismic data, three-dimensional elastic dislocation modeling, and archaeological findings we examined the role that earthquakes played in the demise of Harappan settlements. The study focuses on three different geographical regions: Gujarat, the Sarasvati-Ghaggar-Hakra River valley, and the Makran coast of Pakistan. In Gujarat, the fluvial system of the Rann of Kachchh has undergone significant changes. The Rann of Kachchh formed as a delta for three rivers, becoming an inland sea during the time of Alexander the Great, and ultimately a salty marsh. These changes were brought about by a combination of sea level changes, the truncation of the three rivers by tectonic uplift and the deepening of the Rann by earthquake induced subsidence. Events analogous to the 1819 Allah Bund earthquake, which dammed the Puran River for seven years, would have significantly altered the water source for downstream settlements. Data from the recent 2001 Bhuj event shows that Harappan settlements would have suffered considerable shaking damage from an analogous historical event. Archaeological studies to date have found direct evidence for of at least one large earthquake at Dholavira in 2200 BCE. A number of the mature Harappan settlements are located along the dry Sarasvati-Ghaggar-Hakra river system. The decline of these sites coincides with the divergence of the Sarasvati-Ghaggar-Hakra system to the Indus and Ganga river systems. A succession of earthquakes, along with a period of aridity, likely led to the disappearance of the Sarasvati-Ghaggar-Hakra system. Although this region has not had any large earthquakes in historic times, there is archaeological evidence of two large events at the Harappan site of Kalibangan, at 2900 and 2700 BCE. Along the Makran coast two settlements, believed to have been Harappan seaports, are now located tens of kilometers inland. Changes in sea

  13. Antioxidant and Anticancer Activities of Broccoli By-Products from Different Cultivars and Maturity Stages at Harvest

    PubMed Central

    Hwang, Joon-Ho; Lim, Sang-Bin

    2015-01-01

    Many studies on broccoli have analyzed the functional components and their functionality in terms of antioxidant and anticancer activities; however, these studies have focused on the florets of different varieties. Investigation of the functionality of broccoli by-products such as leaves, stems, and leaf stems from different cultivars and harvest dates might be valuable for utilizing waste materials as useful food components. Total phenolics and sulforaphane contents, and antioxidant and anticancer activities were measured in the leaves, leaf stems, and stems of early-maturing (Kyoyoshi), middle-maturing (Myeongil 96), and late-maturing broccoli (SK3-085) at different harvest dates. Total phenolics in the leaves of Kyoyoshi were about 1.8-fold to 12.1-fold higher than those in all of the other cultivars and parts. The sulforaphane content of Kyoyoshi was 2.8-fold higher in the stems than in the florets. Antioxidant activities using 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and oxygen radical absorbance capacity were highest in Kyoyoshi, followed by Myeongil 96 and SK3-085, most notably in the leaves harvested at the immature stage. Inhibition activity of cell growth against the NCI-H1299 cell lines was highest in the leaves of all cultivars in decreasing order of florets, leaf stems, and stems. The leaves harvested in October (nonflowering stage) had the highest inhibition activity, while those harvested in January (mature broccoli) showed the lowest. The results of this study demonstrate that broccoli leaves and stems contain high levels of total phenolics, and high antioxidant and anticancer activities and can provide opportunities for early-maturing broccoli as functional fresh raw vegetables. PMID:25866744

  14. Silk microgels formed by proteolytic enzyme activity.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-09-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study. PMID:23756227

  15. 76 FR 61725 - Agency Information Collection Activities: Case Submission Form, Case Assistance Form; (Form DHS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-05

    ... Federal Register on July 18, 2011 at 76 FR 42129, for a 60-day public comment period. No comments were... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND...-7001), Online Ombudsman Form DHS-7001 AGENCY: Office of the Citizenship and Immigration...

  16. The role of early neural activity in the maturation of turtle retinal function.

    PubMed

    Sernagor, E; Mehta, V

    2001-10-01

    In the developing vertebrate retina, ganglion cells fire spontaneous bursts of action potentials long before the eye becomes exposed to sensory experience at birth. These early bursts are synchronised between neighbouring retinal ganglion cells (RGCs), yielding unique spatiotemporal patterns: 'waves' of activity sweep across large retinal areas every few minutes. Both at retinal and extraretinal levels, these embryonic retinal waves are believed to guide the wiring of the visual system using hebbian mechanisms of synaptic strengthening. In the first part of this review, we recapitulate the evidence for a role of these embryonic spontaneous bursts of activity in shaping developing complex receptive field properties of RGCs in the turtle embryonic retina. We also discuss the role of visual experience in establishing RGC visual functions, and how spontaneous activity and visual experience interact to bring developing receptive fields to maturation. We have hypothesised that the physiological changes associated with development reflect modifications in the dendritic arbours of RGCs, the anatomical substrate of their receptive fields. We demonstrate that there is a temporal correlation between the period of receptive field expansion and that of dendritic growth. Moreover, the immature spontaneous activity contributes to dendritic growth in developing RGCs. Intracellular staining of RGCs reveals, however, that immature receptive fields only rarely show direct correlation with the layout of the corresponding dendritic tree. To investigate the possibility that not only the presence of the spontaneous activity, but even the precise spatiotemporal patterns encoded in retinal waves might contribute to the refinement of retinal neural circuitry, first we must clarify the mechanisms mediating the generation and propagation of these waves across development. In the second part of this review, we present evidence that turtle retinal waves, visualised using calcium imaging

  17. Dendritic morphology, synaptic transmission, and activity of mature granule cells born following pilocarpine-induced status epilepticus in the rat

    PubMed Central

    Gao, Fei; Song, Xueying; Zhu, Dexiao; Wang, Xiaochen; Hao, Aijun; Nadler, J. Victor; Zhan, Ren-Zhi

    2015-01-01

    To understand the potential role of enhanced hippocampal neurogenesis after pilocarpine-induced status epilepticus (SE) in the development of epilepsy, we quantitatively analyzed the geometry of apical dendrites, synaptic transmission, and activation levels of normotopically distributed mature newborn granule cells in the rat. SE in male Sprague-Dawley rats (between 6 and 7 weeks old) lasting for more than 2 h was induced by an intraperitoneal injection of pilocarpine. The complexity, spine density, miniature post-synaptic currents, and activity-regulated cytoskeleton-associated protein (Arc) expression of granule cells born 5 days after SE were studied between 10 and 17 weeks after CAG-GFP retroviral vector-mediated labeling. Mature granule cells born after SE had dendritic complexity similar to that of granule cells born naturally, but with denser mushroom-like spines in dendritic segments located in the outer molecular layer. Miniature inhibitory post-synaptic currents (mIPSCs) were similar between the controls and rats subjected to SE; however, smaller miniature excitatory post-synaptic current (mEPSC) amplitude with a trend toward less frequent was found in mature granule cells born after SE. After maturation, granule cells born after SE did not show denser Arc expression in the resting condition or 2 h after being activated by pentylenetetrazol-induced transient seizure activity than vicinal GFP-unlabeled granule cells. Thus our results suggest that normotopic granule cells born after pilocarpine-induced SE are no more active when mature than age-matched, naturally born granule cells. PMID:26500490

  18. Chemical Compositions and Macrophage Activation of Polysaccharides from Leon's Mane Culinary-Medicinal Mushroom Hericium erinaceus (Higher Basidiomycetes) in Different Maturation Stages.

    PubMed

    Li, Qiao-Zhen; Wu, Di; Chen, Xia; Zhou, Shuai; Liu, Yanfang; Yang, Yan; Cui, Fengjie

    2015-01-01

    We studied the effect of the maturation stage on the chemical compositions and macrophage activation activity of polysaccharides from the culinary-medicinal mushroom Hericium erinaceus. Results showed that total polysaccharides increased, whereas protein content decreased with the maturation stage development of fruiting body. Nine polysaccharide fractions, 3 from each of the maturity stages IV (small fungal spine stage), V (mid-fungal spine stage) and VI (mature), were prepared using the gradient ethanol precipitation method. The polysaccharide fraction HP4A isolated from the maturating-stage (stage IV) fruiting body had a significant difference from the fractions HP5A (stage V) and HP6A (stage VI) in the molecular weight distribution and monosaccharide compositions. Immunostimulating tests revealed that the polysaccharide fraction HP6 isolated from the mature stage (stage VI) fruiting body presented higher macrophage activation activity. Our findings provided important information for the harvest and use of H. erinaceus with higher qualities and functional benefits. PMID:26082983

  19. Matured Hop Bittering Components Induce Thermogenesis in Brown Adipose Tissue via Sympathetic Nerve Activity

    PubMed Central

    Morimoto-Kobayashi, Yumie; Ohara, Kazuaki; Takahashi, Chika; Kitao, Sayoko; Wang, Guanying; Taniguchi, Yoshimasa; Katayama, Mikio; Nagai, Katsuya

    2015-01-01

    Obesity is the principal symptom of metabolic syndrome, which refers to a group of risk factors that increase the likelihood of atherosclerosis. In recent decades there has been a sharp rise in the incidence of obesity throughout the developed world. Iso-α-acids, the bitter compounds derived from hops in beer, have been shown to prevent diet-induced obesity by increasing lipid oxidation in the liver and inhibition of lipid absorption from the intestine. Whereas the sharp bitterness induced by effective dose of iso-α-acids precludes their acceptance as a nutrient, matured hop bittering components (MHB) appear to be more agreeable. Therefore, we tested MHB for an effect on ameliorating diet-induced body fat accumulation in rodents. MHB ingestion had a beneficial effect but, compared to iso-α-acids and despite containing structurally similar compounds, acted via different mechanisms to reduce body fat accumulation. MHB supplementation significantly reduced body weight gain, epididymal white adipose tissue weight, and plasma non-esterified free fatty acid levels in diet-induced obese mice. We also found that uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) was significantly increased in MHB-fed mice at both the mRNA and protein levels. In addition, MHB administration in rats induced the β-adrenergic signaling cascade, which is related to cAMP accumulation in BAT, suggesting that MHB could modulate sympathetic nerve activity innervating BAT (BAT-SNA). Indeed, single oral administration of MHB elevated BAT-SNA in rats, and this elevation was dissipated by subdiaphragmatic vagotomy. Single oral administration of MHB maintained BAT temperature at a significantly higher level than in control rats. Taken together, these findings indicate that MHB ameliorates diet-induced body fat accumulation, at least partly, by enhancing thermogenesis in BAT via BAT-SNA activation. Our data suggests that MHB is a useful tool for developing functional foods or

  20. Aurora-A recruitment and centrosomal maturation are regulated by a Golgi-activated pool of Src during G2

    PubMed Central

    Barretta, Maria Luisa; Spano, Daniela; D'Ambrosio, Chiara; Cervigni, Romina Ines; Scaloni, Andrea; Corda, Daniela; Colanzi, Antonino

    2016-01-01

    The Golgi apparatus is composed of stacks of cisternae laterally connected by tubules to form a ribbon-like structure. At the onset of mitosis, the Golgi ribbon is broken down into discrete stacks, which then undergo further fragmentation. This ribbon cleavage is required for G2/M transition, which thus indicates that a ‘Golgi mitotic checkpoint' couples Golgi inheritance with cell cycle transition. We previously showed that the Golgi-checkpoint regulates the centrosomal recruitment of the mitotic kinase Aurora-A; however, how the Golgi unlinking regulates this recruitment was unknown. Here we show that, in G2, Aurora-A recruitment is promoted by activated Src at the Golgi. Our data provide evidence that Src and Aurora-A interact upon Golgi ribbon fragmentation; Src phosphorylates Aurora-A at tyrosine 148 and this specific phosphorylation is required for Aurora-A localization at the centrosomes. This process, pivotal for centrosome maturation, is a fundamental prerequisite for proper spindle formation and chromosome segregation. PMID:27242098

  1. Antioxidative activity of bound-form phenolics in potato peel.

    PubMed

    Nara, Kazuhiro; Miyoshi, Takayuki; Honma, Tamaki; Koga, Hidenori

    2006-06-01

    Free and bound-form phenolics were isolated from potato (cv. Toyoshiro) flesh and peel. The free and bound-form phenolics in the peel showed high DPPH radical scavenging activity, while those in the flesh showed low activity. The total amount of chlorogenic acid and caffeic acid in the free-form phenolics from the peel was highly correlated with the DPPH radical scavenging activity. Ferulic acid was identified as the active radical scavenging compound in the bound-form phenolics from the peel. The potato peel may therefore offer an effective source of an antioxidative. PMID:16794331

  2. 76 FR 41279 - Agency Information Collection Activities; Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-13

    ... was previously published in the Federal Register on May 4, 2011, at 76 FR 25364, allowing for a 60-day... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities; Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of an Existing Information Collection;...

  3. 75 FR 51093 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-18

    ... on May 12, 2010, at 75 FR 26782, allowing for a 60-day public comment period. USCIS received 2... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of a Currently Approved Information...

  4. Peroxisome proliferator-activated receptor gamma activators affect the maturation of human monocyte-derived dendritic cells.

    PubMed

    Gosset, P; Charbonnier, A S; Delerive, P; Fontaine, J; Staels, B; Pestel, J; Tonnel, A B; Trottein, F

    2001-10-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma ), a member of the nuclear receptor superfamily, has recently been described as a modulator of macrophage functions and as an inhibitor of T cell proliferation. Here, we investigated the role of PPARgamma in dendritic cells (DC), the most potent antigen-presenting cells. We showed that PPARgamma is highly expressed in immature human monocyte-derived DC (MDDC) and that it may affect the immunostimulatory function of MDDC stimulated with lipopolysaccharide (LPS) or via CD40 ligand (CD40L). We found that the synthetic PPARgamma agonist rosiglitazone (as well as pioglitazone and troglitazone) significantly increases on LPS- and CD40L-activated MDDC, the surface expression of CD36 (by 184% and 104%, respectively) and CD86 (by 54% and 48%), whereas it reduces the synthesis of CD80 (by 42% and 42%). Moreover, activation of PPARgamma resulted in a dramatic decreased secretion of the Th1-promoting factor IL-12 in LPS- and CD40L-stimulated cells (by 47% and 62%), while the production of IL-1beta, TNF-alpha, IL-6 and IL-10 was unaffected. Finally, PPARgamma ligands down-modulate the synthesis of IFN-gamma -inducible protein-10 (recently termed as CXCL10) and RANTES (CCL5), both chemokines involved in the recruitment of Th1 lymphocytes (by 49% and 30%), but not the levels of the Th2 cell-attracting chemokines,macrophage-derived chemokine (CCL22) and thymus and activation regulated chemokine (CCL17), in mature MDDC. Taken together, our data suggest that activation of PPARgamma in human DC may have an impact in the orientation of primary and secondary immune responses by favoring type 2 responses. PMID:11592060

  5. In vivo knockdown of basal forebrain p75 neurotrophin receptor stimulates choline acetyltransferase activity in the mature hippocampus.

    PubMed

    Barrett, Graham L; Naim, Timur; Trieu, Jennifer; Huang, Mengjie

    2016-05-01

    This study seeks to determine whether knockdown of basal forebrain p75 neurotrophin receptor (p75(NTR) ) expression elicits increased hippocampal choline acetyltransferase (ChAT) activity in mature animals. Antisense (AS) oligonucleotides (oligos) targeting p75(NTR) were infused into the medial septal area of mature rats continuously for 4 weeks. In all rats, the cannula outlet was placed equidistant between the left and the right sides of the vertical diagonal band of Broca. We tested phosphorothioate (PS), morpholino (Mo), and gapmer (mixed PS/RNA) oligos. Gapmer AS infusions of 7.5 and 22 μg/day decreased septal p75(NTR) mRNA by 34% and 48%, respectively. The same infusions increased hippocampal ChAT activity by 41% and 55%. Increased hippocampal ChAT activity correlated strongly with septal p75(NTR) downregulation in individual rats. Infusions of PS and Mo AS oligos did not downregulate p75(NTR) mRNA or stimulate ChAT activity. These results demonstrate that p75(NTR) can dynamically regulate hippocampal ChAT activity in the mature CNS. They also reveal the different efficacies of three diverse AS oligo chemistries when infused intracerebrally. Among the three types, gapmer oligos worked best. PMID:26864466

  6. Pu-238 fuel form activities, January 1-31, 1983

    SciTech Connect

    Not Available

    1983-03-01

    This monthly report for /sup 238/Pu Fuel Form Activities has two main sections: SRP-PuFF facility and SRL Fuel Form Activities. The program status, budget information, and milestone schedules are discussed in each main section. The Work Breakdown Structure (WBS) for this program is shown. Only one monthly report per year is processed for EDB.

  7. Pu-238 fuel form activities, January 1-31, 1981

    SciTech Connect

    Not Available

    1981-02-01

    This monthly report for /sup 238/Pu Fuel Form Activities has two main sections: SRP-PuFF facility and SRL Fuel Form Activities. The program status, budget information, and milestone schedules are discussed in each main section. The Work Breakdown Structure (WBS) for this program is shown. Only one monthly report per year is processed for EDB.

  8. Pu-238 fuel form activities, January 1-31, 1982

    SciTech Connect

    Not Available

    1982-03-01

    This monthly report for /sup 238/Pu fuel form activities has two main sections: SRP-PuFF facility and SRL fuel form activities. The program status, budget information, and milestone schedules are discussed in each main section. The Work Breakdown Structure (WBS) for this program is shown. Only one monthly report per year is processed for EDB.

  9. Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes.

    PubMed

    Fan, Heng-Yu; Huo, Li-Jun; Meng, Xiao-Qian; Zhong, Zhi-Sheng; Hou, Yi; Chen, Da-Yuan; Sun, Qing-Yuan

    2003-11-01

    Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins

  10. Activation of polyphosphoinositide metabolism at artificial maturation of Patella vulgata oocytes.

    PubMed

    Borg, B; De Renzis, G; Payan, P; Ciapa, B

    1992-01-01

    The metabolism of polyphosphoinositides (PPI) has been investigated during the meiosis reinitiation of the oocytes of a prosobranch mollusk, the limpet Patella vulgata. Meiosis reinitiation which leads to germinal vesicle breakdown (GVBD) and metaphase-1 spindle formation was artificially induced by treating the prophase-blocked oocytes with 10 mM NH4Cl, pH 8.2. This treatment, which results in a rise in intracellular pH, triggered a general increase in polyphosphoinositide synthesis. Determinations of phosphorus content showed that maturation induced a 30 to 50% increase in both phosphatidylinositol (PI) and phosphatidylinositol-1 monophosphate (PIP) concentrations. Incorporations of 32PO4 and [3H]inositol have been measured in three classes of polyphosphoinositides: PI, PIP, and phosphatidylinositol 4,5-bisphosphate (PIP2). By comparing incorporation rates of the radiolabeled precursors into PPI before and after meiosis reinitiation, we found that artificial maturation by ammonia induced a 50-fold increase in the turnover of these lipids. No significant burst of inositol 1,4,5-trisphosphate (IP3) was observed after maturation. We suggest that modifications in PPI metabolism occurring at maturation of Patella oocytes might ensure the formation of an important stock of PPI that would be available for the profuse production of IP3, the messenger responsible for the Ca2+ signal at fertilization. PMID:1309456

  11. Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDK1 and MAP kinase during maturation and fertilization of pig oocytes.

    PubMed

    Ogushi, Sugako; Fulka, Josef; Miyano, Takashi

    2005-10-01

    In amphibian oocytes, it is known that germinal vesicle (GV) materials are essential for sperm head decondensation but not for activation of MPF (CDK1 and cyclin B). However, in large animals, the role of GV materials in maturation and fertilization is not defined. In this study, we prepared enucleated pig oocytes at the GV stage and cultured them to examine the activation and inactivation of CDK1 and MAP kinase during maturation and after electro-activation. Moreover, enucleated GV-oocytes after maturation culture were inseminated or injected intracytoplasmically with spermatozoa to examine their ability to decondense the sperm chromatin. Enucleated oocytes showed similar activation/inactivation patterns of CDK1 and MAP kinase as sham-operated oocytes during maturation and after electro-stimulation or intracytoplasmic sperm injection. During the time corresponding to MI/MII transition of sham-operated oocytes, enucleated oocytes inactivated CDK1. However, penetrating sperm heads in enucleated oocytes did not decondense enough to form male pronuclei. To determine whether the factor(s) involved in sperm head decondensation remains associated with the chromatin after GV breakdown (GVBD), we did enucleation soon after GVBD (corresponding to pro-metaphase I, pMI) to remove only chromosomes. The injected sperm heads in pMI-enucleated oocytes decondensed and formed the male pronuclei. These results suggest that in pig oocytes, GV materials are not required for activation/inactivation of CDK1 and MAP kinase, but they are essential for male pronucleus formation. PMID:16153631

  12. Ovary ecdysteroidogenic hormone activates egg maturation in the mosquito Georgecraigius atropalpus after adult eclosion or a blood meal

    PubMed Central

    Gulia-Nuss, Monika; Eum, Jai-Hoon; Strand, Michael R.; Brown, Mark R.

    2012-01-01

    SUMMARY The rockpool mosquito, Georgecraigius atropalpus, is a facultatively autogenous species that produces its first egg clutch without a blood meal shortly after emergence. Several days after depositing this clutch, females must take a blood meal to produce a second egg clutch. Decapitation of females shortly after emergence or blood ingestion prevents egg maturation. Here, we report that a single injected dose of the neuropeptide ovary ecdysteroidogenic hormone (OEH) fully restored egg maturation in decapitated females in both circumstances. This neuropeptide and two insulin-like peptides (ILPs) are potent gonadotropins in the related yellow fever mosquito, Aedes aegypti. ILP3 was marginally restorative in decapitated G. atropalpus, and ILP4 had no effect. Egg maturation in non- and blood-fed G. atropalpus was dependent on the enzymatic mobilization of amino acids from stored protein or the blood meal for yolk protein (vitellogenin, VG) synthesis and uptake by oocytes. We further show that OEH stimulates serine protease activity in the fat body of newly eclosed females or in the midgut of blood-fed ones, and ecdysteroid hormone production by the ovaries of both females. In contrast, only 20-hydroxyecdysone stimulated VG synthesis in the fat body of non- and blood-fed females. Using RNA interference to knock down expression of the insulin receptor, we found that OEH still fully restored autogenous egg maturation. In summary, our results identify OEH as a primary regulator of egg maturation in both autogenous and blood-fed G. atropalpus females and suggest the shift from blood meal-dependent to blood meal-independent release of OEH is a key factor in the evolution of autogeny in this species. PMID:22811249

  13. Treatment of ongoing autoimmune encephalomyelitis with activated B-cell progenitors maturing into regulatory B cells.

    PubMed

    Korniotis, Sarantis; Gras, Christophe; Letscher, Hélène; Montandon, Ruddy; Mégret, Jérôme; Siegert, Stefanie; Ezine, Sophie; Fallon, Padraic G; Luther, Sanjiv A; Fillatreau, Simon; Zavala, Flora

    2016-01-01

    The influence of signals perceived by immature B cells during their development in bone marrow on their subsequent functions as mature cells are poorly defined. Here, we show that bone marrow cells transiently stimulated in vivo or in vitro through the Toll-like receptor 9 generate proB cells (CpG-proBs) that interrupt experimental autoimmune encephalomyelitis (EAE) when transferred at the onset of clinical symptoms. Protection requires differentiation of CpG-proBs into mature B cells that home to reactive lymph nodes, where they trap T cells by releasing the CCR7 ligand, CCL19, and to inflamed central nervous system, where they locally limit immunopathogenesis through interleukin-10 production, thereby cooperatively inhibiting ongoing EAE. These data demonstrate that a transient inflammation at the environment, where proB cells develop, is sufficient to confer regulatory functions onto their mature B-cell progeny. In addition, these properties of CpG-proBs open interesting perspectives for cell therapy of autoimmune diseases. PMID:27396388

  14. Role of Dendritic Cell Maturity/Costimulation for Generation, Homeostasis, and Suppressive Activity of Regulatory T Cells

    PubMed Central

    Pletinckx, Katrien; Döhler, Anja; Pavlovic, Vladimir; Lutz, Manfred B.

    2011-01-01

    Tolerogenicity of dendritic cells (DCs) has initially been attributed exclusively to immature/resting stages, while mature/activated DCs were considered strictly immunogenic. Later, all different subsets among the myeloid/conventional DCs and plasmacytoid DCs have been shown to bear tolerogenic potential, so that tolerogenicity could not be attributed to a specific subset. Immunosuppressive treatments of immature DC subsets could prevent re-programming into mature DCs or upregulated inhibitory surface markers or cytokines. Furthermore, the different T cell tolerance mechanisms anergy, deletion, immune deviation, and suppression require different quantities and qualities of costimulation by DCs. Since expansion of regulatory T cells (Tregs) has been shown to be promoted best by fully mature DCs the role of CD80/B7-1 and CD86/B7-2 as major costimulatory molecules for Treg biology is under debate. In this review, we discuss the role of these and other costimulatory molecules on myeloid DCs and their ligands CD28 and CD152/CTLA-4 on Tregs for peripheral conversion from naive CD4+ T cells into the major subsets of Foxp3+ Tregs and Foxp3− IL-10+ regulatory type-1 T cells (Tr1) or Tr1-like cells and their role for peripheral maintenance in the steady state and after activation. PMID:22566829

  15. Mos/mitogen-activated protein kinase can induce early meiotic phenotypes in the absence of maturation-promoting factor: a novel system for analyzing spindle formation during meiosis I.

    PubMed Central

    Choi, T; Rulong, S; Resau, J; Fukasawa, K; Matten, W; Kuriyama, R; Mansour, S; Ahn, N; Vande Woude, G F

    1996-01-01

    Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block. Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturation-promoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturation-promoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These studies define important new functions for the Mos/MAPK cascade in mouse oocyte maturation and, under these conditions, reveal novel detail of the early stages of oocyte meiosis I. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643471

  16. Market maturity

    SciTech Connect

    Meade, B.; Bowden, S.; Ellis, M

    1995-02-01

    The power sector in the Philipines provides one of the most mature independent power markets in Asia. Over the past five years, National Power Corp. (NPC), the government owned utility, has actively invited the power sector into power generation. Distribution has remained in the hands of private and rural cooperative utilities. Private utilities have been operating as full requirements customers of NPC while the growth in capacity additions by independent power producers (IPPs) has outpaced NPC`s for the second year in a row. With a recovering economy and regulatory reform proceeding, the outlook for independent power remains strong through the end of the decade. The Philipine Congress is now reviewing draft legislation that will decentralize NPC and begin the process of privatization and market-based reforms throughout the country`s power sector.

  17. Crystal structures of the carbamoylated and cyanated forms of HypE for [NiFe] hydrogenase maturation.

    PubMed

    Tominaga, Taiga; Watanabe, Satoshi; Matsumi, Rie; Atomi, Haruyuki; Imanaka, Tadayuki; Miki, Kunio

    2013-12-17

    Hydrogenase pleiotropically acting protein (Hyp)E plays a role in biosynthesis of the cyano groups for the NiFe(CN)2CO center of [NiFe] hydrogenases by catalyzing the ATP-dependent dehydration of the carbamoylated C-terminal cysteine of HypE to thiocyanate. Although structures of HypE proteins have been determined, until now there has been no structural evidence to explain how HypE dehydrates thiocarboxamide into thiocyanate. Here, we report the crystal structures of the carbamoylated and cyanated forms of HypE from Thermococcus kodakarensis in complex with nucleotides at 1.53- and 1.64-Å resolution, respectively. Carbamoylation of the C-terminal cysteine (Cys338) of HypE by chemical modification is clearly observed in the present structures. In the presence of ATP, the thiocarboxamide of Cys338 is successfully dehydrated into the thiocyanate. In the carbamoylated state, the thiocarboxamide nitrogen atom of Cys338 is close to a conserved glutamate residue (Glu272), but the spatial position of Glu272 is less favorable for proton abstraction. On the other hand, the thiocarboxamide oxygen atom of Cys338 interacts with a conserved lysine residue (Lys134) through a water molecule. The close contact of Lys134 with an arginine residue lowers the pKa of Lys134, suggesting that Lys134 functions as a proton acceptor. These observations suggest that the dehydration of thiocarboxamide into thiocyanate is catalyzed by a two-step deprotonation process, in which Lys134 and Glu272 function as the first and second bases, respectively. PMID:24297906

  18. Form-Focused Discovery Activities in English Classes

    ERIC Educational Resources Information Center

    Ogeyik, Muhlise Cosgun

    2011-01-01

    Form-focused discovery activities allow language learners to grasp various aspects of a target language by contributing implicit knowledge by using discovered explicit knowledge. Moreover, such activities can assist learners to perceive and discover the features of their language input. In foreign language teaching environments, they can be used…

  19. Activated carbon fibers and engineered forms from renewable resources

    DOEpatents

    Baker, Frederick S

    2013-02-19

    A method of producing activated carbon fibers (ACFs) includes the steps of providing a natural carbonaceous precursor fiber material, blending the carbonaceous precursor material with a chemical activation agent to form chemical agent-impregnated precursor fibers, spinning the chemical agent-impregnated precursor material into fibers, and thermally treating the chemical agent-impregnated precursor fibers. The carbonaceous precursor material is both carbonized and activated to form ACFs in a single step. The method produces ACFs exclusive of a step to isolate an intermediate carbon fiber.

  20. Activated carbon fibers and engineered forms from renewable resources

    DOEpatents

    Baker, Frederick S.

    2010-06-01

    A method of producing activated carbon fibers (ACFs) includes the steps of providing a natural carbonaceous precursor fiber material, blending the carbonaceous precursor material with a chemical activation agent to form chemical agent-impregnated precursor fibers, spinning the chemical agent-impregnated precursor material into fibers, and thermally treating the chemical agent-impregnated precursor fibers. The carbonaceous precursor material is both carbonized and activated to form ACFs in a single step. The method produces ACFs exclusive of a step to isolate an intermediate carbon fiber.

  1. Variations in elastaselike esterase activities in human leucocytes during cell maturation.

    PubMed

    Feinstein, G; Janoff, A

    1976-05-01

    Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development. PMID:1265076

  2. High dose CD11c-driven IL15 is sufficient to drive NK cell maturation and anti-tumor activity in a trans-presentation independent manner

    PubMed Central

    Polansky, Julia K.; Bahri, Rajia; Divivier, Mylene; Duitman, Erwin H.; Vock, Christina; Goyeneche-Patino, Diego A.; Orinska, Zane; Bulfone-Paus, Silvia

    2016-01-01

    The common gamma (γc)-chain cytokine interleukin 15 (IL15) is a multifunctional immune-modulator which impacts the generation, maturation and activity of many cell types of the innate, as well as the adaptive immune system, including natural killer (NK) and CD8+ T cells. Using a new series of transgenic mice, we analyzed the in vivo potential of IL15 as an immune-regulator when available at different concentrations or delivery modes, i.e. soluble monomer or complexed to its specific receptor α (Rα)-chain. We have identified distinct effects on selected IL15-responsive populations. While CD8+ T cells required complexed forms of IL15/IL15Rα for full functionality, mature NK populations were rescued in an IL15/IL15Rα-deficient environment by high levels of CD11c-restricted IL15. These IL15-conditions were sufficient to limit tumor formation in a lung metastasis model indicating that the NK cell populations were fully functional. These data underline the potential of “free” IL15 in the absence of Rα-complex as a powerful and specific immuno-modulator, which may be beneficial where selective immune-activation is desired. PMID:26822794

  3. High dose CD11c-driven IL15 is sufficient to drive NK cell maturation and anti-tumor activity in a trans-presentation independent manner.

    PubMed

    Polansky, Julia K; Bahri, Rajia; Divivier, Mylene; Duitman, Erwin H; Vock, Christina; Goyeneche-Patino, Diego A; Orinska, Zane; Bulfone-Paus, Silvia

    2016-01-01

    The common gamma (γc)-chain cytokine interleukin 15 (IL15) is a multifunctional immune-modulator which impacts the generation, maturation and activity of many cell types of the innate, as well as the adaptive immune system, including natural killer (NK) and CD8(+) T cells. Using a new series of transgenic mice, we analyzed the in vivo potential of IL15 as an immune-regulator when available at different concentrations or delivery modes, i.e. soluble monomer or complexed to its specific receptor α (Rα)-chain. We have identified distinct effects on selected IL15-responsive populations. While CD8(+) T cells required complexed forms of IL15/IL15Rα for full functionality, mature NK populations were rescued in an IL15/IL15Rα-deficient environment by high levels of CD11c-restricted IL15. These IL15-conditions were sufficient to limit tumor formation in a lung metastasis model indicating that the NK cell populations were fully functional. These data underline the potential of "free" IL15 in the absence of Rα-complex as a powerful and specific immuno-modulator, which may be beneficial where selective immune-activation is desired. PMID:26822794

  4. Numerically bridging lamellipodial and filopodial activity during cell spreading reveals a potentially novel trigger of focal adhesion maturation.

    PubMed

    Loosli, Y; Vianay, B; Luginbuehl, R; Snedeker, J G

    2012-05-01

    We present a novel approach to modeling cell spreading, and use it to reveal a potentially central mechanism regulating focal adhesion maturation in various cell phenotypes. Actin bundles that span neighboring focal complexes at the lamellipodium-lamellum interface were assumed to be loaded by intracellular forces in proportion to bundle length. We hypothesized that the length of an actin bundle (with the corresponding accumulated force at its adhesions) may thus regulate adhesion maturation to ensure cell mechanical stability and morphological integrity. We developed a model to test this hypothesis, implementing a "top-down" approach to simplify certain cellular processes while explicitly incorporating complexity of other key subcellular mechanisms. Filopodial and lamellipodial activities were treated as modular processes with functional spatiotemporal interactions coordinated by rules regarding focal adhesion turnover and actin bundle dynamics. This theoretical framework was able to robustly predict temporal evolution of cell area and cytoskeletal organization as reported from a wide range of cell spreading experiments using micropatterned substrates. We conclude that a geometric/temporal modeling framework can capture the key functional aspects of the rapid spreading phase and resultant cytoskeletal complexity. Hence the model is used to reveal mechanistic insight into basic cell behavior essential for spreading. It demonstrates that actin bundles spanning nascent focal adhesions such that they are aligned to the leading edge may accumulate centripetal endogenous forces along their length, and could thus trigger focal adhesion maturation in a force-length dependent fashion. We suggest that this mechanism could be a central "integrating" factor that effectively coordinates force-mediated adhesion maturation at the lamellipodium-lamellum interface. PMID:22453759

  5. 9-O-Acetylation of sialomucins: a novel marker of murine CD4 T cells that is regulated during maturation and activation.

    PubMed

    Krishna, M; Varki, A

    1997-06-01

    Terminal sialic acids on cell surface glycoconjugates can carry 9-O-acetyl esters. For technical reasons, it has previously been difficult to determine their precise distribution on different cell types. Using a recombinant soluble form of the Influenza C virus hemagglutinin-esterase as a probe for 9-O-acetylated sialic acids, we demonstrate here their preferential expression on the CD4 T cell lineage in normal B10.A mouse lymphoid organs. Of total thymocytes, 8-10% carry 9-O-acetylation; the great majority of these are the more mature PNA-, HSA-, and TCRhi medullary cells. While low levels of 9-O-acetylation are seen on some CD4/CD8 double positive (DP) and CD8 single positive (SP) cells, high levels are present primarily on 80- 85% of CD4 SP cells. Correlation with CD4 and CD8 levels suggests that 9-O-acetylation appears as an early differentiation marker as cells mature from the DP to the CD4 SP phenotype. This high degree of 9-O-acetylation is also present on 90-95% of peripheral spleen and lymph node CD4 T cells. In contrast, only a small minority of CD8 T cells and B cells show such levels of 9-O-acetylation. Among mature peripheral CD4 T lymphocytes, the highly O-acetylated cells are Mel 14(hi), CD44(lo), and CD45R(exon B)hi, features typical of naive cells. Digestions with trypsin and O-sialoglycoprotease (OSGPase) and ELISA studies of lipid extracts indicate that the 9-O-acetylated sialic acids on peripheral CD4 T cells are predominantly on O-linked mucintype glycoproteins and to a lesser degree, on sialylated glycolipids (gangliosides). In contrast, sialic acids on mucin type molecules of CD8 T cells are not O-acetylated; instead these molecules mask the recognition of O-acetylated gangliosides that seem to be present at similar levels as on CD4 cells. The 9-O-acetylated gangliosides on mouse T cells are not bound by CD60 antibodies, which recognize O-acetylated gangliosides in human T cells. Tethering 9-O-acetylated mucins with the Influenza C probe with

  6. Extraction, quantification, and antioxidant activities of phenolics from pericarp and seeds of bitter melons (Momordica charantia) harvested at three maturity stages (immature, mature, and ripe).

    PubMed

    Horax, Ronny; Hettiarachchy, Navam; Chen, Pengyin

    2010-04-14

    Bitter melon (Momordica charantia) is an exotic vegetable used for consumption and medicinal purposes mainly throughout Asia. Phenolics were extracted from pericarp (fleshy portion) and seeds of bitter melons harvested at three maturation stages (immature, mature, and ripe) using ethanol and water solvent systems. Total phenolic assessment demonstrated 80% of ethanol to be the optimal solvent level to extract phenolics either from pericarp or seed. Main phenolic constituents in the extracts were catechin, gallic acid, gentisic acid, chlorogenic acid, and epicatechin. Free radical scavenging assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH) demonstrated the bitter melon extracts as slow rate free radical scavenging agents. There were low correlations between the total phenolic contents and antiradical power values of the extracts, suggesting a possible interaction among the phenolic constituents occurred. Bitter melon phenolic extracts contain natural antioxidant substances, and could be used as antioxidant agents in suitable food products. PMID:20225855

  7. Nuclei and Microtubule Asters Stimulate Maturation/M Phase Promoting Factor (Mpf) Activation in Xenopus Eggs and Egg Cytoplasmic Extracts

    PubMed Central

    Pérez-Mongiovi, Daniel; Beckhelling, Clare; Chang, Patrick; Ford, Christopher C.; Houliston, Evelyn

    2000-01-01

    Although maturation/M phase promoting factor (MPF) can activate autonomously in Xenopus egg cytoplasm, indirect evidence suggests that nuclei and centrosomes may focus activation within the cell. We have dissected the contribution of these structures to MPF activation in fertilized eggs and in egg fragments containing different combinations of nuclei, centrosomes, and microtubules by following the behavior of Cdc2 (the kinase component of MPF), the regulatory subunit cyclin B, and the activating phosphatase Cdc25. The absence of the entire nucleus–centrosome complex resulted in a marked delay in MPF activation, whereas the absence of the centrosome alone caused a lesser delay. Nocodazole treatment to depolymerize microtubules through first interphase had an effect equivalent to removing the centrosome. Furthermore, microinjection of isolated centrosomes into anucleate eggs promoted MPF activation and advanced the onset of surface contraction waves, which are close indicators of MPF activation and could be triggered by ectopic MPF injection. Finally, we were able to demonstrate stimulation of MPF activation by the nucleus–centriole complex in vitro, as low concentrations of isolated sperm nuclei advanced MPF activation in cycling cytoplasmic extracts. Together these results indicate that nuclei and microtubule asters can independently stimulate MPF activation and that they cooperate to enhance activation locally. PMID:10973988

  8. Transcriptional regulation of vascular cambium activity during the transition from juvenile to mature stages in Cunninghamia lanceolata.

    PubMed

    Xu, Huimin; Cao, Dechang; Feng, Jinling; Wu, Hongyang; Lin, Jinxing; Wang, Yanwei

    2016-08-01

    Cunninghamia lanceolata (Lamb.) Hook., an evergreen conifer distributed in southern China, has been recognized as the most commercially important timber species due to its rapid growth. However, the molecular mechanisms underlying growth alternation due to vascular cambium activity are poorly understood. Here, we used cryosectioning to isolate the vascular cambium tissue of C. lanceolata at three stages, namely, juvenile, transition and mature (3-, 13-, and 35-year-old trees respectively) for transcriptome-wide analysis. Through assembling and annotation of transcripts, 108,767 unigenes and some potential growth-regulated genes were identified. A total of 5213, 4873 and 2541 differentially expressed genes (DEGs) were identified in the three stages. DEGs related to cambial activity, cell division and cell wall modification were detected at various developmental stages of the vascular cambium. In addition, some putative genes involved in plant hormone biosynthesis were also differentially regulated. These results indicate that various cambium-related molecular activities result in alterations in the growth of C. lanceolata, particularly during the transition from juvenile to mature stages. The findings of the present study improve our understanding of cambium development and may aid in studies of the molecular mechanisms of wood production and provide fundamental insights into the establishment of the optimal rotation period for silvicultural trees. PMID:27317969

  9. A Socioanalytic Model of Maturity

    ERIC Educational Resources Information Center

    Hogan, Robert; Roberts, Brent W.

    2004-01-01

    K0 describes a point of view on maturity that departs from earlier treatments in two ways. First, it rejects the popular assumption from humanistic psychology that maturity is a function of self-actualization and stipulates that maturity is related to certain performance capacities--namely, the ability to form lasting relationships and to achieve…

  10. Cortical Thickness Maturation and Duration of Music Training: Health-Promoting Activities Shape Brain Development

    PubMed Central

    Hudziak, James J.; Albaugh, Matthew D.; Ducharme, Simon; Karama, Sherif; Spottswood, Margaret; Crehan, Eileen; Evans, Alan C.; Botteron, Kelly N.

    2014-01-01

    Objective To assess the extent to which playing a musical instrument is associated with cortical thickness development among healthy youths. Method Participants were part of the National Institutes of Health (NIH) Magnetic Resonance Imaging (MRI) Study of Normal Brain Development. This study followed a longitudinal design such that participants underwent MRI scanning and behavioral testing on up to three separate visits, occurring at 2-year intervals. MRI, IQ, and music training data were available for 232 youths (334 scans), ranging from 6–18 years of age. Cortical thickness was regressed against the number of years each youth had played a musical instrument. Next, thickness was regressed against an “Age × Years of Playing” interaction term. Age, gender, total brain volume, and scanner were controlled for in analyses. Participant ID was entered as a random effect to account for within-person dependence. False discovery rate correction was applied (p ≤ 0.05). Results There was no association between thickness and years playing a musical instrument. The “Age × Years of Playing” interaction was associated with thickness in motor, premotor, and supplementary motor cortices, as well as prefrontal and parietal cortices. Follow-up analysis revealed that musical training was associated with an increased rate of thickness maturation. Results were largely unchanged when IQ and handedness were included as covariates. Conclusion Playing a musical instrument was associated with more rapid cortical thickness maturation within areas implicated in motor planning and coordination, visuospatial ability, and emotion and impulse regulation. However, given the quasi-experimental nature of this study, we cannot rule out the influence of confounding variables. PMID:25440305

  11. Voltage-Activated Calcium Channels as Functional Markers of Mature Neurons in Human Olfactory Neuroepithelial Cells: Implications for the Study of Neurodevelopment in Neuropsychiatric Disorders

    PubMed Central

    Solís-Chagoyán, Héctor; Flores-Soto, Edgar; Reyes-García, Jorge; Valdés-Tovar, Marcela; Calixto, Eduardo; Montaño, Luis M.; Benítez-King, Gloria

    2016-01-01

    In adulthood, differentiation of precursor cells into neurons continues in several brain structures as well as in the olfactory neuroepithelium. Isolated precursors allow the study of the neurodevelopmental process in vitro. The aim of this work was to determine whether the expression of functional Voltage-Activated Ca2+ Channels (VACC) is dependent on the neurodevelopmental stage in neuronal cells obtained from the human olfactory epithelium of a single healthy donor. The presence of channel-forming proteins in Olfactory Sensory Neurons (OSN) was demonstrated by immunofluorescent labeling, and VACC functioning was assessed by microfluorometry and the patch-clamp technique. VACC were immunodetected only in OSN. Mature neurons responded to forskolin with a five-fold increase in Ca2+. By contrast, in precursor cells, a subtle response was observed. The involvement of VACC in the precursors’ response was discarded for the absence of transmembrane inward Ca2+ movement evoked by step depolarizations. Data suggest differential expression of VACC in neuronal cells depending on their developmental stage and also that the expression of these channels is acquired by OSN during maturation, to enable specialized functions such as ion movement triggered by membrane depolarization. The results support that VACC in OSN could be considered as a functional marker to study neurodevelopment. PMID:27314332

  12. Pore-forming activity of clostridial binary toxins.

    PubMed

    Knapp, O; Benz, R; Popoff, M R

    2016-03-01

    Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. PMID:26278641

  13. Seizure induces activation of multiple subtypes of neural progenitors and growth factors in hippocampus with neuronal maturation confined to dentate gyrus

    SciTech Connect

    Indulekha, Chandrasekharan L.; Sanalkumar, Rajendran; Thekkuveettil, Anoopkumar; James, Jackson

    2010-03-19

    Adult hippocampal neurogenesis is altered in response to different physiological and pathological stimuli. GFAP{sup +ve}/nestin{sup +ve} radial glial like Type-1 progenitors are considered to be the resident stem cell population in adult hippocampus. During neurogenesis these Type-1 progenitors matures to GFAP{sup -ve}/nestin{sup +ve} Type-2 progenitors and then to Type-3 neuroblasts and finally differentiates into granule cell neurons. In our study, using pilocarpine-induced seizure model, we showed that seizure initiated activation of multiple progenitors in the entire hippocampal area such as DG, CA1 and CA3. Seizure induction resulted in activation of two subtypes of Type-1 progenitors, Type-1a (GFAP{sup +ve}/nestin{sup +ve}/BrdU{sup +ve}) and Type-1b (GFAP{sup +ve}/nestin{sup +ve}/BrdU{sup -ve}). We showed that majority of Type-1b progenitors were undergoing only a transition from a state of dormancy to activated form immediately after seizures rather than proliferating, whereas Type-1a showed maximum proliferation by 3 days post-seizure induction. Type-2 (GFAP{sup -ve}/nestin{sup +ve}/BrdU{sup +ve}) progenitors were few compared to Type-1. Type-3 (DCX{sup +ve}) progenitors showed increased expression of immature neurons only in DG region by 3 days after seizure induction indicating maturation of progenitors happens only in microenvironment of DG even though progenitors are activated in CA1 and CA3 regions of hippocampus. Also parallel increase in growth factors expression after seizure induction suggests that microenvironmental niche has a profound effect on stimulation of adult neural progenitors.

  14. Granulocyte colony-stimulating factor does not enhance phagocytosis or microbicidal activity of human mature polymorphonuclear neutrophils in vitro.

    PubMed Central

    Shimono, N; Okada, K; Takeda, D; Eguchi, K; Misumi, H; Sawae, Y; Niho, Y

    1994-01-01

    The direct effects of human granulocyte colony-stimulating factor (hG-CSF) on mature polymorphonuclear neutrophils (PMNs) in vitro were studied with regard to chemotaxis, superoxide production, and phagocytosis and microbicidal activity against the following viable microorganisms: Staphylococcus aureus, serum-resistant Pseudomonas aeruginosa, and Candida albicans. Recombinant hG-CSF (rhG-CSF) acted as a chemoattractant for human PMNs in a dose-dependent manner. The chemotactic response of PMNs to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was not enhanced by rhG-CSF at any of the concentrations used. rhG-CSF did not induce the generation of superoxide by itself. However, rhG-CSF was able to prime human PMNs and to enhance O2- release stimulated by FMLP in a dose-dependent manner. rhg-CSF did not enhance phagocytosis or killing of the three species of microorganisms by normal PMNs. With PMNs obtained from patients who had hematological disorders or solid tumors, no enhancement of the microbicidal activity was observed in most cases. Microbial killing mediated by PMNs depended on the ratio of PMNs to target organisms. We concluded from these facts that the most important effect of rhG-CSF was to increase the number of the peripheral PMNs and not to enhance the functions of mature PMNs. PMID:8556501

  15. Localized epigenetic silencing of a damage-activated WNT enhancer limits regeneration in mature Drosophila imaginal discs

    PubMed Central

    Harris, Robin E; Setiawan, Linda; Saul, Josh; Hariharan, Iswar K

    2016-01-01

    Many organisms lose the capacity to regenerate damaged tissues as they mature. Damaged Drosophila imaginal discs regenerate efficiently early in the third larval instar (L3) but progressively lose this ability. This correlates with reduced damage-responsive expression of multiple genes, including the WNT genes wingless (wg) and Wnt6. We demonstrate that damage-responsive expression of both genes requires a bipartite enhancer whose activity declines during L3. Within this enhancer, a damage-responsive module stays active throughout L3, while an adjacent silencing element nucleates increasing levels of epigenetic silencing restricted to this enhancer. Cas9-mediated deletion of the silencing element alleviates WNT repression, but is, in itself, insufficient to promote regeneration. However, directing Myc expression to the blastema overcomes repression of multiple genes, including wg, and restores cellular responses necessary for regeneration. Localized epigenetic silencing of damage-responsive enhancers can therefore restrict regenerative capacity in maturing organisms without compromising gene functions regulated by developmental signals. DOI: http://dx.doi.org/10.7554/eLife.11588.001 PMID:26840050

  16. Changes in cellular structures and enzymatic activities during browning of Scots pine callus derived from mature buds.

    PubMed

    Laukkanen, Hanna; Rautiainen, Lea; Taulavuori, Erja; Hohtola, Anja

    2000-04-01

    Visible browning is a typical feature of callus cultures derived from shoot tips of mature Scots pine (Pinus sylvestris L.). Because the ability of callus to regenerate is low, we determined the effect of browning on growth and changes in cellular structure during culture. Striking alterations in cellular structure were detected by LM (light microscopy), EM (electron microscopy) and SEM (scanning electron microscopy). Accumulation of phenolic substances was shown by histochemical staining. Staining for beta-glucosidase activity of soluble proteins that had been subjected to polyacrylamide gel electrophoresis indicated lignification of cells. The measured growth rate of callus was low compared with a hypothetical growth curve. Peroxidase activity increased rapidly soon after the start of the culture period, but especially between the second and third weeks of culture. At this time, the degradation of cell membranes and browning began coincident with the loss of chlorophyll. We conclude that browning is associated with cell disorganization and eventual cell death, making tissue culture of mature pine especially difficult. PMID:12651442

  17. The Influence of Organized Physical Activity (including Gymnastics) on Young Adult Skeletal Traits: Is Maturity Phase Important?

    PubMed Central

    Bernardoni, Brittney; Scerpella, Tamara A.; Rosenbaum, Paula F.; Kanaley, Jill A.; Raab, Lindsay N.; Li, Quefeng; Wang, Sijian; Dowthwaite, Jodi N.

    2015-01-01

    We prospectively evaluated adolescent organized physical activity (PA) as a factor in adult female bone traits. Annual DXA scans accompanied semi-annual records of anthropometry, maturity and PA for 42 participants in this preliminary analysis (criteria: appropriately timed DXA scans at ~1 year pre-menarche [predictor] and ~5 years post-menarche [dependent variable]). Regression analysis evaluated total adolescent inter-scan PA and PA over 3 maturity sub-phases as predictors of young adult bone outcomes: 1) bone mineral content (BMC), geometry and strength indices at non-dominant distal radius and femoral neck; 2) sub-head BMC; 3) lumbar spine BMC. Analyses accounted for baseline gynecological age (years pre- or post-menarche), baseline bone status, adult body size and inter-scan body size change. Gymnastics training was evaluated as a potentially independent predictor, but did not improve models for any outcomes (p<0.07). Pre-menarcheal bone traits were strong predictors of most adult outcomes (semi-partial r2 = 0.21-0.59, p≤0.001). Adult 1/3 radius and sub-head BMC were predicted by both total PA and PA 1-3 years post-menarche (p<0.03). PA 3-5 years post-menarche predicted femoral narrow neck width, endosteal diameter and buckling ratio (p<0.05). Thus, participation in organized physical activity programs throughout middle and high school may reduce lifetime fracture risk in females. PMID:25386845

  18. The Influence of Organized Physical Activity (Including Gymnastics) on Young Adult Skeletal Traits: Is Maturity Phase Important?

    PubMed

    Bernardoni, Brittney; Scerpella, Tamara A; Rosenbaum, Paula F; Kanaley, Jill A; Raab, Lindsay N; Li, Quefeng; Wang, Sijian; Dowthwaite, Jodi N

    2015-05-01

    We prospectively evaluated adolescent organized physical activity (PA) as a factor in adult female bone traits. Annual DXA scans accompanied semiannual records of anthropometry, maturity, and PA for 42 participants in this preliminary analysis (criteria: appropriately timed DXA scans at ~1 year premenarche [predictor] and ~5 years postmenarche [dependent variable]). Regression analysis evaluated total adolescent interscan PA and PA over 3 maturity subphases as predictors of young adult bone outcomes: 1) bone mineral content (BMC), geometry, and strength indices at nondominant distal radius and femoral neck; 2) subhead BMC; 3) lumbar spine BMC. Analyses accounted for baseline gynecological age (years pre- or postmenarche), baseline bone status, adult body size and interscan body size change. Gymnastics training was evaluated as a potentially independent predictor, but did not improve models for any outcomes (p > .07). Premenarcheal bone traits were strong predictors of most adult outcomes (semipartial r2 = .21-0.59, p ≤ .001). Adult 1/3 radius and subhead BMC were predicted by both total PA and PA 1-3 years postmenarche (p < .03). PA 3-5 years postmenarche predicted femoral narrow neck width, endosteal diameter, and buckling ratio (p < .05). Thus, participation in organized physical activity programs throughout middle and high school may reduce lifetime fracture risk in females. PMID:25386845

  19. The Influence of 2-Year Changes in Physical Activity, Maturation, and Nutrition on Adiposity in Adolescent Youth.

    PubMed

    Benítez-Porres, Javier; Alvero-Cruz, José Ramón; Carrillo de Albornoz, Margarita; Correas-Gómez, Lorena; Barrera-Expósito, Jesús; Dorado-Guzmán, Manuel; Moore, Justin B; Carnero, Elvis A

    2016-01-01

    The aim of this longitudinal study was to explore temporal patterns of physical activity (PA) and adiposity in Spanish adolescents. Eighty healthy adolescents were followed over a 2-year period (42 girls and 38 boys). A PA score was estimated using the Physical Activity Questionnaire for Adolescents (PAQ-A). Adiposity was assessed by anthropometric measurements; body mass index (BMI) and fat mass percent (FMP) were calculated using standard equations. Sexual maturity was estimated by percentage of predicted adult stature. Dietary intake was assessed by a self-administered food-frequency questionnaire. Three assessments were performed: September 2011 (S1), 2012 (S2), and 2013 (S3). A repeated-measures ANOVA was conducted to examine temporal changes in PA and FMP and sex change in maturation categories (two factor mixed-design, 2x2x3). A stepwise linear regression was conducted in order to estimate the predictors of FMP change. Significant changes for FMP were found between S1, S2, and S3 (23.41±8.24 vs. 21.89±7.82 vs. 22.05±8.06, p<0.05; respectively); a significant interaction of FMP with sex was observed (F = 4.387, p<0.05 for S2-S3), but not for maturation. PA at S2 was significantly higher than S3 (2.58±0.72 vs. 2.29±0.73, p<0.001). An interaction between PA change and sex was statically significant (F = 4.889, p<0.05 for S2-S3). A reduction in PA was observed after the S2 period without changes in adiposity. In contrast, a significant reduction in FMP was seen between S1 and S2, while PA did not significantly change. There were no significant differences for nutritional variables between S1 and S3, and nutrition was not a determinant of the changes in PA or FMP. Our results suggest that body composition changes observed during adolescence are not driven by changes in PA. Moreover, the interaction analysis suggests that PA behavior is affected by sex, but is not modified by maturation. PMID:27607063

  20. Ongoing intrinsic synchronous activity is required for the functional maturation of CA3-CA1 glutamatergic synapses.

    PubMed

    Huupponen, Johanna; Molchanova, Svetlana M; Lauri, Sari E; Taira, Tomi

    2013-11-01

    Fine-tuning of synaptic connectivity during development is guided by intrinsic activity of the immature networks characteristically consisting of intermittent bursts of synchronous activity. However, the role of synchronous versus asynchronous activity in synapse maturation in the brain is unclear. Here, we have pharmacologically prevented generation of synchronous activity in the immature rat CA3-CA1 circuitry in a manner that preserves unitary activity. Long-term desynchronization of the network resulted in weakening of AMPA-receptor-mediated glutamatergic transmission in CA1 pyramidal cells. This weakening was dependent on protein phosphatases and mGluR activity, associated with an increase in the proportion of silent synapses and a decrease in the protein levels of GluA4 suggesting postsynaptic mechanisms of expression. The findings demonstrate that synchronous activity in the immature CA3-CA1 circuitry is critical for the induction and maintenance of glutamatergic synapses and underscores the importance of temporal activity patterns in shaping the synaptic circuitry during development. PMID:22941723

  1. Relationships of putative endocrine disruptors to human sexual maturation and thyroid activity in youth.

    PubMed

    Schell, Lawrence M; Gallo, Mia V

    2010-02-01

    Endocrine disruption has become a significant human health concern, but is difficult to study outside of the laboratory for several reasons including the multiplicity of exposures, the difficulty in assessing each exposure, and the variety of possible outcomes among human populations. This review summarizes our studies of the relationships of measured persistent organic pollutants (PCBs, p,p'-DDE, HCB and mirex), and heavy metals (lead and mercury), to outcomes directly related to thyroid function and sexual maturation. These studies were conducted in a sample of Native American youth from the Akwesasne Mohawk community. The participants were first studied during puberty (10-16.9 years of age) and then at approximately 18 years of age. Results from these studies show that PCB levels are positively related to TSH and negatively to free T4. Further, these effects are conditioned by breastfeeding history. Anti-thyroid peroxidase antibody levels also are related to PCB levels suggesting elevated risk of autoimmune disease among the exposed. Earlier age at menarche is associated with higher PCB levels while risk of delay is associated with higher lead levels. Some evidence that the timing of exposure produces different effects is presented, and the level of exposure in the participants suggests that effects observed may be relevant to a considerable proportion of the US population. Further investigations are warranted to determine effect thresholds and mechanisms. PMID:19800354

  2. Maturation processing and characterization of streptopain.

    PubMed

    Chen, Chiu-Yueh; Luo, Shih-Chi; Kuo, Chih-Feng; Lin, Yee-Shin; Wu, Jiunn-Jong; Lin, Ming T; Liu, Ching-Chuan; Jeng, Wen-Yih; Chuang, Woei-Jer

    2003-05-01

    Streptopain is a cysteine protease expressed by Streptococcus pyogenes. To study the maturation mechanism of streptopain, wild-type and Q186N, C192S, H340R, N356D and W357A mutant proteins were expressed in Escherichia coli and purified to homogeneity. Proteolytic analyses showed that the maturation of prostreptococcal pyrogenic exotoxin B zymogen (pro-SPE B) involves eight intermediates with a combination of cis- and trans-processing. Based on the sequences of these intermediates, the substrate specificity of streptopain favors a hydrophobic residue at the P2 site. The relative autocatalytic rates of these mutants exhibited the order Q186N > W357A > N356D, C192S, H340R. Interestingly, the N356D mutant containing protease activity could not be converted into the 28-kDa form by autoprocessing. This observation suggested that Asn(356) might involve the cis-processing of the propeptide. In addition, the maturation rates of pro-SPE B with trypsin and plasmin were 10- and 60-fold slower than that with active mature streptopain. These findings indicate that active mature streptopain likely plays the most important role in the maturation of pro-SPE B under physiological conditions. PMID:12621045

  3. Active suppression of intestinal CD4(+)TCRαβ(+) T-lymphocyte maturation during the postnatal period.

    PubMed

    Torow, Natalia; Yu, Kai; Hassani, Kasra; Freitag, Jenny; Schulz, Olga; Basic, Marijana; Brennecke, Anne; Sparwasser, Tim; Wagner, Norbert; Bleich, André; Lochner, Matthias; Weiss, Siegfried; Förster, Reinhold; Pabst, Oliver; Hornef, Mathias W

    2015-01-01

    Priming of the mucosal immune system during the postnatal period substantially influences host-microbial interaction and susceptibility to immune-mediated diseases in adult life. The underlying mechanisms are ill defined. Here we show that shortly after birth, CD4 T cells populate preformed lymphoid structures in the small intestine and quickly acquire a distinct transcriptional profile. T-cell recruitment is independent of microbial colonization and innate or adaptive immune stimulation but requires β7 integrin expression. Surprisingly, neonatal CD4 T cells remain immature throughout the postnatal period under homeostatic conditions but undergo maturation and gain effector function on barrier disruption. Maternal SIgA and regulatory T cells act in concert to prevent immune stimulation and maintain the immature phenotype of CD4 T cells in the postnatal intestine during homeostasis. Active suppression of CD4 T-cell maturation during the postnatal period might contribute to prevent auto-reactivity, sustain a broad TCR repertoire and establish life-long immune homeostasis. PMID:26195040

  4. The durably resistant rice cultivar Digu activates defence gene expression before the full maturation of Magnaporthe oryzae appressorium.

    PubMed

    Li, Weitao; Liu, Ya; Wang, Jing; He, Min; Zhou, Xiaogang; Yang, Chao; Yuan, Can; Wang, Jichun; Chern, Mawsheng; Yin, Junjie; Chen, Weilan; Ma, Bingtian; Wang, Yuping; Qin, Peng; Li, Shigui; Ronald, Pamela; Chen, Xuewei

    2016-04-01

    Rice blast caused by the fungal pathogen Magnaporthe oryzae is one of the most destructive diseases worldwide. Although the rice-M. oryzae interaction has been studied extensively, the early molecular events that occur in rice before full maturation of the appressorium during M. oryzae invasion are unknown. Here, we report a comparative transcriptomics analysis of the durably resistant rice variety Digu and the susceptible rice variety Lijiangxintuanheigu (LTH) in response to infection by M. oryzae (5, 10 and 20 h post-inoculation, prior to full development of the appressorium). We found that the transcriptional responses differed significantly between these two rice varieties. Gene ontology and pathway analyses revealed that many biological processes, including extracellular recognition and biosynthesis of antioxidants, terpenes and hormones, were specifically activated in Digu shortly after infection. Forty-eight genes encoding receptor kinases (RKs) were significantly differentially regulated by M. oryzae infection in Digu. One of these genes, LOC_Os08g10300, encoding a leucine-rich repeat RK from the LRR VIII-2 subfamily, conferred enhanced resistance to M. oryzae when overexpressed in rice. Our study reveals that a multitude of molecular events occur in the durably resistant rice Digu before the full maturation of the appressorium after M. oryzae infection and that membrane-associated RKs play important roles in the early response. PMID:26095454

  5. Characterization of the common acute lymphoblastic leukaemia antigen (CD10) as an activation molecule on mature human B cells.

    PubMed Central

    Kiyokawa, N; Kokai, Y; Ishimoto, K; Fujita, H; Fujimoto, J; Hata, J I

    1990-01-01

    Distinct expression pattern of CD10 molecules during B cell activation was analysed using in vivo and in vitro systems. By two-colour flowcytometrical analysis, CD10 was found to be expressed at a specific stage of in vivo activating B cells. The expression of CD10 during B cell activation appeared to be unique from that of other activation-related B cell antigens including L29, MA6, OKT9 and OKT10. Although the expression of CD10 was associated with that of the activation-related B cell antigens, CD10+ B cells could be separated in the distinct fractions to those expressing other activation-related B cell antigens when fractionated by cell gravity. In particular, certain CD10+ B cells were detected positive for the resting B cell antigen, L30. In vitro studies revealed that CD10+ B cells arose from CD10- B cells at an early step of B cell activation, and disappeared lately when activated by Staphylococcus aureus Cowan I. Collectively, CD10 was an antigen transiently expressed at an early phase of B cell activation process. Expression of CD10 and other antigens on Burkitt's lymphomas (15 cases) was studied next. All cases were CD10+, and 87% (13 cases) were also L30+. In addition, six of CD10+ L30+ cases were L29+. This observation suggested that Burkitt's lymphomas were phenotypically similar to the B cells at an early phase of activation, those expressing CD10 and L30, simultaneously. The present study has dissected a precise expression pattern of CD10 on mature B cell activation in vitro and in vivo, and could be implicated for the histogenesis of one of the poorly characterized B cell lymphoma, namely Burkitt's lymphoma. PMID:2138527

  6. Time to burn (calories)? The impact of retirement on physical activity among mature Americans.

    PubMed

    Kämpfen, Fabrice; Maurer, Jürgen

    2016-01-01

    Physical activity is crucial for maintaining and improving health, especially at advanced ages. While retirement increases the amount of time available for physical activity, there is only limited evidence regarding the causal effect of retirement on recommended levels of physical activity. Addressing this gap in the literature, we use data from the U.S. Health and Retirement Study to estimate the causal impact of retirement on meeting the federal government's 2008 Physical Activity Guidelines for Americans. Using official early and normal retirement ages as instruments for retirement, our causal IV analyses suggest significant positive effects of retirement on meeting the Guidelines. These effects are robust with regard to the treatment of unobserved individual-specific heterogeneity, the measurement of guideline compliance, the definition of retirement and respondents' health insurance status. We also show that the effects of retirement on physical activity are larger for persons with higher levels of education and wealth. PMID:26773282

  7. The Prodomain-bound Form of Bone Morphogenetic Protein 10 Is Biologically Active on Endothelial Cells*

    PubMed Central

    Jiang, He; Salmon, Richard M.; Upton, Paul D.; Wei, Zhenquan; Lawera, Aleksandra; Davenport, Anthony P.; Morrell, Nicholas W.; Li, Wei

    2016-01-01

    BMP10 is highly expressed in the developing heart and plays essential roles in cardiogenesis. BMP10 deletion in mice results in embryonic lethality because of impaired cardiac development. In adults, BMP10 expression is restricted to the right atrium, though ventricular hypertrophy is accompanied by increased BMP10 expression in a rat hypertension model. However, reports of BMP10 activity in the circulation are inconclusive. In particular, it is not known whether in vivo secreted BMP10 is active or whether additional factors are required to achieve its bioactivity. It has been shown that high-affinity binding of the BMP10 prodomain to the mature ligand inhibits BMP10 signaling activity in C2C12 cells, and it was proposed that prodomain-bound BMP10 (pBMP10) complex is latent. In this study, we demonstrated that the BMP10 prodomain did not inhibit BMP10 signaling activity in multiple endothelial cells, and that recombinant human pBMP10 complex, expressed in mammalian cells and purified under native conditions, was fully active. In addition, both BMP10 in human plasma and BMP10 secreted from the mouse right atrium were fully active. Finally, we confirmed that active BMP10 secreted from mouse right atrium was in the prodomain-bound form. Our data suggest that circulating BMP10 in adults is fully active and that the reported vascular quiescence function of BMP10 in vivo is due to the direct activity of pBMP10 and does not require an additional activation step. Moreover, being an active ligand, recombinant pBMP10 may have therapeutic potential as an endothelial-selective BMP ligand, in conditions characterized by loss of BMP9/10 signaling. PMID:26631724

  8. Antioxidant activity in mature branches of ponderosa pine (Pinus ponderosa) under long-term, low concentration ozone exposure

    SciTech Connect

    Benes, S.E.; Murphy, T.M.; Laeuchli, A. ); Anderson, P.D.; Houpis, J.L.J. )

    1990-05-01

    Antioxidant activity (superoxide dismutase, peroxidases and glutathione) is being examined in mature needle tissue of ponderosa pine exposed to elevated levels of ozone (O{sub 3}). Trees used in this study are 8-14 year-old clones produced from buds from a 70 year-old tree grafted onto seedling rootstock. Trees are exposed to O{sub 3} using a newly developed branch exposure chamber (BEC). Ozone treatments are charcoal-filtered, ambient and 2x ambient concentrations. A non-chambered branch will determine the effect of exposure chamber. Superoxide dismutase (SOD) activity measured in current-year needles in February via nitrobluetetrazolium (NBT) reduction was 138.5 {plus minus} 15 (SD) units mg{sup {minus}1} protein. The activity of guaiacol-oxidizing peroxidases was 89 {plus minus} 19 (SD) {Delta}A{sub 470} min{sup {minus}1} mg{sup {minus}1} protein. Ascorbate peroxidase and the ratio of oxidized to reduced glutathione (GSSG/GSH) are also being monitored. Antioxidant activity will be measured monthly across the ozone season (March to November) and during natural ozone episodes. Cellular antioxidant activity will be related to needle photosynthesis, respiration and stomatal conductance measured using a Licor 6200 portable photosynthesis apparatus.

  9. cdc25 is one of the MPM-2 antigens involved in the activation of maturation-promoting factor.

    PubMed Central

    Kuang, J; Ashorn, C L; Gonzalez-Kuyvenhoven, M; Penkala, J E

    1994-01-01

    MPM-2 antigens, a discrete set of phosphoproteins that contain similar phosphoepitopes recognized by the monoclonal antibody MPM-2, are phosphorylated during M-phase induction. Our previous studies suggested that certain MPM-2 antigens are involved in the appearance of maturation-promoting factor (MPF) activity. Because the central mitotic regulator cdc2 kinase has been shown to exhibit MPF activity, we explored the possibility that certain MPM-2 antigens are regulators of cdc2 kinase. We found that MPM-2 binding of its antigens would inhibit the autoamplification of cdc2 kinase in Xenopus oocytes and interfere with cyclin-activation of cdc2 kinase in Xenopus interphase egg extract. Immunodepletion of MPM-2 antigens from cyclin-induced M-phase egg extract caused the inactivation of cdc2 kinase, which was accompanied by an inhibitory phosphorylation of p34cdc2 on Thr 14 and Tyr 15, indicating that at least one MPM-2 antigen is a positive regulator of p34cdc2 dephosphorylation. We then showed that cdc25 from M-phase arrested egg extract is an MPM-2 antigen. These results suggest that phosphorylation of the epitope recognized by MPM-2 may be a crucial event in the activation of cdc25 and that the kinase(s) that phosphorylates this MPM-2 epitope may be an important regulator of cdc2 kinase activation. Images PMID:8019000

  10. Cell-to-Cell Transfer of HIV-1 via Virological Synapses Leads to Endosomal Virion Maturation that Activates Viral Membrane Fusion

    PubMed Central

    Dale, Benjamin M.; McNerney, Gregory P.; Thompson, Deanna L.; Hubner, Wolfgang; de los Reyes, Kevin; Chuang, Frank Y.S.; Huser, Thomas; Chen, Benjamin K.

    2012-01-01

    SUMMARY HIV-1 can infect T cells by cell-free virus or by direct virion transfer between cells through cell contact-induced structures called virological synapses (VS). During VS-mediated infection, virions accumulate within target cell endosomes. We show that after crossing the VS, the transferred virus undergoes both maturation and viral membrane fusion. Following VS transfer, viral membrane fusion occurs with delayed kinetics and transferred virions display reduced sensitivity to patient antisera compared to mature, cell-free virus. Furthermore, particle fusion requires that the transferred virions undergo proteolytic maturation within acceptor cell endosomes, which occurs over several hours. Rapid, live cell confocal microscopy demonstrated that viral fusion can occur in compartments that have moved away from the VS. Thus, HIV particle maturation activates viral fusion in target CD4+ T cell endosomes following transfer across the VS and may represent a pathway by which HIV evades antibody neutralization. PMID:22177560

  11. Activated and unactivated forms of human erythrocyte aldose reductase.

    PubMed Central

    Srivastava, S K; Hair, G A; Das, B

    1985-01-01

    Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates. PMID:3933003

  12. Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation.

    PubMed

    Carlson, Karen-Sue B; Nguyen, Lan; Schwartz, Kat; Lawrence, Daniel A; Schwartz, Bradford S

    2016-01-01

    Tissue-type plasminogen activator (t-PA), initially characterized for its critical role in fibrinolysis, also has key functions in both physiologic and pathologic processes in the CNS. Neuroserpin (NSP) is a t-PA specific serine protease inhibitor (serpin) found almost exclusively in the CNS that regulates t-PA's proteolytic activity and protects against t-PA mediated seizure propagation and blood-brain barrier disruption. This report demonstrates that NSP inhibition of t-PA varies profoundly as a function of pH within the biologically relevant pH range for the CNS, and reflects the stability, rather than the formation of NSP: t-PA acyl-enzyme complexes. Moreover, NSP differentiates between the zymogen-like single chain form (single chain t-PA, sct-PA) and the mature protease form (two chain t-PA, tct-PA) of t-PA, demonstrating different pH profiles for protease inhibition, different pH ranges over which catalytic deacylation occurs, and different pH dependent profiles of deacylation rates for each form of t-PA. NSP's pH dependent inhibition of t-PA is not accounted for by differential acylation, and is specific for the NSP-t-PA serpin-protease pair. These results demonstrate a novel mechanism for the differential regulation of the two forms of t-PA in the CNS, and suggest a potential specific regulatory role for CNS pH in controlling t-PA proteolytic activity. PMID:27378851

  13. Neuroserpin Differentiates Between Forms of Tissue Type Plasminogen Activator via pH Dependent Deacylation

    PubMed Central

    Carlson, Karen-Sue B.; Nguyen, Lan; Schwartz, Kat; Lawrence, Daniel A.; Schwartz, Bradford S.

    2016-01-01

    Tissue-type plasminogen activator (t-PA), initially characterized for its critical role in fibrinolysis, also has key functions in both physiologic and pathologic processes in the CNS. Neuroserpin (NSP) is a t-PA specific serine protease inhibitor (serpin) found almost exclusively in the CNS that regulates t-PA’s proteolytic activity and protects against t-PA mediated seizure propagation and blood–brain barrier disruption. This report demonstrates that NSP inhibition of t-PA varies profoundly as a function of pH within the biologically relevant pH range for the CNS, and reflects the stability, rather than the formation of NSP: t-PA acyl-enzyme complexes. Moreover, NSP differentiates between the zymogen-like single chain form (single chain t-PA, sct-PA) and the mature protease form (two chain t-PA, tct-PA) of t-PA, demonstrating different pH profiles for protease inhibition, different pH ranges over which catalytic deacylation occurs, and different pH dependent profiles of deacylation rates for each form of t-PA. NSP’s pH dependent inhibition of t-PA is not accounted for by differential acylation, and is specific for the NSP-t-PA serpin-protease pair. These results demonstrate a novel mechanism for the differential regulation of the two forms of t-PA in the CNS, and suggest a potential specific regulatory role for CNS pH in controlling t-PA proteolytic activity. PMID:27378851

  14. Mothers' Knowledge of Early Adolescents' Activities following the Middle School Transition and Pubertal Maturation

    ERIC Educational Resources Information Center

    Laird, Robert D.; Marrero, Matthew D.

    2011-01-01

    This study tested a sequential mediation model to determine whether experiences, social cognitions, or parent-adolescent interactional processes account for lower levels of mothers' knowledge of adolescents' whereabouts and activities following early adolescents' transition into middle school (MS) and pubertal development. Cross-sectional data…

  15. Cambial activity related to tree size in a mature silver-fir plantation

    PubMed Central

    Rathgeber, Cyrille B. K.; Rossi, Sergio; Bontemps, Jean-Daniel

    2011-01-01

    Background and Aims Our knowledge about the influences of environmental factors on tree growth is principally based on the study of dominant trees. However, tree social status may influence intra-annual dynamics of growth, leading to differential responses to environmental conditions. The aim was to determine whether within-stand differences in stem diameters of trees belonging to different crown classes resulted from variations in the length of the growing period or in the rate of cell production. Methods Cambial activity was monitored weekly in 2006 for three crown classes in a 40-year-old silver-fir (Abies alba) plantation near Nancy (France). Timings, duration and rate of tracheid production were assessed from anatomical observations of the developing xylem. Key Results Cambial activity started earlier, stopped later and lasted longer in dominant trees than in intermediate and suppressed ones. The onset of cambial activity was estimated to have taken 3 weeks to spread to 90 % of the trees in the stand, while the cessation needed 6 weeks. Cambial activity was more intense in dominant trees than in intermediate and suppressed ones. It was estimated that about 75 % of tree-ring width variability was attributable to the rate of cell production and only 25 % to its duration. Moreover, growth duration was correlated to tree height, while growth rate was better correlated to crown area. Conclusions These results show that, in a closed conifer forest, stem diameter variations resulted principally from differences in the rate of xylem cell production rather than in its duration. Tree size interacts with environmental factors to control the timings, duration and rate of cambial activity through functional processes involving source–sink relationships principally, but also hormonal controls. PMID:21816842

  16. A Longitudinal Examination of the Influence of Maturation on Physical Self-Perceptions and the Relationship with Physical Activity in Early Adolescent Girls

    ERIC Educational Resources Information Center

    Knowles, Ann-Marie; Niven, Ailsa G.; Fawkner, Samantha G.; Henretty, Joan M.

    2009-01-01

    This longitudinal study investigated the influence of maturation on physical self-perceptions and the relationship with physical activity in early adolescent girls (N = 150; mean age = 12.79 plus or minus 0.31). Physical characteristics were measured and participants completed the Physical Activity Questionnaire for Children, the Children and…

  17. Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

    PubMed Central

    Miinalainen, Ilkka J.; Hiltunen, J. Kalervo; Michels, Paul A. M.; Antonenkov, Vasily D.

    2012-01-01

    Background Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (PK+/PCl−∼0.31), while the other two types of channels are slightly selective for cations (PK+/PCl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. Conclusions/Significance These results indicate that the membrane of glycosomes apparently

  18. Viral and host control of cytomegalovirus maturation

    PubMed Central

    Tandon, Ritesh; Mocarski, Edward S.

    2012-01-01

    Maturation in herpesviruses initiates in the nucleus of the infected cell with encapsidation of viral DNA to form nucleocapsids and concludes with envelopment in the cytoplasm to form infectious virions that egress the cell. The entire process of virus maturation is orchestrated by protein-protein interactions and enzymatic activities of viral and host origin. Viral tegument proteins play important roles in maintaining the structural stability of capsids and directing the acquisition of virus envelope. Envelopment occurs at modified host membranes and exploits host vesicular trafficking. In this review, we summarize the current knowledge and concepts in human cytomegalovirus (HCMV) maturation and their parallels in other herpesviruses with an emphasis on viral and host factors regulating this process. PMID:22633075

  19. Thylakoid Membrane Maturation and PSII Activation Are Linked in Greening Synechocystis sp. PCC 6803 Cells1

    PubMed Central

    Barthel, Sandra; Bernát, Gábor; Seidel, Tobias; Rupprecht, Eva; Kahmann, Uwe; Schneider, Dirk

    2013-01-01

    Thylakoid membranes are typical and essential features of both chloroplasts and cyanobacteria. While they are crucial for phototrophic growth of cyanobacterial cells, biogenesis of thylakoid membranes is not well understood yet. Dark-grown Synechocystis sp. PCC 6803 cells contain only rudimentary thylakoid membranes but still a relatively high amount of phycobilisomes, inactive photosystem II and active photosystem I centers. After shifting dark-grown Synechocystis sp. PCC 6803 cells into the light, “greening” of Synechocystis sp. PCC 6803 cells, i.e. thylakoid membrane formation and recovery of photosynthetic electron transport reactions, was monitored. Complete restoration of a typical thylakoid membrane system was observed within 24 hours after an initial lag phase of 6 to 8 hours. Furthermore, activation of photosystem II complexes and restoration of a functional photosynthetic electron transport chain appears to be linked to the biogenesis of organized thylakoid membrane pairs. PMID:23922268

  20. Effect of biomass concentration on methane oxidation activity using mature compost and graphite granules as substrata.

    PubMed

    Xie, S; O'Dwyer, T; Freguia, S; Pikaar, I; Clarke, W P

    2016-10-01

    Reported methane oxidation activity (MOA) varies widely for common landfill cover materials. Variation is expected due to differences in surface area, the composition of the substratum and culturing conditions. MOA per methanotrophic cell has been calculated in the study of natural systems such as lake sediments to examine the inherent conditions for methanotrophic activity. In this study, biomass normalised MOA (i.e., MOA per methanotophic cell) was measured on stabilised compost, a commonly used cover in landfills, and on graphite granules, an inert substratum widely used in microbial electrosynthesis studies. After initially enriching methanotrophs on both substrata, biomass normalised MOA was quantified under excess oxygen and limiting methane conditions in 160ml serum vials on both substrata and blends of the substrata. Biomass concentration was measured using the bicinchoninic acid assay for microbial protein. The biomass normalised MOA was consistent across all compost-to-graphite granules blends, but varied with time, reflecting the growth phase of the microorganisms. The biomass normalised MOA ranged from 0.069±0.006μmol CH4/mg dry biomass/h during active growth, to 0.024±0.001μmol CH4/mg dry biomass/h for established biofilms regardless of the substrata employed, indicating the substrata were equally effective in terms of inherent composition. The correlation of MOA with biomass is consistent with studies on methanotrophic activity in natural systems, but biomass normalised MOA varies by over 5 orders of magnitude between studies. This is partially due to different methods being used to quantify biomass, such as pmoA gene quantification and the culture dependent Most Probable Number method, but also indicates that long term exposure of materials to a supply of methane in an aerobic environment, as can occur in natural systems, leads to the enrichment and adaptation of types suitable for those conditions. PMID:27515185

  1. Synchrony between fruit maturation and effective dispersers' foraging activity increases seed protection against seed predators

    PubMed Central

    Boulay, Raphaël; Carro, Francisco; Soriguer, Ramón C; Cerdá, Xim

    2007-01-01

    The evolution of pollination and seed dispersal mutualisms is conditioned by the spatial and temporal co-occurrence of animals and plants. In the present study we explore the timing of seed release of a myrmecochorous plant (Helleborus foetidus) and ant activity in two populations in southern Spain during 2 consecutive years. The results indicate that fruit dehiscence and seed shedding occur mostly in the morning and correspond to the period of maximum foraging activity of the most effective ant dispersers. By contrast, ant species that do not transport seeds and/or that do not abound near the plants are active either before or after H. foetidus diaspores are released. Experimental analysis of diet preference for three kinds of food shows that effective ant dispersers are mostly scavengers that readily feed on insect corpses and sugars. Artificial seed depots suggest that seeds deposited on the ground out of the natural daily time window of diaspore releasing are not removed by ants and suffer strong predation by nocturnal rodents Apodemus sylvaticus. Nevertheless, important inter-annual variations in rodent populations cast doubts on their real importance as selection agents. We argue that traits allowing synchrony between seed presentation and effective partners may constitute a crucial pre-adaptation for the evolution of plant–animal mutualisms involving numerous animal partners. PMID:17698486

  2. NUCLEAR ACTIVITY IS MORE PREVALENT IN STAR-FORMING GALAXIES

    SciTech Connect

    Rosario, D. J.; Lutz, D.; Berta, S.; Popesso, P.; Genzel, R.; Saintonge, A.; Tacconi, L.; Wuyts, S. E-mail: lutz@mpe.mpg.de E-mail: popesso@mpe.mpg.de E-mail: amelie@mpe.mpg.de E-mail: swuyts@mpe.mpg.de; and others

    2013-07-01

    We explore the question of whether low and moderate luminosity active galactic nuclei (AGNs) are preferentially found in galaxies that are undergoing a transition from active star formation (SF) to quiescence. This notion has been suggested by studies of the UV-optical colors of AGN hosts, which find them to be common among galaxies in the so-called Green Valley, a region of galaxy color space believed to be composed mostly of galaxies undergoing SF quenching. Combining the deepest current X-ray and Herschel/PACS far-infrared (FIR) observations of the two Chandra Deep Fields with redshifts, stellar masses, and rest-frame photometry derived from the extensive and uniform multi-wavelength data in these fields, we compare the rest-frame U - V color distributions and star formation rate distributions of AGNs and carefully constructed samples of inactive control galaxies. The UV-to-optical colors of AGNs are consistent with equally massive inactive galaxies at redshifts out to z {approx} 2, but we show that such colors are poor tracers of SF. While the FIR distributions of both star-forming AGNs and star-forming inactive galaxies are statistically similar, we show that AGNs are preferentially found in star-forming host galaxies, or, in other words, AGNs are less likely to be found in weakly star-forming or quenched galaxies. We postulate that, among X-ray-selected AGNs of low and moderate accretion luminosities, the supply of cold gas primarily determines the accretion rate distribution of the nuclear black holes.

  3. Antimicrobial activity of crude epicarp and seed extracts from mature avocado fruit (Persea americana) of three cultivars.

    PubMed

    Raymond Chia, Teck Wah; Dykes, Gary A

    2010-07-01

    The epicarp and seed of Persea Americana Mill. var. Hass (Lauraceae), Persea Americana Mill. var. Shepard, and Persea americana Mill. var Fuerte cultivars of mature avocados (n = 3) were ground separately and extracted with both absolute ethanol and distilled water. Extracts were analyzed for antimicrobial activity using the microtiter broth microdilution assay against four Gram-positive bacteria, six Gram-negative bacteria, and one yeast. Antimicrobial activity against two molds was determined by the hole plate method. The ethanol extracts showed antimicrobial activity (104.2-416.7 microg/mL) toward both Gram-positive and Gram-negative bacteria (except Escherichia coli), while inhibition of the water extracts was only observed for Listeria monocytogenes (93.8-375.0 microg/mL) and Staphylococcus epidermidis (354.2 microg/mL). The minimum concentration required to inhibit Zygosaccharomyces bailii was 500 microg/mL for the ethanol extracts, while no inhibition was observed for the water extracts. No inhibition by either ethanol or water extracts was observed against Penicillium spp. and Aspergillus flavus. PMID:20645772

  4. HIV-1-derived lentiviral vectors directly activate plasmacytoid dendritic cells, which in turn induce the maturation of myeloid dendritic cells.

    PubMed

    Rossetti, Maura; Gregori, Silvia; Hauben, Ehud; Brown, Brian D; Sergi, Lucia Sergi; Naldini, Luigi; Roncarolo, Maria-Grazia

    2011-02-01

    Lentiviral vectors (LV) can induce type I interferon (IFN I) production from murine plasmacytoid dendritic cells (pDC), but not myeloid (my)DC. Here, we investigated whether this mechanism is conserved in human DC. MyDC and pDC were isolated from peripheral blood and transduced with increasing vector concentrations. Compared with in vitro differentiated monocyte-derived DC, the transduction efficiency of peripheral blood DC was low (ranging from <1% to 45%), with pDC showing the lowest susceptibility to LV transduction. Phenotype and function of myDC were not directly modified by LV transduction; by contrast, pDC produced significant levels of IFN-α and tumor necrosis factor-α. pDC activation was dependent on functional vector particles and was mediated by Toll-like receptor 7/9 triggering. Coculture of myDC with pDC in the presence of LV resulted in myDC activation, with CD86 up-regulation and interleukin-6 secretion. These findings demonstrate that the induction of transgene-specific immunity is triggered by an innate immune response with pDC activation and consequent myDC maturation, a response that closely resembles the one induced by functional viruses. This information is important to design strategies aimed at using LV in humans for gene therapy, where adverse immune responses must be avoided, or for cancer immunotherapy, where inducing immunity is the goal. PMID:20825284

  5. 76 FR 25364 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and From I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-04

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and From I-864W; Extension of an Existing Information Collection; Comment Request. ACTION: 60-Day Notice of Information Collection Under Review; Form I- 864, Affidavit of...

  6. ZEAXANTHIN EPOXIDASE Activity Potentiates Carotenoid Degradation in Maturing Seed1[OPEN

    PubMed Central

    Magallanes-Lundback, Maria; Lipka, Alexander E.; Angelovici, Ruthie; DellaPenna, Dean

    2016-01-01

    Elucidation of the carotenoid biosynthetic pathway has enabled altering the composition and content of carotenoids in various plants, but to achieve desired nutritional impacts, the genetic components regulating carotenoid homeostasis in seed, the plant organ consumed in greatest abundance, must be elucidated. We used a combination of linkage mapping, genome-wide association studies (GWAS), and pathway-level analysis to identify nine loci that impact the natural variation of seed carotenoids in Arabidopsis (Arabidopsis thaliana). ZEAXANTHIN EPOXIDASE (ZEP) was the major contributor to carotenoid composition, with mutants lacking ZEP activity showing a remarkable 6-fold increase in total seed carotenoids relative to the wild type. Natural variation in ZEP gene expression during seed development was identified as the underlying mechanism for fine-tuning carotenoid composition, stability, and ultimately content in Arabidopsis seed. We previously showed that two CAROTENOID CLEAVAGE DIOXYGENASE enzymes, CCD1 and CCD4, are the primary mediators of seed carotenoid degradation, and here we demonstrate that ZEP acts as an upstream control point of carotenoid homeostasis, with ZEP-mediated epoxidation targeting carotenoids for degradation by CCD enzymes. Finally, four of the nine loci/enzymatic activities identified as underlying natural variation in Arabidopsis seed carotenoids also were identified in a recent GWAS of maize (Zea mays) kernel carotenoid variation. This first comparison of the natural variation in seed carotenoids in monocots and dicots suggests a surprising overlap in the genetic architecture of these traits between the two lineages and provides a list of likely candidates to target for selecting seed carotenoid variation in other species. PMID:27208224

  7. Active curcumin nanoparticles formed from a volatile microemulsion template.

    PubMed

    Margulis, K; Srinivasan, S; Ware, M J; Summers, H D; Godin, B; Magdassi, S

    2014-01-01

    We report on biological performance of organic nanoparticles formed by a simple method based on rapid solvent removal from a volatile microemulsion. The particular focus of the study was on testing the suitability of the method for substances soluble in partially water-miscible organic solvents as well as on evaluating the therapeutic activity of the resultant nanoparticles. Curcumin was employed as a model for hydrophobic drug, and, as it is soluble in water-miscible organic solvents, it was successfully incorporated into a new cyclopentanone-water microemulsion system. During rapid solvent removal by spray-drying, the nanometric droplets of the microemulsion were converted into nanoparticles containing amorphous curcumin with the average size of 20.2±3.4 nm, having ζ potential of -36.2 ±1.8 mV. These nanoparticles were dispersible in water and retained the high loading of the active substance. The therapeutic activity of the resulting nanoparticles was demonstrated in a pancreatic cancer cell line Panc-1. The effective concentration for reducing the metabolic activity was found to be 11.5 μM for nanoparticles compared with 19.5 μM for free curcumin. PMID:25485110

  8. The regulator of MAT2 (ROM2) protein binds to early maturation promoters and represses PvALF-activated transcription.

    PubMed Central

    Chern, M S; Bobb, A J; Bustos, M M

    1996-01-01

    The regulation of maturation (MAT)- and late embryogenesis (LEA)-specific gene expression in dicots involves factors related to ABI3, a seed-specific component of the abscisic acid signal transduction pathways from Arabidopsis. In French bean (Phaseolus vulgaris), the ABI3-like factor, PvALF, activates transcription from MAT promoters of phytohemagglutinin (DLEC2) and beta-phaseolin (PHS beta) genes. We describe the regulator of MAT2 (ROM2) as a basic leucine zipper (bZIP) DNA binding protein that recognizes motifs with symmetric (ACGT) and asymmetric (ACCT) core elements present in both MAT promoters. ROM2 antagonizes trans-activation of the DLEC2 promoter by PvALF in transient expression assays. Repression was abolished by mutations that prevented binding of ROM2 to the DLEC2 seed enhancer region. Moreover, a hybrid protein composed of a PvALF activation domain and the DNA binding and dimerization domain of ROM2 activated gene expression, indicating that ROM2 recognizes the DLEC2 enhancer in vivo; consequently, ROM2 functions as a DNA binding site-dependent repressor. Supershift analysis of nuclear proteins, using a ROM2-specific antibody, revealed an increase in ROM2 DNA binding activity during seed desiccation. A corresponding increase in ROM2 mRNA coincided with the period when DLEC2 mRNA levels declined in embryos. These results demonstrate developmental regulation of the ROM2 repressor and point to a role for this factor in silencing DLEC2 transcription during late embryogenesis. PMID:8742714

  9. Effect of maturity at harvest on lycopene content and antioxidant activity in 'Vanessa' personal-sized watermelon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stage of maturity at harvest of fresh fruit has a profound effect on marketing and storage of a number of small to medium sized horticultural commodities. In many instances there are established USDA grades and maturity indices to assist growers and the produce industry alike. However, in the ...

  10. Antibacterial Activity of Blue Light against Nosocomial Wound Pathogens Growing Planktonically and as Mature Biofilms

    PubMed Central

    Thwaite, Joanne E.; Burt, Rebecca; Laws, Thomas R.; Raguse, Marina; Moeller, Ralf; Webber, Mark A.; Oppenheim, Beryl A.

    2016-01-01

    ABSTRACT The blue wavelengths within the visible light spectrum are intrinisically antimicrobial and can photodynamically inactivate the cells of a wide spectrum of bacteria (Gram positive and negative) and fungi. Furthermore, blue light is equally effective against both drug-sensitive and -resistant members of target species and is less detrimental to mammalian cells than is UV radiation. Blue light is currently used for treating acnes vulgaris and Helicobacter pylori infections; the utility for decontamination and treatment of wound infections is in its infancy. Furthermore, limited studies have been performed on bacterial biofilms, the key growth mode of bacteria involved in clinical infections. Here we report the findings of a multicenter in vitro study performed to assess the antimicrobial activity of 400-nm blue light against bacteria in both planktonic and biofilm growth modes. Blue light was tested against a panel of 34 bacterial isolates (clinical and type strains) comprising Acinetobacter baumannii, Enterobacter cloacae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Enterococcus faecium, Klebsiella pneumoniae, and Elizabethkingia meningoseptica. All planktonic-phase bacteria were susceptible to blue light treatment, with the majority (71%) demonstrating a ≥5-log10 decrease in viability after 15 to 30 min of exposure (54 J/cm2 to 108 J/cm2). Bacterial biofilms were also highly susceptible to blue light, with significant reduction in seeding observed for all isolates at all levels of exposure. These results warrant further investigation of blue light as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications. IMPORTANCE Blue light shows great promise as a novel decontamination strategy for the nosocomial environment, as well as additional wider decontamination applications (e.g., wound closure during surgery). This warrants further

  11. DEVELOPMENT QUALIFICATION AND DISPOSAL OF AN ALTERNATIVE IMMOBILIZED LOW-ACTIVITY WASTE FORM AT THE HANFORD SITE

    SciTech Connect

    SAMS TL; EDGE JA; SWANBERG DJ; ROBBINS RA

    2011-01-13

    Demonstrating that a waste form produced by a given immobilization process is chemically and physically durable as well as compliant with disposal facility acceptance criteria is critical to the success of a waste treatment program, and must be pursued in conjunction with the maturation of the waste processing technology. Testing of waste forms produced using differing scales of processing units and classes of feeds (simulants versus actual waste) is the crux of the waste form qualification process. Testing is typically focused on leachability of constituents of concern (COCs), as well as chemical and physical durability of the waste form. A principal challenge regarding testing immobilized low-activity waste (ILAW) forms is the absence of a standard test suite or set of mandatory parameters against which waste forms may be tested, compared, and qualified for acceptance in existing and proposed nuclear waste disposal sites at Hanford and across the Department of Energy (DOE) complex. A coherent and widely applicable compliance strategy to support characterization and disposal of new waste forms is essential to enhance and accelerate the remediation of DOE tank waste. This paper provides a background summary of important entities, regulations, and considerations for nuclear waste form qualification and disposal. Against this backdrop, this paper describes a strategy for meeting and demonstrating compliance with disposal requirements emphasizing the River Protection Project (RPP) Integrated Disposal Facility (IDF) at the Hanford Site and the fluidized bed steam reforming (FBSR) mineralized low-activity waste (LAW) product stream.

  12. The activity and copy number of mitochondrial DNA in ovine oocytes throughout oogenesis in vivo and during oocyte maturation in vitro

    PubMed Central

    Cotterill, Matthew; Harris, Sarah E.; Collado Fernandez, Esther; Lu, Jianping; Huntriss, John D.; Campbell, Bruce K.; Picton, Helen M.

    2013-01-01

    Mitochondria are responsible for the production of ATP, which drives cellular metabolic and biosynthetic processes. This is the first study to quantify the mtDNA copy number across all stages of oogenesis in a large monovulatory species, it includes assessment of the activity of mitochondria in germinal vesicle (GV) and metaphase II (MII) oocytes through JC1 staining. Primordial to early antral follicles (n = 249) were isolated from the sheep ovarian cortex following digestion at 37°C for 1 h and all oocytes were disaggregated from their somatic cells. Germinal vesicle oocytes (n = 133) were aspirated from 3- to 5-mm diameter antral follicles, and mature MII oocytes (n = 71) were generated following in vitro maturation (IVM). The mtDNA copy number in each oocyte was quantified using real-time PCR and showed a progressive, but variable increase in the amount of mtDNA in oocytes from primordial follicles (605 ± 205, n = 8) to mature MII oocytes (744 633 ± 115 799, n = 13; P < 0.05). Mitochondrial activity (P > 0.05) was not altered during meiotic progression from GV to MII during IVM. The observed increase in the mtDNA copy number across oogenesis reflects the changing ATP demands needed to orchestrate cytoskeletal and cytoplasmic reorganization during oocyte growth and maturation and the need to fuel the resumption of meiosis in mature oocytes following the pre-ovulatory gonadotrophin surge. PMID:23468533

  13. Vector-encoded Helicobacter pylori neutrophil-activating protein promotes maturation of dendritic cells with Th1 polarization and improved migration.

    PubMed

    Ramachandran, Mohanraj; Jin, Chuan; Yu, Di; Eriksson, Fredrik; Essand, Magnus

    2014-09-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor involved in H. pylori infection. Both HP-NAP protein and oncolytic viruses encoding HP-NAP have been suggested as immunotherapeutic anticancer agents and adjuvants for vaccination but with little known about its mode of action to activate adaptive immunity. Dendritic cells (DCs) are key players in bridging innate and adaptive immune responses, and in this study we aim to evaluate the effect of HP-NAP on DC maturation, migration, and induction of adaptive immune response. Maturation markers CD83, CD80, CD86, HLA-DR, CD40, and CCR7 were upregulated on human DCs after treatment with supernatants from HP-NAP adenovirus-infected cells. HP-NAP-activated DCs had a Th1 cytokine secretion profile, with high IL-12 and relatively low IL-10 secretion, and migrated toward CCL19. Ag-specific T cells were efficiently expanded by Ag-presenting HP-NAP-activated DCs, which is an important property of functionally mature DCs. Furthermore, intradermal injections of HP-NAP-encoding adenovirus in C57BL/6 mice enhanced resident DC migration to draining lymph nodes, which was verified by imaging lymph nodes by two-photon microscopy and by phenotyping migrating cells by flow cytometry. In conclusion, therapeutic effects of HP-NAP are mediated by maturation of DCs and subsequent activation of Ag-specific T cells in addition to provoking innate immunity. PMID:25049358

  14. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  15. Efficient stereoselective synthesis of 2-acetamido-1,2-dideoxyallonojirimycin (DAJNAc) and sp(2)-iminosugar conjugates: Novel hexosaminidase inhibitors with discrimination capabilities between the mature and precursor forms of the enzyme.

    PubMed

    de la Fuente, Alex; Rísquez-Cuadro, Rocío; Verdaguer, Xavier; García Fernández, José M; Nanba, Eiji; Higaki, Katsumi; Ortiz Mellet, Carmen; Riera, Antoni

    2016-10-01

    Due to their capacity to inhibit hexosaminidases, 2-acetamido-1,2-dideoxy-iminosugars have been widely studied as potential therapeutic agents for various diseases. An efficient stereoselective synthesis of 2-acetamido-1,2-dideoxyallonojirimycin (DAJNAc), the most potent inhibitor of human placenta β-N-acetylglucosaminidase (β-hexosaminidase) among the epimeric series, is here described. This novel procedure can be easily scaled up, providing enough material for structural modifications and further biological tests. Thus, two series of sp(2)-iminosugar conjugates derived from DAJNAc have been prepared, namely monocyclic DAJNAc-thioureas and bicyclic 2-iminothiazolidines, and their glycosidase inhibitory activity evaluated. The data evidence the utmost importance of developing diversity-oriented synthetic strategies allowing optimization of electrostatic and hydrophobic interactions to achieve high inhibitory potencies and selectivities among isoenzymes. Notably, strong differences in the inhibition potency of the compounds towards β-hexosaminidase from human placenta (mature) or cultured fibroblasts (precursor form) were encountered. The ensemble of data suggests that the ratio between them, and not the inhibition potency towards the placenta enzyme, is a good indication of the chaperoning potential of TaySachs disease-associated mutant hexosaminidase. PMID:26564401

  16. Optically active surfaces formed by ion implantation and thermal treatment

    SciTech Connect

    Gea, L.A.; Boatner, L.A.; Evans, H.M.; Zuhr, R.

    1996-08-01

    Embedded VO{sub 2} precipitates have been formed in single-crystal sapphire by the ion co-implantation of vanadium and oxygen and subsequent thermal annealing. The embedded VO{sub 2} particles have been shown to exhibit an optical switching behavior that is comparable to that of continuous thin films. In this work, the mechanisms of formation of these optically active particles are investigated. It is shown that precipitation of the vanadium dioxide phase is favored when the thermal treatment is performed on an ion-damaged but still crystalline (rather than amorphized) Al{sub 2}O{sub 3} substrate. The best optical switching behavior is observed in this case, and this behavior is apparently correlated with a more-favorable dispersion of VO{sub 2} small particles inside the matrix.

  17. Chemical Fingerprints of Star Forming Regions and Active Galaxies

    NASA Astrophysics Data System (ADS)

    Pérez-Beaupuits, Juan-Pablo

    2010-10-01

    This thesis is devoted to the study of the physical conditions of the interstellar medium (ISM) in active galactic nuclei (AGNs) and Galactic star-forming regions, using mostly single-dish millimeter observations. I first study the excitation conditions of dense gas in a group of Seyfert galaxies using radiative transfer models (Chapter 2). I then study the galaxy NGC 1068, and try to distinguish signatures of the contributions from the AGN and the starburst ring by incorporating observations of high-J transitions of dense gas tracers (Chapter 3). Later, I venture into the mid-infrared spectral region to study different aspects of the AGN and starburst components in the galaxy NGC 4945 (Chapter 4). In Chapter 5 I delve into theoretical aspects of the dynamical evolution of gas in an AGN torus. I use a 3D hydrodynamic simulation with chemical abundances driven by X-rays. The aim is to understand the effects of X-ray irradiation by the AGN on the temperature, formation and destruction of the molecular gas. I finally explore a Galactic star-forming region, the Omega Nebula, with high resolution single dish observations, to study the properties of the warm gas and to constrain chemical models (Chapters 6 and 7).

  18. 75 FR 16492 - Agency Information Collection Activities: Form G-28, and Form G-28I, Revision of an Existing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-01

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form G-28, and Form G- 28I, Revision of an Existing Information Collection; Comment Request ACTION: 60-Day Notice of Information Collection under Review: Form G- 28, Notice of Entry of Appearance as Attorney or...

  19. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner

    PubMed Central

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  20. ENDOCRINE-DISRUPTING CHEMICALS: PREPUBERTAL EXPOSURES AND EFFECTS ON SEXUAL MATURATION AND THYROID ACTIVITY IN THE FEMALE RAT. A FOCUS ON THE EDSTAC RECOMMENDATIONS

    EPA Science Inventory

    Endocrine-disrupting chemicals: prepubertal exposures and effects on sexual maturation and thyroid activity in the female rat. A focus on the EDSTAC recommendations.

    Goldman JM, Laws SC, Balchak SK, Cooper RL, Kavlock RJ.

    Reproductive Toxicology Division, National H...

  1. Physical Activity, Physical Self-Concept, and Health-Related Quality of Life of Extreme Early and Late Maturing Adolescent Girls

    ERIC Educational Resources Information Center

    Cumming, Sean P.; Sherar, Lauren B.; Smart, Joanna E. Hunter; Rodrigues, Aristides M. M.; Standage, Martyn; Gillison, Fiona B.; Malina, Robert M.

    2012-01-01

    In this study we tested for differences in physical activity (PA), physical self-concept, and health-related quality of life (HRQoL) between the least and most biologically mature adolescent females within their respective chronological and academic year groups. A total of 222 British female adolescents aged 10 to 14 years (X[bar] age = 12.7…

  2. Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

    PubMed

    Park, Joohyun; Cho, Oh Yeon; Kim, Jung Ah; Chang, Sunghoe

    2016-01-01

    Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity. PMID:27534442

  3. The SPS100 gene of Saccharomyces cerevisiae is activated late in the sporulation process and contributes to spore wall maturation.

    PubMed Central

    Law, D T; Segall, J

    1988-01-01

    We previously described the use of a differential hybridization screen of a genomic DNA library of Saccharomyces cerevisiae to identify sporulation-specific (SPS) genes (A. Percival-Smith and J. Segall, Mol. Cell. Biol. 4:142-150, 1984). This initial screen identified 14 SPS genes that are first expressed 6 to 8 h after transfer of cells to sporulation medium. Accumulation of transcripts corresponding to these genes becomes maximal at 8 to 12 h of sporulation, the time at which meiotic events are nearing completion, and by 15 h of sporulation, transcript levels are beginning to decrease. In the present study two additional SPS genes, first expressed at 12 h of sporulation, were isolated. The steady-state level of transcripts corresponding to these two genes, termed SPS100 and SPS101, remains unchanged from 15 to 35 h, a time coincident with spore wall maturation. The nature of the putative 34.2-kilodalton protein encoded by the SPS100 gene is consistent with its being a component of the glycoprotein matrix of the spore wall; the protein contains a potential signal sequence and cleavage site and numerous sites for potential glycosylation. A MATa sps100/MAT alpha sps100 strain was found to be indistinguishable from the wild-type strain when assessed for efficiency of ascus formation and spore viability. However, a more detailed analysis of the mutant strain revealed that the SPS100 gene product serves a protective role during the early stages of spore wall formation. The time at which resistance to ether could first be detected in developing spores was delayed by 5 h in the mutant strain relative to the wild-type strain. This phenotype is presumably a reflection of a defect in spore wall maturation. This study has confirmed that temporally distinct classes of sporulation-specific genes are sequentially activated during the process of meiosis and spore formation and has shown that the SPS100 gene, identified on the basis of its developmental-specific expression

  4. ROLE OF AMPK THOUGHOUT MEIOTIC MATURATION IN THE MOUSE OOCYTE: EVIDENCE FOR PROMOTION OF POLAR BODY FORMATION AND SUPPRESSION OF PREMATURE ACTIVATION

    PubMed Central

    Downs, Stephen M.; Ya, Ru; Davis, Christopher

    2014-01-01

    This study was conducted to assess the role of AMPK in regulating meiosis in mouse oocytes from the germinal vesicle stage to metaphase II. Exposure of mouse cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) during spontaneous maturation in vitro to AMPK-activating agents resulted in augmentation of the rate and frequency of polar body formation. Inhibitors of AMPK had an opposite, inhibitory effect. In addition, the AMPK inhibitor, compound C (Cmpd C) increased the frequency of oocyte activation. The stimulatory action of the AMPK-activating agent, AICAR, and the inhibitory action of Cmpd C were diminished if exposure was delayed, indicating an early action of AMPK on polar body formation. The frequency of spontaneous and Cmpd C-induced activation in CEO was reduced as the period of hormonal priming was increased, and AMPK stimulation eliminated the activation response. Immunostaining of oocytes with antibody to active AMPK revealed an association of active kinase with chromatin, spindle poles and midbody during maturation. Immunolocalization of the α1 catalytic subunit of AMPK showed an association with condensed chromatin and the meiotic spindle, but not in the spindle poles or midbody; α2 stained only diffusely throughout the oocyte. These data suggest that AMPK is involved in a regulatory capacity throughout maturation and helps promote the completion of meiosis while suppressing premature activation. PMID:20830737

  5. c-mos and cdc2 Cooperate in the Translational Activation of Fibroblast Growth Factor Receptor-1 during Xenopus Oocyte Maturation

    PubMed Central

    Culp, Patricia A.; Musci, Thomas J.

    1999-01-01

    During oocyte maturation in Xenopus, previously quiescent maternal mRNAs are translationally activated at specific times. We hypothesized that the translational recruitment of individual messages is triggered by particular cellular events and investigated the potential for known effectors of the meiotic cell cycle to activate the translation of the FGF receptor-1 (XFGFR) maternal mRNA. We found that both c-mos and cdc2 activate the translation of XFGFR. However, although oocytes matured by injection of recombinant cdc2/cyclin B translate normal levels of XFGFR protein, c-mos depletion reduces the level of XFGFR protein induced by cdc2/cyclin B injection. In oocytes blocked for cdc2 activity, injection of mos RNA induced low levels of XFGFR protein, independent of MAPK activity. Through the use of injected reporter RNAs, we show that the XFGFR 3′ untranslated region inhibitory element is completely derepressed by cdc2 alone. In addition, we identified a new inhibitory element through which both mos and cdc2 activate translation. We found that cdc2 derepresses translation in the absence of polyadenylation, whereas mos requires poly(A) extension to activate XFGFR translation. Our results demonstrate that mos and cdc2, in addition to functioning as key regulators of the meiotic cell cycle, cooperate in the translational activation of a specific maternal mRNA during oocyte maturation. PMID:10564256

  6. A Functional Interplay between Human Immunodeficiency Virus Type 1 Protease Residues 77 and 93 Involved in Differential Regulation of Precursor Autoprocessing and Mature Protease Activity

    PubMed Central

    Counts, Christopher J.; Ho, P. Shing; Donlin, Maureen J.; Tavis, John E.; Chen, Chaoping

    2015-01-01

    HIV-1 protease (PR) is a viral enzyme vital to the production of infectious virions. It is initially synthesized as part of the Gag-Pol polyprotein precursor in the infected cell. The free mature PR is liberated as a result of precursor autoprocessing upon virion release. We previously described a model system to examine autoprocessing in transfected mammalian cells. Here, we report that a covariance analysis of miniprecursor (p6*-PR) sequences derived from drug naïve patients identified a series of amino acid pairs that vary together across independent viral isolates. These covariance pairs were used to build the first topology map of the miniprecursor that suggests high levels of interaction between the p6* peptide and the mature PR. Additionally, several PR-PR covariance pairs are located far from each other (>12 Å Cα to Cα) relative to their positions in the mature PR structure. Biochemical characterization of one such covariance pair (77–93) revealed that each residue shows distinct preference for one of three alkyl amino acids (V, I, and L) and that a polar or charged amino acid at either of these two positions abolishes precursor autoprocessing. The most commonly observed 77V is preferred by the most commonly observed 93I, but the 77I variant is preferred by other 93 variances (L, V, or M) in supporting precursor autoprocessing. Furthermore, the 77I93V covariant enhanced precursor autoprocessing and Gag polyprotein processing but decreased the mature PR activity. Therefore, both covariance and biochemical analyses support a functional association between residues 77 and 93, which are spatially distant from each other in the mature PR structure. Our data also suggests that these covariance pairs differentially regulate precursor autoprocessing and the mature protease activity. PMID:25893662

  7. Crocin and geniposide profiles and radical scavenging activity of gardenia fruits (Gardenia jasminoides Ellis) from different cultivars and at the various stages of maturation.

    PubMed

    Chen, Yang; Zhang, Hao; Li, Yi-Xin; Cai, Le; Huang, Juan; Zhao, Can; Jia, Lin; Buchanan, Ryan; Yang, Ting; Jiang, Li-Juan

    2010-06-01

    The major components of gardenia fruits are geniposide and water soluble pigment crocins. In this study, we investigate crocins and geniposide profiles of gardenia fruits from different cultivars and at the various stages of maturation. DPPH scavenging activity of gardenia fruits from different cultivars and at the various stages of fruit maturation was also assayed. Quantitative determination of crocins in the gardenia at the various stages of maturation revealed a significant increase when ripening. However, geniposide content was negatively correlated with ripening stages. A significant difference was observed when comparing crocin content of different gardenia from various cultivars and geniposide content also showed marked variety. Current study indicated no relationship between crocin and geniposide content in gardenia fruits at the various stages of maturation and DPPH radical scavenging activity. Data showed that, although crocins feature markedly less DPPH scavenging activity than gardenia ethanol extract, total crocin content of gardenias collected in various cultivars correlate, to a certain degree, with radical scavenging effects of the Chinese traditional medicine (r=0.75). PMID:19815056

  8. The Etv1 transcription factor activity-dependently downregulates a set of genes controlling cell growth and differentiation in maturing cerebellar granule cells.

    PubMed

    Okazawa, Makoto; Abe, Haruka; Nakanishi, Shigetada

    2016-05-13

    In the early postnatal period, cerebellar granule cells exhibit an activity-dependent downregulation of a set of immaturation genes involved in cell growth and migration and are shifted to establishment of a mature network formation. Through the use of a granule cell culture and both pharmacological and RNA interference (siRNA) analyses, the present investigation revealed that the downregulation of these immaturation genes is controlled by strikingly unified signaling mechanisms that operate sequentially through the stimulation of AMPA and NMDA receptors, tetrodotoxin-sensitive Na(+) channels and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). This signaling cascade induces the Etv1 transcription factor, and knockdown of Etv1 by a siRNA technique prevented this activity-dependent downregulation of immaturation genes. Thus, taken into consideration the mechanism that controls the upregulation of maturation genes involved in synaptic formation, these results indicate that Etv1 orchestrates the activity-dependent regulation of both maturation and immaturation genes in developing granule cells and plays a key role in specifying the identity of mature granule cells in the cerebellum. PMID:27059140

  9. Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity.

    PubMed Central

    Rosé, J R; Babé, L M; Craik, C S

    1995-01-01

    The human immunodeficiency virus type 1 (HIV-1) protease is the enzyme required for processing of the Gag and Gag-Pol polyproteins to yield mature, infectious virions. Although the complete absence of proteolytic activity prevents maturation, the level of activity sufficient for maturation and subsequent infectivity has not been determined. Amino acid substitutions that reduce catalytic activity without affecting substrate recognition have been engineered into the active site of the HIV-1 protease. The catalytic efficiency (kcat) of the HIV-1 protease is decreased 4-fold when threonine 26 is replaced by serine (T26S) and approximately 50-fold when alanine 28 is replaced by serine (A28S). Genes containing these mutations were cloned into a proviral vector for analysis of their effects on virion maturation and infectivity. The results show that virions containing the T26S protease variant, in which only 25% of the protease is active, are very similar to wild-type virions, although slight reductions in infectivity are observed. Virions containing the A28S protease variant are not infectious, even though a limited amount of polyprotein processing does occur. There appears to be a linear correlation between the level of protease activity and particle infectivity. Our observations suggest that a threshold of protease activity exists between a 4-fold and 50-fold reduction, below which processing is insufficient to yield infectious particles. Our data also suggest that a reduction of protease activity by 50-fold or greater is sufficient to prevent the formation of infectious particles. PMID:7535864

  10. Increased expression with differential subcellular location of cytidine deaminase APOBEC3G in human CD4(+) T-cell activation and dendritic cell maturation.

    PubMed

    Oliva, Harold; Pacheco, Rodrigo; Martinez-Navio, José M; Rodríguez-García, Marta; Naranjo-Gómez, Mar; Climent, Núria; Prado, Carolina; Gil, Cristina; Plana, Montserrat; García, Felipe; Miró, José M; Franco, Rafael; Borras, Francesc E; Navaratnam, Naveenan; Gatell, José M; Gallart, Teresa

    2016-08-01

    APOBEC3G (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G; A3G) is an innate defense protein showing activity against retroviruses and retrotransposons. Activated CD4(+) T cells are highly permissive for HIV-1 replication, whereas resting CD4(+) T cells are refractory. Dendritic cells (DCs), especially mature DCs, are also refractory. We investigated whether these differences could be related to a differential A3G expression and/or subcellular distribution. We found that A3G mRNA and protein expression is very low in resting CD4(+) T cells and immature DCs, but increases strongly following T-cell activation and DC maturation. The Apo-7 anti-A3G monoclonal antibody (mAb), which was specifically developed, confirmed these differences at the protein level and disclosed that A3G is mainly cytoplasmic in resting CD4(+) T cells and immature DCs. Nevertheless, A3G translocates to the nucleus in activated-proliferating CD4(+) T cells, yet remaining cytoplasmic in matured DCs, a finding confirmed by immunoblotting analysis of cytoplasmic and nuclear fractions. Apo-7 mAb was able to immunoprecipitate endogenous A3G allowing to detect complexes with numerous proteins in activated-proliferating but not in resting CD4(+) T cells. The results show for the first time the nuclear translocation of A3G in activated-proliferating CD4(+) T cells. PMID:26987686

  11. Proper activation of MafA is required for optimal differentiation and maturation of pancreatic β-cells.

    PubMed

    El Khattabi, Ilham; Sharma, Arun

    2015-12-01

    A key therapeutic approach for the treatment of Type 1 diabetes (T1D) is transplantation of functional islet β-cells. Despite recent advances in generating stem cell-derived glucose-responsive insulin(+) cells, their further maturation to fully functional adult β-cells still remains a daunting task. Conquering this hurdle will require a better understanding of the mechanisms driving maturation of embryonic insulin(+) cells into adult β-cells, and the implementation of that knowledge to improve current differentiation protocols. Here, we will review our current understanding of β-cell maturation, and discuss the contribution of key β-cell transcription factor MafA, to this process. The fundamental importance of MafA in regulating adult β-cell maturation and function indicates that enhancing MafA expression may improve the generation of definitive β-cells for transplantation. Additionally, we suggest that the temporal control of MafA induction at a specific stage of β-cell differentiation will be the next critical challenge for achieving optimum maturation of β-cells. PMID:26696512

  12. Visualization of RelB expression and activation at the single-cell level during dendritic cell maturation in Relb-Venus knock-in mice.

    PubMed

    Seki, Takao; Yamamoto, Mami; Taguchi, Yuu; Miyauchi, Maki; Akiyama, Nobuko; Yamaguchi, Noritaka; Gohda, Jin; Akiyama, Taishin; Inoue, Jun-ichiro

    2015-12-01

    RelB is activated by the non-canonical NF-κB pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb(Venus/Venus) mice developed without any abnormalities observed in the Relb(-/-) mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb(Venus/Venus) DC analyses revealed that DCs consist of RelB(-), RelB(low) and RelB(high) populations. The RelB(high) population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB(low) population was in the cytoplasm. Although both the RelB(low) and RelB(-) populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB(low) than in the RelB(-) splenic conventional DCs. Taken together, our results identify the RelB(low) population as a possible novel intermediate maturation stage of cDCs and the Relb(Venus/Venus) mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-κB pathway. PMID:26115685

  13. Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form

    PubMed Central

    Creanga, Adrian; Glenn, Thomas D.; Mann, Randall K.; Saunders, Adam M.; Talbot, William S.; Beachy, Philip A.

    2012-01-01

    Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action. PMID:22677548

  14. Transcriptional Landscape of Cardiomyocyte Maturation

    PubMed Central

    Uosaki, Hideki; Cahan, Patrick; Lee, Dong I.; Wang, Songnan; Miyamoto, Matthew; Fernandez, Laviel; Kass, David A.; Kwon, Chulan

    2015-01-01

    SUMMARY Decades of progress in developmental cardiology has advanced our understanding of the early aspects of heart development, including cardiomyocyte (CM) differentiation. However, control of CM maturation which is subsequently required to generate adult myocytes, remains elusive. Here, we analyzed over 200 microarray datasets from early embryonic to adult hearts and identified a large number of genes whose expression shifts gradually and continuously during maturation. We generated an atlas of integrated gene expression, biological pathways, transcriptional regulators, and gene regulatory networks (GRNs), which show discrete sets of key transcriptional regulators and pathways activated or suppressed during CM maturation. We developed a GRN-based program named MatStatCM that indexes CM maturation status. MatStatCM reveals that pluripotent stem cell-derived CMs mature early in culture, but are arrested at the late embryonic stage with aberrant regulation of key transcription factors. Our study provides a foundation for understanding CM maturation. PMID:26586429

  15. Newly synthesized protein(s) must associate with p34cdc2 to activate MAP kinase and MPF during progesterone-induced maturation of Xenopus oocytes.

    PubMed Central

    Nebreda, A R; Gannon, J V; Hunt, T

    1995-01-01

    The meiotic maturation of Xenopus oocytes triggered by progesterone requires new protein synthesis to activate both maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAP kinase). Injection of mRNA encoding mutant p34cdc2 (K33R) that can bind cyclins but lacks protein kinase activity strongly inhibited progesterone-induced activation of both MPF and MAP kinase in Xenopus oocytes. Similar results were obtained by injection of GST-p34cdc2 K33R protein or by injection of a monoclonal antibody (A17) against p34cdc2 that blocks its activation by cyclins. Both the dominant-negative p34cdc2 and monoclonal antibody A17 blocked the accumulation of p39mos and activation of MAP kinase in response to progesterone, as well as blocking the appearance of MPF, although they did not inhibit the translation of p39mos mRNA. These results suggest that: (i) activation of free p34cdc2 by newly made proteins, probably cyclin(s), is normally required for the activation of both MPF and MAP kinase by progesterone in Xenopus oocytes; (ii) the activation of translation of cyclin mRNA normally precedes, and does not require either MPF or MAP kinase activity; and (iii) de novo synthesis and accumulation of p39mos is probably both necessary and sufficient for the activation of MAP kinase in response to progesterone. Images PMID:8521817

  16. Epidermal growth factor-mediated mitogen-activated protein kinase3/1 pathway is conducive to in vitro maturation of sheep oocytes.

    PubMed

    Ni, Hemin; Sheng, Xihui; Cui, Xu; Gu, Meichao; Liu, Yunhai; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2015-01-01

    Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo's capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10⁻⁴ mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10-6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10⁻⁴ mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10⁻⁶ mol/l U0126 group and 42.6% in 10⁻⁴ mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10⁻⁴ mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes. PMID:25799554

  17. Epidermal Growth Factor-Mediated Mitogen-Activated Protein Kinase3/1 Pathway Is Conducive to In Vitro Maturation of Sheep Oocytes

    PubMed Central

    Cui, Xu; Gu, Meichao; Liu, Yunhai; Qi, Xiaolong; Xing, Shuhan; Guo, Yong

    2015-01-01

    Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10−4 mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10−6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10−4 mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10−6 mol/l U0126 group and 42.6% in 10−4 mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10−4 mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes. PMID:25799554

  18. 76 FR 30738 - Agency Information Collection Activities: Form G-845 and Form G-845 Supplement, Revision of a...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... the Federal Register on February 22, 2011, at 76 FR 9805, allowing for a 60-day public comment period... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form G-845 and Form G- 845 Supplement, Revision of a Currently Approved Information Collection; Comment Request...

  19. Kinetics of mature T-cell development in the thymus

    SciTech Connect

    Egerton, M.; Scollay, R.; Shortman, K. )

    1990-04-01

    We have reexamined the balance between cell birth, cell maturation, and cell death in the thymus by labeling dividing thymocytes and their progeny in vivo with (3H)-thymidine, isolating clearly defined subpopulations by fluorescence-activated cell sorting, and determining the distribution of label by autoradiography. When mature thymocytes were precisely defined (as CD4+CD8- CD3+ or CD4-CD8+ CD3+) and separated from immature single positives (CD4+CD8- CD3- and CD4-CD8+ CD3-), a lag was observed in the rate of entry of (3H)thymidine into mature cells. Thus, many of the mature thymocytes appear to derive from a small nondividing cortical thymocyte pool, rather than originating directly from the earliest dividing CD4+CD8+ blasts. There was little evidence for cell division during or after mature thymocyte formation, suggesting a one-for-one differentiation from cortical cells rather than selective clonal expansion. The rate of production of mature single positive thymocytes agreed closely with estimates of the rate of export of mature T cells from the thymus and was only 3% of the rate of production of double-positive cortical thymocytes. This was compatible with a stringent selection process and extensive intrathymic cell death and suggested that no extensive negative selection occurred after the mature cells were formed.

  20. Helicobacter pylori inhibits dendritic cell maturation via interleukin-10-mediated activation of the signal transducer and activator of transcription 3 pathway.

    PubMed

    Rizzuti, David; Ang, Michelle; Sokollik, Christiane; Wu, Ted; Abdullah, Majd; Greenfield, Laura; Fattouh, Ramzi; Reardon, Colin; Tang, Michael; Diao, Jun; Schindler, Christian; Cattral, Mark; Jones, Nicola L

    2015-01-01

    Helicobacter pylori infects the human gastric mucosa causing a chronic infection that is the primary risk factor for gastric cancer development. Recent studies demonstrate that H. pylori promotes tolerogenic dendritic cell (DC) development indicating that this bacterium evades the host immune response. However, the signaling pathways involved in modulating DC activation during infection remain unclear. Here, we report that H. pylori infection activated the signal transducer and activator of transcription 3 (STAT3) pathway in murine bone marrow-derived DCs (BMDCs) and splenic DCs isolated ex vivo. Isogenic cagA-, cagE-, vacA- and urease-mutants exhibited levels of phosphoSTAT3 that were comparable to in the wild-type (WT) parent strain. H. pylori-infected BMDCs produced increased immunosuppressive IL-10, which activated STAT3 in an autocrine/paracrine fashion. Neutralization of IL-10 prevented H. pylori-mediated STAT3 activation in both BMDCs and splenic DCs. In addition, anti-IL-10 treatment of infected H. pylori-BMDCs was associated with increased CD86 and MHC II expression and enhanced proinflammatory IL-1β cytokine secretion. Finally, increased CD86 and MHC II expression was detected in H. pylori-infected STAT3 knockout DCs when compared to WT controls. Together, these results demonstrate that H. pylori infection induces IL-10 secretion in DCs, which activates STAT3, thereby modulating DC maturation and reducing IL-1β secretion. These findings identify a host molecular mechanism by which H. pylori can manipulate the innate immune response to potentially favor chronic infection and promote carcinogenesis. PMID:25412627

  1. Heterogeneous activity of immature and mature cells of the murine monocyte-macrophage lineage derived from different anatomical districts against yeast-phase Candida albicans.

    PubMed Central

    Decker, T; Lohmann-Matthes, M L; Baccarini, M

    1986-01-01

    Mature mononuclear phagocytes have been receiving much attention as effectors of spontaneous candidacidal activity, although with controversial results due to differences in the effector populations and the methods used in different laboratories. We here systematically compare the fungistatic activity of immature and mature cells of the murine macrophage series. The results show that nonadherent, nonphagocytic precursor cells (isolated either [90% purity] from bone marrow liquid cultures or from the organs of mice in which inflammatory conditions had been elicited in vivo) exerted a strong extracellular candidastatic activity. In contrast, mature macrophages, either obtained from different anatomical areas (spleen, liver, lung, peritoneal cavity) or matured in vitro from the precursor populations, displayed striking heterogeneity in their ability to inhibit the growth of Candida albicans, depending on the anatomical site they were derived from. Lymphokine activation did not alter the fungistatic pattern of the untreated cells. The different macrophage populations behaved very differently also in the production of reactive oxygen intermediates (ROI) in response to phagocytosis of C. albicans. The amounts of ROI generated, however, showed no correlation with candidastatic ability. Low levels of candidastatic activity exerted by resident peritoneal macrophages (good ROI producers) were inhibited by catalase, whereas high levels of growth inhibition by Kupffer cells (poor ROI producers) after 8 h of assay were hardly influenced by the enzyme. Our data suggest the existence of two different effector mechanisms in macrophage-mediated C. albicans growth inhibition, a rather inefficient ROI-dependent one, and a second, very efficient oxygen-independent mechanism. The implications of these findings are discussed. PMID:3533781

  2. A GC-MS study of the volatile organic composition of straw and oyster mushrooms during maturity and its relation to antioxidant activity.

    PubMed

    Zhang, Zhuo-Min; Wu, Wen-Wei; Li, Gong-Ke

    2008-09-01

    Mushrooms are very popular in the market for their nutritional and medicinal use. Mushroom volatiles are not only an important factor in the flavor, but also contain many antioxidant compounds. Antioxidant activity is a very important property for disease prevention. The volatile compositional characteristics of straw mushrooms (Volvariella volvacea [Bull. ex Fr.] Sing.) and oyster mushrooms (Pleurotus ostreatus [Jacq. ex Fr.] Kummer) during maturity and the mushroom antioxidant activity related to the non-volatiles and volatiles are studied by a chromatographic method in combination with a spectrophotometric method. The volatile compounds of straw and oyster mushrooms are sampled and identified by a combination sampling method, including headspace solid phase microextraction and steam distillation, followed by gas chromatography-mass spectrometry detection. Among all the volatile compounds identified, 1-octen-3-ol and 3-octanone are the two main compounds with the highest amounts in the volatile compositions of straw and oyster mushrooms. During maturity time of the straw mushrooms, the unsaturated 1-octen-3-ol peak area is reduced, whereas the saturated 3-octanone peak area is increased. However, during normal maturity time of oyster mushrooms, the peak areas of 1-octen-3-ol and 3-octanone remain at the same level. 1-Octen-3-ol has a different antioxidant activity from 3-octanone. Combining the results of antioxidant experiments of water extract and main volatile components by the use of a phosphomolybdenum spectrophotometric method, the conclusion is drawn that oyster mushrooms might possess stronger antioxidant activities than straw mushrooms. PMID:18796224

  3. The Essay: Theory and Pedagogy for an Active Form.

    ERIC Educational Resources Information Center

    Heilker, Paul

    Calling for a radical reexamination of the traditional foundation of composition instruction--the thesis/support form, this book argues that the essay, with its informality, conversational tone, meditative mood, and integration of form and content, is better suited to developmental, epistemological, ideological, and feminist rhetorical…

  4. Enterotoxigenicity of Mature 45-Kilodalton and Processed 35-Kilodalton Forms of Hemagglutinin Protease Purified from a Cholera Toxin Gene-Negative Vibrio cholerae Non-O1, Non-O139 Strain

    PubMed Central

    Ghosh, A.; Saha, D. R.; Hoque, K. M.; Asakuna, M.; Yamasaki, S.; Koley, H.; Das, S. S.; Chakrabarti, M. K.; Pal, A.

    2006-01-01

    Cholera toxin gene-negative Vibrio cholerae non-O1, non-O139 strain PL-21 is the etiologic agent of cholera-like syndrome. Hemagglutinin protease (HAP) is one of the major secretory proteins of PL-21. The mature 45-kDa and processed 35-kDa forms of HAP were purified in the presence and absence of EDTA from culture supernatants of PL-21. Enterotoxigenicities of both forms of HAP were tested in rabbit ileal loop (RIL), Ussing chamber, and tissue culture assays. The 35-kDa HAP showed hemorrhagic fluid response in a dose-dependent manner in the RIL assay. Histopathological examination of 20 μg of purified protease-treated rabbit ileum showed the presence of erythrocytes and neutrophils in the upper part of the villous lamina propria. Treatment with 40 μg of protease resulted in gross damage of the villous epithelium with inflammation, hemorrhage, and necrosis. The 35-kDa form of HAP, when added to the lumenal surface of rat ileum loaded in an Ussing chamber, showed a decrease in the intestinal short-circuit current and a cell rounding effect on HeLa cells. The mature 45-kDa form of HAP showed an increase in intestinal short-circuit current in an Ussing chamber and a cell distending effect on HeLa cells. These results show that HAP may play a role in the pathogenesis of PL-21. PMID:16622232

  5. Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo.

    PubMed

    Alam, Md Rowshon; Majumdar, Alokes; Thazhathveetil, Arun Kalliat; Liu, Su-Ting; Liu, Ji-Lan; Puri, Nitin; Cuenoud, Bernard; Sasaki, Shigeki; Miller, Paul S; Seidman, Michael M

    2007-09-01

    We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences. PMID:17691818

  6. Supplementation with spermine during in vitro maturation of porcine oocytes improves early embryonic development after parthenogenetic activation and somatic cell nuclear transfer.

    PubMed

    Jin, J X; Lee, S; Khoirinaya, C; Oh, A; Kim, G A; Lee, B C

    2016-03-01

    Spermine plays an important role in protection from reactive oxygen species (ROS) in bacteria, yeast, and mammalian cells, but there are few studies on the effects of spermine on porcine oocyte maturation and subsequent embryo development. The aim of this study was to determine the effects of spermine on in vitro maturation (IVM) of porcine oocytes and their developmental competence after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). We evaluated nuclear maturation, intracellular glutathione (GSH), and ROS levels in oocytes, and their subsequent embryonic development, as well as gene expression in mature oocytes, cumulus cells, and PA blastocysts. After treatment with various concentrations of spermine in IVM culture medium, there was no significant difference in nuclear maturation rate. However, spermine treatment groups (10- 500 µM) showed significantly increased intracellular GSH levels and decreased ROS levels compared to the control ( < 0.05). Furthermore, 10 µM spermine supported significantly higher blastocyst formation rates after PA than the control group ( < 0.05). According to the optimal condition from the PA results, we investigated the effects of 10 µM spermine on SCNT, and it also significantly improved blastocyst formation rates compared with the control group ( < 0.05). In evaluating the effects of 10 µM spermine on gene expression, there was significantly lower expression of a proapoptotic gene () and higher expression of an antiapoptotic gene () in cumulus cells ( < 0.05). was increased in spermine-treated oocytes. Levels of transcription for and were significantly increased in PA blastocysts. In conclusion, 10 µM spermine supplementation during IVM improved the development of porcine PA and SCNT embryos by increasing intracellular GSH, scavenging ROS levels, and regulating gene expression. PMID:27065258

  7. STYPu fuel form activities, March 1-September 30, 1985

    SciTech Connect

    Not Available

    1986-01-01

    The SRP portion of this report summarizes production STYPuO2 fuel forms for use in radioisotopic thermoelectric generators (RTG's) in the Plutonium Fuel Form (PuFF) Facility at the Savannah River Plant. The PuFF Facility began producing iridium-encapsulated, 62.5-watt STYPuO2 right circular cylinders for GPHS (General Purpose Heat Source) RTG's in June 1980; this program was completed in December 1983. The PuFF Facility has been placed in a production readiness mode of operation pending funding of additional heat source programs.

  8. 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.

  9. A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets

    PubMed Central

    Nuovo, Gerard J; Elton, Terry S; Nana-Sinkam, Patrick; Volinia, Stefano; Croce, Carlo M; Schmittgen, Thomas D

    2009-01-01

    There are relatively few protocols described for the in situ detection of microRNA (miRNA) and they often use cryostat sections, signal amplification and hybridization or washes of 50−60 °C. This protocol describes in situ miRNA detection that can be done in paraffin-embedded, formalin-fixed tissue. Detection of the miRNA precursors can be done by RT in situ PCR, which can theoretically detect one copy per cell. The key variable for the RT in situ PCR protocol is optimal protease digestion, which is then followed by overnight DNase digestion and target specific incorporation of the reported nucleotide into the amplified cDNA. Detection of mature miRNAs is achieved by in situ hybridization with locked nucleic acid probes. This part of the protocol involves a brief protease digestion, followed by an overnight hybridization, short low stringency wash and detection of the labeled probe. The key variables for this method include probe concentration and stringency conditions. Each miRNA in situ method takes 1 d. The final step of the protocol involves colabeling by immunohistochemistry for the putative target of the miRNA, which is done after the in situ hybridization step and takes a few hours. PMID:19131963

  10. Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

    SciTech Connect

    Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko; Chiba, Joe

    2009-09-11

    We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventional DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.

  11. NF-E2, FLI1 and RUNX1 collaborate at areas of dynamic chromatin to activate transcription in mature mouse megakaryocytes

    PubMed Central

    Zang, Chongzhi; Luyten, Annouck; Chen, Justina; Liu, X. Shirley; Shivdasani, Ramesh A.

    2016-01-01

    Mutations in mouse and human Nfe2, Fli1 and Runx1 cause thrombocytopenia. We applied genome-wide chromatin dynamics and ChIP-seq to determine these transcription factors’ (TFs) activities in terminal megakaryocyte (MK) maturation. Enhancers with H3K4me2-marked nucleosome pairs were most enriched for NF-E2, FLI and RUNX sequence motifs, suggesting that this TF triad controls much of the late MK program. ChIP-seq revealed NF-E2 occupancy near previously implicated target genes, whose expression is compromised in Nfe2-null cells, and many other genes that become active late in MK differentiation. FLI and RUNX were also the motifs most enriched near NF-E2 binding sites and ChIP-seq implicated FLI1 and RUNX1 in activation of late MK, including NF-E2-dependent, genes. Histones showed limited activation in regions of single TF binding, while enhancers that bind NF-E2 and either RUNX1, FLI1 or both TFs gave the highest signals for TF occupancy and H3K4me2; these enhancers associated best with genes activated late in MK maturation. Thus, three essential TFs co-occupy late-acting cis-elements and show evidence for additive activity at genes responsible for platelet assembly and release. These findings provide a rich dataset of TF and chromatin dynamics in primary MK and explain why individual TF losses cause thrombopocytopenia. PMID:27457419

  12. NF-E2, FLI1 and RUNX1 collaborate at areas of dynamic chromatin to activate transcription in mature mouse megakaryocytes.

    PubMed

    Zang, Chongzhi; Luyten, Annouck; Chen, Justina; Liu, X Shirley; Shivdasani, Ramesh A

    2016-01-01

    Mutations in mouse and human Nfe2, Fli1 and Runx1 cause thrombocytopenia. We applied genome-wide chromatin dynamics and ChIP-seq to determine these transcription factors' (TFs) activities in terminal megakaryocyte (MK) maturation. Enhancers with H3K4me2-marked nucleosome pairs were most enriched for NF-E2, FLI and RUNX sequence motifs, suggesting that this TF triad controls much of the late MK program. ChIP-seq revealed NF-E2 occupancy near previously implicated target genes, whose expression is compromised in Nfe2-null cells, and many other genes that become active late in MK differentiation. FLI and RUNX were also the motifs most enriched near NF-E2 binding sites and ChIP-seq implicated FLI1 and RUNX1 in activation of late MK, including NF-E2-dependent, genes. Histones showed limited activation in regions of single TF binding, while enhancers that bind NF-E2 and either RUNX1, FLI1 or both TFs gave the highest signals for TF occupancy and H3K4me2; these enhancers associated best with genes activated late in MK maturation. Thus, three essential TFs co-occupy late-acting cis-elements and show evidence for additive activity at genes responsible for platelet assembly and release. These findings provide a rich dataset of TF and chromatin dynamics in primary MK and explain why individual TF losses cause thrombopocytopenia. PMID:27457419

  13. Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

    PubMed Central

    Zavašnik-Bergant, Tina; Bergant Marušič, Martina

    2016-01-01

    Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii. PMID:26960148

  14. Mechanically driven activation of polyaniline into its conductive form.

    PubMed

    Baytekin, Bilge; Baytekin, H Tarik; Grzybowski, Bartosz A

    2014-07-01

    Mechanical treatment of polymers produces surface cations and anions which, as demonstrated here for the first time, can drive chemical reactions. In particular, it is shown that such a mechanical treatment transforms nonconductive polyaniline into its conductive form. These results provide a mechanical means of patterning conductive polymers and also coating small polymer objects with conductive polyaniline films preventing accumulation of static electricity. PMID:24824971

  15. Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin.

    PubMed

    Handley, Lindsey D; Treuheit, Nicholas A; Venkatesh, Varun J; Komives, Elizabeth A

    2015-11-01

    Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation. PMID:26468766

  16. Forming a Learning Culture to Promote Fracture Prevention Activities

    ERIC Educational Resources Information Center

    Hjalmarson, Helene V.; Strandmark, Margaretha

    2012-01-01

    Purpose: The purpose of this paper is to explore interprofessional experiences of incorporating fracture prevention activities in clinical practice inspired by an empowerment approach. Design/methodology/approach: Data collection consisted primarily of focus groups interviews, systematized and analyzed by the grounded theory method. The study took…

  17. Leptin promotes fetal lung maturity and upregulates SP-A expression in pulmonary alveoli type-II epithelial cells involving TTF-1 activation.

    PubMed

    Chen, Hui; Zhang, Jian-Ping; Huang, Hui; Wang, Zhen-Hua; Cheng, Rui; Cai, Wei-Bin

    2013-01-01

    The placental hormone leptin has important functions in fetal and neonatal growth, and prevents depressed respiration in leptin-deficient mice. The effect of leptin on respiratory distress suffered by low birth weight and premature infants has been studied. However, it is unclear how leptin enhances lung maturity in the fetus and ameliorates neonatal respiratory distress. In the present study, we found that antenatal treatment with leptin for 2 d significantly enhanced the relative alveolus area and improved the maturity of fetal lungs in a rat model of fetal growth restriction (FGR). Mean birth weight and lung wet weight were higher in the leptin-treated group than in the PBS-treated group, indicating promotion of fetal growth. Leptin upregulated the intracellular expression and extracellular secretion of surfactant protein (SP) A in type-II alveolar epithelial cells (AECs) in vivo and in vitro. Dual positive effects of leptin were found on protein expression and transcriptional activity of thyroid transcription factor-1 (TTF-1), a nuclear transcription essential for branching morphogenesis of the lung and expression of SP-A in type-II AECs. Knockdown of TTF-1 by RNA interference indicated that TTF-1 may play a vital role in leptin-induced SP-A expression. These results suggest that leptin may have great therapeutic potential for the treatment of FGR, and leptin-mediated SP-A induction and lung maturity of the fetus are TTF-1 dependent. PMID:23894445

  18. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  19. Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

    PubMed Central

    de la Fuente-Núñez, César; Mansour, Sarah C.; Wang, Zhejun; Jiang, Lucy; Breidenstein, Elena B.M.; Elliott, Melissa; Reffuveille, Fany; Speert, David P.; Reckseidler-Zenteno, Shauna L.; Shen, Ya; Haapasalo, Markus; Hancock, Robert E.W.

    2014-01-01

    Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients. PMID:26221537

  20. An 11-kDa form of human immunodeficiency virus protease expressed in Escherichia coli is sufficient for enzymatic activity.

    PubMed Central

    Graves, M C; Lim, J J; Heimer, E P; Kramer, R A

    1988-01-01

    In order to define the protease domain of human immunodeficiency virus 1, various regions of the pol open reading frame were cloned and expressed in Escherichia coli. Antiserum directed against the conserved retroviral protease active site was used to identify pol precursor and processed species containing the presumed protease domain. The smallest product that accumulates is about 11 kDa as measured by NaDodSO4/PAGE. This size agrees with that predicted from the presence in this region of two Phe-Pro sequences, which is one of the cleavage sites recognized by HIV protease. DNA encoding only the predicted 11-kDa protein was cloned, bypassing the need for autoprocessing, and the protein was expressed to a high level in E. coli. This form is active as demonstrated by its ability to specifically cleave protease-deficient pol protein in vivo in E. coli. Extracts of E. coli containing the 11-kDa protease also process human immunodeficiency virus gag substrates in vitro. These results demonstrate that the 11-kDa protease is sufficient for enzymatic activity and are consistent with a major role for this form in virus maturation. Images PMID:3282230

  1. Proliferative activity of preovulatory follicles and newly formed corpora lutea in cycling rats from late prooestrus to early oestrus

    PubMed Central

    GAYTÁN, FRANCISCO; BELLIDO, CARMEN; MORALES, CONCEPCIÓN; AGUILAR, ENRIQUE; SÁNCHEZ-CRIADO, JOSÉ EUGENIO

    1997-01-01

    Ovaries from adult cycling rats were studied from 1600 h on the day of prooestrus to 0700 h on the day of oestrus in order to relate the cyclic hormonal changes to the proliferative activity of preovulatory and postovulatory (i.e. newly-formed corpora lutea) follicles. Proliferative activity was studied by the immunohistochemical demonstration of DNA-incorporated 5-bromodeoxyuridine (BrdU). The proliferative activity of granulosa cells (GC) in large preovulatory follicles showed a centripetal pattern and decreased during prooestrus, reaching a minimum at 2100 h. However, a proliferative wave was found in the GC of preovulatory follicles at 0200 h on the day of oestrus and in those of newly-formed corpora lutea at 0700 h on the day of oestrus. These results suggest that the granulosa cells of preovulatory follicles show maturational changes that followed a different pattern, depending on their location within the follicle, and that the proliferative wave found from 0200 to 0700 h on oestrus is important for the establishment of the number of steroidogenic cells in the cyclic corpus luteum. PMID:9418999

  2. Active Curved Polymers Form Vortex Patterns on Membranes

    NASA Astrophysics Data System (ADS)

    Denk, Jonas; Huber, Lorenz; Reithmann, Emanuel; Frey, Erwin

    2016-04-01

    Recent in vitro experiments with FtsZ polymers show self-organization into different dynamic patterns, including structures reminiscent of the bacterial Z ring. We model FtsZ polymers as active particles moving along chiral, circular paths by Brownian dynamics simulations and a Boltzmann approach. Our two conceptually different methods point to a generic phase behavior. At intermediate particle densities, we find self-organization into vortex structures including closed rings. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation.

  3. Active Curved Polymers Form Vortex Patterns on Membranes.

    PubMed

    Denk, Jonas; Huber, Lorenz; Reithmann, Emanuel; Frey, Erwin

    2016-04-29

    Recent in vitro experiments with FtsZ polymers show self-organization into different dynamic patterns, including structures reminiscent of the bacterial Z ring. We model FtsZ polymers as active particles moving along chiral, circular paths by Brownian dynamics simulations and a Boltzmann approach. Our two conceptually different methods point to a generic phase behavior. At intermediate particle densities, we find self-organization into vortex structures including closed rings. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation. PMID:27176542

  4. 75 FR 21013 - Agency Information Collection Activities: Form N-644; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-22

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-644... Collection Under Review; Form N- 644, Application for Posthumous Citizenship; OMB Control No. 1615-0059. The..., USCIS will be evaluating whether to revise the Form N-644. Should USCIS decide to revise Form N-644...

  5. 76 FR 21913 - Agency Information Collection Activities: Form N-644; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-19

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-644... Collection Under Review: Form N- 644, Application for Posthumous Citizenship; OMB Control No. 1615-0059. The..., USCIS will be evaluating whether to revise the Form N-644. Should USCIS decide to revise Form N-644...

  6. 75 FR 51095 - Agency Information Collection Activities: Form N-336; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-18

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-336... Collection under Review; Form N- 336, Request for Hearing on a Decision in Naturalization Proceedings Under... the Form N-336. Should USCIS decide to revise Form N-336 we will advise the public when we publish...

  7. Heat of Hydration of Low Activity Cementitious Waste Forms

    SciTech Connect

    Nasol, D.

    2015-07-23

    During the curing of secondary waste grout, the hydraulic materials in the dry mix react exothermally with the water in the secondary low-activity waste (LAW). The heat released, called the heat of hydration, can be measured using a TAM Air Isothermal Calorimeter. By holding temperature constant in the instrument, the heat of hydration during the curing process can be determined. This will provide information that can be used in the design of a waste solidification facility. At the Savannah River National Laboratory (SRNL), the heat of hydration and other physical properties are being collected on grout prepared using three simulants of liquid secondary waste generated at the Hanford Site. From this study it was found that both the simulant and dry mix each had an effect on the heat of hydration. It was also concluded that the higher the cement content in the dry materials mix, the greater the heat of hydration during the curing of grout.

  8. Identification of biotransformation products of citalopram formed in activated sludge.

    PubMed

    Beretsou, Vasiliki G; Psoma, Aikaterini K; Gago-Ferrero, Pablo; Aalizadeh, Reza; Fenner, Kathrin; Thomaidis, Nikolaos S

    2016-10-15

    Citalopram (CTR) is a worldwide highly consumed antidepressant which has demonstrated incomplete removal by conventional wastewater treatment. Despite its global ubiquitous presence in different environmental compartments, little is known about its behaviour and transformation processes during wastewater treatment. The present study aims to expand the knowledge on fate and transformation of CTR during the biological treatment process. For this purpose, batch reactors were set up to assess biotic, abiotic and sorption losses of this compound. One of the main objectives of the study was the identification of the formed transformation products (TPs) by applying suspect and non-target strategies based on liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). The complementary use of reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) for the identification of polar TPs, and the application of in-house developed quantitative structure-retention relationship (QSRR) prediction models, in addition to the comprehensive evaluation of the obtained MS/MS spectra, provided valuable information to support identification. In total, fourteen TPs were detected and thirteen of them were tentatively identified. Four compounds were confirmed (N-desmethylCTR, CTR amide, CTR carboxylic acid and 3-oxo-CTR) through the purchase of the corresponding reference standard. Probable structures based on diagnostic evidence were proposed for the additional nine TPs. Eleven TPs are reported for the first time. A transformation pathway for the biotransformation of CTR was proposed. The presence of the identified TPs was assessed in real wastewater samples through retrospective analysis, resulting in the detection of five compounds. Finally, the potential ecotoxicological risk posed by CTR and its TPs to different trophic levels of aquatic organisms was evaluated by means of risk quotients. PMID:27459150

  9. Wound healing activity of topical application forms based on ayurveda.

    PubMed

    Datta, Hema Sharma; Mitra, Shankar Kumar; Patwardhan, Bhushan

    2011-01-01

    The traditional Indian medicine-Ayurveda, describes various herbs, fats, oils and minerals with anti-aging as well as wound healing properties. With aging, numerous changes occur in skin, including decrease in tissue cell regeneration, decrease in collagen content, loss of skin elasticity and mechanical strength. We prepared five topical anti-aging formulations using cow ghee, flax seed oil, Phyllanthus emblica fruits, Shorea robusta resin, Yashada bhasma as study materials. For preliminary efficacy evaluation of the anti-aging activity we chose excision and incision wound healing animal models and studied the parameters including wound contraction, collagen content and skin breaking strength which in turn is indicative of the tissue cell regeneration capacity, collagenation capacity and mechanical strength of skin. The group treated with the formulations containing Yashada bhasma along with Shorea robusta resin and flax seed oil showed significantly better wound contraction (P < .01), higher collagen content (P < .05) and better skin breaking strength (P < .01) as compared to control group; thus proposing them to be effective prospective anti-aging formulations. PMID:19252191

  10. Wound Healing Activity of Topical Application Forms Based on Ayurveda

    PubMed Central

    Datta, Hema Sharma; Mitra, Shankar Kumar; Patwardhan, Bhushan

    2011-01-01

    The traditional Indian medicine—Ayurveda, describes various herbs, fats, oils and minerals with anti-aging as well as wound healing properties. With aging, numerous changes occur in skin, including decrease in tissue cell regeneration, decrease in collagen content, loss of skin elasticity and mechanical strength. We prepared five topical anti-aging formulations using cow ghee, flax seed oil, Phyllanthus emblica fruits, Shorea robusta resin, Yashada bhasma as study materials. For preliminary efficacy evaluation of the anti-aging activity we chose excision and incision wound healing animal models and studied the parameters including wound contraction, collagen content and skin breaking strength which in turn is indicative of the tissue cell regeneration capacity, collagenation capacity and mechanical strength of skin. The group treated with the formulations containing Yashada bhasma along with Shorea robusta resin and flax seed oil showed significantly better wound contraction (P < .01), higher collagen content (P < .05) and better skin breaking strength (P < .01) as compared to control group; thus proposing them to be effective prospective anti-aging formulations. PMID:19252191

  11. [L forms of Staphylococcus aureus. Behavior of coagulase, hemolytic and desoxyribonuclease activities and antibiotic sensitivity].

    PubMed

    Loschiavo, F; Giarrizzo, S

    1977-01-01

    L Forms derived from strains of coagulase positive Staphylococcus aureus, have, on the whole, preserved their DNAsic, haemolitic and coagulastic activities. L. forms showed high resistence to antibiotics acting on the bacterial cell-wall. The sensibility to other antibiotics was, roughly, analogous for the L forms as well as for the bacterial strains ones, with the exception of the clortetraciclin and the diidrostreptomicin, ehich proved to be comparatively more active on the L forms. PMID:614141

  12. Loss of Mitochondrial Malate Dehydrogenase Activity Alters Seed Metabolism Impairing Seed Maturation and Post-Germination Growth in Arabidopsis.

    PubMed

    Sew, Yun Shin; Ströher, Elke; Fenske, Ricarda; Millar, A Harvey

    2016-06-01

    Mitochondrial malate dehydrogenase (mMDH; EC 1.1.1.37) has multiple roles; the most commonly described is its catalysis of the interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. The roles of mMDH in Arabidopsis (Arabidopsis thaliana) seed development and germination were investigated in mMDH1 and mMDH2 double knockout plants. A significant proportion of mmdh1mmdh2 seeds were nonviable and developed only to torpedo-shaped embryos, indicative of arrested seed embryo growth during embryogenesis. The viable mmdh1mmdh2 seeds had an impaired maturation process that led to slow germination rates as well as retarded post-germination growth, shorter root length, and decreased root biomass. During seed development, mmdh1mmdh2 showed a paler green phenotype than the wild type and exhibited deficiencies in reserve accumulation and reduced final seed biomass. The respiration rate of mmdh1mmdh2 seeds was significantly elevated throughout their maturation, consistent with the previously reported higher respiration rate in mmdh1mmdh2 leaves. Mutant seeds showed a consistently higher content of free amino acids (branched-chain amino acids, alanine, serine, glycine, proline, and threonine), differences in sugar and sugar phosphate levels, and lower content of 2-oxoglutarate. Seed-aging assays showed that quiescent mmdh1mmdh2 seeds lost viability more than 3 times faster than wild-type seeds. Together, these data show the important role of mMDH in the earliest phases of the life cycle of Arabidopsis. PMID:27208265

  13. Lateralization, maturation, and anteroposterior topography in the lateral habenula revealed by ZIF268/EGR1 immunoreactivity and labeling history of neuronal activity.

    PubMed

    Ichijo, Hiroyuki; Hamada, Michito; Takahashi, Satoru; Kobayashi, Makoto; Nagai, Takeharu; Toyama, Tomoko; Kawaguchi, Masahumi

    2015-06-01

    We report habenular lateralization in a simple transgenic mouse model used for labeling a facet of neuronal activity history. A transgenic construct comprised of a zif268/egr1 immediate-early gene promoter and a gene for normal Venus fluorescent protein with a membrane tag converted promoter activity into long-life fluorescent proteins, which was thought to describe a facet of neuronal activity history by summing neuronal activity. In addition to mapping the immediate-early gene-immunopositive cells, this method helped demonstrate the functionality of the lateral habenular nucleus (LHb). During postnatal development, the LHb was activated between postnatal days 10 and 16. The water-immersion restraint stress also activated the LHb over a similar period. LHb activation was functionally lateralized, but had no directional bias at the population level. Moreover, the posterior LHb was activated in the early stage after the stress, while the anterior LHb was activated in the later stage. Our results indicate lateralization, maturation, and anteroposterior topography of the LHb during postnatal development and the stress response. PMID:25637311

  14. Constitutive nuclear factor-kappaB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes by controlling the expression of distinct Bcl-2 family proteins.

    PubMed

    Bureau, Fabrice; Vanderplasschen, Alain; Jaspar, Fabrice; Minner, Frédéric; Pastoret, Paul-Pierre; Merville, Marie-Paule; Bours, Vincent; Lekeux, Pierre

    2002-05-15

    Constitutive nuclear factor kappaB (NF-kappaB) activity protects quiescent mature immune cells from spontaneous apoptosis. Here, we examined whether NF-kappaB exerts its antiapoptotic function in these cells through the control of Bcl-2 family proteins. Specific pharmacologic inhibitors of NF-kappaB were used to achieve total NF-kappaB inactivation in quiescent human blood lymphocytes, granulocytes, and monocytes. NF-kappaB inhibition induced drastic lymphocyte and granulocyte apoptosis, but only moderate monocyte apoptosis. T- and B-cell apoptosis was slow and associated with a gradual down-regulation of the prosurvival Bcl-2 family proteins Bcl-x(L) and Bcl-2, respectively. By contrast, granulocyte apoptosis was fast and accompanied by a rapid cellular accumulation of Bcl-x(S), the proapoptotic Bcl-x isoform that is generated from alternative splicing of the bcl-x pre-mRNA. Finally, antisense bcl-x(L) and bcl-2 knockdown in T and B cells, respectively, and induction of Bcl-x(S) expression in granulocytes through antisense oligonucleotide-mediated redirection of bcl-x pre-mRNA splicing were sufficient to induce significant apoptosis in these cells. Taken together, these results reveal that basal NF-kappaB activity preserves homeostasis of quiescent mature lymphocytes and granulocytes through regulation of distinct members of the Bcl-2 family. This study sheds light on the constitutive mechanisms by which NF-kappaB maintains defense integrity. PMID:11986224

  15. Roles of the signal peptide and mature domains in the secretion and maturation of the neutral metalloprotease from Streptomyces cacaoi.

    PubMed Central

    Chang, S C; Su, M H; Lee, Y H

    1997-01-01

    The neutral metalloprotease (Npr) of Streptomyces cacaoi is synthesized as a prepro-Npr precursor form consisting of a secretory signal peptide, a propeptide and the mature metalloprotease. The maturation of Npr occurs extracellularly via an autoproteolytic processing of the secreted pro-Npr. The integrity of the propeptide is essential for the formation of mature active Npr but not for its secretion [Chang, Chang and Lee (1994) J. Biol. Chem. 269, 3548-3554]. In this study we investigated whether the secretion and maturation of Npr require the integrity of its signal peptide region and mature protease domain. Five signal peptide mutants were generated, including the substitution mutations at the positively charged region (mutant IR6LE), the central hydrophobic region (mutants GI19EL and G19N), the boundary of the hydrophobic core-cleavage region (mutant P30L) and at the residues adjacent to the signal peptidase cleavage site (mutant YA33SM). All these lesions delayed the export of Npr to the growth medium and also resulted in a 2-10-fold decrease in Npr export. The most severe effect was noted in mutants GI19EL and P30L. When these signal peptide mutations were fused separately with the propeptide lacking the Npr mature domain, the secretory defect on the propeptide was also observed, and this impairment was again more severely expressed in mutants GI19EL and P30L. Thus the Npr signal peptide seems to have more constraints on the hydrophobic core region and at the proline residue within the boundary of the hydrophobic core-cleavage site. Deletion mutations within the C-terminal mature protease domain that left its active site intact still blocked the proteolytic processing of mutant precursor forms of pro-Npr, although their secretions were unaffected. These results, together with our previous findings, strongly suggest that the signal peptide of Npr plays a pivotal role in the secretion of both Npr and the propeptide, but not in the maturation of Npr. On the

  16. Meiotic maturation of incompetent prepubertal sheep oocytes is induced by paracrine factor(s) released by gonadotropin-stimulated oocyte-cumulus cell complexes and involves mitogen-activated protein kinase activation.

    PubMed

    Cecconi, Sandra; Mauro, Annunziata; Capacchietti, Giulia; Berardinelli, Paolo; Bernabò, Nicola; Di Vincenzo, Anna Rita; Mattioli, Mauro; Barboni, Barbara

    2008-01-01

    In this study, sheep oocyte-cumulus cell complexes (OCC) derived from medium (M) antral follicles (M-OCC) were in vitro matured alone or in coculture with OCC derived from small (S) antral follicles (S-OCC) to investigate the contribution of cumulus cells (CC) and oocytes to the process of oocyte meiotic maturation and cumulus expansion (CE). Experiments were conducted with or without gonadotropins (FSH/LH). Regardless of culture conditions, about 12% of S-oocytes reached the metaphase II stage, and S-CC showed a low degree of CE. In contrast, both maturational processes were significantly stimulated by gonadotropins in M-OCC. However, about 48% of S-oocytes progressed to metaphase II, and S-CC expanded after coculture with gonadotropin-stimulated M-OCC and M-CC but not with mural granulosa cells. Both maturational processes were inhibited when S-OCC were cocultured with M-denuded oocytes, or when S-denuded oocytes were cocultured with M-CC. The capacity of these paracrine factor(s) to activate the MAPK pathway in somatic and germ cells of S-complexes was investigated. It was found that MAPK kinase/MAPK phosphorylation levels in M-OCC but not in S-OCC were significantly increased by gonadotropins, first in CC and later in the oocytes. Kinase phosphorylations were activated only in S-oocytes cocultured with M-OCC or M-CC. These results demonstrate that soluble factors specifically produced by M-CC are capable to induce meiotic maturation and CE in S-complexes by acting via CC. These factors can induce MAPK activation only in S-oocytes, whose meiotic arrest could be due to the inability of surrounding CC to respond to gonadotropin stimulation. PMID:17884935

  17. Re-splicing of mature mRNA in cancer cells promotes activation of distant weak alternative splice sites

    PubMed Central

    Kameyama, Toshiki; Suzuki, Hitoshi; Mayeda, Akila

    2012-01-01

    Transcripts of the human tumor susceptibility gene 101 (TSG101) are aberrantly spliced in many cancers. A major aberrant splicing event on the TSG101 pre-mRNA involves joining of distant alternative 5′ and 3′ splice sites within exon 2 and exon 9, respectively, resulting in the extensive elimination of the mRNA. The estimated strengths of the alternative splice sites are much lower than those of authentic splice sites. We observed that the equivalent aberrant mRNA could be generated from an intron-less TSG101 gene expressed ectopically in breast cancer cells. Remarkably, we identified a pathway-specific endogenous lariat RNA consisting solely of exonic sequences, predicted to be generated by a re-splicing between exon 2 and exon 9 on the spliced mRNA. Our results provide evidence for a two-step splicing pathway in which the initial constitutive splicing removes all 14 authentic splice sites, thereby bringing the weak alternative splice sites into close proximity. We also demonstrate that aberrant multiple-exon skipping of the fragile histidine triad (FHIT) pre-mRNA in cancer cells occurs via re-splicing of spliced FHIT mRNA. The re-splicing of mature mRNA can potentially generate mutation-independent diversity in cancer transcriptomes. Conversely, a mechanism may exist in normal cells to prevent potentially deleterious mRNA re-splicing events. PMID:22675076

  18. Composition and topology of activity cliff clusters formed by bioactive compounds.

    PubMed

    Stumpfe, Dagmar; Dimova, Dilyana; Bajorath, Jürgen

    2014-02-24

    The assessment of activity cliffs has thus far mostly focused on compound pairs, although the majority of activity cliffs are not formed in isolation but in a coordinated manner involving multiple active compounds and cliffs. However, the composition of coordinated activity cliff configurations and their topologies are unknown. Therefore, we have identified all activity cliff configurations formed by currently available bioactive compounds and analyzed them in network representations where activity cliff configurations occur as clusters. The composition, topology, frequency of occurrence, and target distribution of activity cliff clusters have been determined. A limited number of large cliff clusters with unique topologies were identified that were centers of activity cliff formation. These clusters originated from a small number of target sets. However, most clusters were of small to moderate size. Three basic topologies were sufficient to describe recurrent activity cliff cluster motifs/topologies. For example, frequently occurring clusters with star topology determined the scale-free character of the global activity cliff network and represented a characteristic activity cliff configuration. Large clusters with complex topology were often found to contain different combinations of basic topologies. Our study provides a first view of activity cliff configurations formed by currently available bioactive compounds and of the recurrent topologies of activity cliff clusters. Activity cliff clusters of defined topology can be selected, and from compounds forming the clusters, SAR information can be obtained. The SAR information of activity cliff clusters sharing a/one specific activity and topology can be compared. PMID:24437577

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  7. Dance as a Fitness Activity: The Impact of Teaching Style and Dance Form.

    ERIC Educational Resources Information Center

    Fromel, Karel; Vasendova, Jana; Stratton, Gareth; Pangrazi, Robert P.

    2002-01-01

    Analyzed the amount of activity, intensity of activity, and attitudes of participants in Czech high school physical education classes taught using different teaching styles and dance forms. Measurements of heart rate and dance intensity and student surveys indicated that teaching style and dance form significantly impacted the intensity and volume…

  8. Role of pro-brain-derived neurotrophic factor (proBDNF) to mature BDNF conversion in activity-dependent competition at developing neuromuscular synapses

    PubMed Central

    Je, H. Shawn; Yang, Feng; Ji, Yuanyuan; Nagappan, Guhan; Hempstead, Barbara L.; Lu, Bai

    2012-01-01

    Formation of specific neuronal connections often involves competition between adjacent axons, leading to stabilization of the active terminal, while retraction of the less active ones. The underlying molecular mechanisms remain unknown. We show that activity-dependent conversion of pro–brain-derived neurotrophic factor (proBDNF) to mature (m)BDNF mediates synaptic competition. Stimulation of motoneurons triggers proteolytic conversion of proBDNF to mBDNF at nerve terminals. In Xenopus nerve–muscle cocultures, in which two motoneurons innervate one myocyte, proBDNF-p75NTR signaling promotes retraction of the less active terminal, whereas mBDNF–tyrosine-related kinase B (TrkB) p75NTR (p75 neurotrophin receptor) facilitates stabilization of the active one. Thus, proBDNF and mBDNF may serve as potential “punishment” and “reward” signals for inactive and active terminals, respectively, and activity-dependent conversion of proBDNF to mBDNF may regulate synapse elimination. PMID:23019376

  9. Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion

    PubMed Central

    Sun, Wei; Hu, Wei; Xu, Ruijuan; Jin, Junfei; Szulc, Zdzislaw M.; Zhang, Guofeng; Galadari, Sehamuddin H.; Obeid, Lina M.; Mao, Cungui

    2009-01-01

    The polypeptide core of the integrin β1 subunit (β1) is glycosylated sequentially in the endoplasmic reticulum and the Golgi complex to form β1 precursor and mature β1, respectively. The β1 precursor to mature β1 conversion, termed β1 maturation, regulates the cell surface levels and function of β1-containing integrins, β1 integrins. Here we demonstrate that the human alkaline ceramidase 2 (ACER2), a Golgi enzyme, regulates β1 maturation by controlling the generation of sphingosine. ACER2 overexpression inhibited β1 maturation, thus leading to a decrease in the levels of mature β1 in T-REx HeLa cells, whereas RNA interference-mediated knockdown of ACER2 enhanced β1 maturation in MCF-7 cells. ACER2 overexpression decreased the cell surface levels of β1 integrins, thus inhibiting cell adhesion to fibronectin or collagen, whereas ACER2 knockdown has the opposite effects. Treatment with all-trans retinoic acid (ATRA) increased both the expression of ACER2 and the generation of sphingosine in HeLa cells and inhibited β1 maturation. ACER2 knockdown attenuated the inhibitory effects of ATRA on both β1 maturation and cell adhesion. In contrast, treatment with phorbol myristate acetate (PMA), a protein kinase C activator, decreased the expression of ACER2 and sphingosine in T-REx HeLa cells, thus enhancing β1 maturation. ACER2 overexpression inhibited the stimulatory effects of PMA on both β1 maturation and cell adhesion. These results suggest that the ACER2/sphingosine pathway plays an important role in regulating β1 maturation and cell adhesion mediated by β1 integrins.—Sun, W., Hu, W., Xu, R., Jin, J., Szulc, Z. M., Zhang, G., Galadari, S. H., Obeid, L. M, Mao, C. Alkaline ceramidase 2 regulates β1 integrin maturation and cell adhesion. PMID:18945876

  10. In vitro activity of Rutaceae species against the trypomastigote form of Trypanosoma cruzi.

    PubMed

    Mafezoli, J; Vieira, P C; Fernandes, J B; da Silva, M F; de Albuquerque, S

    2000-11-01

    The activity of crude plant extracts of nine species of Rutaceae against the trypomastigote form of Trypanosoma cruzi was evaluated at 4 mg/ml. Thirty-two crude extracts were tested and eight of them showed significant activity (>80%). The most active extract was obtained from the stems of Pilocarpus spicatus (97.3%). Fractionation of the active crude extracts provided 25 fractions which were tested against the trypomastigote form of T. cruzi at 2 mg/ml. Of these six showed significant activity (>80%). The most active fractions (100%) were obtained from the leaves of Almeidea coerulea (butanol fraction) and Conchocarpus inopinatus (dichloromethane fraction). PMID:11025175

  11. Extracellular ATP inhibits chloride channels in mature mammalian skeletal muscle by activating P2Y1 receptors.

    PubMed

    Voss, Andrew A

    2009-12-01

    ATP is released from skeletal muscle during exercise, a discovery dating back to 1969. Surprisingly, few studies have examined the effects of extracellular ATP on mature mammalian skeletal muscle. This electrophysiological study examined the effects of extracellular ATP on fully innervated rat levator auris longus using two intracellular microelectrodes. The effects of ATP were determined by measuring the relative changes of miniature endplate potentials (mEPPs) and voltage responses to step current pulses in individual muscle fibres. Exposure to ATP (20 microm) prolonged the mEPP falling phase by 31 +/- 7.5% (values +/- s.d., n = 3 fibres). Concurrently, the input resistance increased by 31 +/- 2.0% and the time course of the voltage responses increased by 59 +/- 3.0%. Analogous effects were observed using 2 and 5 microm ATP, and on regions distal from the neuromuscular junction, indicating that physiologically relevant levels of ATP enhanced electrical signalling over the entire muscle fibre. The effects of extracellular ATP were blocked by 200 microm anthracene-9-carboxylic acid, a chloride channel inhibitor, and reduced concentrations of extracellular chloride, indicating that ATP inhibited chloride channels. A high affinity agonist for P2Y receptors, 2-methylthioadenosine-5-O-diphosphate (2MeSADP), induced similar effects to ATP with an EC(50) of 160 +/- 30 nm. The effects of 250 nm2MeSADP were blocked by 500 nmMRS2179, a specific P2Y(1) receptor inhibitor, suggesting that ATP acts on P2Y(1) receptors to inhibit chloride channels. The inhibition of chloride channels by extracellular ATP has implications for muscle excitability and fatigue, and the pathophysiology of myotonias. PMID:19805741

  12. cis9, trans11-Conjugated Linoleic Acid Differentiates Mouse 3T3-L1 Preadipocytes into Mature Small Adipocytes through Induction of Peroxisome Proliferator-activated Receptor γ.

    PubMed

    Sakuma, Satoru; Nishioka, Yuki; Imanishi, Ryohta; Nishikawa, Kenji; Sakamoto, Hirotada; Fujisawa, Junji; Wada, Koichiro; Kamisaki, Yoshinori; Fujimoto, Yohko

    2010-09-01

    Dietary conjugated linoleic acid (CLA) has been reported to exhibit a number of therapeutic effects in animal models and patients, such as anti-hypertensive, anti-hyperlipidemic, anti-arteriosclerotic, anti-carcinogenic, and anti-diabetic effects. However, the underlying mechanism is not well-characterized. In the present study, the effects of cis(c)9, trans(t)11-CLA on the differentiation of mouse 3T3-L1 preadipocytes into mature adipocytes were examined. Treatment with c9, t11-CLA in the presence of insulin, dexamethasone, and 3-isobutyl-1-methyl-xanthine (differentiation cocktail) significantly stimulated the accumulation of triacylglycerol. The microscopic observation of cells stained by Oil Red O demonstrated that c9, t11-CLA increases the amount and proportion of small mature adipocytes secreting adiponectin, a benign adipocytokine, when compared to the differentiation cocktail alone. Furthermore, c9, t11-CLA increased bioactive peroxisome proliferator-activated receptor γ (PPARγ) levels in a nuclear extract of 3T3-L1 cells, suggesting the enhancing effect of this fatty acid on the nuclear transmission of PPARγ, a master regulator of adipocyte differentiation, in 3T3-L1 cells. These results suggest that the therapeutic effects of c9, t11-CLA on lifestyle-related diseases are partially due to the enhanced formation of small adipocytes from preadipocytes via PPARγ stimulation. PMID:20838573

  13. Neurite Outgrowth of Mature Retinal Ganglion Cells and PC12 Cells Requires Activity of CK1δ and CK1ε

    PubMed Central

    Bischof, Joachim; Müller, Adrienne; Fänder, Miriam

    2011-01-01

    Mature retinal ganglion cells (RGCs) do not normally regenerate severed axons after optic nerve injury and show only little neurite outgrowth in culture. However, RGCs can be transformed into an active regenerative state after lens injury (LI) enabling these neurons to regrow axons in vitro and in vivo. In the current study we investigated the role of CK1δ and CK1ε activity in neurite outgrowth of LI stimulated RGCs and nerve growth factor (NGF) stimulated PC12 cells, respectively. In both cell types CK1δ and ε were localized in granular particles aligned at microtubules in neurites and growth cones. Although LI treatment did not measurably affect the expression of CK1δ and ε, it significantly elevated the specific kinase activity in the retina. Similarly, CK1δ/ε specific kinase activity was also elevated in NGF treated PC12 cells compared with untreated controls. Neurite extension in PC12 cells was associated with a change in the activity of CK1δ C-terminal targeting kinases, suggesting that activity of these kinases might be necessary for neurite outgrowth. Pharmacological inactivation of CK1δ and ε markedly compromised neurite outgrowth of both, PC12 cells and LI stimulated RGCs in a concentration dependent manner. These data provide evidence for a so far unknown, but essential role of CK1 isoforms in neurite growth. PMID:21698236

  14. The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

    PubMed Central

    Choi, T; Fukasawa, K; Zhou, R; Tessarollo, L; Borror, K; Resau, J; Vande Woude, G F

    1996-01-01

    Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692939

  15. Fibrils Colocalize Caspase-3 with Procaspase-3 to Foster Maturation*

    PubMed Central

    Zorn, Julie A.; Wolan, Dennis W.; Agard, Nicholas J.; Wells, James A.

    2012-01-01

    Most proteases are expressed as inactive precursors, or zymogens, that become activated by limited proteolysis. We previously identified a small molecule, termed 1541, that dramatically promotes the maturation of the zymogen, procaspase-3, to its mature form, caspase-3. Surprisingly, compound 1541 self-assembles into nanofibrils, and localization of procaspase-3 to the fibrils promotes activation. Here, we interrogate the biochemical mechanism of procaspase-3 activation on 1541 fibrils in addition to proteogenic amyloid-β(1–40) fibrils. In contrast to previous reports, we find no evidence that procaspase-3 alone is capable of self-activation, consistent with its fate-determining role in executing apoptosis. In fact, mature caspase-3 is >107-fold more active than procaspase-3, making this proenzyme a remarkably inactive zymogen. However, we also show that fibril-induced colocalization of trace amounts of caspase-3 or other initiator proteases with procaspase-3 dramatically stimulates maturation of the proenzyme in vitro. Thus, similar to known cellular signaling complexes, these synthetic or natural fibrils can serve as platforms to concentrate procaspase-3 for trans-activation by upstream proteases. PMID:22872644

  16. The distal upstream promoter in Ly49 genes, Pro1, is active in mature NK cells and T cells, does not require TATA boxes, and displays enhancer activity1

    PubMed Central

    Gays, Frances; Taha, Sally; Brooks, Colin G.

    2016-01-01

    Missing self recognition of MHC class I molecules is mediated in murine species through the stochastic expression of CD94/NKG2 and Ly49 receptors on NK cells. Previous studies have suggested that the stochastic expression of Ly49 receptors is achieved through the use of an alternate upstream promoter, designated Pro1, that is active only in immature NK cells, and operates via the mutually exclusive binding of transcription initiation complexes to closely opposed forward and reverse TATA boxes, forward transcription being transiently required to activate the downstream promoters, Pro2/Pro3, that are subsequently responsible for transcription in mature NK cells. Here we report that Pro1 transcripts are not restricted to immature NK cells but are also found in mature NK cells and T cells, and that Pro1-fragments display strong promoter activity in mature NK cell and T cell lines as well as in immature NK cells. However, the strength of promoter activity in vitro does not correlate well with Ly49 expression in vivo and forward promoter activity is generally weak or undetectable, suggesting that components outside of Pro1 are required for efficient forward transcription. Indeed, conserved sequences immediately upstream and downstream of the core Pro1 region were found to inhibit or enhance promoter activity. Most surprisingly, promoter activity does not require either the forward or reverse TATA boxes, but is instead dependent on residues in the largely invariant central region of Pro1. Importantly, Pro1 displays strong enhancer activity suggesting that this may be its principal function in vivo. PMID:25926675

  17. Activity of Ocimum basilicum, Ocimum canum, and Cymbopogon citratus essential oils against Plasmodium falciparum and mature-stage larvae of Anopheles funestus s.s.

    PubMed

    Akono Ntonga, Patrick; Baldovini, Nicolas; Mouray, Elisabeth; Mambu, Lengo; Belong, Philippe; Grellier, Philippe

    2014-01-01

    The biological activities of essential oils from three plants grown in Cameroon: Ocimum basilicum, Ocimum canum, and Cymbopogon citratus were tested against Plasmodium falciparum and mature-stage larvae of Anopheles funestus. Gas chromatography and gas chromatography - mass spectrometry analyses showed that the main compounds are geranial, 1,8-cineole and linalool in C. citratus, O. canum and O. basilicum, respectively. Larvicidal tests carried out according to the protocol recommended by the World Health Organization showed that the essential oil of leaves of C. citratus is the most active against larvae of An. funestus (LC50 values = 35.5 ppm and 34.6 ppm, respectively, for larval stages III and IV after 6 h of exposure). Besides, the in vitro anti-plasmodial activity evaluated by the radioisotopic method showed that the C. citratus oil is the most active against P. falciparum, with an IC50 value of 4.2 ± 0.5 μg/mL compared with O. canum (20.6 ± 3.4 μg/mL) and O. basilicum (21 ± 4.6 μg/mL). These essential oils can be recommended for the development of natural biocides for fighting the larvae of malaria vectors and for the isolation of natural products with anti-malarial activity. PMID:24995776

  18. Activity of Ocimum basilicum, Ocimum canum, and Cymbopogon citratus essential oils against Plasmodium falciparum and mature-stage larvae of Anopheles funestus s.s.

    PubMed Central

    Akono Ntonga, Patrick; Baldovini, Nicolas; Mouray, Elisabeth; Mambu, Lengo; Belong, Philippe; Grellier, Philippe

    2014-01-01

    The biological activities of essential oils from three plants grown in Cameroon: Ocimum basilicum, Ocimum canum, and Cymbopogon citratus were tested against Plasmodium falciparum and mature-stage larvae of Anopheles funestus. Gas chromatography and gas chromatography – mass spectrometry analyses showed that the main compounds are geranial, 1,8-cineole and linalool in C. citratus, O. canum and O. basilicum, respectively. Larvicidal tests carried out according to the protocol recommended by the World Health Organization showed that the essential oil of leaves of C. citratus is the most active against larvae of An. funestus (LC50 values = 35.5 ppm and 34.6 ppm, respectively, for larval stages III and IV after 6 h of exposure). Besides, the in vitro anti-plasmodial activity evaluated by the radioisotopic method showed that the C. citratus oil is the most active against P. falciparum, with an IC50 value of 4.2 ± 0.5 μg/mL compared with O. canum (20.6 ± 3.4 μg/mL) and O. basilicum (21 ± 4.6 μg/mL). These essential oils can be recommended for the development of natural biocides for fighting the larvae of malaria vectors and for the isolation of natural products with anti-malarial activity. PMID:24995776

  19. Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes.

    PubMed

    Meng, Dongheng; Dai, Meihong; Xu, Bi-Lin; Zhao, Zhong-Shu; Liang, Xiaoliang; Wang, Mingqiu; Tang, Xiao-Feng; Tang, Bing

    2016-01-01

    Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75- and 45-kDa mature forms involves both hetero- and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations. PMID:26497454

  20. Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes

    PubMed Central

    Meng, Dongheng; Dai, Meihong; Xu, Bi-Lin; Zhao, Zhong-Shu; Liang, Xiaoliang; Wang, Mingqiu; Tang, Xiao-Feng

    2015-01-01

    Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75- and 45-kDa mature forms involves both hetero- and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations. PMID:26497454

  1. 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…

  2. Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogencially activated and fertilized embryos.

    PubMed

    Martin, Jacinta H; Bromfield, Elizabeth G; Aitken, R John; Lord, Tessa; Nixon, Brett

    2016-09-01

    These data document the vitality of mature mouse oocytes (Metaphase II (MII)) and early stage embryos (zygotes) following exposure to the genotoxic chemotherapeutic agent, etoposide, in combination with PSC833, a selective inhibitor of permeability glycoprotein. They also illustrate the vitality of parthenogencially activated and fertilized embryos following incubation with the calcium chelator BAPTA-AM (1,2-Bis(2-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester)), cycloheximide (an antibiotic that is capable of inhibiting protein synthesis), and hydrogen peroxide (a potent reactive oxygen species). Finally, they present evidence that permeability glycoprotein is not represented in the proteome of mouse spermatozoa. Our interpretation and discussion of these data feature in the article "Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes" (Martin et al., in press) [1]. PMID:27547800

  3. Muscarinic Receptor Activation Affects Pulmonary Artery Contractility in Sheep: The Impact of Maturation and Chronic Hypoxia on Endothelium-Dependent and Endothelium-Independent Function.

    PubMed

    Giang, Michael; Papamatheakis, Demosthenes G; Nguyen, Dan; Paez, Ricardo; Blum Johnston, Carla; Kim, Joon; Brunnell, Alexander; Blood, Quintin; Goyal, Ravi; Longo, Lawrence D; Wilson, Sean M

    2016-06-01

    Giang, Michael, Demosthenes G. Papamatheakis, Dan Nguyen, Ricardo Paez, Carla Blum Johnston, Joon Kim, Alexander Brunnell, Quintin Blood, Ravi Goyal, Lawrence D. Longo, and Sean M. Wilson. Muscarinic receptor activation affects pulmonary artery contractility in sheep: the impact of maturation and chronic hypoxia on endothelium-dependent and endothelium-independent function. High Alt Med Biol. 17:122-132, 2015.-Muscarinic receptor activation in the pulmonary vasculature can cause endothelium-dependent vasodilation and smooth muscle-dependent vasoconstriction. Chronic hypoxia (CH) can modify both of these responses. This study aimed to assess the combined influence of CH and maturation on endothelium-dependent and endothelium-independent muscarinic-induced vasoreactivity. This was accomplished by performing wire myography on endothelium-intact or endothelium-disrupted pulmonary arterial rings isolated from normoxic or CH fetal and adult sheep. In endothelium-intact arteries, vasodilation was evaluated using cumulative bradykinin doses in phenylephrine and carbachol precontracted pulmonary arterial segments; and vasoconstriction was examined using cumulative doses of carbachol following bradykinin predilation. Effects of nonselective (atropine) and selective M1 (pirenzepine), M2 (AFDX116), and M3 (4-DAMP and Dau5884) muscarinic receptor antagonists were assessed in disrupted arteries. In normoxic arteries, bradykinin relaxation was twofold greater in the adult compared to fetus, while carbachol contraction was fourfold greater. In adult arteries, CH increased bradykinin relaxation and carbachol contraction. In vessels with intact endothelium, maturation and CH augmented maximal response and efficacy for carbachol constriction and bradykinin relaxation. Approximately 50%-80% of adult normoxic and CH endothelium-disrupted arteries contracted to acetylcholine, while ∼50% of fetal normoxic and ∼10% of CH arteries responded. Atropine reduced carbachol

  4. Heme oxygenase-1 inhibits basophil maturation and activation but promotes its apoptosis in T helper type 2-mediated allergic airway inflammation.

    PubMed

    Zhong, Wenwei; Di, Caixia; Lv, Jiajia; Zhang, Yanjie; Lin, Xiaoliang; Yuan, Yufan; Lv, Jie; Xia, Zhenwei

    2016-03-01

    The anti-inflammatory role of heme oxygenase-1 (HO-1) has been studied extensively in many disease models including asthma. Many cell types are anti-inflammatory targets of HO-1, such as dendritic cells and regulatory T cells. In contrast to previous reports that HO-1 had limited effects on basophils, which participate in T helper type 2 immune responses and antigen-induced allergic airway inflammation, we demonstrated in this study, for the first time, that the up-regulation of HO-1 significantly suppressed the maturation of mouse basophils, decreased the expression of CD40, CD80, MHC-II and activation marker CD200R on basophils, blocked DQ-ovalbumin uptake and promoted basophil apoptosis both in vitro and in vivo, leading to the inhibition of T helper type 2 polarization. These effects of HO-1 were mimicked by exogenous carbon monoxide, which is one of the catalytic products of HO-1. Furthermore, adoptive transfer of HO-1-modified basophils reduced ovalbumin-induced allergic airway inflammation. The above effects of HO-1 can be reversed by the HO-1 inhibitor Sn-protoporphyrin IX. Moreover, conditional depletion of basophils accompanying hemin treatment further attenuated airway inflammation compared with the hemin group, indicating that the protective role of HO-1 may involve multiple immune cells. Collectively, our findings demonstrated that HO-1 exerted its anti-inflammatory function through suppression of basophil maturation and activation, but promotion of basophil apoptosis, providing a possible novel therapeutic target in allergic asthma. PMID:26879758

  5. Cooperative Activity of GABP with PU.1 or C/EBPε Regulates Lamin B Receptor Gene Expression, Implicating Their Roles in Granulocyte Nuclear Maturation.

    PubMed

    Malu, Krishnakumar; Garhwal, Rahul; Pelletier, Margery G H; Gotur, Deepali; Halene, Stephanie; Zwerger, Monika; Yang, Zhong-Fa; Rosmarin, Alan G; Gaines, Peter

    2016-08-01

    Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted E-twenty-six (ETS) factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils. PMID:27342846

  6. Calcium current activation kinetics in isolated pyramidal neurones of the Ca1 region of the mature guinea-pig hippocampus.

    PubMed

    Kay, A R; Wong, R K

    1987-11-01

    1. Neurones were isolated from the CA1 region of the guinea-pig hippocampus and subjected to the whole-cell mode of voltage clamping, to determine the kinetics of voltage-gated Ca2+ channel activation. 2. Isolated neurones had an abbreviated morphology, having lost most of the distal dendritic tree during the isolation procedure. The electrical compactness of the cells facilitates voltage clamp analysis. 3. Block of sodium and potassium currents revealed a persistent current activated on depolarization above -40 mV, which inactivated slowly when the intracellular medium contained EGTA. The current was blocked by Co2+ and Cd2+, augmented by increases in Ca2+ and could be carried by Ba2+, suggesting that the current is borne by Ca2+. 4. Steady-state activation of the Ca2+ current was found to be well described by the Boltzman equation raised to the second power. 5. The open channel's current-voltage (I-V) relationship rectified in the inward direction and was consistent with the constant-field equation. 6. The kinetics of Ca2+ current onset followed m2 kinetics throughout the range of its activation. Tail current kinetics were in accord with this model. A detailed Hodgkin-Huxley model was derived, defining the activation of this current. 7. The kinetics of the currents observed in this regionally and morphologically defined class of neurones were consistent with the existence of a single kinetic class of channels. PMID:2451732

  7. 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.

  8. 75 FR 30098 - Reports, Forms and RecordKeeping Requirements; Agency Information Collection Activity Under OMB...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-28

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-19

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-09

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    2011-04-12

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    2010-11-30

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    2011-04-12

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  1. 75 FR 52539 - Agency Information Collection Activities: Form I-777, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-26

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-28

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    2010-06-09

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    2011-03-03

    ... information collection was previously published in the Federal Register on December 14, 2010, at 75 FR 77890... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form G-1145... Information Collection Under Review: Form G- 1145, E-Notification of Application/Petition Acceptance;...

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    2011-08-09

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    2012-06-07

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  18. 75 FR 57049 - Agency Information Collection Activities: Form I-777, Application for Replacement of Northern...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-17

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  19. 75 FR 10298 - Agency Information Collection Activities: Form I-929; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-05

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-929; Extension of an Existing Information Collection; Comment Request ACTION: 30 Day Notice of Information Collection Under Review; Form I-929,...

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-02-24

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  1. 78 FR 52824 - Proposed Information Collection (Bowel and Bladder Care Billing Form) Activity: Comment Request

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-26

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  2. 76 FR 41282 - Agency Information Collection Activities: Form I-363, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-13

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY Citizenship and Immigration Services Agency Information Collection Activities: Form I-363, Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice of Information Collection Under Review: Form I-...

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-19

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  4. 75 FR 35822 - Agency Information Collection Activities: Form I-363; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-06-23

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  5. 78 FR 73875 - Agency Information Collection Activities: Canadian Border Boat Landing Permit (CBP Form I-68)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-12-09

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Canadian Border Boat Landing Permit (CBP Form I-68) AGENCY: U.S. Customs and Border Protection (CBP), Department of Homeland... requirement concerning the Canadian Border Boat Landing Permit (Form I-68). This request for comment is...

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-05

    ... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Canadian Border Boat Landing Permit (CBP Form I-68) AGENCY: U.S. Customs and Border Protection (CBP), Department of Homeland... concerning the Canadian Border Boat Landing Permit (Form I- ] 68). This request for comment is being...

  7. Effect of chemical form of selenium on tissue glutathione peroxidase activity in developing rats

    NASA Technical Reports Server (NTRS)

    Lane, Helen W.; Strength, Ralph; Johnson, Janet; White, Marguerite T.

    1991-01-01

    The hypothesis that the stage of development of rats may affect the availability of various forms of selenium for the activity of glutathione peroxidase (GSHPx) in the rat was experimentally investigated. One experiment evaluated the availability of selenium as selenite or selenomethionine for GSPHx activity during three developmental states in rats: fetus and 7-day old and 14-day old nursing pups. In all tissues studied, GSHPx activity was highest in the 14-day-old pups whose dams were in the selenomethionine group. Rat pups given intraperitoneal selenite had higher liver and kidney GSHPx activity than pups given the same amount of selenium as intraperitoneal selenomethionine. In a second experiment, all dams were fed the same basal diet and pups were weaned to diets containing one of two levels of selenium and one of three forms of selenium (selenite, selenomethionine, or selenocystine). The results also supported the hypothesis these dietary forms of selenium are differentially available for GSHPx activity.

  8. Sulfate reducing bacterial community and in situ activity in mature fine tailings analyzed by real time qPCR and microsensor.

    PubMed

    Liu, Hong; Tan, Shuying; Yu, Tong; Liu, Yang

    2016-06-01

    Sulfate reducing bacteria (SRB) play significant roles in anaerobic environments in oil sands mature fine tailings (MFTs). Hydrogen sulfide (H2S) is produced during the biological sulfate reduction process. The production of toxic H2S is one of the concerns because it may hinder the landscape remediation efficiency of oil sands tailing ponds. In present study, the in situ activity and the community structure of SRB in MFT and gypsum amended MFT in two settling columns were investigated. Combined techniques of H2S microsensor and dissimilatory sulfite reductase β-subunit (dsrB) genes-based real time quantitative polymerase chain reaction (qPCR) were applied to detect the in situ H2S and the abundance of SRB. A higher diversity of SRB and more H2S were observed in gypsum amended MFT than that in MFT, indicating a higher sulfate reduction activity in gypsum amended MFT; in addition, the activity of SRB varied as depth in both MFT and gypsum amended MFT: the deeper the more H2S produced. Long-term plans for tailings management can be assessed more wisely with the information provided in this study. PMID:27266310

  9. Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts.

    PubMed

    Pitari, Maria Rita; Rossi, Marco; Amodio, Nicola; Botta, Cirino; Morelli, Eugenio; Federico, Cinzia; Gullà, Annamaria; Caracciolo, Daniele; Di Martino, Maria Teresa; Arbitrio, Mariamena; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2015-09-29

    miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3'UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD. PMID:26160841

  10. Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts

    PubMed Central

    Pitari, Maria Rita; Rossi, Marco; Amodio, Nicola; Botta, Cirino; Morelli, Eugenio; Federico, Cinzia; Gullà, Annamaria; Caracciolo, Daniele; Di Martino, Maria Teresa; Arbitrio, Mariamena; Giordano, Antonio; Tagliaferri, Pierosandro; Tassone, Pierfrancesco

    2015-01-01

    miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3′UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD. PMID:26160841

  11. Amyloid-β(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia.

    PubMed

    Paranjape, Geeta S; Terrill, Shana E; Gouwens, Lisa K; Ruck, Benjamin M; Nichols, Michael R

    2013-03-01

    Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer's disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1-42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1-42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1-42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia. PMID:23242692

  12. Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia

    PubMed Central

    Paranjape, Geeta S.; Terrill, Shana E.; Gouwens, Lisa K.; Ruck, Benjamin M.; Nichols, Michael R.

    2012-01-01

    Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1–42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia. PMID:23242692

  13. Delta Inulin Adjuvant Enhances Plasmablast Generation, Expression of Activation-Induced Cytidine Deaminase and B-Cell Affinity Maturation in Human Subjects Receiving Seasonal Influenza Vaccine

    PubMed Central

    Li, Lei; Honda-Okubo, Yoshikazu; Li, Connie; Sajkov, Dimitar; Petrovsky, Nikolai

    2015-01-01

    There is a major need for new adjuvants to improve the efficacy of seasonal and pandemic influenza vaccines. Advax is a novel polysaccharide adjuvant based on delta inulin that has been shown to enhance the immunogenicity of influenza vaccine in animal models and human clinical trials. To better understand the mechanism for this enhancement, we sought to assess its effect on the plasmablast response in human subjects. This pilot study utilised cryopreserved 7 day post-vaccination (7dpv) peripheral blood mononuclear cell samples obtained from a subset of 25 adult subjects from the FLU006-12 trial who had been immunized intramuscularly with a standard dose of 2012 trivalent inactivated influenza vaccine (TIV) alone (n=9 subjects) or combined with 5mg (n=8) or 10mg (n=8) of Advax adjuvant. Subjects receiving Advax adjuvant had increased 7dpv plasmablasts, which in turn exhibited a 2-3 fold higher rate of non-silent mutations in the B-cell receptor CDR3 region associated with higher expression of activation-induced cytidine deaminase (AID), the major enzyme controlling BCR affinity maturation. Together, these data suggest that Advax adjuvant enhances influenza immunity in immunized subjects via multiple mechanisms including increased plasmablast generation, AID expression and CDR3 mutagenesis resulting in enhanced BCR affinity maturation and increased production of high avidity antibody. How Advax adjuvant achieves these beneficial effects on plasmablasts remains the subject of ongoing investigation. Trial Registration Australia New Zealand Clinical Trials Register ACTRN12612000709842 https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=362709 PMID:26177480

  14. Differential activity of stanniocalcin in male and female fresh water teleost Mastacembelus armatus (Lacepede) during gonadal maturation.

    PubMed

    Verma, Sushant Kumar; Alim, Abdul

    2014-01-01

    The present study was carried out to analyze the differences in the activity of hormone stanniocalcin (STC) between male and female fishes of Mastacembelus armatus during their gonadal cycle. A large variation in nuclear diameter of cells of corpuscles of Stannius (CS) were recorded in relation to testicular cycle as well as ovarian cycle which indicates that the cellular activity varied with different phases of reproductive cycle in both male and female fish. Similar changes in nuclear diameter of CS cells were also observed after 17alpha-methyltestosterone administration in males and 17 β-estradiol administrations in females. A positive correlation was observed between plasma STC levels, gonadosomatic index (GSI) and the sex steroids in both sexes, suggesting that STC has a role in the processes involved in gonadal development. In addition females showed remarkable changes in plasma calcium level during gonadal cycle while no such change for males were observed. In females the plasma calcium level estimated during different phases of reproductive cycle indicates positive correlation between plasma level of calcium and gonad growth. Thus hyperactivity of CS cells was noted in both male and female fishes during gonadal cycle along with the differences in the activity of STC as well. In female it may act as hypocalcemic factor and bring the level of calcium to normal which increases during preparatory and pre spawning phases to fulfill the increased demand of calcium for vitellogenesis. However data of male fishes indicated that plasma STC concentration varied widely during gonadal cycle but showed no consistent relationship to plasma calcium level. PMID:25003342

  15. Hemolytic complement activity and concentrations of its third component during maturation of the immune response in colostrum-deprived foals.

    PubMed

    Bernoco, M M; Liu, I K; Willits, N H

    1994-07-01

    Six foals were deprived of colostrum for the first 36 hours after birth and, instead, received reconstituted powdered milk. Five control foals suckled their dams naturally. Blood samples were obtained from all the foals after birth and at approximately weekly intervals until at least 5.5 months of age. Sera were analyzed for hemolytic complement activity, complement component C3, and correlating IgG concentration. Hemolytic complement (P = 0.0145) and C3 (P = 0.0002) values were significantly higher in colostrum-deprived foals (CDF) than in naturally nursed foals at 2 to 5 days of age. In addition, significantly (P = 0.0149) higher IgG concentration was found in CDF than in naturally nursed foals between 3 and 5.5 months of age. It was concluded that the observed high complement activity in CDF within 2 to 5 days of age may provide an alternative in immune defense for IgG-deprived foals after failure of colostral transfer. PMID:7978631

  16. Differential Expression of Extracellular Lipase and Protease Activities of Mycelial and Yeast Forms in Malassezia furfur.

    PubMed

    Juntachai, Weerapong; Kajiwara, Susumu

    2015-10-01

    Malassezia furfur is a dimorphic yeast that is part of the human skin microflora. This fungus is a pathogen of a certain skin diseases, such as pityriasis versicolor, and in rare cases causes systemic infection in neonates. However, the role of dimorphism in the pathogenicity remains unclear. A modified induction medium (IM) was successfully able to induce mycelial growth of M. furfur under both solid and liquid condition. Filamentous elements with branching hyphae were observed when cultured in the IM. Furthermore, addition of bovine fetus serum into the liquid IM did not promote hyphal formation; on the contrary, it retrograded hyphae to the yeast form. Plate-washing assay showed that M. furfur hyphae did not possess the ability of invasive growth. Secretory proteins from both yeast and hyphal forms were isolated, and lipase and protease activities were analyzed. Intriguingly, the hyphal form showed higher activities than those of the yeast form, particularly the protease activity. PMID:26173769

  17. Comparing Two Forms of Concept Map Critique Activities to Facilitate Knowledge Integration Processes in Evolution Education

    ERIC Educational Resources Information Center

    Schwendimann, Beat A.; Linn, Marcia C.

    2016-01-01

    Concept map activities often lack a subsequent revision step that facilitates knowledge integration. This study compares two collaborative critique activities using a Knowledge Integration Map (KIM), a form of concept map. Four classes of high school biology students (n?=?81) using an online inquiry-based learning unit on evolution were assigned…

  18. Cathepsin B activity has a crucial role in the developmental competence of bovine cumulus-oocyte complexes exposed to heat shock during in vitro maturation.

    PubMed

    Balboula, A Z; Yamanaka, K; Sakatani, M; Kawahara, M; Hegab, A O; Zaabel, S M; Takahashi, M

    2013-10-01

    Cathepsin B was found to be correlated inversely with the quality of bovine oocytes and embryos. The aims of this study were to evaluate i) the relationship between heat shock during in vitro maturation (IVM) of bovine cumulus-oocyte complexes (COCs) and cathepsin B activity in relation to apoptosis and ii) the effect of supplementation of cathepsin B inhibitor (E-64) during IVM of heat-shocked COCs on embryonic development. After IVM at 38.5 °C for 22 h (control group) or at 38.5 °C for 5 h followed by 41 °C for 17 h (heat shock group) either with or without 1 μM E-64, activities and protein expression of cathepsin B and caspase 3 were evaluated as well as TUNEL staining. After IVF, developmental rate, total cell number, and the percentage of apoptotic cells in blastocysts were evaluated on day 8 (day 0, IVF day). Heat-shocked IVM COCs showed significantly high activities and expressions of both cathepsin B, and caspase 3 accompanied by a significant increase in number of TUNEL-positive cells. Addition of E-64 significantly decreased the activities of cathepsin B and caspase 3, and TUNEL-positive cells in heat-shocked IVM COCs. Moreover, addition of 1 μM E-64 during IVM under heat shock conditions significantly improved both developmental competence and quality of the produced embryos. These results indicate that heat shock induction of cathepsin B is associated with apoptosis of COCs, and inhibition of cathepsin B activity can improve the developmental competence of heat-shocked COCs during IVM. PMID:23898216

  19. 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…

  20. Mycobacterium tuberculosis PE25/PPE41 protein complex induces activation and maturation of dendritic cells and drives Th2-biased immune responses.

    PubMed

    Chen, Wei; Bao, Yige; Chen, Xuerong; Burton, Jeremy; Gong, Xueli; Gu, Dongqing; Mi, Youjun; Bao, Lang

    2016-04-01

    Mycobacterium tuberculosis evades innate host immune responses by parasitizing macrophages and causes significant morbidity and mortality around the world. A mycobacterial antigen that can activate dendritic cells (DCs) and elicit effective host innate immune responses will be vital to the development of an effective TB vaccine. The M. tuberculosis genes PE25/PPE41 encode proteins which have been associated with evasion of the host immune response. We constructed a PE25/PPE41 complex gene via splicing by overlapping extension and expressed it successfully in E. coli. We investigated whether this protein complex could interact with DCs to induce effective host immune responses. The PE25/PPE41 protein complex induced maturation of isolated mouse DCs in vitro, increasing expression of cell surface markers (CD80, CD86 and MHC-II), thereby promoting Th2 polarization via secretion of pro-inflammatory cytokines IL-4 and IL-10. In addition, PE25/PPE41 protein complex-activated DCs induced proliferation of mouse CD4(+) and CD8(+) T cells, and a strong humoral response in immunized mice. The sera of five TB patients were also highly reactive to this antigen. These findings suggest that interaction of the PE25/PPE41 protein complex with DCs may be of great immunological significance. PMID:26318856

  1. An Orange Ripening Mutant Links Plastid NAD(P)H Dehydrogenase Complex Activity to Central and Specialized Metabolism during Tomato Fruit Maturation[C][W

    PubMed Central

    Nashilevitz, Shai; Melamed-Bessudo, Cathy; Izkovich, Yinon; Rogachev, Ilana; Osorio, Sonia; Itkin, Maxim; Adato, Avital; Pankratov, Ilya; Hirschberg, Joseph; Fernie, Alisdair R.; Wolf, Shmuel; Usadel, Björn; Levy, Avraham A.; Rumeau, Dominique; Aharoni, Asaph

    2010-01-01

    In higher plants, the plastidial NADH dehydrogenase (Ndh) complex supports nonphotochemical electron fluxes from stromal electron donors to plastoquinones. Ndh functions in chloroplasts are not clearly established; however, its activity was linked to the prevention of the overreduction of stroma, especially under stress conditions. Here, we show by the characterization of OrrDs, a dominant transposon-tagged tomato (Solanum lycopersicum) mutant deficient in the NDH-M subunit, that this complex is also essential for the fruit ripening process. Alteration to the NDH complex in fruit changed the climacteric, ripening-associated metabolites and transcripts as well as fruit shelf life. Metabolic processes in chromoplasts of ripening tomato fruit were affected in OrrDs, as mutant fruit were yellow-orange and accumulated substantially less total carotenoids, mainly β-carotene and lutein. The changes in carotenoids were largely influenced by environmental conditions and accompanied by modifications in levels of other fruit antioxidants, namely, flavonoids and tocopherols. In contrast with the pigmentation phenotype in mature mutant fruit, OrrDs leaves and green fruits did not display a visible phenotype but exhibited reduced Ndh complex quantity and activity. This study therefore paves the way for further studies on the role of electron transport and redox reactions in the regulation of fruit ripening and its associated metabolism. PMID:20571113

  2. Zona pellucida birefringence correlates with developmental capacity of bovine oocytes classified by maturational environment, COC morphology and G6PDH activity.

    PubMed

    Held, Eva; Mertens, Eva-Maria; Mohammadi-Sangcheshmeh, Abdollah; Salilew-Wondim, Dessie; Besenfelder, Urban; Havlicek, Vitezslav; Herrler, Andreas; Tesfaye, Dawit; Schellander, Karl; Hölker, Michael

    2012-01-01

    In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence. PMID:22541545

  3. Differential cellulolytic activity of native-form and C-terminal tagged-form cellulase derived from coptotermes formosanus and expressed in E. coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The endogenous cellulase gene (CfEG3a) of Coptotermes formosanus, an economically important pest termite, was cloned and overexpressed in both native form (nCfEG) and C-terminal His-tagged form (tCfEG) in E.coli. Both forms of recombinant cellulases showed hydrolytic activity on cellulosic substrate...

  4. Induction of maturation and activation of human dendritic cells: A mechanism underlying the beneficial effect of Viscum album as complimentary therapy in cancer

    PubMed Central

    Elluru, Sri Ramulu; van Huyen, Jean-Paul Duong; Delignat, Sandrine; Kazatchkine, Michel D; Friboulet, Alain; Kaveri, Srini V; Bayry, Jagadeesh

    2008-01-01

    Background Viscum album (VA) preparations have been used as a complimentary therapy in cancer. In addition to their cytotoxic properties, they have also been shown to have immunostimulatory properties. In the present study, we examine the hypothesis that the VA preparations induce activation of human DC that facilitates effective tumor regression. Methods Four day old monocyte-derived immature DCs were treated with VA Qu Spez at 5, 10 and 15 μg/ml for 48 hrs. The expression of surface molecules was analyzed by flow cytometry. The ability of Qu Spez-educated DC to stimulate T cells was analyzed by allogeneic mixed lymphocyte reaction and activation of Melan-A/MART-1-specific M77-80 CD8+T cells. Cytokines in cell free culture supernatant was analyzed by cytokine bead array assay. Results VA Qu Spez stimulated DCs presented with increased expression of antigen presenting molecule HLA-DR and of co-stimulatory molecules CD40, CD80 and CD86. The VA Qu Spez also induced the secretion of inflammatory cytokines IL-6 and IL-8. Further, Qu Spez-educated DC stimulated CD4+T cells in a allogeneic mixed lymphocyte reaction and activated melanoma antigen Melan-A/MART-1-specific M77-80 CD8+T cells as evidenced by increased secretion of TNF-α and IFNγ. Conclusion The VA preparations stimulate the maturation and activation of human DCs, which may facilitate anti-tumoral immune responses. These results should assist in understanding the immunostimulatory properties of VA preparations and improving the therapeutic strategies. PMID:18533025

  5. The effects of feedback lighting on the circadian rhythm of locomotor activity and the reproductive maturation of the male Djungarian hamster (Phodopus sungorus)

    NASA Technical Reports Server (NTRS)

    Ferraro, J. S.

    1988-01-01

    The non-parametric model of entrainment suggests that brief pulses of light, delivered between dusk and dawn can simulate the phasing effects of full photoperiods or even constant light (LL). Feedback lighting (LDFB) is a lighting condition where individual animals, otherwise in constant darkness (DD), are exposed to light in response to a monitored behavior. The specific purpose of this type of illumination is to expose the circadian cycle to light only during the subjective night. LDFB has been used to support this hypothesis in several species of nocturnal rodents and one species of diurnal primate by producing similar free-running periods in LDFB as in LL. This lighting condition has also been used to test the hypothesis that exposing the subjective night to even short duration light pulses will maintain reproductive function in long day breeders. In the Syrian hamster (Mesocricetus auratus), however, LDFB is not as photostimulatory as LL despite extensive light exposure during the subjective night. In the experiments presented here, a group of immature male Djungarian hamsters (Phodopus sungorus) were placed in individual light-tight sound attenuated chambers where they had free access to food, water and an activity wheel. The animals were exposed to one of four lighting conditions [DD, LL, LDFB or a neighbor control of feedback lighting (LDFB NC)] for approximately 30 days shortly after weaning. LDFB NC is a lighting condition where a neighbor control hamster receives the identical lighting regime as a paired animal exposing itself to LDFB, yet the neighbor has no control over it. A fifth group was exposed to a light-dark cycle of 16 hours of light and 8 hours of dark (LD16:8). This group was housed in cages in a colony room and did not have access to a running wheel. The free-running periods of the locomotor activity rhythms for hamsters exposed to LDFB and LL were not similar, unlike the results for rats, Syrian hamsters, mice, monkeys and even mature

  6. The effects of feedback lighting on the circadian rhythm of locomotor activity and the reproductive maturation of the male Djungarian hamster (Phodopus sungorus).

    PubMed

    Ferraro, J S

    1988-01-01

    The non-parametric model of entrainment suggests that brief pulses of light, delivered between dusk and dawn can simulate the phasing effects of full photoperiods or even constant light (LL). Feedback lighting (LDFB) is a lighting condition where individual animals, otherwise in constant darkness (DD), are exposed to light in response to a monitored behavior. The specific purpose of this type of illumination is to expose the circadian cycle to light only during the subjective night. LDFB has been used to support this hypothesis in several species of nocturnal rodents and one species of diurnal primate by producing similar free-running periods in LDFB as in LL. This lighting condition has also been used to test the hypothesis that exposing the subjective night to even short duration light pulses will maintain reproductive function in long day breeders. In the Syrian hamster (Mesocricetus auratus), however, LDFB is not as photostimulatory as LL despite extensive light exposure during the subjective night. In the experiments presented here, a group of immature male Djungarian hamsters (Phodopus sungorus) were placed in individual light-tight sound attenuated chambers where they had free access to food, water and an activity wheel. The animals were exposed to one of four lighting conditions [DD, LL, LDFB or a neighbor control of feedback lighting (LDFB NC)] for approximately 30 days shortly after weaning. LDFB NC is a lighting condition where a neighbor control hamster receives the identical lighting regime as a paired animal exposing itself to LDFB, yet the neighbor has no control over it. A fifth group was exposed to a light-dark cycle of 16 hours of light and 8 hours of dark (LD16:8). This group was housed in cages in a colony room and did not have access to a running wheel. The free-running periods of the locomotor activity rhythms for hamsters exposed to LDFB and LL were not similar, unlike the results for rats, Syrian hamsters, mice, monkeys and even mature

  7. Presence of Rheumatoid Factor during Chronic HCV Infection Is Associated with Expansion of Mature Activated Memory B-Cells that Are Hypo-Responsive to B-Cell Receptor Stimulation and Persist during the Early Stage of IFN Free Therapy

    PubMed Central

    Reyes-Avilés, Elane; Kostadinova, Lenche; Rusterholtz, Anne; Cruz-Lebrón, Angelica; Falck-Ytter, Yngve; Anthony, Donald D.

    2015-01-01

    Approximately half of those with chronic hepatitis C virus (HCV) infection have circulating rheumatoid factor (RF), and a portion of these individuals develop cryoglobulinemic vasculitis. B cell phenotype/function in relation to RF in serum has been unclear. We examined B cell subset distribution, activation state (CD86), cell cycle state (Ki67), and ex-vivo response to BCR, TLR9 and TLR7/8 stimulation, in chronic HCV-infected donors with or without RF, and uninfected donors. Mature-activated B-cells of HCV-infected donors had lower CD86 expression compared to uninfected donors, and in the presence of RF they also showed reduced CD86 expression in response to BCR and TLR9 stimulation. Additionally, mature activated memory B cells of HCV RF+ donors less commonly expressed Ki67+ than HCV RF- donors, and did not proliferate as well in response to BCR stimulation. Proportions of mature-activated B cells were enhanced, while naïve B-cells were lower in the peripheral blood of HCV-RF+ compared to RF- and uninfected donors. None of these parameters normalize by week 8 of IFN free direct acting antiviral (DAA) therapy in HCV RF+ donors, while in RF- donors, mature activated B cell proportions did normalize. These data indicate that while chronic HCV infection alone results in a lower state of activation in mature activated memory B cells, the presence of RF in serum is associated with a more pronounced state of unresponsiveness and an overrepresentation of these B cells in the blood. This phenotype persists at least during the early time window after removal of HCV from the host. PMID:26649443

  8. A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80.

    PubMed Central

    Parthun, M R; Jaehning, J A

    1992-01-01

    The GAL4 activator and GAL80 repressor proteins regulate the expression of yeast genes in response to galactose. A complex of the two proteins isolated from glucose-grown cells is inactive in an in vitro transcription reaction but binds DNA and blocks activation by the GAL4-VP16 chimeric activator. The complex purified from galactose-grown cells contains a mixture of phosphorylated and unphosphorylated forms of GAL4. The galactose-induced form of GAL4 activates in vitro transcription to levels similar to those seen with GAL4-VP16. The induced GAL4 complex is indistinguishable in size and apparent shape from the uninduced complex, consistent with a continued association with GAL80. These results confirm in vivo analyses that correlate GAL4 phosphorylation with galactose induction and support a model of transcriptional activation that does not require GAL80 dissociation. Images PMID:1406674

  9. Microarray Analysis Reveals Increased Transcriptional Repression and Reduced Metabolic Activity but Not Major Changes in the Core Apoptotic Machinery during Maturation of Sympathetic Neurons

    PubMed Central

    Raba, Mikk; Palgi, Jaan; Lehtivaara, Maria; Arumäe, Urmas

    2016-01-01

    Postnatal maturation of the neurons whose main phenotype and basic synaptic contacts are already established includes neuronal growth, refinement of synaptic contacts, final steps of differentiation, programmed cell death period (PCD) etc. In the sympathetic neurons, postnatal maturation includes permanent end of the PCD that occurs with the same time schedule in vivo and in vitro suggesting that the process could be genetically determined. Also many other changes in the neuronal maturation could be permanent and thus based on stable changes in the genome expression. However, postnatal maturation of the neurons is poorly studied. Here we compared the gene expression profiles of immature and mature sympathetic neurons using Affymetrix microarray assay. We found 1310 significantly up-regulated and 1151 significantly down-regulated genes in the mature neurons. Gene ontology analysis reveals up-regulation of genes related to neuronal differentiation, chromatin and epigenetic changes, extracellular factors and their receptors, and cell adhesion, whereas many down-regulated genes were related to metabolic and biosynthetic processes. We show that termination of PCD is not related to major changes in the expression of classical genes for apoptosis or cell survival. Our dataset is deposited to the ArrayExpress database and is a valuable source to select candidate genes in the studies of neuronal maturation. As an example, we studied the changes in the expression of selected genes Igf2bp3, Coro1A, Zfp57, Dcx, and Apaf1 in the young and mature sympathetic ganglia by quantitative PCR and show that these were strongly downregulated in the mature ganglia. PMID:27013977

  10. In situ pore-forming alginate hydrogel beads loaded with in situ formed nano-silver and their catalytic activity.

    PubMed

    Wang, Qin; Liu, Shanshan; Wang, Hong; Yang, Yajiang

    2016-05-14

    An aqueous mixture of sodium carbonate (Na2CO3) and sodium alginate (Na-ALG) was added dropwise into an aqueous solution of Ca(NO3)2, leading to the formation of calcium alginate (Ca-ALG) hydrogel beads. Meanwhile Na2CO3 as a pore-forming precursor was transformed in situ into CaCO3 nanoparticles (CaCO3 NPs). SEM images show that CaCO3 NPs aggregates with a size of ∼10 μm were uniformly distributed in the Ca-ALG hydrogel beads. After subsequent erosion using acetic acid, Ca-ALG hydrogel beads with a uniform microporous structure were obtained. The porosity and specific surface area of such in situ pore-formed hydrogel beads are 16 and 14 times higher than those of the beads prepared in the absence of Na2CO3. Additionally, their porous structure can be modulated by varying the amount of Na2CO3. The obtained porous Ca-ALG hydrogel beads were further immersed into an aqueous solution of AgNO3. Under UV irradiation, the Ag(+) ions adsorbed in the Ca-ALG were in situ reduced to Ag nanoparticles (Ag NPs). SEM and TEM images show that Ag NPs with a size of ∼10 nm were uniformly distributed in the matrix of the hydrogel beads. The loading amount of Ag in the beads can also be modulated by varying the amount of Na2CO3. Furthermore, the resultant Ca-ALG beads loaded with Ag NPs (Ag/Ca-ALG) were used as catalysts. Their catalytic activity was evaluated by using the reduction reaction of 4-nitrophenol as a model reaction. The rate constant of the reaction in the presence of dry porous Ag/Ca-ALG beads was found to be 36 times higher than that in the presence of beads prepared in the absence of Na2CO3. Such a high catalytic efficiency can be attributed to their porous structure and consequent high Ag-loading capacity. PMID:27093592

  11. Control of spine maturation and pruning through proBDNF synthesized and released in dendrites.

    PubMed

    Orefice, Lauren L; Shih, Chien-Cheng; Xu, Haifei; Waterhouse, Emily G; Xu, Baoji

    2016-03-01

    Excess synapses formed during early postnatal development are pruned over an extended period, while the remaining synapses mature. Synapse pruning is critical for activity-dependent refinement of neuronal connections and its dysregulation has been found in neurodevelopmental disorders such as autism spectrum disorders; however, the mechanism underlying synapse pruning remains largely unknown. As dendritic spines are the postsynaptic sites for the vast majority of excitatory synapses, spine maturation and pruning are indicators for maturation and elimination of these synapses. Our previous studies have found that dendritically localized mRNA for brain-derived neurotrophic factor (BDNF) regulates spine maturation and pruning. Here we investigated the mechanism by which dendritic Bdnf mRNA, but not somatically restricted Bdnf mRNA, promotes spine maturation and pruning. We found that neuronal activity stimulates both translation of dendritic Bdnf mRNA and secretion of its translation product mainly as proBDNF. The secreted proBDNF promotes spine maturation and pruning, and its effect on spine pruning is in part mediated by the p75(NTR) receptor via RhoA activation. Furthermore, some proBDNF is extracellularly converted to mature BDNF and then promotes maturation of stimulated spines by activating Rac1 through the TrkB receptor. In contrast, translation of somatic Bdnf mRNA and the release of its translation product mainly as mature BDNF are independent of action potentials. These results not only reveal a biochemical pathway regulating synapse pruning, but also suggest that BDNF synthesized in the soma and dendrites is released through distinct secretory pathways. PMID:26705735

  12. A Novel Mechanism of Lysosomal Acid Sphingomyelinase Maturation

    PubMed Central

    Jenkins, Russell W.; Idkowiak-Baldys, Jolanta; Simbari, Fabio; Canals, Daniel; Roddy, Patrick; Riner, Clarke D.; Clarke, Christopher J.; Hannun, Yusuf A.

    2011-01-01

    Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn2+)-independent lysosomal aSMase (L-SMase) and a Zn2+-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn2+-activated proenzyme to a mature 65 kDa, Zn2+-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn2+-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same Mr as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function. PMID:21098024

  13. Cone Early Maturity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hop cone early maturity is thought to be caused by diffuse infections of cone, just prior to harvest, by Podosphaera macularis. The disease is best managed by limiting the amount of leaf infection by P. macularis prior to bloom. The yield and quality reductions associated with Hop cone early matur...

  14. Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity.

    PubMed

    Senthilkumar, Velusamy; Busby, Stephen J W; Gunasekaran, Paramasamy; Senthikumar, Velusamy; Bushby, Stephen J W

    2003-10-01

    Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues. PMID:14584923

  15. Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

    PubMed Central

    Carroll-Portillo, Amanda; Cannon, Judy L.; te Riet, Joost; Holmes, Anna; Kawakami, Yuko; Kawakami, Toshiaki; Cambi, Alessandra

    2015-01-01

    Mast cells (MCs) produce soluble mediators such as histamine and prostaglandins that are known to influence dendritic cell (DC) function by stimulating maturation and antigen processing. Whether direct cell–cell interactions are important in modulating MC/DC function is unclear. In this paper, we show that direct contact between MCs and DCs occurs and plays an important role in modulating the immune response. Activation of MCs through FcεRI cross-linking triggers the formation of stable cell–cell interactions with immature DCs that are reminiscent of the immunological synapse. Direct cellular contact differentially regulates the secreted cytokine profile, indicating that MC modulation of DC populations is influenced by the nature of their interaction. Synapse formation requires integrin engagement and facilitates the transfer of internalized MC-specific antigen from MCs to DCs. The transferred material is ultimately processed and presented by DCs and can activate T cells. The physiological outcomes of the MC–DC synapse suggest a new role for intercellular crosstalk in defining the immune response. PMID:26304724

  16. [Value of different serological tests in the diagnosis of various forms of active ocular toxoplasmosis].

    PubMed

    Prost, M; Umiński, J

    1992-01-01

    The analysis of results of indirect immunofluorescence and direct agglutination reaction in patients with various forms of ocular toxoplasmosis showed that these reactions have a limited value in diagnostics of this condition. One can define a sure diagnosis of active ocular toxoplasmosis when the titre of these reactions are higher than 512. The ELISA IgM reaction is specific for the active form of this disease. The sensitivity of this reaction amounts 43-50%. In cases of iridocyclitis the results of the serological tests were similar to those of the control group; this confirms the hypothesis that the inflammation is evoked by an allergic reaction. PMID:1635369

  17. The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4alpha activities and introduces a PKA phosphorylation site in its DNA-binding domain.

    PubMed

    Oxombre, Bénédicte; Kouach, Mostafa; Moerman, Ericka; Formstecher, Pierre; Laine, Bernard

    2004-11-01

    HNF4alpha (hepatocyte nuclear factor 4alpha) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic beta-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4alpha gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115-->Ser) HNF4alpha mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4alpha-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic beta-cell lines, this mutation resulted in strong impairments of HNF4alpha transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1alpha, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115-->Glu) mutation mimicking phosphorylation reduced HNF4alpha DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4alpha function. PMID:15233628

  18. The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4α activities and introduces a PKA phosphorylation site in its DNA-binding domain

    PubMed Central

    2004-01-01

    HNF4α (hepatocyte nuclear factor 4α) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic β-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4α gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115→Ser) HNF4α mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4α-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic β-cell lines, this mutation resulted in strong impairments of HNF4α transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1α, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115→Glu) mutation mimicking phosphorylation reduced HNF4α DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4α function. PMID:15233628

  19. Stimulators of AMP-activated kinase (AMPK) inhibit seawater- but not cAMP-induced oocyte maturation in a marine worm: Implications for interactions between cAMP and AMPK signaling.

    PubMed

    Stricker, Stephen A; Swiderek, Lee; Nguyen, Thanh

    2010-06-01

    Previous studies have shown that elevations in intraoocytic cAMP prevent mammalian oocytes from maturing, whereas cAMP degradation allows these oocytes to begin maturation, as evidenced by the onset of oocyte nuclear disassembly (="germinal vesicle breakdown", GVBD). Moreover, such cAMP degradation not only reduces cAMP levels but also generates AMP, which in turn can stimulate AMP-activated kinase (AMPK), a well-documented inducer of GVBD in mice. Alternatively, in some marine invertebrates, intraoocytic cAMP triggers, rather than blocks, GVBD, and whether AMPK up- or downregulates maturation in these species has not been tested. Thus, AMPK was monitored in the nemertean worm Cerebratulus during GVBD stimulated by seawater (SW) or cAMP elevators. In oocytes lacking surrounding follicle cells, AMPK activity was initially elevated in immature oocytes but subsequently reduced during SW- or cAMP-induced GVBD, given that the catalytic alpha-subunit of AMPK in maturing oocytes displayed a decreased stimulatory phosphorylation at T172 and an increased inhibitory phosphorylation at S485/491. Accordingly, AMPK-mediated phosphorylation of acetyl-CoA carboxylase, a known target of active AMPK, also declined during maturation. Moreover, treatments with either ice-cold calcium-free seawater (CaFSW) or AMPK agonists dissolved in SW maintained AMPK activity and inhibited GVBD. Conversely, adding cAMP elevators to CaFSW- or SW-solutions of AMPK activators restored GVBD while promoting S485/491 phosphorylation and AMPK deactivation. Collectively, such findings not only demonstrate for the first time that intraoocytic AMPK can block GVBD in the absence of surrounding follicle cells, but these results also provide evidence for a novel GVBD-regulating mechanism involving AMPK deactivation by cAMP-mediated S485/491 phosphorylation. PMID:20336704

  20. Structural and functional maturation of the fetal human choriocapillaris

    PubMed Central

    Baba, Takayuki; Grebe, Rhonda; Hasegawa, Takuya; Bhutto, Imran; Merges, Carol; McLeod, D. Scott; Lutty, Gerard A.

    2016-01-01

    Purpose The purpose of this study was to examine the structural and functional maturation of the choriocapillaris (CC). We sought to determine when fenestrations formed, pericytes invest the capillaries and endothelial cells became functional. Methods Immunohistochemistry was performed on cryopreserved sections of embryonic/fetal human eyes from 7 to 22 weeks gestation (WG) using antibodies against PAL-E, PV-1 (fenestrations), carbonic anhydrase IV (CA IV), eNOS, and alpha smooth muscle actin (αSMA) and NG2 (two pericyte markers) and endothelial cell (EC) markers (CD34, CD31). Alkaline phosphatase (APase) enzymatic activity was demonstrated by enzyme histochemistry. Transmission electron microscopy (TEM) was performed on 11, 14, 16 and 22 WG eyes. Adult human eyes were used as positive controls. Results All EC markers were present in CC by 7 WG. PAL-E, CA IV and eNOS immunoreactivities and APase activity were present in CC by 7–9 WG. TEM analysis demonstrated how structurally immature this vasculature was even at 11 WG: no basement membrane, absence of pericytes, and poorly formed lumens that were filled with filopodia. The few fenestrations that were observed were often present within the luminal space in the filopodia. Contiguous fenestrations and significant PV-1 were not observed until 21–22 WG. αSMA was prominent at 22 WG and the maturation of pericytes was confirmed by TEM. Conclusions It appears that EC and their precursors have several mature functional characteristics well before they are structurally mature. Although EC make tight junctions early in development, contiguous fenestrations and mature pericytes occur much later in development. PMID:19264887

  1. Functional Specificity of the Visual Word Form Area: General Activation for Words and Symbols but Specific Network Activation for Words

    ERIC Educational Resources Information Center

    Reinke, Karen; Fernandes, Myra; Schwindt, Graeme; O'Craven, Kathleen; Grady, Cheryl L.

    2008-01-01

    The functional specificity of the brain region known as the Visual Word Form Area (VWFA) was examined using fMRI. We explored whether this area serves a general role in processing symbolic stimuli, rather than being selective for the processing of words. Brain activity was measured during a visual 1-back task to English words, meaningful symbols…

  2. High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene

    PubMed Central

    Brinch-Pedersen, Henrik

    2013-01-01

    The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a. PMID:23918958

  3. Process for forming a homogeneous oxide solid phase of catalytically active material

    DOEpatents

    Perry, Dale L.; Russo, Richard E.; Mao, Xianglei

    1995-01-01

    A process is disclosed for forming a homogeneous oxide solid phase reaction product of catalytically active material comprising one or more alkali metals, one or more alkaline earth metals, and one or more Group VIII transition metals. The process comprises reacting together one or more alkali metal oxides and/or salts, one or more alkaline earth metal oxides and/or salts, one or more Group VIII transition metal oxides and/or salts, capable of forming a catalytically active reaction product, in the optional presence of an additional source of oxygen, using a laser beam to ablate from a target such metal compound reactants in the form of a vapor in a deposition chamber, resulting in the deposition, on a heated substrate in the chamber, of the desired oxide phase reaction product. The resulting product may be formed in variable, but reproducible, stoichiometric ratios. The homogeneous oxide solid phase product is useful as a catalyst, and can be produced in many physical forms, including thin films, particulate forms, coatings on catalyst support structures, and coatings on structures used in reaction apparatus in which the reaction product of the invention will serve as a catalyst.

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  18. 76 FR 28128 - Reports, Forms, and Record Keeping Requirements; Agency Information Collection Activity Under OMB...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-13

    ... comment period was published on September 13, 2010 (75 FR 55627-55628). DATES: Comments must be submitted... National Highway Traffic Safety Administration Reports, Forms, and Record Keeping Requirements; Agency Information Collection Activity Under OMB Review AGENCY: National Highway Traffic Safety Administration...

  19. 75 FR 52541 - Agency Information Collection Activities: Form I-243, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-26

    ... published in the Federal Register on June 9, 2010, at 75 FR 32799, allowing for a 60-day public comment... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-243, Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice...

  20. 76 FR 69275 - Agency Information Collection Activities: Form I-192, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-08

    ... the Federal Register on August 12, 2011, at 76 FR 50239, allowing for a 60- day public comment period... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-192, Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice...

  1. 77 FR 23734 - Agency Information Collection Activities: Form I-361, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-20

    ..., 2012, at 77 FR 9259, allowing for a 60-day public comment period. USCIS did not receive any comments... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-361, Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice...

  2. 75 FR 52541 - Agency Information Collection Activities: Form I-865, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-26

    ... was previously published in the Federal Register on June 9, 2010, at 75 FR 32801, allowing for a 60... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-865, Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice...

  3. 76 FR 43335 - Agency Information Collection Activities: Form I-765, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-20

    ... collection was previously published in the Federal Register on April 19, 2011, at 76 FR 21912 allowing for a... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-765, Extension of a Currently Approved Information Collection; Comment Request ACTION: 30-Day Notice...

  4. 40 CFR 761.205 - Notification of PCB waste activity (EPA Form 7710-53).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Notification of PCB waste activity (EPA Form 7710-53). 761.205 Section 761.205 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT POLYCHLORINATED BIPHENYLS (PCBs) MANUFACTURING, PROCESSING, DISTRIBUTION IN COMMERCE, AND USE PROHIBITIONS...

  5. 75 FR 57480 - Agency Information Collection Activities: Customs Declaration (Form 6059B)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-21

    ... SECURITY U.S. Customs And Border Protection Agency Information Collection Activities: Customs Declaration (Form 6059B) AGENCY: U.S. Customs and Border Protection (CBP), Department of Homeland Security. ACTION... the Customs Declaration. This request for comment is being made pursuant to the Paperwork...

  6. 75 FR 71452 - Agency Information Collection Activities: Customs Declaration (Form 6059B)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-23

    ... published in the Federal Register (75 FR 57480) on September 21, 2010, allowing for a 60-day comment period... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Customs Declaration (Form 6059B) AGENCY: U.S. Customs and Border Protection, Department of Homeland Security. ACTION:...

  7. 76 FR 11805 - Agency Information Collection Activities: Form I-134, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-03

    ... published in the Federal Register on December 14, 2010, at 75 FR 77891, allowing for a 60-day public comment... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-134... Department of Homeland Security, U.S. Citizenship and Immigration Services (USCIS) will be submitting...

  8. 75 FR 71451 - Agency Information Collection Activities: Form N-470, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-23

    ... information collection was previously published in the Federal Register on August 18, 2010, at 75 FR 51096... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-470... Control No. 1615-0056. The Department of Homeland Security, U.S. Citizenship and Immigration...

  9. 76 FR 75892 - Agency Information Collection Activities: Form I-690; Revision of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... collection notice was previously published in the Federal Register on August 19, 2011, at 76 FR 51996... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-690... 1615-0032. The Department of Homeland Security, U.S. Citizenship and Immigration Services (USCIS)...

  10. 75 FR 12249 - Agency Information Collection Activities: Form I-134, Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-03-15

    ... the Federal Register on November 12, 2009, at 74 FR 58302 allowing for a 60-day public comment period... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-134... Homeland Security, U.S. Citizenship and Immigration Services (USCIS) has submitted the...

  11. 75 FR 65500 - Agency Information Collection Activities: Form I-360, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-25

    ... information collection was previously published in the Federal Register on June 30, 2010, at 75 FR 37820... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-360... Control No. 1615-0020. The Department of Homeland Security, U.S. Citizenship and Immigration...

  12. 76 FR 37059 - Agency Information Collection Activities: Proposed Collection; Comment Request-Form FNS-339...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-24

    ... Food and Nutrition Service Agency Information Collection Activities: Proposed Collection; Comment Request--Form FNS-339, Federal-State Supplemental Nutrition Program(s) Agreement AGENCY: Food and Nutrition Service, USDA. ACTION: Notice. SUMMARY: In accordance with the Paperwork Reduction Act of...

  13. Chemical forms of selenium affect glutatione peroxidase activity in human Caco-2 cell model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The bioavailability of selenium (Se) is complicated because there are multiple naturally occurring chemical forms of this element in nature. Assessing the ability of a Se source to restore GPX1 activity in laboratory animals and humans is the most commonly used method. To search for an alternative (...

  14. 75 FR 5099 - Agency Information Collection Activities: Form N-648, Revision of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-02-01

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-648... No. 1615-0060. ] The Department of Homeland Security, U.S. Citizenship and Immigration Services... practical utility; (2) Evaluate the accuracy of the agency's estimate of the burden of the collection...

  15. 78 FR 28820 - Commission Information Collection Activities (FERC Form 80); Comment Request; Revision

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-16

    ... same docket, which was subsequently published in the Federal Register on 4/25/2013 (78 FR 24402). SUMMARY: In compliance with the requirements of the Paperwork Reduction Act of 1995, 44 U.S.C. 3506(c)(2... Energy Regulatory Commission Commission Information Collection Activities (FERC Form 80); Comment...

  16. 78 FR 45919 - Commission Information Collection Activities (FERC Form 80); Comment Request; Extension

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-30

    ... the Federal Register (78 FR 28820, 5/16/2013) requesting public comments. FERC received no comments on... Energy Regulatory Commission Commission Information Collection Activities (FERC Form 80); Comment Request... the requirements of the Paperwork Reduction Act of 1995, 44 U.S.C. 3507(a)(1)(D), the Federal...

  17. 77 FR 41796 - Agency Information Collection Activities: Monthly Report on Naturalization Papers, Form N-4...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-07-16

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Monthly Report on Naturalization Papers, Form N-4; Extension of a Currently Approved Information Collection; Comment Request ACTION: 60-Day Notice of...

  18. 77 FR 13136 - Agency Information Collection Activities: Form I-134; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-03-05

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-134; Extension of an Existing Information Collection; Comment Request ACTION: 30-Day Notice of Information Collection Under Review: Affidavit of...

  19. Relationship between phosphorus forms and phosphatase activity in soils amended with poultry manure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Up to 80% of the phosphorus (P) in poultry manure (PM) can be present in organic forms that require mineralization via phosphatase enzymes prior to plant utilization. To determine the correlation between soil P distribution and phosphatase activity we sequentially extracted two Maine soils amended...

  20. 78 FR 46597 - Agency Information Collection Activities: Comment Request for the Production Estimate (2 Forms)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-08-01

    ....S. Geological Survey Agency Information Collection Activities: Comment Request for the Production...). SUPPLEMENTARY INFORMATION: I. Abstract This collection is needed to provide data on mineral production for... OMB Control Number: 1028-0065. Form Numbers: 9-4042-A and 9-4124-A. Title: Production Estimate....

  1. 76 FR 11807 - Agency Information Collection Activities: Form N-565, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-03

    ... 75 FR 80835, allowing for a 60-day public comment period. USCIS did not receive any comments for this... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-565... Document; OMB Control No. 1615-0091. * * * * * The Department of Homeland Security, U.S. Citizenship...

  2. 75 FR 51094 - Agency Information Collection Activities: Form I-363, Extension of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-18

    .... The information collection was previously published in the Federal Register on June 23, 2010, at 75 FR... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-363... Amerasian; OMB Control No. 1615-0022. The Department of Homeland Security, U.S. Citizenship and...

  3. 75 FR 3245 - Agency Information Collection Activities: Aircraft/Vessel Report (Form I-92)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-20

    ... published in the Federal Register (74 FR 54839) on October 23, 2009, allowing for a 60-day comment period... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Aircraft/Vessel... approval in accordance with the Paperwork Reduction Act: Aircraft/ Vessel Report (Form I-92). This is...

  4. 76 FR 2917 - Agency Information Collection Activities: Canadian Border Boat Landing Permit (CBP Form I-68)

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-18

    ... proposed information collection was previously published in the Federal Register (75 FR 61508) on October 5... SECURITY U.S. Customs and Border Protection Agency Information Collection Activities: Canadian Border Boat Landing Permit (CBP Form I-68) AGENCY: U.S. Customs and Border Protection, Department of Homeland...

  5. PROCESS FOR THE PRODUCTION OF AN ACTIVATED FORM OF UO$sub 2$

    DOEpatents

    Polissar, M.J.

    1957-09-24

    A process for producing a highly active form of UO/sub 2/ characterized both by rapid oxidation in air and by rapid chlorination with CCl/sub 4/ vapor at an elevated temperature is reported. In accordance with the process, commercial UO/sub 2/, is subjected to a series of oxidation-reduction operations to produce a form of UC/sub 2/ of enhanced reactivity. By treatimg commercial UO/sub 2/ at a temperature between 335 and 485 deg C with methane, then briefly with an oxygen containing gas and followimg this by a second treatment with a methane containing gas, the original relatively stable charge of UO/sub 2/ will be transformed into an active form of UO/sub 2/.

  6. Formation scheme and antioxidant activity of a novel Maillard pigment, pyrrolothiazolate, formed from cysteine and glucose.

    PubMed

    Noda, Kyoko; Terasawa, Naoko; Murata, Masatsune

    2016-06-15

    We recently identified 6-hydroxy-3[R],7a[S]-dimethyl-7-oxo-2,3-dihydropyrrolo[2,1-b]thiazole-3-calboxylic acid, a novel pyrrolothiazole derivative carrying a carboxy group and named pyrrolothiazolate, as a Mallard pigment formed from l-cysteine and d-glucose. Here we described the formation of its enantiomer, the plausible formation scheme of pyrrolothiazolate, and its antioxidant activity. When d-cysteine was used instead of l-cysteine in the reaction mixture, the enantiomer of pyrrolothiazolate was obtained. The carbon at position 1 of glucose was incorporated into two methyl groups of pyrrolothiazolate. The pigment was considered to be formed through 1-deoxyglucosone (1-DG). The dehydrated isomer of 1-DG would be condensed with the thiol and amino groups of cysteine. This condensate was dehydrated and cyclized to form pyrrolothiazolate. This compound was an antioxidant showing radical scavenging activity. PMID:26987433

  7. Activated RSC-nucleosome complex and persistently altered form of the nucleosome.

    PubMed

    Lorch, Y; Cairns, B R; Zhang, M; Kornberg, R D

    1998-07-10

    RSC, an abundant, essential chromatin-remodeling complex, related to SWI/SNF complex, binds nucleosomes and naked DNA with comparable affinities, as shown by gel shift analysis. The RSC-nucleosome complex is converted in the presence of ATP to a slower migrating form. This activated complex exhibits greatly increased susceptibility to endo- and exonucleases but retains a full complement of histones. Activation persists in the absence of ATP, and on removal of RSC, the nucleosome is released in an altered form, with a diminished electrophoretic mobility, greater sedimentation rate, and marked instability at elevated ionic strength. The reaction is reversible in the presence of RSC and ATP, with conversion of the altered form back to the nucleosome. PMID:9674424

  8. Heterogeneity of Catalase in Maturing and Germinated Cotton Seeds 1

    PubMed Central

    Kunce, Christine M.; Trelease, Richard N.

    1986-01-01

    To investigate possible charge and size heterogeneity of catalase (EC 1.11.1.6) in cotton (Gossypium hirsutum L. cv Deltapine 62), extracts of cotyledons from different developmental ages were subjected to nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing. Special precautions (e.g. fresh homogenates, reducing media) were necessary to prevent artefacts due to enzyme modification during extraction and storage. When the gels were stained for enzyme activity, two distinct electrophoretic forms of catalase were resolved in extracts of maturing and mature cotton seeds. In germinated seeds, three additional cathodic forms were detected revealing a total of five electrophoretic variants. In green cotyledons, the two anodic forms characteristic of ungerminated seeds were less active; whereas, the most cathodic form was predominant. All forms of catalase were found in isolated glyoxysomes. Corresponding electrophoretic patterns were found on Western blots probed with anticatalase serum; no immunoreactive, catalytically inactive forms were detected. Western blots of sodium dodecyl sulfate-polyacrylamide gels revealed only one immunoreactive (55 kilodaltons) polypeptide in cotton extracts of all developmental ages. Results from isoelectric focusing and Ferguson plots indicate that the electrophoretic variants of catalase are charge isomers with a molecular weight of approximately 230,000. Images Fig. 1 Fig. 2 Fig. 3 Fig. 6 Fig. 7 PMID:16664956

  9. 76 FR 31972 - Agency Information Collection Activities: Form I-508 and Form I-508F, Extension of a Currently...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-02

    ..., Exemptions and Immunities; OMB Control No. 1615-0025. The Department of Homeland Security, U.S. Citizenship..., Privileges, Exemptions and Immunities. (3) Agency form number, if any, and the applicable component of...

  10. Binary Toxin Subunits of Lysinibacillus sphaericus Are Monomeric and Form Heterodimers after In Vitro Activation

    PubMed Central

    Choong, Yeu Khai; Torres, Jaume; Boonserm, Panadda

    2016-01-01

    The binary toxin from Lysinibacillus sphaericus has been successfully used for controlling mosquito-transmitted diseases. An activation step shortens both subunits BinA and BinB before their interaction with membranes and internalization in midgut cells, but the precise role of this activation step is unknown. Herein, we show conclusively using three orthogonal biophysical techniques that protoxin subunits form only monomers in aqueous solution. However, in vitro activated toxins readily form heterodimers. This oligomeric state did not change after incubation of these heterodimers with detergent. These results are consistent with the evidence that maximal toxicity in mosquito larvae is achieved when the two subunits, BinA and BinB, are in a 1:1 molar ratio, and directly link proteolytic activation to heterodimerization. Formation of a heterodimer must thus be necessary for subsequent steps, e.g., interaction with membranes, or with a suitable receptor in susceptible mosquito species. Lastly, despite existing similarities between BinB C-terminal domain with domains 3 and 4 of pore-forming aerolysin, no aerolysin-like SDS-resistant heptameric oligomers were observed when the activated Bin subunits were incubated in the presence of detergents or lipidic membranes. PMID:27341696

  11. Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens

    SciTech Connect

    Hagvall, Lina; Baron, Jens Malte; Boerje, Anna; Weidolf, Lars; Merk, Hans; Karlberg, Ann-Therese

    2008-12-01

    Contact sensitization is caused by low molecular weight compounds which penetrate the skin and bind to protein. In many cases, these compounds are activated to reactive species, either by autoxidation on exposure to air or by metabolic activation in the skin. Geraniol, a widely used fragrance chemical, is considered to be a weak allergen, although its chemical structure does not indicate it to be a contact sensitizer. We have shown that geraniol autoxidizes and forms allergenic oxidation products. In the literature, it is suggested but not shown that geraniol could be metabolically activated to geranial. Previously, a skin-like CYP cocktail consisting of cutaneous CYP isoenzymes, was developed as a model system to study cutaneous metabolism. In the present study, we used this system to investigate CYP-mediated activation of geraniol. In incubations with the skin-like CYP cocktail, geranial, neral, 2,3-epoxygeraniol, 6,7-epoxygeraniol and 6,7-epoxygeranial were identified. Geranial was the main metabolite formed followed by 6,7-epoxygeraniol. The allergenic activities of the identified metabolites were determined in the murine local lymph node assay (LLNA). Geranial, neral and 6,7-epoxygeraniol were shown to be moderate sensitizers, and 6,7-epoxygeranial a strong sensitizer. Of the isoenzymes studied, CYP2B6, CYP1A1 and CYP3A5 showed high activities. It is likely that CYP1A1 and CYP3A5 are mainly responsible for the metabolic activation of geraniol in the skin, as they are expressed constitutively at significantly higher levels than CYP2B6. Thus, geraniol is activated through both autoxidation and metabolism. The allergens geranial and neral are formed via both oxidation mechanisms, thereby playing a large role in the sensitization to geraniol.

  12. [Biophysical aspects of biological activity structure--strain calcium carbonat in micellar form].

    PubMed

    Ivanov, V I; Stekhin, A A; Iakovleva, G V; Savostikova, O N; Alekseeva, A V; P'ianzina, I P

    2013-01-01

    Results of the study of electrochemical and structural state of phase of associated water in the solutions of structurally stressed calcium carbonate in the micellar form are reported. On the base on the comparison of structural--physical changes of activated water with the data on the activity of bioluminiscentic "Ecolyum" microorganisms in their noncontact activation the electronic mechanism of the effect of activated water on cellular metabolism is substantiated The use of "Micellate of calcium" possessing non-contact electron-donor action on cellular structures was shown to permit to compensate the deficit of electrons and thereby to restore the activities of reductases and iron-containing peptides required for the production of regulatory ROS and alteration in redox state of the intracellular environment. PMID:24624817

  13. Data Product Maturity

    Atmospheric Science Data Center

    2013-03-25

    ... document, maturity levels are provided separately for each scientific data set (SDS) included with the data files. The data product ... indiscriminate use of these data products as the basis for research findings, journal publications, and/or presentations.   ...

  14. Integration of active pharmaceutical ingredient solid form selection and particle engineering into drug product design.

    PubMed

    Ticehurst, Martyn David; Marziano, Ivan

    2015-06-01

    This review seeks to offer a broad perspective that encompasses an understanding of the drug product attributes affected by active pharmaceutical ingredient (API) physical properties, their link to solid form selection and the role of particle engineering. While the crucial role of active pharmaceutical ingredient (API) solid form selection is universally acknowledged in the pharmaceutical industry, the value of increasing effort to understanding the link between solid form, API physical properties and drug product formulation and manufacture is now also being recognised. A truly holistic strategy for drug product development should focus on connecting solid form selection, particle engineering and formulation design to both exploit opportunities to access simpler manufacturing operations and prevent failures. Modelling and predictive tools that assist in establishing these links early in product development are discussed. In addition, the potential for differences between the ingoing API physical properties and those in the final product caused by drug product processing is considered. The focus of this review is on oral solid dosage forms and dry powder inhaler products for lung delivery. PMID:25677227

  15. Dopaminergic Toxin 1-Methyl-4-Phenylpyridinium, Proteins α-Synuclein and Glia Maturation Factor Activate Mast Cells and Release Inflammatory Mediators.

    PubMed

    Kempuraj, Duraisamy; Thangavel, Ramasamy; Yang, Evert; Pattani, Sagar; Zaheer, Smita; Santillan, Donna A; Santillan, Mark K; Zaheer, Asgar

    2015-01-01

    Parkinson's disease (PD) is characterized by the presence of Lewy bodies and degeneration of dopaminergic neurons. 1-methyl-4-phenylpyridinium (MPP+), a metabolite of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and Lewy body component α-synuclein activates glia in PD pathogenesis. Mast cells and glia maturation factor (GMF) are implicated in neuroinflammatory conditions including Multiple Sclerosis. However, the role of mast cells in PD is not yet known. We have analyzed the effect of recombinant GMF, MPP+, α-synuclein and interleukin-33 (IL-33) on mouse bone marrow-derived cultured mast cells (BMMCs), human umbilical cord blood-derived cultured mast cells (hCBMCs) and mouse brain-derived cultured astrocytes by quantifying cytokines/chemokines released using ELISA or by detecting the expression of co-stimulatory molecules CD40 and CD40L by flow cytometry. GMF significantly released chemokine (C-C motif) ligand 2 (CCL2) from BMMCs but its release was reduced in BMMCs from GMF knockout mice. GMF, α-synuclein and MPP+ released IL-1β, β-hexosaminidase from BMMCs, and IL-8 from hCBMCs. GMF released CCL5, and IL-33- induced the expression of GMF from hCBMCs. Novel GMF expression was detected in hCBMCs and BMMCs by immunocytochemistry. GMF released tumor necrosis factor-alpha (TNF-α) from mouse astrocytes, and this release was greater in BMMC- astrocyte coculture than in individual cultures. Flow cytometry results showed increased IL-33 expression by GMF and MPP+, and GMF-induced CD40 expression in astrocytes. Proinflammatory mediator release by GMF, MPP+ and α-synuclein, as well as GMF expression by mast cells indicate a potential therapeutic target for neurodegenerative diseases including PD. PMID:26275153

  16. Additive-dominance genetic model analyses for late-maturity alpha-amylase activity in a bread wheat factorial crossing population.

    PubMed

    Rasul, Golam; Glover, Karl D; Krishnan, Padmanaban G; Wu, Jixiang; Berzonsky, William A; Ibrahim, Amir M H

    2015-12-01

    Elevated level of late maturity α-amylase activity (LMAA) can result in low falling number scores, reduced grain quality, and downgrade of wheat (Triticum aestivum L.) class. A mating population was developed by crossing parents with different levels of LMAA. The F2 and F3 hybrids and their parents were evaluated for LMAA, and data were analyzed using the R software package 'qgtools' integrated with an additive-dominance genetic model and a mixed linear model approach. Simulated results showed high testing powers for additive and additive × environment variances, and comparatively low powers for dominance and dominance × environment variances. All variance components and their proportions to the phenotypic variance for the parents and hybrids were significant except for the dominance × environment variance. The estimated narrow-sense heritability and broad-sense heritability for LMAA were 14 and 54%, respectively. High significant negative additive effects for parents suggest that spring wheat cultivars 'Lancer' and 'Chester' can serve as good general combiners, and that 'Kinsman' and 'Seri-82' had negative specific combining ability in some hybrids despite of their own significant positive additive effects, suggesting they can be used as parents to reduce LMAA levels. Seri-82 showed very good general combining ability effect when used as a male parent, indicating the importance of reciprocal effects. High significant negative dominance effects and high-parent heterosis for hybrids demonstrated that the specific hybrid combinations; Chester × Kinsman, 'Lerma52' × Lancer, Lerma52 × 'LoSprout' and 'Janz' × Seri-82 could be generated to produce cultivars with significantly reduced LMAA level. PMID:26403988

  17. 1,25-Dihydroxyvitamin D3 and extracellular calcium promote mineral deposition via NPP1 activity in a mature osteoblast cell line MLO-A5.

    PubMed

    Yang, Dongqing; Turner, Andrew G; Wijenayaka, Asiri R; Anderson, Paul H; Morris, Howard A; Atkins, Gerald J

    2015-09-01

    While vitamin D supplementation is common, the anabolic mechanisms that improve bone status are poorly understood. Under standard mineralising conditions including media ionised calcium of 1.1 mM, 1,25-dihydroxyvitamin D3 (1,25D) enhanced differentiation and mineral deposition by the mature osteoblast/pre-osteocyte cell line, MLO-A5. This effect was markedly increased with a higher ionised calcium level (1.5 mM). Gene expression analyses revealed that 1,25D-induced mineral deposition was associated with induction of Enpp1 mRNA, coding for nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) and NPP1 protein levels. Since MLO-A5 cells express abundant alkaline phosphatase that was not further modified by 1,25D treatment or exposure to increased calcium, this finding suggested that the NPP1 production of pyrophosphate (PPi) may provide alkaline phosphatase with substrate for the generation of inorganic phosphate (Pi). Consistent with this, co-treatment with Enpp1 siRNA or a NPP1 inhibitor, PPADS, abrogated 1,25D-induced mineral deposition. These data demonstrate that 1,25D stimulates osteoblast differentiation and mineral deposition, and interacts with the extracellular calcium concentration. 1,25D regulates Enpp1 expression, which presumably, in the context of adequate tissue non-specific alkaline phosphatase activity, provides Pi to stimulate mineralisation. Our findings suggest a mechanism by which vitamin D with adequate dietary calcium can improve bone mineral status. PMID:26054750

  18. Gonadotropin-dependent oocyte maturational competence requires activation of the protein kinase A pathway and synthesis of RNA and protein in ovarian follicles of Nibe, Nibea mitsukurii (Teleostei, Sciaenidae)

    USGS Publications Warehouse

    Yoshizaki, G.; Shusa, M.; Takeuchi, T.; Patino, R.

    2002-01-01

    Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage; whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH [17,20??-dihydroxy-4-pregnen-3-one or 17,20??,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.

  19. [Effect of different nitrogen forms and ratio on growth and active ingredient content of Platycodon grandiflorum].

    PubMed

    Duan, Yun-jing; Wang, Kang-cai; Niu, Ling-hui; Li, Ke; Su, Yun-yun

    2015-10-01

    To providing evidence about nitrogen adequate application of Platycodon grandiflorum, the pot culture experiment was conducted to study the effect of nitrogen on the growth, physiological metabolism and the quality of P. grandiflorum. The activity of NR, GS and SOD, POD and CAT were determined. And the nitrate and ammonium nitrogen content, photosynthetic characteristics, active components of P. grandiflorum were determined. The results showed that the nitrate nitrogen content and P. biomass reached its maximum value, when NH4(+)-N/NO3(-) -N was 0: 100, the activity of NR. The activity of GS was the highest at the NH4(+) -N/NO3(-) -N ratio of 25:75 and ammonium nitrogen content was the highest at 75:25. The activity of SOD decreased and then increased with the increasing of NO3(-) -N. At the NH4(+) -N/NO3(-) -N ratio of 25: 75, the activity of CAT had its maximum value and the content of MDA had the minimum value. At the same time, the content of platycodon D was the highest at this treatment. The studies had shown that different nitrogen forms and ratio had a significant effect on the characteristics of photosynthetic physiology, nitrogen metabolism and resistance adjustment, growth and the quality of P. grandiflorum. The NH4(+) -N/NO3(-) -N ratio of 25: 75 was a suitable ratio of nitrogen forms for the growth of P. Grandiflorum and accumulating the content of platycodon D. PMID:26975097

  20. An ELISA method detecting the active form of suPAR.

    PubMed

    Zhou, Xiaolei; Xu, Mingming; Huang, Hailong; Mazar, Andrew; Iqbal, Zafar; Yuan, Cai; Huang, Mingdong

    2016-11-01

    Urokinase plasminogen activator receptor (uPAR) exists in a number of formats in human plasma, including soluble uPAR (suPAR) and uPAR fragments. We developed an ELISA method to detect specifically the active form suPAR, which binds to its natural ligand uPA. The intra CV and inter CV of this ELISA assay is 8.5% and 9.6% respectively, and the assay can recover 99.74% of added recombinant suPAR from 10% plasma. This assay is quite sensitive, capable of detecting down to 15pg/ml of suPAR, and can measure suPAR concentrations in the range of 0.031-8ng/ml with high linear relationship. Plasma samples from pregnant women were also measured for the active form of suPAR with this assay, giving an averaged level of 1.39ng/ml, slightly higher than the level of pooled plasma from healthy donors (0.96ng/ml). This study demonstrates the feasibility to measure the active form of suPAR, which will likely have value in clinical applications. PMID:27591605

  1. Crystallization and preliminary analysis of active nitroalkane oxidase in three crystal forms.

    PubMed

    Nagpal, Akanksha; Valley, Michael P; Fitzpatrick, Paul F; Orville, Allen M

    2004-08-01

    Nitroalkane oxidase (NAO), a flavoprotein cloned and purified from Fusarium oxysporum, catalyzes the oxidation of neutral nitroalkanes to the corresponding aldehydes or ketones, with the production of H2O2 and nitrite. In this paper, the crystallization and preliminary X-ray data analysis of three crystal forms of active nitroalkane oxidase are described. The first crystal form belongs to a trigonal space group (either P3(1)21 or P3(2)21, with unit-cell parameters a = b = 103.8, c = 487.0 A) and diffracts to at least 1.6 A resolution. Several data sets were collected using 2theta and kappa geometry in order to obtain a complete data set to 2.07 A resolution. Solvent-content and Matthews coefficient analysis suggests that crystal form 1 contains two homotetramers per asymmetric unit. Crystal form 2 (P2(1)2(1)2(1); a = 147.3, b = 153.5, c = 169.5 A) and crystal form 3 (P3(1) or P3(2); a = b = 108.9, c = 342.5 A) are obtained from slightly different conditions and also contain two homotetramers per asymmetric unit, but have different solvent contents. A three-wavelength MAD data set was collected from selenomethionine-enriched NAO (SeMet-NAO) in crystal form 3 and will be used for phasing. PMID:15272176

  2. 76 FR 46781 - Commission Information Collection Activities (FERC Form 1 and FERC Form 1F); Comment Request...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-03

    ... to providing this information, such as administrative costs and the cost for information technology... 5 1 116 580 Total 243,438 The total estimated annual cost burden to respondents on the FERC Form 1 is $12,385,758 (242,858 hours x $51/hour \\3\\). The average cost per respondent is $59,262. \\3\\...

  3. 76 FR 53144 - Agency Information Collection Activities: Form I-508 and Form I-508F, Extension of a Currently...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-25

    ... information collection was previously published in the Federal Register on June 2, 2011, at 76 FR 31972..., Exemptions and Immunities. The Department of Homeland Security, U.S. Citizenship and Immigration Services..., Privileges, Exemptions and Immunities. (3) Agency form number, if any, and the applicable component of...

  4. Cyclotron production of ``very high specific activity'' platinum radiotracers in No Carrier Added form

    NASA Astrophysics Data System (ADS)

    Birattari, C.; Bonardi, M.; Groppi, F.; Gini, L.; Gallorini, M.; Sabbioni, E.; Stroosnijder, M. F.

    2001-12-01

    At the "Radiochemistry Laboratory" of Accelerators and Applied Superconductivity Laboratory, LASA, several production and quality assurance methods for short-lived and high specific activity radionuclides, have been developed. Presently, the irradiations are carried out at the Scanditronix MC40 cyclotron (K=38; p, d, He-4 and He-3) of JRC-Ispra, Italy, of the European Community, while both chemical purity and specific activity determination are carried out at the TRIGA MARK II research reactor of University of Pavia and at LASA itself. In order to optimize the irradiation conditions for platinum radiotracer production, both thin- and thick-target excitation function of natOs(α,xn) nuclear reactions were measured. A very selective radiochemical separation to obtain Pt radiotracers in No Carrier Added form, has been developed. Both real specific activity and chemical purity of radiotracer, have been determined by neutron activation analysis and atomic absorption spectrometry. An Isotopic Dilution Factor (IDF) of the order of 50 is achieved.

  5. Linear Closed-form Solution and Finite-element Analysis of an Active Tensegrity Unit

    NASA Astrophysics Data System (ADS)

    Kmeť, Stanislav; Platko, Peter

    2012-11-01

    Results of the linear closed form solution of an active or adaptive tensegrity unit, as well as its numerical analysis using finite element method are presented in the paper. The shape of the unit is an octahedral cell with a square base and it is formed by thirteen members (four bottom and four top cables, four edge struts and one central strut). The central strut is designed as an actuator that allows for an adjustment of the shape of the unit which leads to changes of tensile forces in the cables. Due to the diagonal symmetry of the 3D tensegrity unit the closed-form analysis is based on the 2D solution of the equivalent planar biconvex cable system with one central strut under a vertical point load.

  6. Determination of the active form of the tetranuclear copper sulfur cluster in nitrous oxide reductase.

    PubMed

    Johnston, Esther M; Dell'Acqua, Simone; Ramos, Susana; Pauleta, Sofia R; Moura, Isabel; Solomon, Edward I

    2014-01-15

    N2OR has been found to have two structural forms of its tetranuclear copper active site, the 4CuS Cu(Z)* form and the 4Cu2S Cu(Z) form. EPR, resonance Raman, and MCD spectroscopies have been used to determine the redox states of these sites under different reductant conditions, showing that the Cu(Z)* site accesses the 1-hole and fully reduced redox states, while the Cu(Z) site accesses the 2-hole and 1-hole redox states. Single-turnover reactions of N2OR for Cu(Z) and Cu(Z)* poised in these redox states and steady-state turnover assays with different proportions of Cu(Z) and Cu(Z)* show that only fully reduced Cu(Z)* is catalytically competent in rapid turnover with N2O. PMID:24364717

  7. Active heterodimers are formed from human DNA topoisomerase II alpha and II beta isoforms.

    PubMed Central

    Biersack, H; Jensen, S; Gromova, I; Nielsen, I S; Westergaard, O; Andersen, A H

    1996-01-01

    DNA topoisomerase II is a nuclear enzyme essential for chromosome dynamics and DNA metabolism. In mammalian cells, two genetically and biochemically distinct topoisomerase II forms exist, which are designated topoisomerase II alpha and topoisomerase II beta. In our studies of human topoisomerase II, we have found that a substantial fraction of the enzyme exists as alpha/beta heterodimers in HeLa cells. The ability to form heterodimers was verified when human topoisomerases II alpha and II beta were coexpressed in yeast and investigated in a dimerization assay. Analysis of purified heterodimers shows that these enzymes maintain topoisomerase II specific catalytic activities. The natural existence of an active heterodimeric subclass of topoisomerase II merits attention whenever topoisomerases II alpha and II beta function, localization, and cell cycle regulation are investigated. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8710863

  8. Role of drop distortion in enhancing the lightning activity in clouds formed over cities

    NASA Astrophysics Data System (ADS)

    Bhalwankar, Rohini; Kamra, A. K.

    2013-03-01

    Atmospheric pollutants can modify the electrification and lightning activity in thunderclouds. Laboratory simulation experiments show that distortion of water drops is more when drops are formed from water polluted with Sulfate/Nitrate salts than that from distilled water and the difference in distortions is more in a higher electric field. Further, the polluted water drops falling in a horizontal electric field can trigger a discharge on their surface and the discharge can propagate as a streamer in lower electric fields as compared to that from distilled water drops. The difference in electrical conductivities of polluted and unpolluted water drops is most likely the key factor for manifestation of these differences. It is proposed that the enhanced distortion of polluted drops coupled with the change in their characteristics to trigger and propagate a discharge in lower electric fields may significantly contribute to the enhancement of lightning activity observed in clouds formed over big cities.

  9. Effect of the Active Site D25N Mutation on the Structure, Stability and Ligand Binding of the Mature HIV-1 Protease

    SciTech Connect

    Sayer, Jane M.; Liu, Fengling; Ishima, Rieko; Weber, Irene T.; Louis, John M.

    2008-09-03

    All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR{sub D25N}) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 {+-} 0.09 {mu}m) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR{sub D25N} in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 C, respectively. Only minimal differences are observed in high resolution crystal structures of PR{sub D25N} complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH{sub 2} (RPB), as compared with PR{center_dot}DRV and PR{center_dot}RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T{sub m} is smaller for PR{sub D25N} (6.2 C) than for PR (8.7 C). The T{sub m} of PR{sub D25N}{center_dot}DRV increases by only 3 C relative to free PR{sub D25N}, as compared with a 22 C increase for PR{center_dot}DRV, and the mutation increases the ligand dissociation constant of PR{sub D25N}{center_dot}DRV by a factor of {approx}10{sup 6} relative to PR{center_dot}DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

  10. Protease-Activated Pore-Forming Peptides for the Treatment and Imaging of Prostate Cancer

    PubMed Central

    LeBeau, Aaron M.; Denmeade, Samuel R.

    2015-01-01

    A common hallmark of cancers with highly aggressive phenotypes is increased proteolysis in the tumor and the surrounding microenvironment. Prostate cancer has a number of proteases uniquely associated with it that may play various important roles in disease progression. In this report, we utilize the peritumoral proteolytic activity of prostate cancer to activate engineered peptide constructs for the treatment and noninvasive imaging of prostate cancer. Using a modular "propeptide" approach, a cationic diastereomeric pore-forming peptide domain was linked to an inactivating acidic peptide domain. The inactivating acidic peptide domain was engineered to be a cleavable substrate for the secreted serine protease prostate-specific antigen (PSA) or the transmembrane metalloprotease prostate-specific membrane antigen (PSMA). The propeptides were then evaluated in a direct comparison study. Both the PSA and PSMA activated propeptides were found to be cytotoxic to prostate cancer cells in vitro. In vivo, however, treatment of LNCaP and CWR22Rv1 xenografts with the PSMA propeptide resulted in a pronounced cytostatic effect when compared with xenografts treated with the PSA propeptide or the cationic diastereomeric peptide alone. The PSMA activated propeptide also proved to be an effective optical imaging probe in vivo when labeled with a near-infrared fluorophore. These data suggest that protease-activated pore-forming peptides could potentially be used for both imaging and treating prostate cancer. PMID:25537662

  11. Antimicrobial and Antioxidant Activity of Chitosan/Hydroxypropyl Methylcellulose Film-Forming Hydrosols Hydrolyzed by Cellulase.

    PubMed

    Zimoch-Korzycka, Anna; Bobak, Łukasz; Jarmoluk, Andrzej

    2016-01-01

    The aim of this study was to evaluate the impact of cellulase (C) on the biological activity of chitosan/hydroxypropyl methylcellulose (CH/HPMC) film-forming hydrosols. The hydrolytic activity of cellulase in two concentrations (0.05% and 0.1%) was verified by determination of the progress of polysaccharide hydrolysis, based on viscosity measurement and reducing sugar-ends assay. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging effect, the ferric reducing antioxidant power (FRAP), and microbial reduction of Pseudomonas fluorescens, Yersinia enterocolitica, Bacillus cereus, and Staphylococcus aureus were studied. During the first 3 h of reaction, relative reducing sugar concentration increased progressively, and viscosity decreased rapidly. With increasing amount of enzyme from 0.05% to 0.1%, the reducing sugar concentration increased, and the viscosity decreased significantly. The scavenging effect of film-forming solutions was improved from 7.6% at time 0 and without enzyme to 52.1% for 0.1% cellulase after 20 h of reaction. A significant effect of cellulase addition and reaction time on antioxidant power of the tested film-forming solutions was also reported. Film-forming hydrosols with cellulase exhibited a bacteriostatic effect on all tested bacteria, causing a total reduction. PMID:27608008

  12. The chemokine system in diverse forms of macrophage activation and polarization.

    PubMed

    Mantovani, Alberto; Sica, Antonio; Sozzani, Silvano; Allavena, Paola; Vecchi, Annunciata; Locati, Massimo

    2004-12-01

    Plasticity and functional polarization are hallmarks of the mononuclear phagocyte system. Here we review emerging key properties of different forms of macrophage activation and polarization (M1, M2a, M2b, M2c), which represent extremes of a continuum. In particular, recent evidence suggests that differential modulation of the chemokine system integrates polarized macrophages in pathways of resistance to, or promotion of, microbial pathogens and tumors, or immunoregulation, tissue repair and remodeling. PMID:15530839

  13. Validity and Reliability of International Physical Activity Questionnaire-Short Form in Chinese Youth

    ERIC Educational Resources Information Center

    Wang, Chao; Chen, Peijie; Zhuang, Jie

    2013-01-01

    Purpose: The psychometric profiles of the widely used International Physical Activity Questionnaire-Short Form (IPAQ-SF) in Chinese youth have not been reported. The purpose of this study was to examine the validity and reliability of the IPAQ-SF using a sample of Chinese youth. Method: One thousand and twenty-one youth (M[subscript age] = 14.26 ±…

  14. Mechanistic insights into the first Lygus-active β-pore forming protein.

    PubMed

    Jerga, Agoston; Chen, Danqi; Zhang, Chunfen; Fu, Jinping; Kouadio, Jean-Louis K; Wang, Yanfei; Duff, Stephen M G; Howard, Jennifer E; Rydel, Timothy J; Evdokimov, Artem G; Ramaseshadri, Parthasarathy; Evans, Adam; Bolognesi, Renata; Park, Yoonseong; Haas, Jeffrey A

    2016-06-15

    The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active β-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the β-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered β-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality. PMID:27001423

  15. 77 FR 9259 - Agency Information Collection Activities: Forms G-1041 and G-1041A, Extension of a Currently...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-16

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Forms G-1041... Notice of Information Collection Under Review: Forms G- 1041 and G-1041A, Genealogy Index Search Request... evaluating whether to revise forms G-1041 and G-1041A. Should USCIS decide to revise forms G- 1041 and...

  16. MAVS Forms Functional Prion-Like Aggregates To Activate and Propagate Antiviral Innate Immune Response

    PubMed Central

    Hou, Fajian; Sun, Lijun; Zheng, Hui; Skaug, Brian; Jiang, Qiu-Xing; Chen, Zhijian J.

    2011-01-01

    SUMMARY In response to viral infection, RIG-I–like RNA helicases bind to viral RNA and activate the mitochondrial protein MAVS, which in turn activates the transcription factors IRF3 and NF-κB to induce type-I interferons. We have previously shown that RIG-I binds to unanchored lysine-63 (K63) polyubiquitin chains and that this binding is important for MAVS activation; however, the mechanism underlying MAVS activation is not understood. Here we show that viral infection induces the formation of very large MAVS aggregates, which potently activate IRF3 in the cytosol. We find that a fraction of recombinant MAVS protein forms fibrils capable of activating IRF3. Remarkably, the MAVS fibrils behave like prions and effectively convert endogenous MAVS into functional aggregates. We also show that, in the presence of K63 ubiquitin chains, RIG-I catalyzes the conversion of MAVS on the mitochondrial membrane to prion-like aggregates. These results suggest that a prion-like conformational switch of MAVS activates and propagates the antiviral signaling cascade. PMID:21782231

  17. Brain activation patterns resulting from learning letter forms through active self-production and passive observation in young children

    PubMed Central

    Kersey, Alyssa J.; James, Karin H.

    2013-01-01

    Although previous literature suggests that writing practice facilitates neural specialization for letters, it is unclear if this facilitation is driven by the perceptual feedback from the act of writing or the actual execution of the motor act. The present study addresses this issue by measuring the change in BOLD signal in response to hand-printed letters, unlearned cursive letters, and cursive letters that 7-year-old children learned actively, by writing, and passively, by observing an experimenter write. Brain activation was assessed using fMRI while perceiving letters—in both cursive and manuscript forms. Results showed that active training led to increased recruitment of the sensori-motor network associated with letter perception as well as the insula and claustrum, but passive observation did not. This suggests that perceptual networks for newly learned cursive letters are driven by motor execution rather than by perceptual feedback. PMID:24069007

  18. Theoretical studies of chemical reactivity of metabolically activated forms of aromatic amines toward DNA.

    PubMed

    Shamovsky, Igor; Ripa, Lena; Blomberg, Niklas; Eriksson, Leif A; Hansen, Peter; Mee, Christine; Tyrchan, Christian; O'Donovan, Mike; Sjö, Peter

    2012-10-15

    The metabolism of aromatic and heteroaromatic amines (ArNH₂) results in nitrenium ions (ArNH⁺) that modify nucleobases of DNA, primarily deoxyguanosine (dG), by forming dG-C8 adducts. The activated amine nitrogen in ArNH⁺ reacts with the C8 of dG, which gives rise to mutations in DNA. For the most mutagenic ArNH₂, including the majority of known genotoxic carcinogens, the stability of ArNH⁺ is of intermediate magnitude. To understand the origin of this observation as well as the specificity of reactions of ArNH⁺ with guanines in DNA, we investigated the chemical reactivity of the metabolically activated forms of ArNH₂, that is, ArNHOH and ArNHOAc, toward 9-methylguanine by DFT calculations. The chemical reactivity of these forms is determined by the rate constants of two consecutive reactions leading to cationic guanine intermediates. The formation of ArNH⁺ accelerates with resonance stabilization of ArNH⁺, whereas the formed ArNH⁺ reacts with guanine derivatives with the constant diffusion-limited rate until the reaction slows down when ArNH⁺ is about 20 kcal/mol more stable than PhNH⁺. At this point, ArNHOH and ArNHOAc show maximum reactivity. The lowest activation energy of the reaction of ArNH⁺ with 9-methylguanine corresponds to the charge-transfer π-stacked transition state (π-TS) that leads to the direct formation of the C8 intermediate. The predicted activation barriers of this reaction match the observed absolute rate constants for a number of ArNH⁺. We demonstrate that the mutagenic potency of ArNH₂ correlates with the rate of formation and the chemical reactivity of the metabolically activated forms toward the C8 atom of dG. On the basis of geometric consideration of the π-TS complex made of genotoxic compounds with long aromatic systems, we propose that precovalent intercalation in DNA is not an essential step in the genotoxicity pathway of ArNH₂. The mechanism-based reasoning suggests rational design strategies to

  19. A comparison of the efficiency of G protein activation by ligand-free and light-activated forms of rhodopsin.

    PubMed Central

    Melia, T J; Cowan, C W; Angleson, J K; Wensel, T G

    1997-01-01

    Activation of the photoreceptor G protein transducin (Gt) by opsin, the ligand-free form of rhodopsin, was measured using rod outer segment membranes with densities of opsin and Gt similar to those found in rod cells. When GTPgammaS was used as the activating nucleotide, opsin catalyzed transducin activation with an exponential time course with a rate constant k(act) on the order of 2 x 10(-3)s(-1). Comparison under these conditions to activation by flash-generated metarhodopsin II (MII) revealed that opsin- and R*-catalyzed activation showed similar kinetics when MII was present at a surface density approximately 10(-6) lower than that of opsin. Thus, in contrast to some previous reports, we find that the catalytic potency of opsin is only approximately 10(-6) that of MII. In the presence of residual retinaldehyde-derived species present in membranes treated with hydroxylamine after bleaching, the apparent k(act) observed was much higher than that for opsin, suggesting a possible explanation for previous reports of more efficient activation by opsin. These results are important for considering the possible role of opsin in the diverse phenomena in which it has been suggested to play a key role, such as bleaching desensitization and retinal degeneration induced by continuous light or vitamin A deprivation. PMID:9414230

  20. Implications for the active form of human insulin based on the structural convergence of highly active hormone analogues

    PubMed Central

    Jiráček, Jiří; Žáková, Lenka; Antolíková, Emília; Watson, Christopher J.; Turkenburg, Johan P.; Dodson, Guy G.; Brzozowski, Andrzej M.

    2010-01-01

    Insulin is a key protein hormone that regulates blood glucose levels and, thus, has widespread impact on lipid and protein metabolism. Insulin action is manifested through binding of its monomeric form to the Insulin Receptor (IR). At present, however, our knowledge about the structural behavior of insulin is based upon inactive, multimeric, and storage-like states. The active monomeric structure, when in complex with the receptor, must be different as the residues crucial for the interactions are buried within the multimeric forms. Although the exact nature of the insulin’s induced-fit is unknown, there is strong evidence that the C-terminal part of the B-chain is a dynamic element in insulin activation and receptor binding. Here, we present the design and analysis of highly active (200–500%) insulin analogues that are truncated at residue 26 of the B-chain (B26). They show a structural convergence in the form of a new β-turn at B24-B26. We propose that the key element in insulin’s transition, from an inactive to an active state, may be the formation of the β-turn at B24-B26 associated with a trans to cis isomerisation at the B25-B26 peptide bond. Here, this turn is achieved with N-methylated L-amino acids adjacent to the trans to cis switch at the B25-B26 peptide bond or by the insertion of certain D-amino acids at B26. The resultant conformational changes unmask previously buried amino acids that are implicated in IR binding and provide structural details for new approaches in rational design of ligands effective in combating diabetes. PMID:20133841

  1. Influence of malignant cell clonogenic capacities and position along the maturation pathway on their susceptibility to lymphokine-activated killer cell cytotoxicity.

    PubMed

    Thomas, X; Anglaret, B; Adeleine, P; Maritaz, O; Bailly, M; Fiere, D; Archimbaud, E

    1998-01-01

    In order to investigate the sensitivity of malignant target cells to lysis by LAK cells according to their clonogenic capacities and their position along the maturation pathway, we compared clonogenic and chromium release cytotoxicity assays performed on human hematopoietic cell lines using Effector: Target ratios of 1:1, 3:1, 6:1, 12:1, 24:1, 48:1 and 96:1, and studied the sensitivity of HL-60 and U937 human cell lines after exposure to different factors including GM-CSF, SCF, IFN, Retinoic acid (RA), DMSO, and TPA which are able to recruit cells into the cell cycle or to induce cell differentiation. There was a good correlation between the lysis of the target cells using 51Cr release and the growth inhibition in semisolid medium. The degree of inhibition was significantly higher using the colony growth assay (p = 0.006). Regarding the effects of culturing cell lines with proliferating and differentiating agents on the sensitivity of these cell lines to LAK cytolysis, a correlation was noted between the proliferative response of the U937 cell line and susceptibility to LAK cell lysis (p = 0.01), while results appeared close to significance with HL-60. The most significant effects were a decreased sensitivity of HL-60 to LAK lysis with RA (p < 0.001) and TPA (p < 0.001), and an increased susceptibility of U937 to LAK lysis with GM-CSF (p < 0.0001). In studies planned to investigate whether susceptibility of treated cells to LAK activity was a consequence of a downregulation of adhesion molecules expressed on target cell surface, the proportion of cells expressing adhesion molecules was not significantly changed, except for CD54 expression on HL-60 cells which showed a higher expression, after cells were treated with RA or DMSO. We conclude that clonogenic cells are more sensitive to LAK cell lysis and that cell line sensitivity to LAK cytolysis can be modulated by a variety of agents of potential therapeutic use. The poor correlation between adhesion molecules

  2. Multifunctional porous titanium oxide coating with apatite forming ability and photocatalytic activity on a titanium substrate formed by plasma electrolytic oxidation.

    PubMed

    Akatsu, T; Yamada, Y; Hoshikawa, Y; Onoki, T; Shinoda, Y; Wakai, F

    2013-12-01

    Plasma electrolytic oxidation (PEO) was used to make a multifunctional porous titanium oxide (TiO2) coating on a titanium substrate. The key finding of this study is that a highly crystalline TiO2 coating can be made by performing the PEO in an ammonium acetate (CH3COONH4) solution; the PEO coating was formed by alternating between rapid heating by spark discharges and quenching in the solution. The high crystallinity of the TiO2 led to the surface having multiple functions, including apatite forming ability and photocatalytic activity. Hydroxyapatite formed on the PEO coating when it was soaked in simulated body fluid. The good apatite forming ability can be attributed to the high density of hydroxyl groups on the anatase and rutile phases in the coating. The degradation of methylene blue under ultraviolet radiation indicated that the coating had high photocatalytic activity. PMID:24094199

  3. Special Form Testing of Sealed Source Encapsulation for High-Alpha-Activity Actinide Materials

    SciTech Connect

    Martinez, Oscar A

    2016-01-01

    In the United States all transportation of radioactive material is regulated by the U.S. Department of Transportation (DOT). Beginning in 2008 a new type of sealed-source encapsulation package was developed and tested by Oak Ridge National Laboratory (ORNL). These packages contain high-alpha-activity actinides and are regulated and transported in accordance with the requirements for DOT Class 7 hazardous material. The DOT provides specific regulations pertaining to special form encapsulation designs. The special form designation indicates that the encapsulated radioactive contents have a very low probability of dispersion even when subjected to significant structural events. The special form designs have been shown to simplify the delivery, transport, acceptance, and receipt processes. It is intended for these sealed-source encapsulations to be shipped to various facilities making it very advantageous for them to be certified as special form. To this end, DOT Certificates of Competent Authority (CoCAs) have been sought for the design suitable for containing high-alpha-activity actinide materials. This design consists of the high-alpha-activity material encapsulated within a triangular zirconia canister, referred to as a ZipCan, tile that is then enclosed by a spherical shell. The spherical shell design, with ZipCan tile inside, was tested for compliance with the special form regulations found in 49 CFR 173.469. The spherical enclosure was subjected to 9-m impact, 1 m percussion, and 10-minute thermal tests at the Packaging Evaluation Facility located at the National Transportation Research Center in Knoxville, TN USA and operated by ORNL. Before and after each test, the test units were subjected to a helium leak check and a bubble test. The ZipCan tiles and core were also subjected to the tests required for ISO 2919:2012(E), including a Class IV impact test and heat test and subsequently subjected to helium leakage rate tests [49 CFR 173.469(a)(4)(i)]. The impact

  4. Staphylococcus aureus forms spreading dendrites that have characteristics of active motility.

    PubMed

    Pollitt, Eric J G; Crusz, Shanika A; Diggle, Stephen P

    2015-01-01

    Staphylococcus aureus is historically regarded as a non-motile organism. More recently it has been shown that S. aureus can passively move across agar surfaces in a process called spreading. We re-analysed spreading motility using a modified assay and focused on observing the formation of dendrites: branching structures that emerge from the central colony. We discovered that S. aureus can spread across the surface of media in structures that we term 'comets', which advance outwards and precede the formation of dendrites. We observed comets in a diverse selection of S. aureus isolates and they exhibit the following behaviours: (1) They consist of phenotypically distinct cores of cells that move forward and seed other S. aureus cells behind them forming a comet 'tail'; (2) they move when other cells in the comet tail have stopped moving; (3) the comet core is held together by a matrix of slime; and (4) the comets etch trails in the agar as they move forwards. Comets are not consistent with spreading motility or other forms of passive motility. Comet behaviour does share many similarities with a form of active motility known as gliding. Our observations therefore suggest that S. aureus is actively motile under certain conditions. PMID:26680153

  5. Staphylococcus aureus forms spreading dendrites that have characteristics of active motility

    PubMed Central

    Pollitt, Eric J. G.; Crusz, Shanika A.; Diggle, Stephen P.

    2015-01-01

    Staphylococcus aureus is historically regarded as a non-motile organism. More recently it has been shown that S. aureus can passively move across agar surfaces in a process called spreading. We re-analysed spreading motility using a modified assay and focused on observing the formation of dendrites: branching structures that emerge from the central colony. We discovered that S. aureus can spread across the surface of media in structures that we term ‘comets’, which advance outwards and precede the formation of dendrites. We observed comets in a diverse selection of S. aureus isolates and they exhibit the following behaviours: (1) They consist of phenotypically distinct cores of cells that move forward and seed other S. aureus cells behind them forming a comet ‘tail’; (2) they move when other cells in the comet tail have stopped moving; (3) the comet core is held together by a matrix of slime; and (4) the comets etch trails in the agar as they move forwards. Comets are not consistent with spreading motility or other forms of passive motility. Comet behaviour does share many similarities with a form of active motility known as gliding. Our observations therefore suggest that S. aureus is actively motile under certain conditions. PMID:26680153

  6. Adaptor Protein Complex 2 (AP-2) Mediated, Clathrin Dependent Endocytosis, And Related Gene Activities, Are A Prominent Feature During Maturation Stage Amelogenesis

    PubMed Central

    LACRUZ, Rodrigo S.; BROOKES, Steven J.; WEN, Xin; JIMENEZ, Jaime M.; VIKMAN, Susanna; HU, Ping; WHITE, Shane N.; LYNGSTADAAS, S. Petter; OKAMOTO, Curtis T.; SMITH, Charles E.; PAINE, Michael L.

    2012-01-01

    Molecular events defining enamel matrix removal during amelogenesis are poorly understood. Early reports have suggested that adaptor proteins (AP) participate in ameloblast-mediated endocytosis. Enamel formation involves the secretory and maturation stages, with an increase in resorptive function during the latter. Here, using real time PCR, we show that the expression of clathrin and adaptor protein subunits are up-regulated in maturation stage rodent enamel organ cells. AP-2 is the most up-regulated of the four distinct adaptor protein complexes. Immunolocalization confirms the presence of AP-2 and clathrin in ameloblasts with strongest reactivity at the apical pole. These data suggest that the resorptive functions of enamel cells involve AP-2 mediated, clathrin dependent endocytosis, thus implying the likelihood of a specific membrane-bound receptor(s) of enamel matrix protein debris. The mRNA expression of other endocytosis-related gene products is also up-regulated during maturation including: lysosomal-associated membrane protein 1 (Lamp1), cluster of differentiation 63 and 68 (Cd63 and Cd68), ATPase, H+ transporting, lysosomal V0 subunit D2 (Atp6v0d2), ATPase, H+ transporting, lysosomal V1 subunit B2 (Atp6v1b2), chloride channel, voltage-sensitive 7 (Clcn7) and cathepsin K (Ctsk). Immunohistological data confirms the expression of a number of these proteins in maturation stage ameloblasts. The enamel of Cd63-null mice was also examined. Despite increased mRNA and protein expression in the enamel organ during maturation, the enamel of Cd63-null mice appeared normal. This may suggest inherent functional redundancies between Cd63 and related gene products, such as Lamp1 and Cd68. Ameloblast-like LS8 cells treated with the enamel matrix protein complex Emdogain® showed up-regulation of AP-2 and clathrin subunits, further supporting the existence of a membrane-bound receptor regulated pathway for the endocytosis of enamel matrix proteins. These data together

  7. From enzyme maturation to synthetic chemistry: the case of hydrogenases.

    PubMed

    Artero, Vincent; Berggren, Gustav; Atta, Mohamed; Caserta, Giorgio; Roy, Souvik; Pecqueur, Ludovic; Fontecave, Marc

    2015-08-18

    prepare a hybrid system, displaying remarkable structural similarities to an [FeFe]-hydrogenase, and we show here for the first time that it is catalytically active for proton reduction. This system is based on the combination of HydF, a protein involved in the maturation of [FeFe]-hydrogenase (HydA), and a close mimic of the active site of this class of enzymes. Moreover, the synthetic [Fe2(adt)(CO)4(CN)2](2-) (adt(2-)= aza-propanedithiol) mimic, alone or within a HydF hybrid system, was shown to be able to maturate and activate a form of HydA itself lacking its diiron active site. We discuss the exciting perspectives this "synthetic maturation" opens regarding the "invention" of novel hydrogenases by the chemists. PMID:26165393

  8. [CELLS FORM AND THEIR SENSITIVITY TO LYTIC ACTIVITY OF NATURAL KILLER CELLS UNDER THE ANTIOXIDANT ACTION].

    PubMed

    Kirpichnikova, K M; Petrov, Yu P; Filatova, N A; Gamaley, I A

    2015-01-01

    The present paper is an attempt to estimate the influence of cell surface morphology changes to functional activity under the effect of antioxidant, N-acetylcysteine (NAC), and alpha-lipoic asid (ALA). Two experimental parameters were used to characterize transformed fibroblasts 3T3-SV40 status. The functional one was the cell sensitivity to lysis by natural killer (NK) mouse splenocytes, and morphology index (cell form index) was a cell area. We showed that addition of NAC or ALA to the cell medium caused fast decrease of cell area and changes of cell form. On the other hand, their sensitivity to lysis NK cells gradually and significantly decreased. Then we compared NAC or ALA effect with the effects of other substances, which were non-antioxidants but caused cell responses which concurred with of antioxidants, at least partly. They were: latrunculin B, desorganizing actin filaments (as both antioxidants), OTZ reducing ROS level in the cell (as NAC), BSO (inhibitor of glutathione synthesis), increasing ROS level in the cell (as ALA), antibodies to gelatinases, MMP-2 and MMP-9 inactivating their activities (as both antioxidants). The results obtained showed a correlation between changes of morphology index and functional activity, sensitivity to lysis by NK cells. We suppose that geometry of cell surface might be a functional indicator of cell reaction to the antioxidant. PMID:26591569

  9. Antifungal activity and pore-forming mechanism of astacidin 1 against Candida albicans.

    PubMed

    Choi, Hyemin; Lee, Dong Gun

    2014-10-01

    In a previous report, a novel antibacterial peptide astacidin 1 (FKVQNQHGQVVKIFHH) was isolated from hemocyanin of the freshwater crayfish Pacifastacus leniusculus. In this study, the antifungal activity and mechanism of astacidin 1 were evaluated. Astacidin 1 exhibited antifungal activity against Candida albicans, Trichosporon beigelii, Malassezia furfur, and Trichophyton rubrum. Also, astacidin 1 had fungal cell selectivity in human erythrocytes without causing hemolysis. To understand the antifungal mechanism, membrane studies were done against C. albicans and T. beigelii. Flow cytometric analysis and K(+) measurement showed membrane damage, resulting in membrane permeabilization and K(+) release-induced membrane depolarization. Furthermore, the calcein leakage from liposomes mimicking C. albicans membrane demonstrated that the membrane-active action was driven by pore-forming mechanism. Live cell imaging using fluorescein isothiocyanate-labeled dextrans of various sizes suggested that the radii of pores formed in the C. albicans membrane were 1.4-2.3 nm. Therefore, the present study suggests that astacidin 1 exerts its antifungal effect by damaging the fungal membrane via pore formation. PMID:24955933

  10. Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.

    PubMed Central

    Thompson, S D; Laver, W G; Murti, K G; Portner, A

    1988-01-01

    As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features. Images PMID:2846877

  11. Glutathione peroxidase activity and chemical forms of selenium in tissues of rats given selenite or selenomethionine

    SciTech Connect

    Beilstein, M.A.; Whanger, P.D.

    1988-05-01

    Glutathione peroxidase (GPx) activity and deposition of selenium (Se) were examined in tissues of rats given dietary Se for 7 wk as either selenite or selenomethionine (SeMet) with 75Se radiotracer of the same chemical form. On the basis of Se:75Se ratio, all tissues of the rats fed selenite were equilibrated with the dietary source, but tissues of the SeMet fed animals maintained a ratio of Se:75Se greater than the dietary ratio. Deposition of dietary Se and 75Se was higher in most tissues of rats fed SeMet. Muscle 75Se was the largest single tissue pool of 75Se in both groups accounting for one-third of recovered 75Se in the rats fed selenite, and one-half of recovered 75Se in the rats fed SeMet. Tissue GPx activities were not different between the two dietary groups. The proportion of Se as GPx in tissues was highest in erythrocytes of the rats fed selenite (.81) and lowest in testes and epididymides of the rats fed SeMet (.009). The proportion of Se present in cytosolic GPx was consistently higher in tissues of rats fed selenite. Erythrocytes of the rats fed SeMet had more 75Se associated with hemoglobin, and muscle cytosols of the rats fed selenite had more 75Se associated with the G-protein. The proportion of 75Se as SeMet determined by ion exchange chromatography of tissue hydrolysates was higher in tissues of rats fed SeMet (highest in muscle and hemoglobin, 70%, and lowest in testes, 16%). In contrast, selenocysteine was the predominant form of Se present in tissues of rats given selenite. These results indicate that the form of Se administered will influence the form in the tissues, the percentage of Se with GPx and the body burden of Se.

  12. 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…

  13. Waste recycling by vermicomposting: Maturity and quality assessment via dehydrogenase enzyme activity, lignin, water soluble carbon, nitrogen, phosphorous and other indicators.

    PubMed

    Alidadi, Hossein; Hosseinzadeh, Ahmad; Najafpoor, Ali Asghar; Esmaili, Habibollah; Zanganeh, Jafar; Dolatabadi Takabi, Maryam; Piranloo, Fardin Ghasemy

    2016-11-01

    Present study aims to examine the dynamics of maturation and qualification indicators in various vermicompost treatments and selection of the best treatment along with best maturation time in this regard. In this empirical study, dynamics of chemical (pH, electrical conductivity (EC), total nitrogen (TN), phosphorous, lignin, water soluble carbon (WSC), C/N, NH4/NO3) and biological (dehydrogenase enzyme (DEH) and DEH/WSC) properties were investigated in four various treatments, including various ratios of compost produced from municipal solid waste (MSW) and carbonaceous materials (50:50, 70:30, 85:15 and 100:0) over 100 days. Results showed a significant fluctuation in EC, DEH and DEH/WSC proportions over the process. In addition, a noticeable increase was observed for the dynamics of TN, phosphorous and lignin. In contrast, the C/N, NH4/NO3 and WSC values gradually decreased during the process. Moreover, it was observed that the length of 75 days for the process is an appropriate time for maturation of all treatments. However, the first and second treatments resulted in better outcomes compared with the other types of treatments. From the point of view of quality obtained vermicompost was nitrogen enriched product in all treatments. Whereas, for the phosphorous elements this method is appropriate for the first treatment only. PMID:27472049

  14. Activation of the hole-forming toxin aerolysin by extracellular processing.

    PubMed Central

    Howard, S P; Buckley, J T

    1985-01-01

    A precursor-product relationship between aerolysin and a protein with a higher molecular weight was observed in culture supernatants of Aeromonas hydrophila. The larger protein was isolated by ammonium sulfate precipitation and ion-exchange and hydroxyapatite chromatography and compared with purified aerolysin. It was at least 250 times less hemolytic than aerolysin. Both proteins had the same amino acid sequence at the amino terminus. Cyanogen bromide fragments obtained from the two were identical except that each protein contained one unique fragment, and the fragment from the larger protein was 2,500 daltons larger than the fragment obtained from aerolysin. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin. The results indicate that aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus. Images PMID:3891735

  15. [Study of the Sporothrix schenkii (yeast forms) extract. Electrophoretic and immunoelectrophoretic analyses: characterization of enzymatic activities].

    PubMed

    Walbaum, S; Duriez, T; Dujardin, L; Biguet, J

    1978-07-28

    An extract from living yeast forms of S. schenckii was prepared. The yeasts originated from a shake culture in B.H.I. broth (Difco) incubated for 3 days at 35 degrees C in darkness; they were harvested, washed and disrupted with glass beads in a model MSK Braun mechanical cell homogenizer; a freezing-thawing was added to improve the extract. After electrophoretic separation in agarose gel, the extract's components were characterized by their enzymic activity; with this technique, 30 bands were revealed. These enzymic activities were also investigated on the antigenic fractions of the extract revealed by a rabbit hyperimmunserum: 16 among 22 immunoprecipitates are identified by their catalytic properties. Study of the earliest precipitating antibodies (appearing-order and enzymic caracterization) in rabbits just immunized completes this work. How to ameliorate the quality of the extract by culture and extraction conditions is also specified. PMID:692628

  16. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  17. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  18. Structure, function and dynamics in adenovirus maturation

    DOE PAGESBeta

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  19. Signaling through C/EBP homologous protein and death receptor 5 and calpain activation differentially regulate THP-1 cell maturation-dependent apoptosis induced by Shiga toxin type 1.

    PubMed

    Lee, Moo-Seung; Cherla, Rama P; Lentz, Erin K; Leyva-Illades, Dinorah; Tesh, Vernon L

    2010-08-01

    Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity. PMID:20515924

  20. Phenotypic and functional activation of hyporesponsive KIRnegNKG2Aneg human NK-cell precursors requires IL12p70 provided by Poly(I:C)-matured monocyte-derived dendritic cells.

    PubMed

    Curran, Shane A; Romano, Emanuela; Kennedy, Michael G; Hsu, Katharine C; Young, James W

    2014-10-01

    A functionally responsive natural killer (NK)-cell repertoire requires the acquisition of inhibitory NKG2A and killer immunoglobulin-like receptors (KIR) through pathways that remain undefined. Functional donor NK cells expressing KIRs for non-self class I MHC ligands contribute to a positive outcome after allogeneic hematopoietic stem cell transplantation (alloHSCT) by targeting HLA-matched recipient leukemic cells. Insofar as circulating donor conventional dendritic cells (DC) reconstitute with comparable kinetics with donor NK cells after alloHSCT, we used hyporesponsive KIRnegNKG2Aneg precursor cells to evaluate how specific DC subtypes generate a functionally active NK-cell repertoire. Both monocyte-derived DCs (moDC) and Langerhans-type DCs (LC) induce KIRnegNKG2Aneg precursor cells to express the inhibitory receptors NKG2A and KIR, without requiring cell proliferation. Poly(I:C)-matured moDCs significantly augmented the expression of NKG2A, but not KIR, in an IL12p70-dependent manner. Although all DC-stimulated KIRnegNKG2Aneg cells were able to acquire cytolytic activity against class I MHC-negative targets, the ability to secrete IFNγ was restricted to cells that were stimulated by IL12p70-producing, poly(I:C)-matured moDCs. This critical ability of poly(I:C)-matured moDCs to provide IL12p70 to developing KIRnegNKG2Aneg precursors results in a dom4inant, multifunctional, NKG2Apos NK-cell population that is capable of both cytolysis and IFNγ production. Poly(I:C)-matured moDCs are, therefore, the most effective conventional DC subtype for generating a functionally competent NK-cell repertoire by an IL12p70-dependent mechanism. PMID:25023628

  1. Role of activation in alveolar macrophage-mediated suppression of the plaque-forming cell response.

    PubMed Central

    Mbawuike, I N; Herscowitz, H B

    1988-01-01

    Alveolar macrophages (AM) are highly suppressive of the in vitro plaque-forming cell (PFC) response of spleen cells obtained from mice primed with sheep erythrocytes. Comparison of macrophage populations obtained from disparate anatomical sites revealed that although in both cases there was a cell-concentration-dependent suppression of the PFC response, resident AM or AM activated as a result of intravenous injection of Mycobacterium bovis BCG were equally suppressive at the doses examined. Although there was a similar dose-dependent suppression with peritoneal macrophages, BCG-activated cells were more suppressive of the PFC response than were resident cells. In contrast, splenic macrophages at comparable concentrations were not at all suppressive. Resident AM exhibited significantly lower levels of 5'-nucleotidase activity than did resident peritoneal macrophages. Macrophage-mediated suppression of the in vitro PFC response could not be attributed to the release of toxic oxygen metabolites (H2O2, O2- ,and .OH) or prostaglandins, since the addition of catalase, superoxide dismutase, 2-mercaptoethanol, or indomethacin did not completely reverse suppression. These results suggest that the lung microenvironment may maintain AM in an activated state which contributes to their potential immunoregulatory functions. PMID:2830191

  2. Theoretical study of the phototoxicity of naproxen and the active form of nabumetone.

    PubMed

    Musa, Klefah A K; Eriksson, Leif A

    2008-10-30

    Density functional theory using the hybrid functional B3LYP has been employed in order to study the mechanisms of photoinduced decomposition of the closely related nonsteroidal anti-inflammatory drugs naproxen (NP) and 6-methoxy-2-naphthylacetic acid (MNAA; the active form of nabumetone). The photochemical properties and computed energies of various species obtained in this study show that both drugs dominate in their deprotonated forms at physiological pH. The deprotonated acids are unable to decarboxylate from their excited singlets; instead, they decarboxylate from their first excited triplet states with high efficiency, overcoming energy barriers less than 3 and 1 kcal/mol for MNAA and NP, respectively. The ultraviolet and visible spectra of the neutral, deprotonated, and decarboxylated moieties of MNAA and NP are more-or-less similar but with higher probabilites (oscillator strength) for the latter. This fact, as well as the higher reactivity of NP, is explained in terms of the electron-donating effect of the additional methyl group present in NP. Singlet oxygen, superoxide radical anion, and corresponding peroxyl radical species are expected to be formed in different steps throughout the proposed photodegradation pathways of both drugs, which give rise to their effects on biomolecules, for example, lipid peroxidation. PMID:18834087

  3. Pore-forming activity and structural autoinhibition of the gasdermin family.

    PubMed

    Ding, Jingjin; Wang, Kun; Liu, Wang; She, Yang; Sun, Qi; Shi, Jianjin; Sun, Hanzi; Wang, Da-Cheng; Shao, Feng

    2016-07-01

    Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 10–14 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases. PMID:27281216

  4. Trypanocidal activity of the essential oils in their conventional and nanoemulsion forms: in vitro tests.

    PubMed

    Baldissera, Matheus D; Da Silva, Aleksandro S; Oliveira, Camila B; Zimmermann, Carine E P; Vaucher, Rodrigo A; Santos, Roberto C V; Rech, Virginia C; Tonin, Alexandre A; Giongo, Janice L; Mattos, Cristiane B; Koester, Letícia; Santurio, Janio M; Monteiro, Silvia G

    2013-07-01

    The aim of this study was to investigate the susceptibility in vitro of Trypanosoma evansi to the essential oils of andiroba (Carapa guaianensis) and aroeira (Schinus molle), in their conventional and nanostructured forms. For that, pure oils at concentrations of 0.5%, 1.0% and 2.0% were used. A negative control (untreated) and a positive control (diminazene aceturate 0.5%) were used as comparative parameters. Later, the same tests were performed, using nanoemulsions oils at concentrations of 0.5% and 1.0%. The tests were carried out in triplicates and the numbers of parasites were quantified on 1, 3 and 6 h from onset of the study. A dose-dependent reduction in the number of parasites to the forms of two oils tested was observed after 1 h. The concentration of parasites was significantly reduced at low concentrations after 3 h, as well as at 6 h no alive parasites were observed for the essential oils tested. Ours findings indicate, for the first time, that oils of andiroba and aroeira (in their conventional and nanoemulsion forms) have high activity against T. evansi in vitro, leading to the suggestion that these oils may be applied as an alternative treatment for this disease. PMID:23562884

  5. The TCP1γ subunit of Leishmania donovani forms a biologically active homo-oligomeric complex.

    PubMed

    Bhaskar; Mitra, Kalyan; Kuldeep, Jitendra; Siddiqi, Mohammad Imran; Goyal, Neena

    2015-12-01

    Chaperonins are a class of molecular chaperons that encapsulate nascent or stress-denatured proteins and assist their intracellular assembly and folding in an ATP-dependent manner. The ubiquitous eukaryotic chaperonin, TCP1 ring complex is a hetero-oligomeric complex comprising two rings, each formed of eight subunits that may have distinct substrate recognition and ATP hydrolysis properties. In Leishmania, only the TCP1γ subunit has been cloned and characterized. It exhibited differential expression at various growth stages of promastigotes. In the present study, we expressed the TCP1γ subunit in Escherichia coli to investigate whether it forms chaperonin-like complexes and plays a role in protein folding. LdTCP1γ formed high-molecular-weight complexes within E. coli cells as well as in Leishmania cell lysates. The recombinant protein is arranged into two back-to-back rings of seven subunits each, as predicted by homology modelling and observed by negative staining electron microscopy. This morphology is consistent with that of the oligomeric double-ring group I chaperonins found in mitochondria. The LdTCP1γ homo-oligomeric complex hydrolysed ATP, and was active as assayed by luciferase refolding. Thus, the homo-oligomer performs chaperonin reactions without partner subunit(s). Further, co-immunoprecipitation studies revealed that LdTCP1γ interacts with actin and tubulin proteins, suggesting that the complex may have a role in maintaining the structural dynamics of the cytoskeleton of parasites. PMID:26395202

  6. A problem with estimating the pseudo- activation energy of kerogen thermal maturation from Connan's time-temperature relation in oil genesis.

    USGS Publications Warehouse

    Barker, C.E.

    1984-01-01

    Connan's time-temperature relation in oil genesis as derived from first-order reaction kinetics is algebraically correct, but its application to natural petroleum generation is invalidated by the assumption that the ratio of initial kerogen concentration to degraded kerogen concentration is constant from deposition to the initiation of intense oil generation. The ratio can only remain constant if no reaction is occurring and, therefore, Connan's data on 'reaction time' in petroleum generation (assumed to be the age of the sediment) only measures the time elapsed since the system formed. Thus, the widely cited pseudo-activation energy of 11-14 kcal/mole computed from Connan's equation for the start of oil generation from kerogen is meaningless.-Author

  7. Localization of the Clostridium difficile Cysteine Protease Cwp84 and Insights into Its Maturation Process▿

    PubMed Central

    ChapetónMontes, Diana; Candela, Thomas; Collignon, Anne; Janoir, Claire

    2011-01-01

    Clostridium difficile is a nosocomial pathogen involved in antibiotic-associated diarrhea. C. difficile expresses a cysteine protease, Cwp84, which has been shown to degrade some proteins of the extracellular matrix and play a role in the maturation of the precursor of the S-layer proteins. We sought to analyze the localization and the maturation process of this protease. Two identifiable forms of the protease were found to be associated in the bacteria: a form of ∼80 kDa and a cleaved one of 47 kDa, identified as the mature protease. They were found mainly in the bacterial cell surface fractions and weakly in the extracellular fraction. The 80-kDa protein was noncovalently associated with the S-layer proteins, while the 47-kDa form was found to be tightly associated with the underlying cell wall. Our data supported that the anchoring of the Cwp84 47-kDa form is presumably due to a reassociation of the secreted protein. Moreover, we showed that the complete maturation of the recombinant protein Cwp8430-803 is a sequential process beginning at the C-terminal end, followed by one or more cleavages at the N-terminal end. The processing sites of recombinant Cwp84 are likely to be residues Ser-92 and Lys-518. No proteolytic activity was detected with the mature recombinant protease Cwp8492-518 (47 kDa). In contrast, a fragment including the propeptide (Cwp8430-518) displayed proteolytic activity on azocasein and fibronectin. These results showed that Cwp84 is processed essentially at the bacterial cell surface and that its different forms may display different proteolytic activities. PMID:21784932

  8. Dynamin Forms a Src Kinase–sensitive Complex with Cbl and Regulates Podosomes and Osteoclast Activity

    PubMed Central

    Bruzzaniti, Angela; Neff, Lynn; Sanjay, Archana; Horne, William C.; De Camilli, Pietro; Baron, Roland

    2005-01-01

    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption. PMID:15872089

  9. Dynamin forms a Src kinase-sensitive complex with Cbl and regulates podosomes and osteoclast activity.

    PubMed

    Bruzzaniti, Angela; Neff, Lynn; Sanjay, Archana; Horne, William C; De Camilli, Pietro; Baron, Roland

    2005-07-01

    Podosomes are highly dynamic actin-containing adhesion structures found in osteoclasts, macrophages, and Rous sarcoma virus (RSV)-transformed fibroblasts. After integrin engagement, Pyk2 recruits Src and the adaptor protein Cbl, forming a molecular signaling complex that is critical for cell migration, and deletion of any molecule in this complex disrupts podosome ring formation and/or decreases osteoclast migration. Dynamin, a GTPase essential for endocytosis, is also involved in actin cytoskeleton remodeling and is localized to podosomes where it has a role in actin turnover. We found that dynamin colocalizes with Cbl in the actin-rich podosome belt of osteoclasts and that dynamin forms a complex with Cbl in osteoclasts and when overexpressed in 293VnR or SYF cells. The association of dynamin with Cbl in osteoclasts was decreased by Src tyrosine kinase activity and we found that destabilization of the dynamin-Cbl complex involves the recruitment of Src through the proline-rich domain of Cbl. Overexpression of dynamin increased osteoclast bone resorbing activity and migration, whereas overexpression of dynK44A decreased osteoclast resorption and migration. These studies suggest that dynamin, Cbl, and Src coordinately participate in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton, leading to changes in osteoclast adhesion, migration, and resorption. PMID:15872089

  10. A transcriptionally active form of TFIIIC is modified in poliovirus-infected HeLa cells.

    PubMed Central

    Clark, M E; Dasgupta, A

    1990-01-01

    In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This inhibition is due primarily to the reduction in transcriptional activity of the pol III transcription factor TFIIIC in poliovirus-infected cells. However, the specific binding of TFIIIC to the VAI gene B-box sequence, as assayed by DNase I footprinting, is not altered by poliovirus infection. We have used gel retardation analysis to analyze TFIIIC-DNA complexes formed in nuclear extracts prepared from mock- and poliovirus-infected cells. In mock-infected cell extracts, two closely migrating TFIIIC-containing complexes, complexes I and II, were detected in the gel retardation assay. The slower migrating complex, complex I, was absent in poliovirus-infected cell extracts, and an increase occurred in the intensity of the faster-migrating complex (complex II). Also, in poliovirus-infected cell extracts, a new, rapidly migrating complex, complex III, was formed. Complex III may have been the result of limited proteolysis of complex I or II. These changes in TFIIIC-containing complexes in poliovirus-infected cell extracts correlated kinetically with the decrease in TFIIIC transcriptional activity. Complexes I, II, and III were chromatographically separated; only complex I was transcriptionally active and specifically restored pol III transcription when added to poliovirus-infected cell extracts. Acid phosphatase treatment partially converted complex I to complex II but did not affect the binding of complex II or III. Dephosphorylation and limited proteolysis of TFIIIC are discussed as possible mechanisms for the inhibition of pol III-mediated transcription by poliovirus. Images PMID:2204807

  11. Extensive Post-translational Modification of Active and Inactivated Forms of Endogenous p53*

    PubMed Central

    DeHart, Caroline J.; Chahal, Jasdave S.; Flint, S. J.; Perlman, David H.

    2014-01-01

    The p53 tumor suppressor protein accumulates to very high concentrations in normal human fibroblasts infected by adenovirus type 5 mutants that cannot direct assembly of the viral E1B 55-kDa protein-containing E3 ubiquitin ligase that targets p53 for degradation. Despite high concentrations of nuclear p53, the p53 transcriptional program is not induced in these infected cells. We exploited this system to examine select post-translational modifications (PTMs) present on a transcriptionally inert population of endogenous human p53, as well as on p53 activated in response to etoposide treatment of normal human fibroblasts. These forms of p53 were purified from whole cell lysates by means of immunoaffinity chromatography and SDS-PAGE, and peptides derived from them were subjected to nano-ultra-high-performance LC-MS and MS/MS analyses on a high-resolution accurate-mass MS platform (data available via ProteomeXchange, PXD000464). We identified an unexpectedly large number of PTMs, comprising phosphorylation of Ser and Thr residues, methylation of Arg residues, and acetylation, ubiquitinylation, and methylation of Lys residues—for example, some 150 previously undescribed modifications of p53 isolated from infected cells. These modifications were distributed across all functional domains of both forms of the endogenous human p53 protein, as well as those of an orthologous population of p53 isolated from COS-1 cells. Despite the differences in activity, including greater in vitro sequence-specific DNA binding activity exhibited by p53 isolated from etoposide-treated cells, few differences were observed in the location, nature, or relative frequencies of PTMs on the two populations of human p53. Indeed, the wealth of PTMs that we have identified is consistent with a far greater degree of complex, combinatorial regulation of p53 by PTM than previously anticipated. PMID:24056736

  12. FUNCTION FOLLOWS FORM: ACTIVATION OF SHAPE & FUNCTION FEATURES DURING OBJECT IDENTIFICATION

    PubMed Central

    Yee, Eiling; Huffstetler, Stacy; Thompson-Schill, Sharon L.

    2011-01-01

    Most theories of semantic memory characterize knowledge of a given object as comprising a set of semantic features. But how does conceptual activation of these features proceed during object identification? We present the results of a pair of experiments that demonstrate that object recognition is a dynamically unfolding process in which function follows form. We used eye movements to explore whether activating one object’s concept leads to the activation of others that share perceptual (shape) or abstract (function) features. Participants viewed four-picture displays and clicked on the picture corresponding to a heard word. In critical trials, the conceptual representation of one of the objects in the display was similar in shape or function (i.e., its purpose) to the heard word. Importantly, this similarity was not apparent in the visual depictions (e.g., for the target “frisbee,” the shape-related object was a triangular slice of pizza – a shape that a frisbee cannot take); preferential fixations on the related object were therefore attributable to overlap of the conceptual representations on the relevant features. We observed relatedness effects for both shape and function, but shape effects occurred earlier than function effects. We discuss the implications of these findings for current accounts of the representation of semantic memory. PMID:21417543

  13. Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks

    PubMed Central

    Ammann, Christoph G.; Fille, Manfred; Hausdorfer, Johann; Nogler, Michael

    2014-01-01

    Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies. PMID:24492358

  14. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores.

    PubMed

    Liu, Xing; Zhang, Zhibin; Ruan, Jianbin; Pan, Youdong; Magupalli, Venkat Giri; Wu, Hao; Lieberman, Judy

    2016-07-01

    Inflammatory caspases (caspases 1, 4, 5 and 11) are activated in response to microbial infection and danger signals. When activated, they cleave mouse and human gasdermin D (GSDMD) after Asp276 and Asp275, respectively, to generate an N-terminal cleavage product (GSDMD-NT) that triggers inflammatory death (pyroptosis) and release of inflammatory cytokines such as interleukin-1β. Cleavage removes the C-terminal fragment (GSDMD-CT), which is thought to fold back on GSDMD-NT to inhibit its activation. However, how GSDMD-NT causes cell death is unknown. Here we show that GSDMD-NT oligomerizes in membranes to form pores that are visible by electron microscopy. GSDMD-NT binds to phosphatidylinositol phosphates and phosphatidylserine (restricted to the cell membrane inner leaflet) and cardiolipin (present in the inner and outer leaflets of bacterial membranes). Mutation of four evolutionarily conserved basic residues blocks GSDMD-NT oligomerization, membrane binding, pore formation and pyroptosis. Because of its lipid-binding preferences, GSDMD-NT kills from within the cell, but does not harm neighbouring mammalian cells when it is released during pyroptosis. GSDMD-NT also kills cell-free bacteria in vitro and may have a direct bactericidal effect within the cytosol of host cells, but the importance of direct bacterial killing in controlling in vivo infection remains to be determined. PMID:27383986

  15. In Vitro Antimicrobial Activity of Ethanolic Extract of Polish Propolis against Biofilm Forming Staphylococcus epidermidis Strains

    PubMed Central

    Wojtyczka, Robert D.; Kępa, Małgorzata; Idzik, Danuta; Kabała-Dzik, Agata; Wąsik, Tomasz J.

    2013-01-01

    The aim of the presented study was to examine the antimicrobial activity of ethanol extract of Polish propolis (EEPP) against biofilm-forming CoNS strains in vitro. Our results revealed that EEPP displayed varying degrees of activity against CoNS with MIC values ranging from 1.56 to 0.78 mg/mL. The average MIC was 1.13 ± 0.39 mg/mL while calculated MIC50 and MIC90 values were 0.78 mg/mL and 1.56 mg/mL, respectively. The biofilm formation ability by all tested S. epidermidis strains was inhibited at EEPP concentrations ranging from 0.39 to 1.56 mg/mL. The degree of reduction of AlamarBlue was directly associated with the proliferation of S. epidermidis strains. The increased proliferation of S. epidermidis strains was observed after 12 and 24 hours of incubation in the presence of EEPP concentrations ranging from 0.025 to 0.39 mg/mL. These results suggest that antimicrobial activities of EEPP against S. epidermidis expressed as the reduction of bacterial growth, reduction of biofilm formation ability, and the intensity of proliferation were significantly affected by incubation time and EEPP concentration used as well as the interactions between these factors. PMID:23662143

  16. [Relationship between the oxidation-reduction system of astrocytes with production of active forms of oxygen].

    PubMed

    Semenkova, G N; Kvacheva, Z B; Obydennikova, S V; Cherenkevich, S N; Titov, L P

    1998-01-01

    Cells of neuroglia--the astrocytes are of interest from the point of view of their participation in phagocytosis. Phagocyte ability to generate active oxygen forms (AOF) as used as the basic criterion of the estimation of their functional activity. For the purpose to clear up molecular and cellular mechanisms of phagocytosis a study of astrocyte redox-systems, participating in production of AOF, was undertaken. Registration of AOF in astrocytes was carried out using a method of luminol-dependent chemiluminescence. Primary culture of guinea pig astrocytes was used. Spontaneous chemiluminescence of low intensity was found for the astrocytes at the presence of luminol. The destruction of the cells was accompanied by a significant growth of the intensity of spontaneous chemiluminescence. Suspension of endocutosis inductors, particle of latex and phytohemagglutinin, added to astrocytes did not result in formation of AOF, characteristic for other cells, possessing phagocytosis. It was established, that addition of hydrogen peroxide destroys astrocytes at the presence of luminol and gives rise to the emission. Chemiluminescence was not observed in similar experiments with intact cells. A conclusion was made that inside astrocytes there are structures, which show peroxidase-like activity. PMID:9848214

  17. Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads.

    PubMed

    Rando, R F; Ojwang, J; Elbaggari, A; Reyes, G R; Tinder, R; McGrath, M S; Hogan, M E

    1995-01-27

    An oligonucleotide (I100-15) composed of only deoxyguanosine and thymidine was able to inhibit human immunodeficiency virus type-1 (HIV-1) in culture assay systems. I100-15 did not block virus entry into cells but did reduce viral-specific transcripts. As assessed by NMR and polyacrylamide gel methods, I100-15 appears to form a structure in which two stacked guanosine tetrads are connected by three two-base long loops. Structure/activity experiments indicated that formation of intramolecular guanosine tetrads was necessary to achieve maximum antiviral activity. The single deoxyguanosine nucleotide present in each loop was found to be extremely important for the overall antiviral activity. The toxicity of I100-15 was determined to be well above the 50% effective dose (ED50) in culture which yielded a high therapeutic index (> 100). The addition of a cholesterol moiety to the 3' terminus of I100-15 (I100-23) reduced the ED50 value to less than 50 nM (from 0.12 microM for I100-15) and increased the duration of viral suppression to greater than 21 days (versus 7-10 days for I100-15) after removal of the drug from infected cell cultures. The favorable therapeutic index of such molecules coupled with the prolonged suppression of HIV-1, suggest that such compounds further warrant investigation as potential therapeutic agents. PMID:7829511

  18. Analysis of Gpr126 function defines distinct mechanisms controlling the initiation and maturation of myelin

    PubMed Central

    Glenn, Thomas D.; Talbot, William S.

    2013-01-01

    In peripheral nerves, Schwann cells form the myelin sheath, which allows the efficient propagation of action potentials along axons. The transcription factor Krox20 regulates the initiation of myelination in Schwann cells and is also required to maintain mature myelin. The adhesion G protein-coupled receptor (GPCR) Gpr126 is essential for Schwann cells to initiate myelination, but previous studies have not addressed the role of Gpr126 signaling in myelin maturation and maintenance. Through analysis of Gpr126 in zebrafish, we define two distinct mechanisms controlling the initiation and maturation of myelin. We show that gpr126 mutant Schwann cells elaborate mature myelin sheaths and maintain krox20 expression for months, provided that the early signaling defect is bypassed by transient elevation of cAMP. At the onset of myelination, Gpr126 and protein kinase A (PKA) function as a switch that allows Schwann cells to initiate krox20 expression and myelination. After myelination is initiated, krox20 expression is maintained and myelin maturation proceeds independently of Gpr126 signaling. Transgenic analysis indicates that the Krox20 cis-regulatory myelinating Schwann cell element (MSE) becomes active at the onset of myelination and that this activity is dependent on Gpr126 signaling. Activity of the MSE declines after initiation, suggesting that other elements are responsible for maintaining krox20 expression in mature nerves. We also show that elevated cAMP does not initiate myelination in the absence of functional Neuregulin 1 (Nrg1) signaling. These results indicate that the mechanisms regulating the initiation of myelination are distinct from those mediating the maturation and maintenance of myelin. PMID:23804499

  19. Correlation of primate red nucleus discharge with muscle activity during free-form arm movements.

    PubMed

    Miller, L E; van Kan, P L; Sinkjaer, T; Andersen, T; Harris, G D; Houk, J C

    1993-09-01

    1. We recorded from 239 neurons located in the magnocellular division of the red nucleus of four alert macaque monkeys. At the same time, we recorded electromyographic (EMG) signals from as many as twenty electrodes chronically implanted on muscles of the shoulder, arm, forearm and hand. We recorded EMG signals for periods ranging from several months to a year. 2. The monkeys were trained to perform three free-form food retrieval tasks, each of which activated all of the recorded muscles and most of the neurons. The 'prehension' task required simply that the monkey grasp a piece of food from a fixed point in space. The 'barrier' task required the monkey to reach around a small barrier to obtain the food, and the 'Kluver' task required that food be removed from small holes. During the prehension task, we found approximately equal numbers of neurons that were strongly active while the hand was being moved toward the target (70% of units), and while the food was being grasped (60%). Relatively few units were active as the hand was returned to the mouth (15%). 3. Data files of 1-2 min duration were collected while the monkey performed a single behavioural task. Whenever possible, we recorded files for all three tasks from each neuron. For each file we calculated long time-span analog cross-correlations (+/- 1.28 s) between instantaneous neuronal firing rate and each of the full-wave rectified, low-pass filtered EMG signals. We used the peak correlation and the time of the peak as two summary measures of the functional relation between modulation of neuronal activity and EMG. 4. The magnitude of the strongest correlations was between 0.4 and 0.5 (normalized to a perfect correlation of +/- 1.0). Distal muscles were the most frequently correlated, and extensors were more frequently correlated than flexors. For all monkeys, the lags for well correlated muscles were distributed broadly about a uni-modal value near 0 ms. Eighty five per cent of the correlations larger than

  20. ESR dating of barite in sulphide deposits formed by the sea-floor hydrothermal activities.

    PubMed

    Toyoda, Shin; Fujiwara, Taisei; Uchida, Ai; Ishibashi, Jun-ichiro; Nakai, Shun'ichi; Takamasa, Asako

    2014-06-01

    Barite is a mineral newly found to be practically useful for electron spin resonance (ESR) dating of sulphide deposits formed by the sea-floor hydrothermal activities. The recent studies for the properties of the ESR dating signal in barite are summarised in the present paper as well as the formulas for corrections for accurate dose-rate estimation are developed including the dose-rate conversion factors, shape correction for gamma-ray dose and decay of (226)Ra. Although development of the techniques for ESR dating of barite has been completed, further comparative studies with other dating techniques such as U-Th and (226)Ra-(210)Pb dating are necessary for the technique to be widely used. PMID:24795384