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Sample records for accessory sec protein

  1. Differential Localization of the Streptococcal Accessory Sec Components and Implications for Substrate Export

    PubMed Central

    Yen, Yihfen T.; Cameron, Todd A.; Bensing, Barbara A.; Seepersaud, Ravin; Zambryski, Patricia C.

    2013-01-01

    The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation. PMID:23204472

  2. ATPase activity of Mycobacterium tuberculosis SecA1 and SecA2 proteins and its importance for SecA2 function in macrophages.

    PubMed

    Hou, Jie M; D'Lima, Nadia G; Rigel, Nathan W; Gibbons, Henry S; McCann, Jessica R; Braunstein, Miriam; Teschke, Carolyn M

    2008-07-01

    The Sec-dependent translocation pathway that involves the essential SecA protein and the membrane-bound SecYEG translocon is used to export many proteins across the cytoplasmic membrane. Recently, several pathogenic bacteria, including Mycobacterium tuberculosis, were shown to possess two SecA homologs, SecA1 and SecA2. SecA1 is essential for general protein export. SecA2 is specific for a subset of exported proteins and is important for M. tuberculosis virulence. The enzymatic activities of two SecA proteins from the same microorganism have not been defined for any bacteria. Here, M. tuberculosis SecA1 and SecA2 are shown to bind ATP with high affinity, though the affinity of SecA1 for ATP is weaker than that of SecA2 or Escherichia coli SecA. Amino acid substitution of arginine or alanine for the conserved lysine in the Walker A motif of SecA2 eliminated ATP binding. We used the SecA2(K115R) variant to show that ATP binding was necessary for the SecA2 function of promoting intracellular growth of M. tuberculosis in macrophages. These results are the first to show the importance of ATPase activity in the function of accessory SecA2 proteins.

  3. Protein export through the bacterial Sec pathway.

    PubMed

    Tsirigotaki, Alexandra; De Geyter, Jozefien; Šoštaric, Nikolina; Economou, Anastassios; Karamanou, Spyridoula

    2017-01-01

    The general secretory (Sec) pathway comprises an essential, ubiquitous and universal export machinery for most proteins that integrate into, or translocate through, the plasma membrane. Sec exportome polypeptides are synthesized as pre-proteins that have cleavable signal peptides fused to the exported mature domains. Recent advances have re-evaluated the interaction networks of pre-proteins with chaperones that are involved in pre-protein targeting from the ribosome to the SecYEG channel and have identified conformational signals as checkpoints for high-fidelity targeting and translocation. The recent structural and mechanistic insights into the channel and its ATPase motor SecA are important steps towards the elucidation of the allosteric crosstalk that mediates secretion. In this Review, we discuss recent biochemical, structural and mechanistic insights into the consecutive steps of the Sec pathway - sorting and targeting, translocation and release - in both co-translational and post-translational modes of export. The architecture and conformational dynamics of the SecYEG channel and its regulation by ribosomes, SecA and pre-proteins are highlighted. Moreover, we present conceptual models of the mechanisms and energetics of the Sec-pathway dependent secretion process in bacteria.

  4. Accessory proteins for heterotrimeric G-proteins in the kidney

    PubMed Central

    Park, Frank

    2015-01-01

    Heterotrimeric G-proteins play a fundamentally important role in regulating signal transduction pathways in the kidney. Accessory proteins are being identified as direct binding partners for heterotrimeric G-protein α or βγ subunits to promote more diverse mechanisms by which G-protein signaling is controlled. In some instances, accessory proteins can modulate the signaling magnitude, localization, and duration following the activation of cell membrane-associated receptors. Alternatively, accessory proteins complexed with their G-protein α or βγ subunits can promote non-canonical models of signaling activity within the cell. In this review, we will highlight the expression profile, localization and functional importance of these newly identified accessory proteins to control the function of select G-protein subunits under normal and various disease conditions observed in the kidney. PMID:26300785

  5. The Sec translocon mediated protein transport in prokaryotes and eukaryotes.

    PubMed

    Denks, Kärt; Vogt, Andreas; Sachelaru, Ilie; Petriman, Narcis-Adrian; Kudva, Renuka; Koch, Hans-Georg

    2014-01-01

    Protein transport via the Sec translocon represents an evolutionary conserved mechanism for delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sec translocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria. It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membrane of Bacteria where it constitutes a channel that can be activated by multiple partner proteins. These partner proteins determine the mechanism of polypeptide movement across the channel. During SRP-dependent co-translational targeting, the ribosome threads the nascent protein directly into the Sec channel. This pathway is in Bacteria mainly dedicated for membrane proteins but in Eukaryotes also employed by secretory proteins. The alternative pathway, leading to post-translational translocation across the Sec translocon engages an ATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratcheting mechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesis is also assisted by additional proteins at the lateral gate of SecY/Sec61α and in the lumen of the endoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enables the Sec complex to transport a vast array of substrates. In this review we summarize recent biochemical and structural information on the prokaryotic and eukaryotic Sec translocons and we describe the remarkably complex interaction network of the Sec complexes.

  6. HIV-1 Accessory Proteins: Vpu and Vif

    PubMed Central

    Andrew, Amy; Strebel, Klaus

    2014-01-01

    HIV-1 Vif and Vpu are accessory factors involved in late stages of viral replication. Vif regulates viral infectivity by preventing virion incorporation of APOBEC3G and other members of the family of cytidine deaminases, while Vpu causes degradation of CD4 and promotes virus release by functionally inactivating the host factor BST-2. This chapter described techniques used for the characterization of Vif and Vpu and their functional interaction with host factors. Many of the techniques are, however, applicable to the functional analysis of other viral proteins. PMID:24158820

  7. Two alternative binding mechanisms connect the protein translocation Sec71/Sec72 complex with heat shock proteins.

    PubMed

    Tripathi, Arati; Mandon, Elisabet C; Gilmore, Reid; Rapoport, Tom A

    2017-03-12

    The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum (ER) membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71/Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71/Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the ER membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome- associated nascent polypeptide chains also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71/72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.

  8. Accessories to the crime: recent advances in HIV accessory protein biology.

    PubMed

    Gramberg, Thomas; Sunseri, Nicole; Landau, Nathaniel R

    2009-02-01

    Recent advances in understanding the roles of the lentiviral accessory proteins have provided fascinating insight into the molecular biology of the virus and uncovered previously unappreciated innate immune mechanisms by which the host defends itself. HIV-1 and other lentiviruses have developed accessory proteins that counterattack the antiviral defenses in a sort of evolutionary battle. The virus is remarkably adept at co-opting cellular degradative pathways to destroy the protective proteins. This review focuses on recent advances in understanding three of the accessory proteins-virion infectivity factor (Vif), viral protein R (Vpr), and viral protein U (Vpu)-that target different restriction factors to ensure virus replication. These proteins may provide promising targets for the development of novel classes of antiretroviral drugs.

  9. Decatransin, a new natural product inhibiting protein translocation at the Sec61/SecYEG translocon.

    PubMed

    Junne, Tina; Wong, Joanne; Studer, Christian; Aust, Thomas; Bauer, Benedikt W; Beibel, Martin; Bhullar, Bhupinder; Bruccoleri, Robert; Eichenberger, Jürg; Estoppey, David; Hartmann, Nicole; Knapp, Britta; Krastel, Philipp; Melin, Nicolas; Oakeley, Edward J; Oberer, Lukas; Riedl, Ralph; Roma, Guglielmo; Schuierer, Sven; Petersen, Frank; Tallarico, John A; Rapoport, Tom A; Spiess, Martin; Hoepfner, Dominic

    2015-03-15

    A new cyclic decadepsipeptide was isolated from Chaetosphaeria tulasneorum with potent bioactivity on mammalian and yeast cells. Chemogenomic profiling in S. cerevisiae indicated that the Sec61 translocon complex, the machinery for protein translocation and membrane insertion at the endoplasmic reticulum, is the target. The profiles were similar to those of cyclic heptadepsipeptides of a distinct chemotype (including HUN-7293 and cotransin) that had previously been shown to inhibit cotranslational translocation at the mammalian Sec61 translocon. Unbiased, genome-wide mutagenesis followed by full-genome sequencing in both fungal and mammalian cells identified dominant mutations in Sec61p (yeast) or Sec61α1 (mammals) that conferred resistance. Most, but not all, of these mutations affected inhibition by both chemotypes, despite an absence of structural similarity. Biochemical analysis confirmed inhibition of protein translocation into the endoplasmic reticulum of both co- and post-translationally translocated substrates by both chemotypes, demonstrating a mechanism independent of a translating ribosome. Most interestingly, both chemotypes were found to also inhibit SecYEG, the bacterial Sec61 translocon homolog. We suggest 'decatransin' as the name for this new decadepsipeptide translocation inhibitor.

  10. Nonlethal sec71-1 and sec72-1 mutations eliminate proteins associated with the Sec63p-BiP complex from S. cerevisiae.

    PubMed Central

    Fang, H; Green, N

    1994-01-01

    The sec71-1 and sec72-1 mutations were identified by a genetic assay that monitored membrane protein integration into the endoplasmic reticulum (ER) membrane of the yeast Saccharomyces cerevisiae. The mutations inhibited integration of various chimeric membrane proteins and translocation of a subset of water soluble proteins. In this paper we show that SEC71 encodes the 31.5-kDa transmembrane glycoprotein (p31.5) and SEC72 encodes the 23-kDa protein (p23) of the Sec63p-BiP complex. SEC71 is therefore identical to SEC66 (HSS1), which was previously shown to encode p31.5. DNA sequence analyses reveal that sec71-1 cells contain a nonsense mutation that removes approximately two-thirds of the cytoplasmic C-terminal domain of p31.5. The sec72-1 mutation shifts the reading frame of the gene encoding p23. Unexpectedly, the sec71-1 mutant lacks p31.5 and p23. Neither mutation is lethal, although sec71-1 cells exhibit a growth defect at 37 degrees C. These results show that p31.5 and p23 are important for the trafficking of a subset of proteins to the ER membrane. Images PMID:7841522

  11. Membrane protein insertion and assembly by the bacterial holo-translocon SecYEG–SecDF–YajC–YidC

    PubMed Central

    Komar, Joanna; Alvira, Sara; Schulze, Ryan J.; Martin, Remy; Lycklama a Nijeholt, Jelger A.; Lee, Sarah C.; Dafforn, Tim R.; Deckers-Hebestreit, Gabriele; Berger, Imre; Schaffitzel, Christiane; Collinson, Ian

    2016-01-01

    Protein secretion and membrane insertion occur through the ubiquitous Sec machinery. In this system, insertion involves the targeting of translating ribosomes via the signal recognition particle and its cognate receptor to the SecY (bacteria and archaea)/Sec61 (eukaryotes) translocon. A common mechanism then guides nascent transmembrane helices (TMHs) through the Sec complex, mediated by associated membrane insertion factors. In bacteria, the membrane protein ‘insertase’ YidC ushers TMHs through a lateral gate of SecY to the bilayer. YidC is also thought to incorporate proteins into the membrane independently of SecYEG. Here, we show the bacterial holo-translocon (HTL) — a supercomplex of SecYEG–SecDF–YajC–YidC — is a bona fide resident of the Escherichia coli inner membrane. Moreover, when compared with SecYEG and YidC alone, the HTL is more effective at the insertion and assembly of a wide range of membrane protein substrates, including those hitherto thought to require only YidC. PMID:27435098

  12. Nucleotide sequence of the secA gene and secA(Ts) mutations preventing protein export in Escherichia coli.

    PubMed Central

    Schmidt, M G; Rollo, E E; Grodberg, J; Oliver, D B

    1988-01-01

    The DNA sequence of the secA gene, essential for protein export in Escherichia coli, was determined and found to encode a hydrophilic protein of 901 amino acid residues with a predicted molecular weight of 101,902, consistent with its previously determined size and subcellular location. Sequence analysis of 9 secA(Ts) mutations conferring general protein export and secA regulatory defects revealed that these mutations were clustered in three specific regions within the first 170 amino acid residues of the SecA protein and were the result of single amino acid changes predicted to be severely disruptive of protein structure and function. The DNA sequence immediately upstream of secA was shown to encode a previously inferred gene, gene X. Sequence analysis of a conditionally lethal amber mutation, am109, previously inferred to be located proximally in the secA gene, revealed that it was located distally in gene X and was conditionally lethal due to its polar effect on secA expression. This and additional evidence are presented indicating that gene X and secA are cotranscribed. Images PMID:2841285

  13. SEC-Translocon Dependent Extracytoplasmic Proteins of Candidatus Liberibacter asiaticus

    PubMed Central

    Prasad, Samiksha; Xu, Jin; Zhang, Yunzeng; Wang, Nian

    2016-01-01

    Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide. HLB is associated with three species of the phloem-limited, gram-negative, fastidious α-proteobacteria: Candidatus Liberibacter asiaticus (Las), Ca. L. americanus (Lam), and Ca. L. africanus (Laf) with Las being the most widespread species. Las has not been cultured in artificial media, which has greatly hampered our efforts to understand its virulence mechanisms. Las contains a complete Sec-translocon, which has been suggested to transport Las proteins including virulence factors into the extracytoplasmic milieu. In this study, we characterized the Sec-translocon dependent, signal peptide containing extracytoplasmic proteins of Las. A total of 166 proteins of Las-psy62 strain were predicted to contain signal peptides targeting them out of the cell cytoplasm via the Sec-translocon using LipoP, SigalP 3.0, SignalP 4.1, and Phobius. We also predicated SP containing extracytoplasmic proteins for Las-gxpsy and Las-Ishi-1, Lam, Laf, Ca. L. solanacearum (Lso), and L. crescens (Lcr). For experimental validation of the predicted extracytoplasmic proteins, Escherichia coli based alkaline phosphatase (PhoA) gene fusion assays were conducted. A total of 86 out of the 166 predicted Las proteins were experimentally validated to contain signal peptides. Additionally, Las-psy62 lepB (CLIBASIA_04190), the gene encodes signal peptidase I, was able to partially complement the amber mutant of lepB of E. coli. This work will contribute to the identification of Sec-translocon dependent effector proteins of Las, which might be involved in virulence of Las. PMID:28066334

  14. Accessory proteins of SARS-CoV and other coronaviruses.

    PubMed

    Liu, Ding Xiang; Fung, To Sing; Chong, Kelvin Kian-Long; Shukla, Aditi; Hilgenfeld, Rolf

    2014-09-01

    The huge RNA genome of SARS coronavirus comprises a number of open reading frames that code for a total of eight accessory proteins. Although none of these are essential for virus replication, some appear to have a role in virus pathogenesis. Notably, some SARS-CoV accessory proteins have been shown to modulate the interferon signaling pathways and the production of pro-inflammatory cytokines. The structural information on these proteins is also limited, with only two (p7a and p9b) having their structures determined by X-ray crystallography. This review makes an attempt to summarize the published knowledge on SARS-CoV accessory proteins, with an emphasis on their involvement in virus-host interaction. The accessory proteins of other coronaviruses are also briefly discussed. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses" (see Introduction by Hilgenfeld and Peiris (2013)).

  15. Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation

    PubMed Central

    Allen, William John; Corey, Robin Adam; Oatley, Peter; Sessions, Richard Barry; Radford, Sheena E; Tuma, Roman; Collinson, Ian

    2016-01-01

    The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids. DOI: http://dx.doi.org/10.7554/eLife.15598.001 PMID:27183269

  16. SecA Cotranslationally Interacts with Nascent Substrate Proteins In Vivo

    PubMed Central

    Jamshad, Mohammed; Hanmer, Ruby; Schibich, Daniela; Döring, Kristina; Marcomini, Isabella; Kramer, Günter; Bukau, Bernd

    2016-01-01

    ABSTRACT SecA is an essential component of the Sec machinery in bacteria, which is responsible for transporting proteins across the cytoplasmic membrane. Recent work from our laboratory indicates that SecA binds to ribosomes. Here, we used two different approaches to demonstrate that SecA also interacts with nascent polypeptides in vivo and that these polypeptides are Sec substrates. First, we photo-cross-linked SecA to ribosomes in vivo and identified mRNAs that copurify with SecA. Microarray analysis of the copurifying mRNAs indicated a strong enrichment for proteins containing Sec-targeting sequences. Second, we used a 2-dimensional (2-D) gel approach to analyze radioactively labeled nascent polypeptides that copurify with SecA, including maltose binding protein, a well-characterized SecA substrate. The interaction of SecA with nascent chains was not strongly affected in cells lacking SecB or trigger factor, both of which also interact with nascent Sec substrates. Indeed, the ability of SecB to interact with nascent chains was disrupted in strains in which the interaction between SecA and the ribosome was defective. Analysis of the interaction of SecA with purified ribosomes containing arrested nascent chains in vitro indicates that SecA can begin to interact with a variety of nascent chains when they reach a length of ∼110 amino acids, which is considerably shorter than the length required for interaction with SecB. Our results suggest that SecA cotranslationally recognizes nascent Sec substrates and that this recognition could be required for the efficient delivery of these proteins to the membrane-embedded Sec machinery. IMPORTANCE SecA is an ATPase that provides the energy for the translocation of proteins across the cytoplasmic membrane by the Sec machinery in bacteria. The translocation of most of these proteins is uncoupled from protein synthesis and is frequently described as “posttranslational.” Here, we show that SecA interacts with nascent Sec

  17. SEC/reversed-phase separation of E. coli proteins.

    PubMed

    Apffell, Alex

    2010-05-01

    Although many chromatographic modes can be coupled for the multidimensional separation of a complex mixture, a very favorable combination is that of size-exclusion chromatography (SEC) and reversed-phase chromatography. The separation mechanisms are largely orthogonal and the mobile phases are compatible. The use of a retentive second dimension allows trapping of specific fractions from the SEC separation and transferring them to the reversed-phase separation. One of the advantages of multidimensional chromatography is that it is scalable in terms of automation. In a completely manual system, fractions eluting from a first dimension can be manually collected and injected into a second separation dimension. In an automated system, fraction transfer can be accomplished through automated valving. The following protocol illustrates this approach, with both a manual method and an external column switching method integrated into an automated high-performance liquid chromatography (HPLC) method. As an illustration, soluble proteins from Escherichia coli are separated.

  18. Analysis of polypeptide movement in the SecY channel during SecA-mediated protein translocation.

    PubMed

    Erlandson, Karl J; Or, Eran; Osborne, Andrew R; Rapoport, Tom A

    2008-06-06

    In bacteria most secretory proteins are transported across the plasma membrane by the interplay of the ATPase SecA with the translocation channel formed by the SecY complex; SecA uses cycles of ATP hydrolysis to "push" consecutive segments of a polypeptide substrate through the channel. Here we have addressed the mechanism of this process by following the fate of stalled translocation intermediates. These were generated by using a polypeptide substrate containing a bulky disulfide-bonded loop, thus preventing the final residues from passing through the channel. Protease protection experiments showed that the intermediates were stable in the presence of ATP and could complete translocation once the block was removed. The translocation intermediate was also stable when SecA associated with ATPgammaS, a poorly hydrolyzable ATP analog, or ADP plus AlF(4), which mimics the transition state during ATP hydrolysis. In contrast, when SecA was in its ADP-bound state, the translocating polypeptide moved back into the cytosol, as indicated by the disappearance of the protected fragment. Backsliding was not significantly altered by deletion of the plug domain, a short helix in the center of the SecY channel, but it was slowed down when changes were introduced into the pore ring, the constriction of the hourglass-shaped channel. In all cases, backsliding was significantly slower than forward translocation. Together, these data suggest that SecA binds the polypeptide chain in its ATP state and releases it in the ADP state. The channel itself does not bind the polypeptide chain but provides "friction" that minimizes backsliding when ADP-bound SecA resets to "grab" the next segment of the substrate.

  19. Position-dependent effects of polylysine on Sec protein transport.

    PubMed

    Liang, Fu-Cheng; Bageshwar, Umesh K; Musser, Siegfried M

    2012-04-13

    The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.

  20. Morphology and protein patterns of honey bee drone accessory glands.

    PubMed

    Cruz-Landim, Carminda da; Dallacqua, Rodrigo Pires

    2005-09-30

    We used light and transmission electron microscopy to examine the morphology of the accessory glands of immature and mature adult males of Apis mellifera L. We also made an electrophoretic analysis of the protein content of the mature gland. The glands of the immature male actively secrete a mucous substance that can be seen in the lumen of the gland of the mature male. This secretion stains with mercury bromophenol blue and with periodic acid-Schiff reaction, which stain glyconjugates. The protein content was higher in the lumen secretion than in the gland wall extracts. The electrophoresis patterns of the wall extracts were different from those of the secretion found in the gland lumen.

  1. Localization of lipid raft proteins to the plasma membrane is a major function of the phospholipid transfer protein Sec14.

    PubMed

    Curwin, Amy J; Leblanc, Marissa A; Fairn, Gregory D; McMaster, Christopher R

    2013-01-01

    The Sec14 protein domain is a conserved tertiary structure that binds hydrophobic ligands. The Sec14 protein from Saccharomyces cerevisiae is essential with studies of S. cerevisiae Sec14 cellular function facilitated by a sole temperature sensitive allele, sec14(ts). The sec14(ts) allele encodes a protein with a point mutation resulting in a single amino acid change, Sec14(G266D). In this study results from a genome-wide genetic screen, and pharmacological data, provide evidence that the Sec14(G266D) protein is present at a reduced level compared to wild type Sec14 due to its being targeted to the proteosome. Increased expression of the sec14(ts) allele ameliorated growth arrest, but did not restore the defects in membrane accumulation or vesicular transport known to be defective in sec14(ts) cells. We determined that trafficking and localization of two well characterized lipid raft resident proteins, Pma1 and Fus-Mid-GFP, were aberrant in sec14(ts) cells. Localization of both lipid raft proteins was restored upon increased expression of the sec14(ts) allele. We suggest that a major function provided by Sec14 is trafficking and localization of lipid raft proteins.

  2. Multicopper manganese oxidase accessory proteins bind Cu and heme.

    PubMed

    Butterfield, Cristina N; Tao, Lizhi; Chacón, Kelly N; Spiro, Thomas G; Blackburn, Ninian J; Casey, William H; Britt, R David; Tebo, Bradley M

    2015-12-01

    Multicopper oxidases (MCOs) catalyze the oxidation of a diverse group of metal ions and organic substrates by successive single-electron transfers to O2 via four bound Cu ions. MnxG, which catalyzes MnO2 mineralization by oxidizing both Mn(II) and Mn(III), is unique among multicopper oxidases in that it carries out two energetically distinct electron transfers and is tightly bound to accessory proteins. There are two of these, MnxE and MnxF, both approximately 12kDa. Although their sequences are similar to those found in the genomes of several Mn-oxidizing Bacillus species, they are dissimilar to those of proteins with known function. Here, MnxE and MnxF are co-expressed independent of MnxG and are found to oligomerize into a higher order stoichiometry, likely a hexamer. They bind copper and heme, which have been characterized by electron paramagnetic resonance (EPR), X-ray absorption spectroscopy (XAS), and UV-visible (UV-vis) spectrophotometry. Cu is found in two distinct type 2 (T2) copper centers, one of which appears to be novel; heme is bound as a low-spin species, implying coordination by two axial ligands. MnxE and MnxF do not oxidize Mn in the absence of MnxG and are the first accessory proteins to be required by an MCO. This may indicate that Cu and heme play roles in electron transfer and/or Cu trafficking.

  3. The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

    SciTech Connect

    Nielsen, Anders Lade

    2009-10-23

    Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

  4. A single Sec61-complex functions as a protein-conducting channel.

    PubMed

    Kalies, Kai-Uwe; Stokes, Vivica; Hartmann, Enno

    2008-12-01

    During cotranslational translocation of proteins into the endoplasmic reticulum (ER) translating ribosomes bind to Sec61-complexes. Presently two models exist how these membrane protein complexes might form protein-conducting channels. While electron microscopic data suggest that a ring-like structure consisting of four Sec61-complexes build the channel, the recently solved crystal structure of a homologous bacterial protein complex led to the speculation that the actual tunnel is formed by just one individual Sec61-complex. Using protease protection assays together with quantitative immunoblotting we directly examined the structure of mammalian protein-conducting channels. We found that in native ER-membranes one single Sec61alpha-molecule is preferentially protected by a membrane bound ribosome, both, in the presence and absence of nascent polypeptides. In addition we present evidence that the nascent polypeptide destabilizes the ring-like translocation apparatus formed by four Sec61-complexes. Moreover, we found that after solubilization of ER-membranes a single Sec61-complex is sufficient to protect the nascent polypeptide chain against added proteases. Finally, we could show that this single Sec61-complex allows the movement of the nascent chain, when it has been released from the ribosome by puromycin treatment. Collectively, our data suggest that the active protein-conducting channel in the ER is formed by a single Sec61-complex.

  5. Cell-Free Synthesis of SecYEG Translocon as the Fundamental Protein Transport Machinery

    NASA Astrophysics Data System (ADS)

    Matsubayashi, Hideaki; Kuruma, Yutetsu; Ueda, Takuya

    2014-12-01

    The cell membrane has many indispensable functions for sustaining cell alive besides a role as merely outer envelope. The most of such functions are implemented by membrane embedded proteins that are emerged through the membrane integration machinery, SecYEG translocon. Here, we synthesized SecYEG by expressing the corresponding gene in vitro to study the process of functionalization of the cell membrane.

  6. Crystal structure of a substrate-engaged SecY protein-translocation channel.

    PubMed

    Li, Long; Park, Eunyong; Ling, JingJing; Ingram, Jessica; Ploegh, Hidde; Rapoport, Tom A

    2016-03-17

    Hydrophobic signal sequences target secretory polypeptides to a protein-conducting channel formed by a heterotrimeric membrane protein complex, the prokaryotic SecY or eukaryotic Sec61 complex. How signal sequences are recognized is poorly understood, particularly because they are diverse in sequence and length. Structures of the inactive channel show that the largest subunit, SecY or Sec61α, consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces lipid. The cytoplasmic funnel is empty, while the extracellular funnel is filled with a plug domain. In bacteria, the SecY channel associates with the translating ribosome in co-translational translocation, and with the SecA ATPase in post-translational translocation. How a translocating polypeptide inserts into the channel is uncertain, as cryo-electron microscopy structures of the active channel have a relatively low resolution (~10 Å) or are of insufficient quality. Here we report a crystal structure of the active channel, assembled from SecY complex, the SecA ATPase, and a segment of a secretory protein fused into SecA. The translocating protein segment inserts into the channel as a loop, displacing the plug domain. The hydrophobic core of the signal sequence forms a helix that sits in a groove outside the lateral gate, while the following polypeptide segment intercalates into the gate. The carboxy (C)-terminal section of the polypeptide loop is located in the channel, surrounded by residues of the pore ring. Thus, during translocation, the hydrophobic segments of signal sequences, and probably bilayer-spanning domains of nascent membrane proteins, exit the lateral gate and dock at a specific site that faces the lipid phase.

  7. Secretory COPII Protein SEC31B Is Required for Pollen Wall Development1[OPEN

    PubMed Central

    Zhao, Bingchun; Shi, Haidan; Wang, Wanlei; Liu, Xiaoyu; Gao, Hui; Wang, Xiaoxiao; Zhang, Yinghui; Yang, Meidi; Li, Rui

    2016-01-01

    The pollen wall protects pollen grains from abiotic and biotic stresses. During pollen wall development, tapetal cells play a vital role by secreting proteins, signals, and pollen wall material to ensure microspore development. But the regulatory mechanism underlying the secretory pathway of the tapetum is largely unknown. Here, we characterize the essential role of the Arabidopsis (Arabidopsis thaliana) COPII protein SECRETORY31B (SEC31B) in pollen wall development and the secretory activity of tapetal cells. The sporophyte-controlled atsec31b mutant exhibits severe pollen and seed abortion. Transmission electron microscopy observation indicates that pollen exine formation in the atsec31b mutant is disrupted significantly. AtSEC31B is a functional COPII protein revealed by endoplasmic reticulum (ER) exit site localization, interaction with AtSEC13A, and retarded ER-Golgi protein trafficking in the atsec31b mutant. A genetic tapetum-specific rescue assay indicates that AtSEC31B functions primarily in the tapetum. Moreover, deletion of AtSEC31B interrupted the formation of the ER-derived tapetosome and altered the location of the ATP-BINDING CASSETTE TRANSPORTER9 protein in the tapetum. Therefore, this work demonstrates that AtSEC31B plays a vital role in pollen wall development by regulating the secretory pathway of the tapetal cells. PMID:27634427

  8. Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic[W][OA

    PubMed Central

    Karnik, Rucha; Grefen, Christopher; Bayne, Robert; Honsbein, Annegret; Köhler, Tim; Kioumourtzoglou, Dimitrios; Williams, Mary; Bryant, Nia J.; Blatt, Michael R.

    2013-01-01

    The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation. PMID:23572542

  9. Protein export by the mycobacterial SecA2 system is determined by the preprotein mature domain.

    PubMed

    Feltcher, Meghan E; Gibbons, Henry S; Ligon, Lauren S; Braunstein, Miriam

    2013-02-01

    At the core of the bacterial general secretion (Sec) pathway is the SecA ATPase, which powers translocation of unfolded preproteins containing Sec signal sequences through the SecYEG membrane channel. Mycobacteria have two nonredundant SecA homologs: SecA1 and SecA2. While the essential SecA1 handles "housekeeping" export, the nonessential SecA2 exports a subset of proteins and is required for Mycobacterium tuberculosis virulence. Currently, it is not understood how SecA2 contributes to Sec export in mycobacteria. In this study, we focused on identifying the features of two SecA2 substrates that target them to SecA2 for export, the Ms1704 and Ms1712 lipoproteins of the model organism Mycobacterium smegmatis. We found that the mature domains of Ms1704 and Ms1712, not the N-terminal signal sequences, confer SecA2-dependent export. We also demonstrated that the lipid modification and the extreme N terminus of the mature protein do not impart the requirement for SecA2 in export. We further showed that the Ms1704 mature domain can be efficiently exported by the twin-arginine translocation (Tat) pathway. Because the Tat system exports only folded proteins, this result implies that SecA2 substrates can fold in the cytoplasm and suggests a putative role of SecA2 in enabling export of such proteins. Thus, the mycobacterial SecA2 system may represent another way that bacteria solve the problem of exporting proteins that can fold in the cytoplasm.

  10. Sec-Secretion and Sortase-Mediated Anchoring of Proteins in Gram-Postive Bacteria

    PubMed Central

    Schneewind, Olaf; Missiakas, Dominique

    2014-01-01

    Signal peptide-driven secretion of precursor proteins directs polypeptides across the plasma membrane of bacteria. Two pathways, Sec- and SRP-dependent, converge at the SecYEG translocon to thread unfolded precursor proteins across the membrane, whereas folded preproteins are routed via the Tat secretion pathway. Gram-positive bacteria lack an outer membrane and are surrounded by a rigid layer of peptidoglycan. Interactions with their environment are mediated by proteins that are retained in the cell wall, often through covalent attachment to the peptidoglycan. In this review, we describe the mechanisms for both Sec-dependent secretion and sortase-dependent assembly of proteins in the envelope of Gram-positive bacteria. PMID:24269844

  11. Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

    PubMed Central

    Heimer, Susan R.; Mobley, Harry L. T.

    2001-01-01

    Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins. PMID:11157956

  12. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    PubMed Central

    Halbedel, Sven; Kawai, Maki; Breitling, Reinhard; Hamoen, Leendert W.

    2014-01-01

    The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localizes to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localization of DivIVA in these mutants. Surprisingly, DivIVA was delocalized, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localization depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localization is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed. PMID:24592260

  13. Carotenoid-binding proteins; accessories to carotenoid function.

    PubMed

    Pilbrow, Jodi; Garama, Daniel; Carne, Alan

    2012-01-01

    Understanding of the widespread biological importance of carotenoids is increasing. Accompanying this is the developing recognition that the interaction of carotenoids with other molecules, such as proteins, is also essential. Here the significance of carotenoid-protein interactions with respect to biological function is reviewed for three well characterised carotenoprotein complexes; crustacyanin, the orange carotenoid protein and glutathione-S-transferase P1. In addition a preliminary report is made on the recent partial purification of an echinenone-binding protein extracted from a New Zealand sea urchin, Evechinus chloroticus.

  14. Functional Anatomy of Phospholipid Binding And Regulation of Phosphoinositide Homeostasis By Proteins of the Sec14 Superfamily

    SciTech Connect

    Schaaf, G.; Ortlund, E.A.; Tyeryar, K.R.; Mousley, C.J.; Ile, K.E.; Garrett, T.A.; Ren, J.; Woolls, M.J.; Raetz, C.R.H.; Redinbo, M.R.; Bankaitis, V.A.

    2009-05-27

    Sec14, the major yeast phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein, regulates essential interfaces between lipid metabolism and membrane trafficking from the trans-Golgi network (TGN). How Sec14 does so remains unclear. We report that Sec14 binds PtdIns and PtdCho at distinct (but overlapping) sites, and both PtdIns- and PtdCho-binding activities are essential Sec14 activities. We further show both activities must reside within the same molecule to reconstitute a functional Sec14 and for effective Sec14-mediated regulation of phosphoinositide homeostasis in vivo. This regulation is uncoupled from PtdIns-transfer activity and argues for an interfacial presentation mode for Sec14-mediated potentiation of PtdIns kinases. Such a regulatory role for Sec14 is a primary counter to action of the Kes1 sterol-binding protein that antagonizes PtdIns 4-OH kinase activity in vivo. Collectively, these findings outline functional mechanisms for the Sec14 superfamily and reveal additional layers of complexity for regulating phosphoinositide homeostasis in eukaryotes.

  15. Identification of novel protein-protein interactions at the cytosolic surface of the Sec63 complex in the yeast ER membrane.

    PubMed

    Willer, Martin; Jermy, Andrew J; Young, Barry P; Stirling, Colin J

    2003-01-30

    Precursors of secretory proteins are targeted to the membrane of the endoplasmic reticulum by specific protein complexes that recognize their signal sequence. All eukaryotic cells investigated so far have been found to possess the signal recognition particle (SRP) that targets the majority of precursors to the translocation machinery. In Saccharomyces cerevisiae a number of proteins are translocated independently of SRP. These precursors rely on a different signal sequence-binding complex, which includes Sec62p, Sec63p, Sec71p and Sec72p. Identifying interactions between individual components of this tetrameric protein complex is important in the understanding of its function. We demonstrate a specific interaction between the only two essential proteins in this complex, Sec62p and Sec63p. Second, we show evidence of homodimerization of Sec72p molecules and further identify the YLR301w gene product as a novel in vivo interacting partner of Sec72p. Finally, we determine the authentic N-terminus of Sec62p and describe interacting subdomains of both Sec62p and Sec63p.

  16. The Nuclear Pore Complex Function of Sec13 Protein Is Required for Cell Survival during Retinal Development*

    PubMed Central

    Niu, Xubo; Hong, Jian; Zheng, Xiaofeng; Melville, David B.; Knapik, Ela W.; Meng, Anming; Peng, Jinrong

    2014-01-01

    Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13sq198 mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13sq198, suggesting that the digestive organ defects observed in sec13sq198 are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13sq198 failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13sq198. Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13sq198. Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis. PMID:24627485

  17. Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein.

    PubMed

    Yoshizawa, Susumu; Karatani, Hajime; Wada, Minoru; Kogure, Kazuhiro

    2012-04-01

    Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490 nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max)  ≈ 490 to λ(max)  ≈ 476 nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472 nm, whereas other Vibrio strains emit light with a peak at around 482 nm. Therefore, we investigated the mechanism underlying this blue shift in V. azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V. azureus.

  18. Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

    PubMed Central

    Ghosh, Ratna; de Campos, Marília K. F.; Huang, Jin; Huh, Seong K.; Orlowski, Adam; Yang, Yuan; Tripathi, Ashutosh; Nile, Aaron; Lee, Hsin-Chieh; Dynowski, Marek; Schäfer, Helen; Róg, Tomasz; Lete, Marta G.; Ahyayauch, Hasna; Alonso, Alicia; Vattulainen, Ilpo; Igumenova, Tatyana I.; Schaaf, Gabriel; Bankaitis, Vytas A.

    2015-01-01

    Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P2 binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen. PMID:25739452

  19. Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi

    PubMed Central

    Kamneva, Olga K.; Poudel, Saroj; Ward, Naomi L.

    2015-01-01

    A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1) SecA ATPase domain re-arrangements in some Planctomycetes, and (2) secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system. PMID:26030905

  20. Proteins Related to the Type I Secretion System Are Associated with Secondary SecA_DEAD Domain Proteins in Some Species of Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi.

    PubMed

    Kamneva, Olga K; Poudel, Saroj; Ward, Naomi L

    2015-01-01

    A number of bacteria belonging to the PVC (Planctomycetes-Verrucomicrobia-Chlamydiae) super-phylum contain unusual ribosome-bearing intracellular membranes. The evolutionary origins and functions of these membranes are unknown. Some proteins putatively associated with the presence of intracellular membranes in PVC bacteria contain signal peptides. Signal peptides mark proteins for translocation across the cytoplasmic membrane in prokaryotes, and the membrane of the endoplasmic reticulum in eukaryotes, by highly conserved Sec machinery. This suggests that proteins might be targeted to intracellular membranes in PVC bacteria via the Sec pathway. Here, we show that canonical signal peptides are significantly over-represented in proteins preferentially present in PVC bacteria possessing intracellular membranes, indicating involvement of Sec translocase in their cellular targeting. We also characterized Sec proteins using comparative genomics approaches, focusing on the PVC super-phylum. While we were unable to detect unique changes in Sec proteins conserved among membrane-bearing PVC species, we identified (1) SecA ATPase domain re-arrangements in some Planctomycetes, and (2) secondary SecA_DEAD domain proteins in the genomes of some Planctomycetes, Verrucomicrobia, Proteobacteria, Nitrospirae and Chlorobi. This is the first report of potentially duplicated SecA in Gram-negative bacteria. The phylogenetic distribution of secondary SecA_DEAD domain proteins suggests that the presence of these proteins is not related to the occurrence of PVC endomembranes. Further genomic analysis showed that secondary SecA_DEAD domain proteins are located within genomic neighborhoods that also encode three proteins possessing domains specific for the Type I secretion system.

  1. Functional characterisation of a TLR accessory protein, UNC93B1, in Atlantic salmon (Salmo salar).

    PubMed

    Lee, P T; Zou, J; Holland, J W; Martin, S A M; Scott, C J W; Kanellos, T; Secombes, C J

    2015-05-01

    Toll-like receptors (TLRs) are indispensable components of the innate immune system, which recognise conserved pathogen associated molecular patterns (PAMPs) and induce a series of defensive immune responses to protect the host. Biosynthesis, localisation and activation of TLRs are dependent on TLR accessory proteins. In this study, we identified the accessory protein, UNC93B1, from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs aided by the conserved gene synteny of genes flanking UNC93B1 in fish, birds and mammals. Phylogenetic analysis showed that salmon UNC93B1 grouped with other vertebrate UNC93B1 molecules, and had highest amino acid identity and similarity to zebrafish UNC93B1. The salmon UNC93B1 gene organisation was also similar in structure to mammalian UNC93B1. Our gene expression studies revealed that salmon UNC93B1 was more highly expressed in spleen, liver and gill tissues but was expressed at a lower level in head kidney tissue in post-smolts relative to parr. Moreover, salmon UNC93B1 mRNA transcripts were up-regulated in vivo in spleen tissue from polyI:C treated salmon and in vitro in polyI:C or IFNγ stimulated Salmon Head Kidney-1 (SHK-1) cells. Initial studies into the functional role of salmon UNC93B1 in fish TLR signalling found that both wild type salmon UNC93B1 and a molecule with a site-directed mutation (H424R) co-immunoprecipitated with salmon TLR19, TLR20a and TLR20d. Overall, these data illustrate the potential importance of UNC93B1 as an accessory protein in fish TLR signalling.

  2. Evidence for an Intrinsic Toxicity of Phosphatidylcholine to Sec14p-dependent Protein Transport from the Yeast Golgi Complex

    PubMed Central

    Xie, Zhigang; Fang, Min; Bankaitis, Vytas A.

    2001-01-01

    Yeast phosphatidylinositol-transfer protein (Sec14p) is essential for Golgi secretory function and cell viability. This requirement of Sec14p is relieved by genetic inactivation of the cytidine diphosphate-choline pathway for phosphatidycholine (PtdCho) biosynthesis. Standard phenotypic analyses indicate that inactivation of the phosphatidylethanolamine (PtdEtn) pathway for PtdCho biosynthesis, however, does not rescue the growth and secretory defects associated with Sec14p deficiency. We now report inhibition of choline uptake from the media reveals an efficient “bypass Sec14p” phenotype associated with PtdEtn-methylation pathway defects. We further show that the bypass Sec14p phenotype associated with PtdEtn-methylation pathway defects resembles other bypass Sec14p mutations in its dependence on phospholipase D activity. Finally, we find that increased dosage of enzymes that catalyze phospholipase D-independent turnover of PtdCho, via mechanisms that do not result in a direct production of phosphatidic acid or diacylglycerol, effect a partial rescue of sec14-1ts-associated growth defects. Taken together, these data support the idea that PtdCho is intrinsically toxic to yeast Golgi secretory function. PMID:11294911

  3. Determination of the Oligomeric State of SecYEG Protein Secretion Channel Complex Using in Vivo Photo- and Disulfide Cross-linking*

    PubMed Central

    Zheng, Zeliang; Blum, Amy; Banerjee, Tithi; Wang, Qianyu; Dantis, Virginia; Oliver, Donald

    2016-01-01

    SecYEG protein of bacteria or Sec61αβγ of eukaryotes is a universally conserved heterotrimeric protein channel complex that accommodates the partitioning of membrane proteins into the lipid bilayer as well as the secretion of proteins to the trans side of the plasma or endoplasmic reticular membrane, respectively. SecYEG function is facilitated by cytosolic partners, mainly a nascent chain-ribosome complex or the SecA ATPase motor protein. Extensive efforts utilizing both biochemical and biophysical approaches have been made to determine whether SecYEG functions as a monomer or a dimer, but such approaches have often generated conflicting results. Here we have employed site-specific in vivo photo-cross-linking or cysteine cross-linking, along with co-immunoprecipitation or SecA footprinting techniques to readdress this issue. Our findings show that the SecY dimer to monomer ratio is relatively constant regardless of whether translocons are actively engaged with protein substrate or not. Under the former conditions the SecY dimer can be captured associated with a translocon-jammed substrate, indicative of SecY dimer function. Furthermore, SecA ATPase can be cross-linked to two copies of SecY when the complex contains a translocation intermediate. Collectively, our results suggest that SecYEG dimers are functional units of the translocon. PMID:26747607

  4. HIV-1 Vpu Accessory Protein Induces Caspase-mediated Cleavage of IRF3 Transcription Factor*

    PubMed Central

    Park, Sang Yoon; Waheed, Abdul A.; Zhang, Zai-Rong; Freed, Eric O.; Bonifacino, Juan S.

    2014-01-01

    Vpu is an accessory protein encoded by HIV-1 that interferes with multiple host-cell functions. Herein we report that expression of Vpu by transfection into 293T cells causes partial proteolytic cleavage of interferon regulatory factor 3 (IRF3), a key transcription factor in the innate anti-viral response. Vpu-induced IRF3 cleavage is mediated by caspases and occurs mainly at Asp-121. Cleavage produces a C-terminal fragment of ∼37 kDa that comprises the IRF dimerization and transactivation domains but lacks the DNA-binding domain. A similar cleavage is observed upon infection of the Jurkat T-cell line with vesicular stomatitis virus G glycoprotein (VSV-G)-pseudotyped HIV-1. Two other HIV-1 accessory proteins, Vif and Vpr, also contribute to the induction of IRF3 cleavage in both the transfection and the infection systems. The C-terminal IRF3 fragment interferes with the transcriptional activity of full-length IRF3. Cleavage of IRF3 under all of these conditions correlates with cleavage of poly(ADP-ribose) polymerase, an indicator of apoptosis. We conclude that Vpu contributes to the attenuation of the anti-viral response by partial inactivation of IRF3 while host cells undergo apoptosis. PMID:25352594

  5. The Sec domain protein Scfd1 facilitates trafficking of ECM components during chondrogenesis.

    PubMed

    Hou, Ningning; Yang, Yuxi; Scott, Ian C; Lou, Xin

    2017-01-01

    Chondrogenesis in the developing skeleton requires transformation of chondrocytes from a simple mesenchymal condensation to cells with a highly enriched extracellular matrix (ECM). This transition is in part accomplished by alterations in the chondrocyte protein transport machinery to cope with both the increased amount and large size of ECM components. In a zebrafish mutagenesis screen to identify genes essential for cartilage development, we uncovered a mutant that disrupts the gene encoding Sec1 family domain containing 1 (scfd1). Homozygous scfd1 mutant embryos exhibit a profound craniofacial abnormality caused by a failure of chondrogenesis. Loss of scfd1 was found to hinder ER to Golgi transport of ECM proteins and is accompanied with activation of the unfolded protein response in chondrocytes. We further demonstrate a conserved role for Scfd1 in differentiation of mammalian chondrocytes, in which loss of either SCFD1 or STX18, a SLY1 interacting t-SNARE, severely impair transport of type II collagen. These results show that the existence of a specific export pathway, mediated by a complex containing SCFD1 and STX18 that plays an essential role in secretion of large ECM proteins during chondrogenesis.

  6. Crystallization and preliminary X-ray diffraction analysis of Sfh3, a member of the Sec14 protein superfamily

    SciTech Connect

    Ren, Jihui; Schaaf, Gabriel; Bankaitis, Vytas A.; Ortlund, Eric A.; Pathak, Manish C.

    2012-03-26

    Sec14 is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the yeast Saccharomyces cerevisiae and is the founding member of the Sec14 protein superfamily. Recent functional data suggest that Sec14 functions as a nanoreactor for PtdCho-regulated presentation of PtdIns to PtdIns kinase to affect membrane trafficking. Extrapolation of this concept to other members of the Sec14 superfamily suggests a mechanism by which a comprehensive cohort of Sec14-like nanoreactors sense correspondingly diverse pools of lipid metabolites. In turn, metabolic information is translated to signaling circuits driven by phosphoinositide metabolism. Sfh3, one of five Sec14 homologs in yeast, exhibits several interesting functional features, including its unique localization to lipid particles and microsomes. This localization forecasts novel regulatory interfaces between neutral lipid metabolism and phosphoinositide signaling. To launch a detailed structural and functional characterization of Sfh3, the recombinant protein was purified to homogeneity, diffraction-quality crystals were produced and a native X-ray data set was collected to 2.2 {angstrom} resolution. To aid in phasing, SAD X-ray diffraction data were collected to 1.93 {angstrom} resolution from an SeMet-labeled crystal at the Southeast Regional Collaborative Access Team at the Advanced Photon Source. Here, the cloning and purification of Sfh3 and the preliminary diffraction of Sfh3 crystals are reported, enabling structural analyses that are expected to reveal novel principles governing ligand binding and functional specificity for Sec14-superfamily proteins.

  7. Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity

    PubMed Central

    Kint, Joeri; Dickhout, Annemiek; Kutter, Jasmin; Maier, Helena J.; Britton, Paul; Koumans, Joseph; Pijlman, Gorben P.; Fros, Jelke J.; Wiegertjes, Geert F.

    2015-01-01

    ABSTRACT The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response. IMPORTANCE In the present study, we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for accessory protein 3a in the resistance against the type I IFN response. We also demonstrate that, in a time-dependent manner, IBV effectively interferes with IFN signaling and that its accessory proteins are dispensable for this activity. This study demonstrates that the gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and it identifies accessory protein 3a as multifaceted antagonist of the avian IFN system. PMID:26401035

  8. Characterization of monoclonal antibodies against feline coronavirus accessory protein 7b.

    PubMed

    Lemmermeyer, Tanja; Lamp, Benjamin; Schneider, Rainer; Ziebuhr, John; Tekes, Gergely; Thiel, Heinz-Jürgen

    2016-02-29

    Feline coronaviruses (FCoVs) encode five accessory proteins termed 3a, 3b, 3c, 7a and 7b of unknown function. These proteins are dispensable for viral replication in vitro but are supposed to play a role in virulence. In the current study, we produced and characterized 7b-specific monoclonal antibodies (mAbs). A recombinant form of the 7b protein was expressed as a fusion protein in Escherichia coli, purified by immobilized metal affinity chromatography and used as immunogen. Two hybridoma lines, 5B6 and 14D8, were isolated that expressed mAbs that recognized 7b proteins of both FCoV serotypes. Using an extensive set of N- and C-terminally truncated 7b proteins expressed in E. coli and a synthetic peptide, the binding sites of mAbs 5B6 and 14D8 were mapped to an 18-residue region that comprises the only potential N-glycosylation site of the FCoV 7b protein. The two mAbs were suitable to detect a 24-kDa protein, which represents the nonglycosylated form of 7b in FCoV-infected cells. We speculate that glycosylation of 7b is part of the viral evasion strategy to prevent an immune response against this antigenic site.

  9. The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

    SciTech Connect

    Kumar, Purnima; Gunalan, Vithiagaran; Liu Boping; Chow, Vincent T.K.; Druce, Julian; Birch, Chris; Catton, Mike; Fielding, Burtram C.; Tan, Yee-Joo; Lal, Sunil K.

    2007-09-30

    Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.

  10. Role of Sec61p in the ER-associated degradation of short-lived transmembrane proteins

    PubMed Central

    Scott, Daniel C.; Schekman, Randy

    2008-01-01

    Misfolded proteins in the endoplasmic reticulum (ER) are identified and degraded by the ER-associated degradation pathway (ERAD), a component of ER quality control. In ERAD, misfolded proteins are removed from the ER by retrotranslocation into the cytosol where they are degraded by the ubiquitin–proteasome system. The identity of the specific protein components responsible for retrotranslocation remains controversial, with the potential candidates being Sec61p, Der1p, and Doa10. We show that the cytoplasmic N-terminal domain of a short-lived transmembrane ERAD substrate is exposed to the lumen of the ER during the degradation process. The addition of N-linked glycan to the N terminus of the substrate is prevented by mutation of a specific cysteine residue of Sec61p, as well as a specific cysteine residue of the substrate protein. We show that the substrate protein forms a disulfide-linked complex to Sec61p, suggesting that at least part of the retrotranslocation process involves Sec61p. PMID:18573918

  11. High-Resolution Genetics Identifies the Lipid Transfer Protein Sec14p as Target for Antifungal Ergolines

    PubMed Central

    Cotesta, Simona; Perruccio, Francesca; Knapp, Britta; Fu, Yue; Studer, Christian; Pries, Verena; Riedl, Ralph; Helliwell, Stephen B.; Petrovic, Katarina T.; Movva, N. Rao; Sanglard, Dominique; Tao, Jianshi; Hoepfner, Dominic

    2016-01-01

    Invasive infections by fungal pathogens cause more deaths than malaria worldwide. We found the ergoline compound NGx04 in an antifungal screen, with selectivity over mammalian cells. High-resolution chemogenomics identified the lipid transfer protein Sec14p as the target of NGx04 and compound-resistant mutations in Sec14p define compound-target interactions in the substrate binding pocket of the protein. Beyond its essential lipid transfer function in a variety of pathogenic fungi, Sec14p is also involved in secretion of virulence determinants essential for the pathogenicity of fungi such as Cryptococcus neoformans, making Sec14p an attractive antifungal target. Consistent with this dual function, we demonstrate that NGx04 inhibits the growth of two clinical isolates of C. neoformans and that NGx04-related compounds have equal and even higher potency against C. neoformans. Furthermore NGx04 analogues showed fungicidal activity against a fluconazole resistant C. neoformans strain. In summary, we present genetic evidence that NGx04 inhibits fungal Sec14p and initial data supporting NGx04 as a novel antifungal starting point. PMID:27855158

  12. Structural mechanism for the regulation of HCN ion channels by the accessory protein TRIP8b

    PubMed Central

    DeBerg, Hannah A.; Bankston, John R.; Rosenbaum, Joel C.; Brzovic, Peter S.; Zagotta, William N.; Stoll, Stefan

    2015-01-01

    Summary Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels underlie the cationic Ih current present in many neurons. The direct binding of cAMP to HCN channels increases the rate and extent of channel opening and results in a depolarizing shift in the voltage dependence of activation. TRIP8b is an accessory protein that regulates the cell surface expression and dendritic localization of HCN channels and reduces the cyclic nucleotide dependence of these channels. Here we use electron paramagnetic resonance (EPR) to show that TRIP8b binds to the apo state of the cyclic nucleotide-binding domain (CNBD) of HCN2 channels without changing the overall domain structure. With EPR and nuclear magnetic resonance (NMR), we locate TRIP8b relative to the HCN channel and identify the binding interface on the CNBD. These data provide a structural framework for understanding how TRIP8b regulates the cyclic nucleotide dependence of HCN channels. PMID:25800552

  13. Crystal Structure of a Truncated Urease Accessory Protein UreF From Helicobacter pylori

    PubMed Central

    Lam, Robert; Romanov, Vladimir; Johns, Kathy; Battaile, Kevin P.; Wu-Brown, Jean; Guthrie, Jennifer L.; Hausinger, Robert P.; Pai, Emil F.; Chirgadze, Nickolay Y.

    2010-01-01

    Urease plays a central role in the pathogenesis of Helicobacter pylori in humans. Maturation of this nickel metalloenzyme in bacteria requires the participation of the accessory proteins UreD (termed UreH in H. pylori), UreF, and UreG which form sequential complexes with the urease apoprotein as well as UreE, a metallochaperone. Here, we describe the crystal structure of C-terminal truncated UreF from H. pylori (residues 1-233), the first UreF structure to be determined, at 1.55 Å resolution using SAD methods. UreF forms a dimer in vitro and adopts an all-helical fold congruent with secondary structure prediction. On the basis of evolutionary conservation analysis, the structure reveals a probable binding surface for interaction with other urease components as well as key conserved residues of potential functional relevance. PMID:20635345

  14. The chemistry of phospholipid binding by the saccharomyces cerevisiae phosphatidylinositol transfer protein sec14p as determined by electron paramagnetic resonance spectroscopy.

    SciTech Connect

    Smirnova, T. I.; Chadwick, T. G.; MacArthur, R.; Poluektov, O.; Song, L.; Ryan, M. M.; Schaaf, G.; Bankaitis, V. A.; Chemistry; North Carolina State Univ.; Florida State Univ.; Univ. of North Carolina

    2006-11-17

    The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

  15. A component of the Sec61 ER protein transporting pore is required for plant susceptibility to powdery mildew.

    PubMed

    Zhang, Wen-Jing; Hanisch, Susanne; Kwaaitaal, Mark; Pedersen, Carsten; Thordal-Christensen, Hans

    2013-01-01

    Biotrophic pathogens, like the powdery mildew fungi, require living plant cells for their growth and reproduction. During infection, a specialized structure called the haustorium is formed by the fungus. The haustorium is surrounded by a plant cell-derived extrahaustorial membrane (EHM). Over the EHM, the fungus obtains nutrients from and secretes effector proteins into the plant cell. In the plant cell these effectors interfere with cellular processes such as pathogen defense and membrane trafficking. However, the mechanisms behind effector delivery are largely unknown. This paper provides a model for and new insights into a putative transfer mechanism of effectors into the plant cell. We show that silencing of the barley Sec61βa transcript results in decreased susceptibility to the powdery mildew fungus. HvSec61βa is a component of both the endoplasmic reticulum (ER) translocon and retrotranslocon pores, the latter being part of the ER-associated protein degradation machinery. We provide support for a model suggesting that the retrotranslocon function of HvSec61βa is required for successful powdery mildew fungal infection. HvSec61βa-GFP and a luminal ER marker were co-localized to the ER, which was found to be in close proximity to the EHM around the haustorial body, but not the haustorial fingers. This differential EHM proximity suggests that the ER, including HvSec61βa, may be actively recruited by the haustorium, potentially to provide efficient effector transfer to the cytosol. Effector transport across this EHM-ER interface may occur by a vesicle-mediated process, while the Sec61 retrotranslocon pore potentially provides an escape route for these proteins to reach the cytosol.

  16. Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori.

    PubMed

    Dong, Zhaoming; Wang, Xiaohuan; Zhang, Yan; Zhang, Liping; Chen, Quanmei; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

    2016-05-01

    Male accessory gland (MAG) and female accessory gland (FAG) of the reproductive system are, respectively, responsible for producing seminal proteins and adhesive proteins during copulation and ovulation. Seminal proteins are ejaculated to female along with sperms, whereas adhesive proteins are excreted along with eggs. Proteins from the male and female reproductive organs are usually indicative of rapid adaptive evolution. Understanding the reproductive isolation and species divergence requires identifying reproduction-related proteins from many different species. Here, we present our proteomic analyses of male and female accessory glands of the silkworm, Bombyx mori. Using LC/MS-MS, we identified 2133 MAG proteins and 1872 FAG proteins. In total, 652 proteins were significant more abundant in the MAG than in the FAG, including growth factors, odorant-binding proteins, enzymes, and proteins of unknown function. Growth factors and odorant-binding proteins are potential signaling molecules, whereas most of proteins of unknown function were found to be Lepidoptera-specific proteins with high evolutionary rates. Microarray experiments and semi-quantitative RT-PCR validated that MAG-specific proteins were expressed exclusively in male moths. Totally, 192 proteins were considered as FAG-specific proteins, including protease inhibitors, enzymes, and other proteins. Protease inhibitors were found to be the most abundant FAG-specific proteins, which may protect eggs from infection by inhibiting pathogen-derived proteases. These results provide comprehensive insights into copulation and oviposition. Moreover, the newly identified Lepidoptera-specific MAG proteins provide useful data for future research on the evolution of reproductive proteins in insects.

  17. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans.

    PubMed

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients' depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host.

  18. Overproduction of a Model Sec- and Tat-Dependent Secretory Protein Elicits Different Cellular Responses in Streptomyces lividans

    PubMed Central

    Gullón, Sonia; Marín, Silvia; Mellado, Rafael P.

    2015-01-01

    Streptomyces lividans is considered an efficient host for the secretory production of homologous and heterologous proteins. To identify possible bottlenecks in the protein production process, a comparative transcriptomic approach was adopted to study cellular responses during the overproduction of a Sec-dependent model protein (alpha-amylase) and a Tat-dependent model protein (agarase) in Streptomyces lividans. The overproduction of the model secretory proteins via the Sec or the Tat route in S. lividans does elicit a different major cell response in the bacterium. The stringent response is a bacterial response to nutrients’ depletion, which naturally occurs at late times of the bacterial cell growth. While the induction of the stringent response at the exponential phase of growth may limit overall productivity in the case of the Tat route, the induction of that response does not take place in the case of the Sec route, which comparatively is an advantage in secretory protein production processes. Hence, this study identifies a potential major drawback in the secretory protein production process depending on the secretory route, and provides clues to improving S. lividans as a protein production host. PMID:26200356

  19. An Accessory Protein Required for Anchoring and Assembly of Amyloid Fibers in B. subtilis Biofilms

    PubMed Central

    Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

    2011-01-01

    Cells within Bacillus subtilis biofilms are held in place by an extracellular matrix that contains cell-anchored amyloid fibers, composed of the amyloidogenic protein TasA. As biofilms age they disassemble because the cells release the amyloid fibers. This release appears to be the consequence of incorporation of D-tyrosine, D-leucine, D-tryptophan and D-methionine into the cell wall. Here, we characterize the in vivo roles of an accessory protein TapA (TasA anchoring/assembly protein; previously YqxM) that serves both to anchor the fibers to the cell wall and to assemble TasA into fibers. TapA is found in discrete foci in the cell envelope and these foci disappear when cells are treated with a mixture of D-amino acids. Purified cell wall sacculi retain a functional form of this anchoring protein such that purified fibers can be anchored to the sacculi in vitro. In addition, we show that TapA is essential for the proper assembly of the fibers. Its absence results in a dramatic reduction in TasA levels and what little TasA is left produces only thin fibers that are not anchored to the cell. PMID:21477127

  20. Mutations in exocyst complex subunit SEC6 gene impaired polar auxin transport and PIN protein recycling in Arabidopsis primary root.

    PubMed

    Tan, Xiaoyun; Feng, Yihong; Liu, Yulong; Bao, Yiqun

    2016-09-01

    Polar auxin transport, which is critical for land plant pattern formation and directional growth, is largely depended on asymmetric distribution of PIN proteins at the plasma membrane (PM). Endocytosis and recycling processes play important roles in regulating PIN protein distribution and abundance at the PM. Two subunits (SEC8, EXO70A1) of exocyst, an octameric vesicle-tethering complex, have been reported to be involved in PIN protein recycling in Arabidopsis. However, the function of exocyst complex in PIN protein recycling and polar auxin transport remains incompletely understood. In this study, we utilized two SEC6 down-regulation mutants (PRsec6-1 and PRsec6-2) to investigate the role of exocyst subunit SEC6 in the primary root development, polar auxin transport and PIN proteins recycling. We found that in PRsec6 mutants: 1. Primary root growth was retarded, and lateral root initiation were compromised. 2. Primary roots were sensitive to exogenous auxin 1-napthalene acetic acid (NAA) but not 2,4-dichlorophenoxy (2.4-D). 3. Recycling of PIN1 and PIN2 proteins from the Brefeldin A (BFA) compartment to the PM was delayed. 4. Vesicles accumulated in the primary root tip cells, especially accumulated in the cytosol closed to the PM. These results further demonstrated that the exocyst complex plays an important role in PIN protein recycling and polar auxin transport in Arabidopsis primary root.

  1. ARC3 is a stromal Z-ring accessory protein essential for plastid division

    PubMed Central

    Maple, Jodi; Vojta, Lea; Soll, Jurgen; Møller, Simon G

    2007-01-01

    In plants, chloroplast division is an integral part of development, and these vital organelles arise by binary fission from pre-existing cytosolic plastids. Chloroplasts arose by endosymbiosis and although they have retained elements of the bacterial cell division machinery to execute plastid division, they have evolved to require two functionally distinct forms of the FtsZ protein and have lost elements of the Min machinery required for Z-ring placement. Here, we analyse the plastid division component accumulation and replication of chloroplasts 3 (ARC3) and show that ARC3 forms part of the stromal plastid division machinery. ARC3 interacts specifically with AtFtsZ1, acting as a Z-ring accessory protein and defining a unique function for this family of FtsZ proteins. ARC3 is involved in division site placement, suggesting that it might functionally replace MinC, representing an important advance in our understanding of the mechanism of chloroplast division and the evolution of the chloroplast division machinery. PMID:17304239

  2. Brain-specific interleukin-1 receptor accessory protein in sleep regulation.

    PubMed

    Taishi, Ping; Davis, Christopher J; Bayomy, Omar; Zielinski, Mark R; Liao, Fan; Clinton, James M; Smith, Dirk E; Krueger, James M

    2012-03-01

    Interleukin (IL)-1β is involved in several brain functions, including sleep regulation. It promotes non-rapid eye movement (NREM) sleep via the IL-1 type I receptor. IL-1β/IL-1 receptor complex signaling requires adaptor proteins, e.g., the IL-1 receptor brain-specific accessory protein (AcPb). We have cloned and characterized rat AcPb, which shares substantial homologies with mouse AcPb and, compared with AcP, is preferentially expressed in the brain. Furthermore, rat somatosensory cortex AcPb mRNA varied across the day with sleep propensity, increased after sleep deprivation, and was induced by somnogenic doses of IL-1β. Duration of NREM sleep was slightly shorter and duration of REM sleep was slightly longer in AcPb knockout than wild-type mice. In response to lipopolysaccharide, which is used to induce IL-1β, sleep responses were exaggerated in AcPb knockout mice, suggesting that, in normal mice, inflammation-mediated sleep responses are attenuated by AcPb. We conclude that AcPb has a role in sleep responses to inflammatory stimuli and, possibly, in physiological sleep regulation.

  3. Using SEC-RPLC and RPLC-CIEF as two-dimensional separation strategies for protein profiling

    PubMed Central

    Simpson, David C.; Ahn, Seonghee; Pasa-Tolic, Ljiljana; Bogdanov, Bogdan; Mottaz, Heather M.; Vilkov, Andrey N.; Anderson, Gordon A.; Lipton, Mary S.; Smith, Richard D.

    2007-01-01

    Bottom-up proteomics (analyzing peptides that result from protein digestion) has demonstrated capability for broad proteome coverage and good throughput. However, due to incomplete sequence coverage, this approach is not ideally suited to the study of modified proteins. The modification complement of a protein can best be elucidated by analyzing the intact protein. Two-dimensional gel electrophoresis, typically coupled with the analysis of peptides that result from in-gel digestion, is the most frequently applied protein separation technique in MS-based proteomics. As an alternative, numerous column-based liquid phase techniques, which are generally more amenable to automation, are being investigated. In this work, the combination of size exclusion chromatography (SEC) fractionation with reversed-phase liquid chromatography (RPLC)-Fourier-transform ion cyclotron resonance (FTICR)-mass spectrometry (MS) is compared with the combination of RPLC fractionation with capillary isoelectric focusing (CIEF)-FTICR-MS for the analysis of the Shewanella oneidensis proteome. SEC-RPLC-FTICR-MS allowed the detection of 297 proteins, as opposed to 166 using RPLC-CIEF-FTICR-MS, indicating that approaches based on LC-MS provide better coverage. However, there were significant differences in the sets of proteins detected and both approaches provide a basis for accurately quantifying changes in protein and modified protein abundances. PMID:16732621

  4. Targeting TGFβ superfamily ligand accessory proteins as novel therapeutics for chronic lung disorders.

    PubMed

    Budd, David C; Holmes, Alan M

    2012-09-01

    Dysregulation of the transforming growth factor β (TGFβ) pathway has been implicated to underlie a number of disease indications including chronic lung disorders such as asthma, chronic obstructive pulmonary disease (COPD), interstitial pneumonias, and pulmonary arterial hypertension (PAH). Consequently, the pharmaceutical industry has devoted significant resources in the pursuit of TGFβ pathway inhibitors that target the cognate type I and II receptors and respective ligands. The progress of these approaches has been painfully slow, due in part to dose-limiting safety issues that result from the antagonism of a pathway that is responsible for regulating many fundamental biological processes including immune surveillance and cardiovascular responses. These disappointments have led many in the field to conclude that modulating the TGFβ pathway for chronic indications with a sufficient safety window using conventional approaches may be extremely difficult to achieve. Here we review the rationale and limitations of the use of TGFβ pathway inhibitors in chronic lung disorders and the possibility of targeting TGFβ superfamily ligand accessory proteins to allow rheostatic regulation of signaling to achieve efficacy while maintaining a sufficient therapeutic index.

  5. Transcriptional activation of melanocortin 2 receptor accessory protein by PPARγ in adipocytes

    SciTech Connect

    Kim, Nam Soo; Kim, Yoon-Jin; Cho, Si Young; Lee, Tae Ryong; Kim, Sang Hoon

    2013-09-27

    Highlights: •MRAP enhanced HSL expression. •ACTH-mediated MRAP reduced glycerol release. •PPARγ induced MRAP expression. •PPARγ bound to the MRAP promoter. -- Abstract: Adrenocorticotropic hormone (ACTH) in rodents decreases lipid accumulation and body weight. Melanocortin receptor 2 (MC2R) and MC2R accessory protein (MRAP) are specific receptors for ACTH in adipocytes. Peroxisome proliferator-activated receptor γ (PPARγ) plays a role in the transcriptional regulation of metabolic pathways such as adipogenesis and β-oxidation of fatty acids. In this study we investigated the transcriptional regulation of MRAP expression during differentiation of 3T3-L1 cells. Stimulation with ACTH affected lipolysis in murine mature adipocytes via MRAP. Putative peroxisome proliferator response element (PPRE) was identified in the MRAP promoter region. In chromatin immunoprecipitation and reporter assays, we observed binding of PPARγ to the MRAP promoter. The mutagenesis experiments showed that the −1209/−1198 region of the MRAP promoter could function as a PPRE site. These results suggest that PPARγ is required for transcriptional activation of the MRAP gene during adipogenesis, which contributes to understanding of the molecular mechanism of lipolysis in adipocytes.

  6. A Critical Role for the GluA1 Accessory Protein, SAP97, in Cocaine Seeking

    PubMed Central

    White, Samantha L; Ortinski, Pavel I; Friedman, Shayna H; Zhang, Lei; Neve, Rachael L; Kalb, Robert G; Schmidt, Heath D; Pierce, R Christopher

    2016-01-01

    A growing body of evidence indicates that the transport of GluA1 subunit-containing calcium-permeable AMPA receptors (CP-AMPARs) to synapses in subregions of the nucleus accumbens promotes cocaine seeking. Consistent with these findings, the present results show that administration of the CP-AMPAR antagonist, Naspm, into the caudal lateral core or caudal medial shell of the nucleus accumbens attenuated cocaine priming-induced reinstatement of drug seeking. Moreover, viral-mediated overexpression of ‘pore dead' GluA1 subunits (via herpes simplex virus (HSV) GluA1-Q582E) in the lateral core or medial shell attenuated the reinstatement of cocaine seeking. The overexpression of wild-type GluA1 subunits (via HSV GluA1-WT) in the medial shell, but not the lateral core, enhanced the reinstatement of cocaine seeking. These results indicate that activation of GluA1-containing AMPARs in subregions of the nucleus accumbens reinstates cocaine seeking. SAP97 and 4.1N are proteins involved in GluA1 trafficking to and stabilization in synapses; SAP97-GluA1 interactions also influence dendritic growth. We next examined potential roles of SAP97 and 4.1N in cocaine seeking. Viral-mediated expression of a microRNA that reduces SAP97 protein expression (HSV miSAP97) in the medial accumbens shell attenuated cocaine seeking. In contrast, a virus that overexpressed a dominant-negative form of a 4.1N C-terminal domain (HSV 4.1N-CTD), which prevents endogenous 4.1N binding to GluA1 subunits, had no effect on cocaine seeking. These results indicate that the GluA1 subunit accessory protein SAP97 may represent a novel target for pharmacotherapeutic intervention in the treatment of cocaine craving. PMID:26149358

  7. PROTEIN KINASE Cα MEDIATES A NOVEL FORM OF PLASTICITY IN THE ACCESSORY OLFACTORY BULB

    PubMed Central

    DONG, C.; GODWIN, D. W.; BRENNAN, P. A.; HEGDE, A. N.

    2009-01-01

    Modification of synapses in the accessory olfactory bulb (AOB) is believed to underlie pheromonal memory that enables mate recognition in mice. The memory, which is acquired with single-trial learning forms only with coincident noradrenergic and glutamatergic inputs to the AOB. The mechanisms by which glutamate and norepinephrine (NE) alter the AOB synapses are not well understood. Here we present results that not only reconcile the earlier, seemingly contradictory, observations on the role of glutamate and NE in changing the AOB synapses, but also reveal novel mechanisms of plasticity. Our studies suggest that initially, glutamate acting at Group II metabotropic receptors and NE acting at α2-adrenergic receptors inhibit N-type and R-type Ca2+ channels in mitral cells via a G-Protein. The N-type and R-type Ca2+ channel inhibition is reversed by activation of α1-adrenergic receptors and protein kinase Cα (PKCα). Based on these results, we propose a hypothetical model for a new kind of synaptic plasticity in the AOB that accounts for the previous behavioral data on pheromonal memory. According to this model, initial inhibition of the Ca2+ channels suppresses the GABAergic inhibitory feedback to mitral cells, causing disinhibition and Ca2+ influx. NE also activates phospholipase C (PLC) through α1-adrenergic receptors generating inositol 1,4,5-trisphosphate and diacylglycerol (DAG). Calcium and DAG together activate protein kinase Cα (PKCα) which switches the disinhibition to increased inhibition of mitral cells. Thus, PKCα is likely to be a coincidence detector integrating glutamate and NE input in the AOB and bridging the short-term signaling to long-term structural changes resulting in enhanced inhibition of mitral cells that is thought to underlie memory formation. PMID:19580852

  8. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination.

    PubMed

    Panizza, Silvia; Mendoza, Marco A; Berlinger, Marc; Huang, Lingzhi; Nicolas, Alain; Shirahige, Katsuhiko; Klein, Franz

    2011-08-05

    Meiotic recombination between homologous chromosomes initiates via programmed DNA double-strand breaks (DSBs), generated by complexes comprising Spo11 transesterase plus accessory proteins. DSBs arise concomitantly with the development of axial chromosome structures, where the coalescence of axis sites produces linear arrays of chromatin loops. Recombining DNA sequences map to loops, but are ultimately tethered to the underlying axis. How and when such tethering occurs is currently unclear. Using ChIPchip in yeast, we show that Spo11-accessory proteins Rec114, Mer2, and Mei4 stably interact with chromosome axis sequences, upon phosphorylation of Mer2 by S phase Cdk. This axis tethering requires meiotic axis components (Red1/Hop1) and is modulated in a domain-specific fashion by cohesin. Loss of Rec114, Mer2, and Mei4 binding correlates with loss of DSBs. Our results strongly suggest that hotspot sequences become tethered to axis sites by the DSB machinery prior to DSB formation.

  9. DNMT3B isoforms without catalytic activity stimulate gene body methylation as accessory proteins in somatic cells.

    PubMed

    Duymich, Christopher E; Charlet, Jessica; Yang, Xiaojing; Jones, Peter A; Liang, Gangning

    2016-04-28

    Promoter DNA methylation is a key epigenetic mechanism for stable gene silencing, but is correlated with expression when located in gene bodies. Maintenance and de novo DNA methylation by catalytically active DNA methyltransferases (DNMT1 and DNMT3A/B) require accessory proteins such as UHRF1 and DNMT3L. DNMT3B isoforms are widely expressed, although some do not have active catalytic domains and their expression can be altered during cell development and tumourigenesis, questioning their biological roles. Here, we show that DNMT3B isoforms stimulate gene body methylation and re-methylation after methylation-inhibitor treatment. This occurs independently of the isoforms' catalytic activity, demonstrating a similar functional role to the accessory protein DNMT3L, which is only expressed in undifferentiated cells and recruits DNMT3A to initiate DNA methylation. This unexpected role for DNMT3B suggests that it might substitute for the absent accessory protein DNMT3L to recruit DNMT3A in somatic cells.

  10. Sequences in gibbon ape leukemia virus envelope that confer sensitivity to HIV-1 accessory protein Vpu.

    PubMed

    Janaka, Sanath Kumar; Lucas, Tiffany M; Johnson, Marc C

    2011-11-01

    HIV-1 efficiently forms pseudotyped particles with many gammaretrovirus glycoproteins, such as Friend murine leukemia virus (F-MLV) Env, but not with the related gibbon ape leukemia virus (GaLV) Env or with a chimeric F-MLV Env with a GaLV cytoplasmic tail domain (CTD). This incompatibility is modulated by the HIV-1 accessory protein Vpu. Because the GaLV Env CTD does not resemble tetherin or CD4, the well-studied targets of Vpu, we sought to characterize the modular sequence in the GaLV Env CTD required for this restriction in the presence of Vpu. Using a systematic mutagenesis scan, we determined that the motif that makes GaLV Env sensitive to Vpu is INxxIxxVKxxVxRxK. This region in the CTD of GaLV Env is predicted to form a helix. Mutations in the CTD that would break this helix abolish sensitivity to Vpu. Although many of these positions can be replaced with amino acids with similar biophysical properties without disrupting the Vpu sensitivity, the final lysine residue is required. This Vpu sensitivity sequence appears to be modular, as the unrelated Rous sarcoma virus (RSV) Env can be made Vpu sensitive by replacing its CTD with the GaLV Env CTD. In addition, F-MLV Env can be made Vpu sensitive by mutating two amino acids in its cytoplasmic tail to make it resemble more closely the Vpu sensitivity motif. Surprisingly, the core components of this Vpu sensitivity sequence are also present in the host surface protein CD4, which is also targeted by Vpu through its CTD.

  11. Characterization of the Klebsiella aerogenes urease accessory protein UreD in fusion with the maltose binding protein.

    PubMed

    Carter, Eric L; Hausinger, Robert P

    2010-05-01

    Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a DeltaureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (> 670 kDa) that bound approximately 2.5 Ni2+ ions (K(d) of approximately 50 microM, where K(d) is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (approximately 4 Zn2+ ions per protomer; K(d) of 5 microM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

  12. Male Age Affects Female Mate Preference, Quantity of Accessory Gland Proteins, and Sperm Traits and Female Fitness in D. melanogaster.

    PubMed

    Rezaei, Abolhasan; Krishna, Mysore Siddaiah; Santhosh, Hassan T

    2015-01-01

    For species in which mating is resource-independent and offspring do not receive parental care, theoretical models of age-based female mate preference predict that females should prefer to mate with older males as they have demonstrated ability to survive. Thus, females should obtain a fitness benefit from mating with older males. However, male aging is often associated with reductions in quantity of sperm. The adaptive significance of age-based mate choice is therefore unclear. Various hypotheses have made conflicting predictions concerning this issue, because published studies have not investigated the effect of age on accessory gland proteins and sperm traits. D. melanogaster exhibits resource-independent mating, and offspring do not receive parental care, making this an appropriate model for studying age-based mate choice. In the present study, we found that D. melanogaster females of all ages preferred to mate with the younger of two competing males. Young males performed significantly greater courtship attempts and females showed least rejection for the same than middle-aged and old males. Young males had small accessory glands that contained very few main cells that were larger than average. Nevertheless, compared with middle-aged or old males, the young males transferred greater quantities of accessory gland proteins and sperm to mated females. As a result, females that mated with young male produced more eggs and progeny than those that mated with older males. Furthermore, mating with young male reduced female's lifespan. These studies indicate that quantity of accessory gland proteins and sperm traits decreased with male age and females obtain direct fitness benefit from mating with preferred young males.

  13. Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

    PubMed Central

    Rabouw, Huib H.; Canton, Javier; Sola, Isabel; Enjuanes, Luis; Bredenbeek, Peter J.; Kikkert, Marjolein; de Groot, Raoul J.; van Kuppeveld, Frank J. M.

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I interferon (IFN-α/β) response, the protein kinase R (PKR)-mediated stress response is being recognized as an important innate response pathway. Upon detecting viral dsRNA, PKR phosphorylates eIF2α, leading to the inhibition of cellular and viral translation and the formation of stress granules (SGs), which are increasingly recognized as platforms for antiviral signaling pathways. It is unknown whether cellular infection by MERS-CoV activates the stress response pathway or whether the virus has evolved strategies to suppress this infection-limiting pathway. Here, we show that cellular infection with MERS-CoV does not lead to the formation of SGs. By transiently expressing the MERS-CoV accessory proteins individually, we identified a role of protein 4a (p4a) in preventing activation of the stress response pathway. Expression of MERS-CoV p4a impeded dsRNA-mediated PKR activation, thereby rescuing translation inhibition and preventing SG formation. In contrast, p4a failed to suppress stress response pathway activation that is independent of PKR and dsRNA. MERS-CoV p4a is a dsRNA binding protein. Mutation of the dsRNA binding motif in p4a disrupted its PKR antagonistic activity. By inserting p4a in a picornavirus lacking its natural PKR antagonist, we showed that p4a exerts PKR antagonistic activity also under infection conditions. However, a recombinant MERS-CoV deficient in p4a expression still suppressed SG formation, indicating the expression of at least one other stress response antagonist. This virus also suppressed the dsRNA-independent stress response pathway. Thus, MERS-CoV interferes with antiviral stress responses using at least two different mechanisms, with p4a

  14. Plasma Levels of Soluble Interleukin 1 Receptor Accessory Protein Are Reduced in Obesity

    PubMed Central

    Attard, Chantal; Kulkarni, Hemant; Cummings, Nik; Diego, Vincent P.; Carless, Melanie A.; Shields, Katherine A.; Johnson, Matthew P.; Kowlessur, Sudhir; Dyer, Thomas D.; Comuzzie, Anthony G.; Almasy, Laura; Zimmet, Paul; Moses, Eric K.; Göring, Harald H. H.; Curran, Joanne E.; Blangero, John; Jowett, Jeremy B. M.

    2014-01-01

    Context: Adipokines actuate chronic, low-grade inflammation through a complex network of immune markers, but the current understanding of these networks is incomplete. The soluble isoform of the IL-1 receptor accessory protein (sIL1RAP) occupies an important position in the inflammatory pathways involved in obesity. The pathogenetic and clinical influences of sIL1RAP are unknown. Objective: The objective of the study was to elucidate whether plasma levels of sIL1RAP are reduced in obesity, using affluent clinical, biochemical, and genetic data from two diverse cohorts. Design, Setting, and Participants: The study was conducted in two cohorts: the San Antonio Family Heart Study (n = 1397 individuals from 42 families) and South Asians living in Mauritius, n = 230). Main Outcome Measures: Plasma sIL1RAP levels were measured using an ELISA. The genetic basis of sIL1RAP levels were investigated using both a large-scale gene expression profiling study and a genome-wide association study. Results: A significant decrease in plasma sIL1RAP levels were observed in obese subjects, even after adjustment for age and sex. The sIL1RAP levels demonstrated a strong inverse association with obesity measures in both populations. All associations were more significant in females. Plasma sIL1RAP levels were significantly heritable, correlated with IL1RAP transcript levels (NM_134470), showed evidence for shared genetic influences with obesity measures and were significantly associated with the rs2885373 single-nucleotide polymorphism (P = 6.7 × 10−23) within the IL1RAP gene. Conclusions: Plasma sIL1RAP levels are reduced in obesity and can potentially act as biomarkers of obesity. Mechanistic studies are required to understand the exact contribution of sIL1RAP to the pathogenesis of obesity. PMID:24915116

  15. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster

    SciTech Connect

    Clark, A.G.; Prout, T.; Harshman, L.G.

    1995-01-01

    Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophila melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female`s reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability to resist being displaced by subsequent sperm. This lack of correlation, and the association of Acp alleles with resisting subsequent sperm only, suggests that different mechanisms mediate the two components of sperm displacement. 36 refs., 4 figs., 7 tabs.

  16. Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

    PubMed

    d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

    2015-04-16

    ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains.

  17. Variation in sperm displacement and its association with accessory gland protein loci in Drosophila melanogaster.

    PubMed

    Clark, A G; Aguadé, M; Prout, T; Harshman, L G; Langley, C H

    1995-01-01

    Genes that influence mating and/or fertilization success may be targets for strong natural selection. If females remate frequently relative to the duration of sperm storage and rate of sperm use, sperm displacement may be an important component of male reproductive success. Although it has long been known that mutant laboratory stocks of Drosophila differ in sperm displacement, the magnitude of the naturally occurring genetic variation in this character has not been systematically quantified. Here we report the results of a screen for variation in sperm displacement among 152 lines of Drosophilia melanogaster that were made homozygous for second and/or third chromosomes recovered from natural populations. Sperm displacement was assayed by scoring the progeny of cn;bw females that had been mated sequentially to cn;bw and tested males in either order. Highly significant differences were seen in both the ability to displace sperm that is resident in the female's reproductive tract and in the ability to resist displacement by subsequent sperm. Most lines exhibited nearly complete displacement, having nearly all progeny sired by the second male, but several lines had as few as half the progeny fathered by the second male. Lines that were identified in the screen for naturally occurring variation in sperm displacement were also characterized for single-strand conformation polymorphisms (SSCP) at seven accessory gland protein (Acp) genes, Glucose dehydrogenase (Gld), and Esterase-6 (Est-6). Acp genes encode proteins that are in some cases known to be transmitted to the female in the seminal fluid and are likely candidates for genes that might mediate the phenomenon of sperm displacement. Significant associations were found between particular Acp alleles at four different loci (Acp26Aa/Ab, Acp29B, Acp36DE and Acp53E) and the ability of males to resist displacement by subsequent sperm. There was no correlation between the ability to displace resident sperm and the ability

  18. Identification and Analysis of Bacterial Protein Secretion Inhibitors Utilizing a SecA-LacZ Reporter Fusion System

    PubMed Central

    Alksne, L. E.; Burgio, P.; Hu, W.; Feld, B.; Singh, M. P.; Tuckman, M.; Petersen, P. J.; Labthavikul, P.; McGlynn, M.; Barbieri, L.; McDonald, L.; Bradford, P.; Dushin, R. G.; Rothstein, D.; Projan, S. J.

    2000-01-01

    Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be ≤128 μg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity. PMID:10817687

  19. The accessory proteins REEP5 and REEP6 refine CXCR1-mediated cellular responses and lung cancer progression.

    PubMed

    Park, Cho Rong; You, Dong-Joo; Park, Sumi; Mander, Sunam; Jang, Da-Eun; Yeom, Su-Cheong; Oh, Seong-Hyun; Ahn, Curie; Lee, Sang Heon; Seong, Jae Young; Hwang, Jong-Ik

    2016-12-14

    Some G-protein-coupled receptors have been reported to require accessory proteins with specificity for proper functional expression. In this study, we found that CXCR1 interacted with REEP5 and REEP6, but CXCR2 did not. Overexpression of REEP5 and REEP6 enhanced IL-8-stimulated cellular responses through CXCR1, whereas depletion of the proteins led to the downregulation of the responses. Although REEPs enhanced the expression of a subset of GPCRs, in the absence of REEP5 and REEP6, CXCR1 was expressed in the plasma membrane, but receptor internalization and intracellular clustering of β-arrestin2 following IL-8 treatment were impaired, suggesting that REEP5 and REEP6 might be involved in the ligand-stimulated endocytosis of CXCR1 rather than membrane expression, which resulted in strong cellular responses. In A549 lung cancer cells, which endogenously express CXCR1, the depletion of REEP5 and REEP6 significantly reduced growth and invasion by downregulating IL-8-stimulated ERK phosphorylation, actin polymerization and the expression of genes related to metastasis. Furthermore, an in vivo xenograft model showed that proliferation and metastasis of A549 cells lacking REEP5 and REEP6 were markedly decreased compared to the control group. Thus, REEP5 and REEP6 could be novel regulators of G-protein-coupled receptor signaling whose functional mechanisms differ from other accessory proteins.

  20. The accessory proteins REEP5 and REEP6 refine CXCR1-mediated cellular responses and lung cancer progression

    PubMed Central

    Park, Cho Rong; You, Dong-Joo; Park, Sumi; Mander, Sunam; Jang, Da-Eun; Yeom, Su-Cheong; Oh, Seong-Hyun; Ahn, Curie; Lee, Sang Heon; Seong, Jae Young; Hwang, Jong-Ik

    2016-01-01

    Some G-protein-coupled receptors have been reported to require accessory proteins with specificity for proper functional expression. In this study, we found that CXCR1 interacted with REEP5 and REEP6, but CXCR2 did not. Overexpression of REEP5 and REEP6 enhanced IL-8-stimulated cellular responses through CXCR1, whereas depletion of the proteins led to the downregulation of the responses. Although REEPs enhanced the expression of a subset of GPCRs, in the absence of REEP5 and REEP6, CXCR1 was expressed in the plasma membrane, but receptor internalization and intracellular clustering of β-arrestin2 following IL-8 treatment were impaired, suggesting that REEP5 and REEP6 might be involved in the ligand-stimulated endocytosis of CXCR1 rather than membrane expression, which resulted in strong cellular responses. In A549 lung cancer cells, which endogenously express CXCR1, the depletion of REEP5 and REEP6 significantly reduced growth and invasion by downregulating IL-8-stimulated ERK phosphorylation, actin polymerization and the expression of genes related to metastasis. Furthermore, an in vivo xenograft model showed that proliferation and metastasis of A549 cells lacking REEP5 and REEP6 were markedly decreased compared to the control group. Thus, REEP5 and REEP6 could be novel regulators of G-protein-coupled receptor signaling whose functional mechanisms differ from other accessory proteins. PMID:27966653

  1. The role of accessory proteins in the replication of feline infectious peritonitis virus in peripheral blood monocytes.

    PubMed

    Dedeurwaerder, Annelike; Desmarets, Lowiese M; Olyslaegers, Dominique A J; Vermeulen, Ben L; Dewerchin, Hannah L; Nauwynck, Hans J

    2013-03-23

    The ability to productively infect monocytes/macrophages is the most important difference between the low virulent feline enteric coronavirus (FECV) and the lethal feline infectious peritonitis virus (FIPV). In vitro, the replication of FECV in peripheral blood monocytes always drops after 12h post inoculation, while FIPV sustains its replication in the monocytes from 45% of the cats. The accessory proteins of feline coronaviruses have been speculated to play a prominent role in virulence as deletions were found to be associated with attenuated viruses. Still, no functions have been ascribed to them. In order to investigate if the accessory proteins of FIPV are important for sustaining its replication in monocytes, replication kinetics were determined for FIPV 79-1146 and its deletion mutants, lacking either accessory protein open reading frame 3abc (FIPV-Δ3), 7ab (FIPV-Δ7) or both (FIPV-Δ3Δ7). Results showed that the deletion mutants FIPV-Δ7 and FIPV-Δ3Δ7 could not maintain their replication, which was in sharp contrast to wt-FIPV. FIPV-Δ3 could still sustain its replication, but the percentage of infected monocytes was always lower compared to wt-FIPV. In conclusion, this study showed that ORF7 is crucial for FIPV replication in monocytes/macrophages, giving an explanation for its importance in vivo, its role in the development of FIP and its conservation in field strains. The effect of an ORF3 deletion was less pronounced, indicating only a supportive role of ORF3 encoded proteins during the infection of the in vivo target cell by FIPVs.

  2. The Pediocin PA-1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin PA-1.

    PubMed

    Oppegård, Camilla; Fimland, Gunnar; Anonsen, Jan Haug; Nissen-Meyer, Jon

    2015-05-19

    Peptides, in contrast to proteins, are generally not large enough to form stable and well-defined three-dimensional structures. However, peptides are still able to form correct disulfide bonds. Using pediocin-like bacteriocins, we have examined how this may be achieved. Some pediocin-like bacteriocins, such as pediocin PA-1 and sakacin P[N24C+44C], have four cysteines. There are three possible ways by which the four cysteines may combine to form two disulfide bonds, and the three variants are expected to be produced in approximately equal amounts if their formation is random. Pediocin PA-1 and sakacin P[N24C+44C] with correct disulfide bonds were the main products when they were secreted by the pediocin PA-1 ABC transporter and accessory protein, but when they were secreted by the corresponding secretion machinery for sakacin A, a pediocin-like bacteriocin with one disulfide bond (two cysteines), peptides with all three possible disulfide bonds were produced in approximately equal amounts. All five cysteines in the pediocin PA-1 ABC transporter and the two cysteines (that form a CxxC motif) in the accessory protein were individually replaced with serines to examine their involvement in disulfide bond formation in pediocin PA-1. The Cys86Ser mutation in the accessory protein caused a 2-fold decrease in the amount of pediocin PA-1 with correct disulfide bonds, while the Cys83Ser mutation nearly abolished the production of pediocin PA-1 and resulted in the production of all three disufide bond variants in equal amounts. The Cys19Ser mutation in the ABC transporter completely abolished secretion of pediocin PA-1, suggesting that Cys19 is in the proteolytic active site and involved in cleaving the prebacteriocin. Replacing the other four cysteines in the ABC transporter with serines caused a slight reduction in the overall amount of secreted pediocin PA-1, but the relative amount with the correct disulfide bonds remained large. These results indicate that the pediocin

  3. Activation of Aplysia ARF6 induces neurite outgrowth and is sequestered by the overexpression of the PH domain of Aplysia Sec7 proteins.

    PubMed

    Jang, Deok-Jin; Jun, Yong-Woo; Shim, Jaehoon; Sim, Su-Eon; Lee, Jin-A; Lim, Chae-Seok; Kaang, Bong-Kiun

    2017-02-01

    ADP-ribosylation factors (ARFs) are small guanosine triphosphatases of the Ras superfamily involved in membrane trafficking and regulation of the actin cytoskeleton. Aplysia Sec7 protein (ApSec7), a guanine nucleotide exchange factor for ARF1 and ARF6, induces neurite outgrowth and plays a key role in 5-hydroxyltryptamine-induced neurite growth and synaptic facilitation in Aplysia sensory-motor synapses. However, the specific role of ARF6 signaling on neurite outgrowth in Aplysia neurons has not been examined. In the present study, we cloned Aplysia ARF6 (ApARF6) and revealed that an overexpression of enhanced green fluorescent protein (EGFP)-fused constitutively active ApARF6 (ApARF6-Q67L-EGFP) could induce neurite outgrowth in Aplysia sensory neurons. Further, we observed that ApARF6-induced neurite outgrowth was inhibited by the co-expression of a Sec7 activity-deficient mutant of ApSec7 (ApSec7-E159K). The pleckstrin homology domain of ApSec7 may bind to active ApARF6 at the plasma membrane and prevent active ApARF6-induced functions, including intracellular vacuole formation in HEK293T cells. The results of the present study suggest that activation of ARF6 signaling could induce neurite outgrowth in Aplysia neurons and may be involved in downstream signaling of ApSec7-induced neurite outgrowth in Aplysia neurons.

  4. Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

    PubMed Central

    Fiester, Steven E.; Nwugo, Chika C.; Penwell, William F.; Neary, John M.; Beckett, Amber C.; Arivett, Brock A.; Schmidt, Robert E.; Geiger, Sarah C.; Connerly, Pamela L.; Menke, Sharon M.; Tomaras, Andrew P.

    2015-01-01

    Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3′ end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2′-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence. PMID:25605767

  5. The Small GTPase MoSec4 Is Involved in Vegetative Development and Pathogenicity by Regulating the Extracellular Protein Secretion in Magnaporthe oryzae

    PubMed Central

    Zheng, Huakun; Chen, Simiao; Chen, Xiaofeng; Liu, Shuyan; Dang, Xie; Yang, Chengdong; Giraldo, Martha C.; Oliveira-Garcia, Ely; Zhou, Jie; Wang, Zonghua; Valent, Barbara

    2016-01-01

    The Rab GTPase proteins play important roles in the membrane trafficking, and consequently protein secretion and development of eukaryotic organisms. However, little is known about the function of Rab GTPases in Magnaporthe oryzae. To further explore the function of Rab GTPases, we deleted the ortholog of the yeast Sec4p protein in M. oryzae, namely MoSEC4. The ΔMosec4 mutant is defective in polarized growth and conidiation, and it displays decreased appressorium turgor pressure and attenuated pathogenicity. Notably, the biotrophic invasive hyphae produced in rice cells are more bulbous and compressed in the ΔMosec4 mutant. Further studies showed that deletion of the MoSEC4 gene resulted in decreased secretion of extracellular enzymes and mislocalization of the cytoplasmic effector PWL2-mCherry-NLS. In accordance with a role in secretion, the GFP-MoSec4 fusion protein mainly accumulates at tips of growing vegetative hyphae. Our results suggest that the MoSec4 protein plays important roles in the secretion of extracellular proteins and consequently hyphal development and pathogenicity in the rice blast fungus. PMID:27729922

  6. Memprot: a program to model the detergent corona around a membrane protein based on SEC-SAXS data.

    PubMed

    Pérez, Javier; Koutsioubas, Alexandros

    2015-01-01

    The application of small-angle X-ray scattering (SAXS) to structural investigations of transmembrane proteins in detergent solution has been hampered by two main inherent hurdles. On the one hand, the formation of a detergent corona around the hydrophobic region of the protein strongly modifies the scattering curve of the protein. On the other hand, free micelles of detergent without a precisely known concentration coexist with the protein-detergent complex in solution, therefore adding an uncontrolled signal. To gain robust structural information on such systems from SAXS data, in previous work, advantage was taken of the online combination of size-exclusion chromatography (SEC) and SAXS, and the detergent corona around aquaporin-0, a membrane protein of known structure, could be modelled. A precise geometrical model of the corona, shaped as an elliptical torus, was determined. Here, in order to better understand the correlations between the corona model parameters and to discuss the uniqueness of the model, this work was revisited by analyzing systematic SAXS simulations over a wide range of parameters of the torus.

  7. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins.

    PubMed

    Suryawanshi, Gajendra W; Hoffmann, Alexander

    2015-12-07

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target

  8. Infectious Bronchitis Coronavirus Limits Interferon Production by Inducing a Host Shutoff That Requires Accessory Protein 5b

    PubMed Central

    Kint, Joeri; Langereis, Martijn A.; Maier, Helena J.; Britton, Paul; van Kuppeveld, Frank J.; Koumans, Joseph; Wiegertjes, Geert F.

    2016-01-01

    ABSTRACT During infection of their host cells, viruses often inhibit the production of host proteins, a process that is referred to as host shutoff. By doing this, viruses limit the production of antiviral proteins and increase production capacity for viral proteins. Coronaviruses from the genera Alphacoronavirus and Betacoronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV), establish host shutoff via their nonstructural protein 1 (nsp1). The Gammacoronavirus and Deltacoronavirus genomes, however, do not encode nsp1, and it has been suggested that these viruses do not induce host shutoff. Here, we show that the Gammacoronavirus infectious bronchitis virus (IBV) does induce host shutoff, and we find that its accessory protein 5b is indispensable for this function. Importantly, we found that 5b-null viruses, unlike wild-type viruses, induce production of high concentrations of type I interferon protein in vitro, indicating that host shutoff by IBV plays an important role in antagonizing the host's innate immune response. Altogether, we demonstrate that 5b is a functional equivalent of nsp1, thereby answering the longstanding question of whether lack of nsp1 in gammacoronaviruses is compensated for by another viral protein. As such, our study is a significant step forward in the understanding of coronavirus biology and closes a gap in the understanding of some IBV virulence strategies. IMPORTANCE Many viruses inhibit protein synthesis by their host cell to enhance virus replication and to antagonize antiviral defense mechanisms. This process is referred to as host shutoff. We studied gene expression and protein synthesis in chicken cells infected with the important poultry pathogen infectious bronchitis virus (IBV). We show that IBV inhibits synthesis of host proteins, including that of type I interferon, a key component of the antiviral response. The IBV-induced host shutoff, however, does not require degradation of host RNA. Furthermore, we

  9. Electrophysiological Characteristics of the LQT2 Syndrome Mutation KCNH2-G572S and Regulation by Accessory Protein KCNE2

    PubMed Central

    Liu, Li; Tian, Jinwen; Lu, Caiyi; Chen, Xi; Fu, Yicheng; Xu, Bin; Zhu, Chao; Sun, Yanmei; Zhang, Yu; Zhao, Ying; Li, Yang

    2016-01-01

    Mutations in hERG cause long QT syndrome type 2 which is characterized by a prolonged QT interval on electrocardiogram and predisposition to life-threatening ventricular tachyarrhythmia, syncope, and sudden death. hERG-G572S induces trafficking defects of hERG channel protein from Golgi to the plasma membrane and results in a dominant negative suppression of hERG current density. As an accessory β subunit, KCNE2 promotes hERG migration from Golgi to cellular membrane. In this study, we investigated the rescue effect of KCNE2 in a G572S mutation of hERG. Transfection was performed into HEK293 cells. Patch clamp technique, western blotting analyses and confocal microscopic examination were used. Results showed that KCNE2 had a significantly enhanced effect on G572S mutation current. The increase of current was largest at KCNH2:KCNE2 of 1:3. Confocal images showed co-expressing G572S and KCNE2 could cause a substantial up-regulated membrane protein (155 kDa) expression. Expression of membrane protein accumulated markedly with increasing ratio of KCNH2:KCNE2. G572S defective mutant could be restored by both KCNE2 and lower temperature (27°C), which suggested that the lower temperature could be the favorable circumstances for the rescue function of KCNE2. In this study, we successfully set up “the action potential” on the HEK 293 cells by genetically engineered to express Kir2.1, Nav1.5, and Kv11.1, wherein on reaching over an excitation threshold by current injection. The results suggested that KCNE2 could shorten action potential duration which was prolonged by G572S. These findings described electrophysiological characteristics of the LQT2 syndrome mutation KCNH2-G572S and regulation by accessory protein KCNE2, and provided a clue about LQT2 and relative rescue mechanism. PMID:28082916

  10. Sec14-like Phosphatidylinositol Transfer Proteins and the Biological Landscape of Phosphoinositide Signaling in Plants

    PubMed Central

    Huang, Jin; Ghosh, Ratna; Bankaitis, Vytas A.

    2017-01-01

    Phosphoinositides and soluble inositol phosphates are essential components of a complex intracellular chemical code that regulates major aspects of lipid signaling in eukaryotes. These involvements span a broad array of biological outcomes and activities, and cells are faced with the problem of how to compartmentalize and organize these various signaling events into a coherent scheme. It is in the arena of how phosphoinositide signaling circuits are integrated and, and how phosphoinositide pools are functionally defined and channeled to privileged effectors, that phosphatidylinositol (PtdIns) transfer proteins (PITPs) are emerging as critical players. As plant systems offer some unique advantages and opportunities for study of these proteins, we discuss herein our perspectives regarding the progress made in plant systems regarding PITP function. We also suggest interesting prospects that plant systems hold for interrogating how PITPs work, particularly in multi-domain contexts, to diversify the biological outcomes for phosphoinositide signaling. PMID:27038688

  11. Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana.

    PubMed

    Zeng, Yonglun; Chung, Kin Pan; Li, Baiying; Lai, Ching Man; Lam, Sheung Kwan; Wang, Xiangfeng; Cui, Yong; Gao, Caiji; Luo, Ming; Wong, Kam-Bo; Schekman, Randy; Jiang, Liwen

    2015-11-17

    Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.

  12. Unique COPII component AtSar1a/AtSec23a pair is required for the distinct function of protein ER export in Arabidopsis thaliana

    PubMed Central

    Zeng, Yonglun; Chung, Kin Pan; Li, Baiying; Lai, Ching Man; Lam, Sheung Kwan; Wang, Xiangfeng; Cui, Yong; Gao, Caiji; Luo, Ming; Wong, Kam-Bo; Schekman, Randy; Jiang, Liwen

    2015-01-01

    Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes. PMID:26578783

  13. Mycobacterium tuberculosis Proteasome Accessory Factor A (PafA) Can Transfer Prokaryotic Ubiquitin-Like Protein (Pup) between Substrates

    PubMed Central

    Zhang, Susan; Burns-Huang, Kristin E.; Janssen, Guido V.; Li, Huilin; Ovaa, Huib; Hedstrom, Lizbeth

    2017-01-01

    ABSTRACT The protein degradation machinery of Mycobacterium tuberculosis includes a proteasome and a ubiquitin-like protein (Pup). Proteasome accessory factor A (PafA) attaches Pup to proteins to target them for degradation by the proteasome. Free Pup is unstable and never observed in extracts of M. tuberculosis, an observation that led us to hypothesize that PafA may need alternative sources of Pup. Here, we show that PafA can move Pup from one proteasome substrate, inositol 1-phosphate synthetase (Ino1), to two different proteins, malonyl coenzyme A (CoA)-acyl carrier protein transacylase (FabD) and lonely guy (Log). This apparent “transpupylation” reaction required a previously unrecognized depupylase activity in PafA, and, surprisingly, this depupylase activity was much more efficient than the activity of the dedicated depupylase Dop (deamidase of Pup). Thus, PafA can potentially use both newly synthesized Pup and recycled Pup to doom proteins for degradation. PMID:28223451

  14. Fast and faster: A designed variant of the B-domain of protein A folds in 3 μsec

    PubMed Central

    Arora, Pooja; Oas, Terrence G.; Myers, Jeffrey K.

    2004-01-01

    We have introduced the mutation glycine 29 to alanine, designed to increase the rate of protein folding, into the B-domain of protein A (BdpA). From NMR lineshape analysis, we find the G29A mutation increases the folding rate constant by threefold; the folding time is 3 μsec. Although wild-type BdpA folds extremely fast, simple-point mutations can still speed up the folding; thus, the folding rate is not evolutionarily maximized. The short folding time of G29A BdpA (the shortest time yet reported) makes it an attractive candidate for an all-atom molecular dynamics simulation that could potentially show a complete folding reaction starting from an extended chain. We also constructed a fluorescent variant of BdpA by mutating phenylalanine 13 to tryptophan, allowing fluorescence-based time-resolved temperature-jump measurements. Temperature jumps and NMR complement each other, and give a very complete picture of the folding kinetics. PMID:15044721

  15. Structure of a D-protein gene and amino-acid sequences of the highly repetitive D-proteins secreted by the accessory glands of the mealworm beetle.

    PubMed

    Paesen, G C; Feng, X; Happ, G M

    1996-04-16

    The D-group proteins form the major component of the proteinaceous secretion of the tubular accessory glands of the yellow mealworm beetle, Tenebrio molitor. In a previous paper, we reported the sequence of two D-protein cDNAs and their inferred translation products. Both proteins contain three highly repetitive domains (A, A' and B). In this paper, we present the cDNA-inferred sequences of 8 more D-proteins, none of which contains an A' domain. We also present the structure of a D-protein gene. Southern analysis suggests that genes coding for an A' domain are relatively rare. Genes with a total of 7 or 8 (A + B domain) repeats seem most common.

  16. Competitive binding of the SecA ATPase and ribosomes to the SecYEG translocon.

    PubMed

    Wu, Zht Cheng; de Keyzer, Jeanine; Kedrov, Alexej; Driessen, Arnold J M

    2012-03-09

    During co-translational membrane insertion of membrane proteins with large periplasmic domains, the bacterial SecYEG complex needs to interact both with the ribosome and the SecA ATPase. Although the binding sites for SecA and the ribosome overlap, it has been suggested that these ligands can interact simultaneously with SecYEG. We used surface plasmon resonance and fluorescence correlation spectroscopy to examine the interaction of SecA and ribosomes with the SecYEG complex present in membrane vesicles and the purified SecYEG complex present in a detergent-solubilized state or reconstituted into nanodiscs. Ribosome binding to the SecYEG complex is strongly stimulated when the ribosomes are charged with nascent chains of the monotopic membrane protein FtsQ. This binding is competed by an excess of SecA, indicating that binding of SecA and ribosomes to SecYEG is mutually exclusive.

  17. Elevated intracellular calcium triggers recruitment of the receptor cross-talk accessory protein calcyon to the plasma membrane.

    PubMed

    Ali, Mohammad Kutub; Bergson, Clare

    2003-12-19

    Calcyon is called a "cross-talk accessory protein" because the mechanism by which it enables the typically Gs-linked D1 dopamine receptor to stimulate intracellular calcium release depends on a priming step involving heterologous Gq-linked G-protein-coupled receptor activation. The details of how priming facilitates the D1R calcium response have yet to be precisely elucidated. The present work shows that calcyon is constitutively localized both in vesicular and plasma membrane compartments within HEK293 cells. In addition, surface biotinylation and luminescence assays revealed that priming stimulates a 2-fold increase in the levels of calcyon expressed on the cell surface and that subsequent D1R activation produces further accumulation of the protein in the plasma membrane. The effects of priming and D1R agonists were blocked by nocodazole implicating microtubules in the delivery of calcyon-containing vesicles to the cell surface. Accumulation of calcyon in the plasma membrane correlated well with increased intracellular calcium levels as thapsigargin mimicked, and 2-aminoethoxydiphenylborane abrogated, the effects of priming. KN-62, an inhibitor of calcium/calmodulin-dependent protein kinase II (CaMKII) also blocked the effects of priming and D1R agonists. Furthermore, expression of constitutively active forms of the kinase bypassed the requirement for priming indicating that CaMKII is a key effector in the Ca2+ and microtubule-dependent delivery of calcyon to the cell surface.

  18. Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes.

    PubMed

    Viaud, Julien; Zeghouf, Mahel; Barelli, Hélène; Zeeh, Jean-Christophe; Padilla, André; Guibert, Bernard; Chardin, Pierre; Royer, Catherine A; Cherfils, Jacqueline; Chavanieu, Alain

    2007-06-19

    Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.

  19. Role of the unfolded protein response pathway in regulation of INO1 and in the sec14 bypass mechanism in Saccharomyces cerevisiae.

    PubMed Central

    Chang, Hak J; Jones, Elizabeth W; Henry, Susan A

    2002-01-01

    INO1, encoding inositol 1-phosphate synthase, is the most highly regulated of a class of genes containing the repeated element, UAS(INO), in their promoters. Transcription of UAS(INO)-containing genes is modulated by the availability of exogenous inositol and by signals generated by alteration of phospholipid metabolism. The unfolded protein response (UPR) pathway also is involved in INO1 expression and the ire1Delta and hac1Delta mutants are inositol auxotrophs. We examined the role of the UPR in transmitting a signal generated in response to inositol deprivation and to alteration of phospholipid biosynthesis created in the sec14(ts) cki1Delta genetic background. We report that the UPR is required for sustained high-level INO1 expression in wild-type strains, but not for transient derepression in response to inositol deprivation. Moreover, the UPR is not required for expression or regulation of INO1 in response to the change in lipid metabolism that occurs in the sec14(ts) cki1Delta genetic background. Thus, the UPR signal transduction pathway is not involved directly in transcriptional regulation of INO1 and other UAS(INO)-containing genes. However, we discovered that inactivation of Sec14p leads to activation of the UPR, and that sec14 cki1 strains exhibit defective vacuolar morphology, suggesting that the mechanism by which the cki1Delta mutation suppresses the growth and secretory defect of sec14 does not fully restore wild-type morphology. Finally, synthetic lethality involving sec14 and UPR mutations suggests that the UPR plays an essential role in survival of sec14 cki1 strains. PMID:12242221

  20. A novel Sec14 phospholipid transfer protein from Nicotiana benthamiana is up-regulated in response to Ralstonia solanacearum infection, pathogen associated molecular patterns and effector molecules and involved in plant immunity.

    PubMed

    Kiba, Akinori; Nakano, Masahito; Vincent-Pope, Patrick; Takahashi, Hirotaka; Sawasaki, Tatsuya; Endo, Yaeta; Ohnishi, Kouhei; Yoshioka, Hirofumi; Hikichi, Yasufumi

    2012-07-01

    To elucidate the molecular mechanisms of plant immune responses, we isolated genes whose expression was regulated by inoculation with Ralstonia solanacearum. Here, we report the characterization of Nicotiana benthamiana belonging to the SEC14-gene superfamily designated as Nicotiana benthamiana SEC14 (NbSEC14). NbSEC14 rescued growth defects and impaired invertase secretion associated with the yeast sec14p temperature-sensitive mutant, while recombinant NbSec14 protein had phospholipids transfer activity. NbSEC14 expression was up-regulated in N. benthamiana leaves after inoculation with virulent or avirulent R. solanacearum. Expression of NbSEC14 was induced by treatment with chitin, flg22, and by Agrobacterium-mediated transient expression of INF1 elicitin, AvrA from R. solanacearum, and co-expression of the capsid protein from Tobacco mild green mosaic virus with its cognate resistance L1 protein. NbSEC14-silenced plants showed accelerated growth of both the virulent and avirulent R. solanacearum as well as acceleration of disease development. This study may provide useful information for the further analysis of the function of plant Sec14 protein homologs in the regulation of plant immune responses.

  1. Bivalent Motif-Ear Interactions Mediate the Association of the Accessory Protein Tepsin with the AP-4 Adaptor Complex.

    PubMed

    Mattera, Rafael; Guardia, Carlos M; Sidhu, Sachdev S; Bonifacino, Juan S

    2015-12-25

    The heterotetrameric (ϵ-β4-μ4-σ4) complex adaptor protein 4 (AP-4) is a component of a non-clathrin coat involved in protein sorting at the trans-Golgi network (TGN). Considerable interest in this complex has arisen from the recent discovery that mutations in each of its four subunits are the cause of a congenital intellectual disability and movement disorder in humans. Despite its physiological importance, the structure and function of this coat remain poorly understood. To investigate the assembly of the AP-4 coat, we dissected the determinants of interaction of AP-4 with its only known accessory protein, the ENTH/VHS-domain-containing protein tepsin. Using a variety of protein interaction assays, we found that tepsin comprises two phylogenetically conserved peptide motifs, [GS]LFXG[ML]X[LV] and S[AV]F[SA]FLN, within its C-terminal unstructured region, which interact with the C-terminal ear (or appendage) domains of the β4 and ϵ subunits of AP-4, respectively. Structure-based mutational analyses mapped the binding site for the [GS]LFXG[ML]X[LV] motif to a conserved, hydrophobic surface on the β4-ear platform fold. Both peptide-ear interactions are required for efficient association of tepsin with AP-4, and for recruitment of tepsin to the TGN. The bivalency of the interactions increases the avidity of tepsin for AP-4 and may enable cross-linking of multiple AP-4 heterotetramers, thus contributing to the assembly of the AP-4 coat. In addition to revealing critical aspects of this coat, our findings extend the paradigm of peptide-ear interactions, previously established for clathrin-AP-1/AP-2 coats, to a non-clathrin coat.

  2. Saturated palmitic acid induces myocardial inflammatory injuries through direct binding to TLR4 accessory protein MD2

    PubMed Central

    Wang, Yi; Qian, Yuanyuan; Fang, Qilu; Zhong, Peng; Li, Weixin; Wang, Lintao; Fu, Weitao; Zhang, Yali; Xu, Zheng; Li, Xiaokun; Liang, Guang

    2017-01-01

    Obesity increases the risk for a number of diseases including cardiovascular diseases and type 2 diabetes. Excess saturated fatty acids (SFAs) in obesity play a significant role in cardiovascular diseases by activating innate immunity responses. However, the mechanisms by which SFAs activate the innate immune system are not fully known. Here we report that palmitic acid (PA), the most abundant circulating SFA, induces myocardial inflammatory injury through the Toll-like receptor 4 (TLR4) accessory protein MD2 in mouse and cell culture experimental models. Md2 knockout mice are protected against PA- and high-fat diet-induced myocardial injury. Studies of cell surface binding, cell-free protein–protein interactions and molecular docking simulations indicate that PA directly binds to MD2, supporting a mechanism by which PA activates TLR4 and downstream inflammatory responses. We conclude that PA is a crucial contributor to obesity-associated myocardial injury, which is likely regulated via its direct binding to MD2. PMID:28045026

  3. Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex

    PubMed Central

    Weninger, Keith; Bowen, Mark E.; Choi, Ucheor B.; Chu, Steven; Brunger, Axel T.

    2010-01-01

    Summary Formation of a binary complex between syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) at the active zone is believed to precede assembly of the ternary SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex that is essential for neurotransmitter release. Despite its importance in models of synaptic neurotransmitter release, this binary complex has been difficult to characterize by bulk methods due to the prevalence of a 2:1 dead-end species. Here we used single molecule fluorescence resonance energy transfer (smFRET) to study the structure and dynamics of the 1:1 syntaxin/SNAP-25 binary complex. The binary complex is conformationally variable with FRET efficiency states often changing on the second timescale. One state corresponds to a parallel three-helix bundle configuration, while other states correspond to configurations with one of the SNAP-25 SNARE domains dissociated. All configurations of the binary complex are rapidly locked into the single three-helix bundle configuration by the addition of synaptobrevin. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex serves as a dynamic acceptor for synaptobrevin binding, and interactions with accessory proteins stabilize this acceptor. In a high protein density cellular environment the syntaxin/SNAP-25 complex is therefore expected to be in the configuration where it can rapidly interact with synaptobrevin so its formation is unlikely a limiting step for SNARE-mediated neurotransmitter release. PMID:18275821

  4. Protein and Peptide Composition of Male Accessory Glands of Apis mellifera Drones Investigated by Mass Spectrometry.

    PubMed

    Gorshkov, Vladimir; Blenau, Wolfgang; Koeniger, Gudrun; Römpp, Andreas; Vilcinskas, Andreas; Spengler, Bernhard

    2015-01-01

    In honeybees, reproductive females usually mate early in their life with more than 10 males in free flight, often within 10 minutes, and then store male gametes for up to five years. Because of the extreme polyandry and mating in free flight special adaptations in males are most likely. We present here the results of an investigation of the protein content of four types of male reproductive glands from the Western honeybee (Apis mellifera) drone, namely seminal vesicles (secretion in ejaculate), as well as bulbus, cornua and mucus glands (secretions for the mating plug). Using high resolution and accuracy mass spectrometry and a combination of database searching and de novo sequencing techniques it was possible to identify 50 different proteins in total, inside all mentioned glands, except in the mucus gland. Most of the proteins are unique for a specific gland type, only one of them (H9KEY1/ATP synthase subunit O) was found in three glands, and 7 proteins were found in two types of glands. The identified proteins represent a wide variety of biological functions and can be assigned to several physiological classes, such as protection, energy generation, maintaining optimal conditions, associated mainly with vesicula seminalis; signaling, cuticle proteins, icarpin and apolipoproteins located mainly in the bulbus and cornua glands; and some other classes. Most of the discovered proteins were not found earlier during investigation of semen, seminal fluid and tissue of reproductive glands of the bee drone. Moreover, we provide here the origin of each protein. Thus, the presented data might shed light on the role of each reproductive gland.

  5. A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration

    PubMed Central

    Al-Ashhab, Motee; Abu-Libdeh, Bassam; Gallon, Matthew; Sharma, Kanchan; Yaacov, Barak; Coulthard, Elizabeth; Caldwell, Maeve A.; Edvardson, Simon; Cullen, Peter J.; Elpeleg, Orly

    2016-01-01

    The composition of the neuronal cell surface dictates synaptic plasticity and thereby cognitive development. This remodeling of the synapses is governed by the endocytic network which internalize transmembrane proteins, then sort them back to the cell surface or carry them to the lysosome for degradation. The multi-protein retromer complex is central to this selection, capturing specific transmembrane proteins and remodeling the cell membrane to form isolated cargo-enriched transport carriers. We investigated a consanguineous family with four patients who presented in infancy with intractable myoclonic epilepsy and lack of psychomotor development. Using exome analysis, we identified a homozygous deleterious mutation in SNX27, which encodes sorting nexin 27, a retromer cargo adaptor. In western analysis of patient fibroblasts, the encoded mutant protein was expressed at an undetectable level when compared with a control sample. The patients’ presentation and clinical course recapitulate that reported for the SNX27 knock-out mouse. Since the cargo proteins for SNX27-mediated sorting include subunits of ionotropic glutamate receptors and endosome-to-cell surface synaptic insertion of AMPA receptors is severely perturbed in SNX27−/− neurons, it is proposed that at least part of the neurological aberrations observed in the patients is attributed to defective sorting of ionotropic glutamate receptors. SNX27 deficiency is now added to the growing list of neurodegenerative disorders associated with retromer dysfunction. PMID:25894286

  6. A Novel CNS-Restricted Isoform of the IL-1R Accessory Protein Modulates Neuronal Responses to IL-1

    PubMed Central

    Smith, Dirk E.; Lipsky, Brian P.; Russell, Chris; Ketchem, Randal R.; Kirchner, Jacqueline; Hensley, Kelly; Boissonneault, Vincent; Plante, Marie-Michèle; Rivest, Serge; Huang, Yangyang; Friedman, Wilma; Sims, John E.

    2014-01-01

    SUMMARY IL-1 has multiple functions in both the periphery and the central nervous system (CNS) and is regulated at many levels. We identified a novel isoform of the IL-1R Accessory Protein (termed AcPb) that is expressed exclusively in the CNS. AcPb interacted with IL-1 and the IL-1 receptor but was unable to mediate canonical IL-1 responses. AcPb expression, however, modulated neuronal gene expression in response to IL-1 treatment in vitro. Animals lacking AcPb demonstrated an intact peripheral IL-1 response and developed experimental autoimmune encephalomyelitis (EAE) similarly to wild type mice. AcPb-deficient mice were instead more vulnerable to local inflammatory challenge in the CNS and suffered enhanced neuronal degeneration as compared to AcP-deficient or wild type mice. These findings implicate AcPb as an additional component of the highly regulated IL-1 system and suggest it may play a role in modulating CNS responses to IL-1 and the interplay between inflammation and neuronal survival. PMID:19481478

  7. SEC23B — EDRN Public Portal

    Cancer.gov

    SEC23B is a member of the SEC23B/SEC24B family. SEC23B is a component of the COPII coat, that covers ER-derived vesicles involved in transport from the endoplasmic reticulum to the Golgi apparatus. By similarity, it has been shown that COPII acts in the cytoplasm to promote the transport of secretory, plasma membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex.

  8. Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18.

    PubMed

    Zick, Michael; Orr, Amy; Schwartz, Matthew L; Merz, Alexey J; Wickner, William T

    2015-05-05

    Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17(L291A,L292A) did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17(F21S,M22S), with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17(F21S,M22S), interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.

  9. Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

    PubMed Central

    Zick, Michael; Orr, Amy; Schwartz, Matthew L.; Merz, Alexey J.; Wickner, William T.

    2015-01-01

    Sec17 [soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17F21S,M22S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17F21S,M22S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion. PMID:25902545

  10. Male Accessory Gland Protein Reduces Egg Laying in a Simultaneous Hermaphrodite

    PubMed Central

    Koene, Joris M.; Sloot, Wiebe; Montagne-Wajer, Kora; Cummins, Scott F.; Degnan, Bernard M.; Smith, John S.; Nagle, Gregg T.; ter Maat, Andries

    2010-01-01

    Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipient's optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined. PMID:20404934

  11. Structure and intracellular targeting of the SARS-coronavirus Orf7a accessory protein.

    PubMed

    Nelson, Christopher A; Pekosz, Andrew; Lee, Chung A; Diamond, Michael S; Fremont, Daved H

    2005-01-01

    The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 A resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded beta sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.

  12. Manipulation of the infectious bronchitis coronavirus genome for vaccine development and analysis of the accessory proteins.

    PubMed

    Cavanagh, Dave; Casais, Rosa; Armesto, Maria; Hodgson, Teri; Izadkhasti, Sousan; Davies, Marc; Lin, Fengsheng; Tarpey, Ian; Britton, Paul

    2007-07-26

    Infectious bronchitis coronavirus (IBV) is the cause of the single most economically costly infectious disease of domestic fowl in the UK--and probably so in many countries that have a developed poultry industry. A major reason for its continued dominance is its existence as many serotypes, determined by the surface spike protein (S), cross-protection being poor. Although controlled to some degree by live and inactivated vaccines, a new generation of IB vaccines is called for. Reverse genetic or 'infectious clone' systems, which allow the manipulation of the IBV genome, are key to this development. New vaccines would ideally be: genetically stable (i.e. maintain a stable attenuated phenotype); administered in ovo; and be flexible with respect to the source of the spike protein gene. Rational attenuation of IBV requires the identification of genes that are simultaneously not essential for replication and whose absence would reduce pathogenicity. Being able to modify a 'core' vaccine strain to make it applicable to a prevailing serotype requires a procedure for doing so, and the demonstration that 'spike-swapping' is sufficient to induce good immunity. We have demonstrated that four small IBV proteins, encoded by genes 3 and 5, are not essential for replication; failure to produce these proteins had little detrimental affect on the titre of virus produced. Our current molecularly cloned IBV, strain Beaudette, is non-pathogenic, so we do not know what effect the absence of these proteins would have on pathogenicity. That said, plaque size and composition of various gene 3/5 recombinant IBVs in cell culture, and reduced output and ciliostasis in tracheal organ cultures, shows that they are less aggressive than the wild-type Beaudette. Consequently these genes remain targets for rational attenuation. We have recently obtained evidence that one or more of the 15 proteins encoded by gene 1 are also determinants of pathogenicity. Hence gene 1 is also a target for rational

  13. Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

    PubMed Central

    Romero, Diego; Vlamakis, Hera; Losick, Richard

    2014-01-01

    Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules. PMID:24488317

  14. Two-ligand priming mechanism for potentiated phosphoinositide synthesis is an evolutionarily conserved feature of Sec14-like phosphatidylinositol and phosphatidylcholine exchange proteins

    PubMed Central

    Huang, Jin; Ghosh, Ratna; Tripathi, Ashutosh; Lönnfors, Max; Somerharju, Pentti; Bankaitis, Vytas A.

    2016-01-01

    Lipid signaling, particularly phosphoinositide signaling, plays a key role in regulating the extreme polarized membrane growth that drives root hair development in plants. The Arabidopsis AtSFH1 gene encodes a two-domain protein with an amino-terminal Sec14-like phosphatidylinositol transfer protein (PITP) domain linked to a carboxy-terminal nodulin domain. AtSfh1 is critical for promoting the spatially highly organized phosphatidylinositol-4,5-bisphosphate signaling program required for establishment and maintenance of polarized root hair growth. Here we demonstrate that, like the yeast Sec14, the AtSfh1 PITP domain requires both its phosphatidylinositol (PtdIns)- and phosphatidylcholine (PtdCho)-binding properties to stimulate PtdIns-4-phosphate [PtdIns(4)P] synthesis. Moreover, we show that both phospholipid-binding activities are essential for AtSfh1 activity in supporting polarized root hair growth. Finally, we report genetic and biochemical evidence that the two-ligand mechanism for potentiation of PtdIns 4-OH kinase activity is a broadly conserved feature of plant Sec14-nodulin proteins, and that this strategy appeared only late in plant evolution. Taken together, the data indicate that the PtdIns/PtdCho-exchange mechanism for stimulated PtdIns(4)P synthesis either arose independently during evolution in yeast and in higher plants, or a suitable genetic module was introduced to higher plants from a fungal source and subsequently exploited by them. PMID:27193303

  15. Olfactory receptor accessory proteins play crucial roles in receptor function and gene choice

    PubMed Central

    Sharma, Ruchira; Ishimaru, Yoshiro; Davison, Ian; Ikegami, Kentaro; Chien, Ming-Shan; You, Helena; Chi, Quiyi; Kubota, Momoka; Yohda, Masafumi; Ehlers, Michael; Matsunami, Hiroaki

    2017-01-01

    Each of the olfactory sensory neurons (OSNs) chooses to express a single G protein-coupled olfactory receptor (OR) from a pool of hundreds. Here, we show the receptor transporting protein (RTP) family members play a dual role in both normal OR trafficking and determining OR gene choice probabilities. Rtp1 and Rtp2 double knockout mice (RTP1,2DKO) show OR trafficking defects and decreased OSN activation. Surprisingly, we discovered a small subset of the ORs are expressed in larger numbers of OSNs despite the presence of fewer total OSNs in RTP1,2DKO. Unlike typical ORs, some overrepresented ORs show robust cell surface expression in heterologous cells without the co-expression of RTPs. We present a model in which developing OSNs exhibit unstable OR expression until they choose to express an OR that exits the ER or undergo cell death. Our study sheds light on the new link between OR protein trafficking and OR transcriptional regulation. DOI: http://dx.doi.org/10.7554/eLife.21895.001 PMID:28262096

  16. Pharmacogenomics of beta-adrenergic receptors and their accessory signaling proteins in heart failure.

    PubMed

    Dorn, Gerald W; Liggett, Stephen B

    2008-12-01

    beta-Adrenergic receptors (betaAR) are widely expressed on cardiovascular cells. Pharmacological stimulation or blockade of betaAR signaling is the therapeutic mainstay in cardiogenic shock, hypertension, ischemia, arrhythmias, and heart failure. Interindividual variability in the response to betaAR agonists and antagonists has prompted examination of variability in the genes encoding betaAR signaling pathway members. Prominent among the genes that have been examined so far in heart failure are the beta(1)AR, beta(2)AR, and G-protein-coupled receptor kinase 5 (GRK5). Each has nonsynonymous polymorphisms that alter amino acid sequence and protein function and regulation in cell-based systems, genetically altered mouse models, or human hearts. Here, we review these phenotypes and results from published clinical studies, with a focus on heart failure pharmacogenomics. Thus far, very few studies have utilized analogous protocols or drugs, and discrepancies in the clinical studies are apparent. A compelling approach is the use of multiple methods to understand the molecular, cellular, and organ phenotypes of a variant and couple these with clinical studies designed to specifically address the relevance of those phenotypes in humans. Undoubtedly, additional loci will be identified, and together, will provide for genetically driven, individualized treatments for heart failure.

  17. Drug Repurposing: Tolfenamic Acid Inactivates PrbP, a Transcriptional Accessory Protein in Liberibacter asiaticus

    PubMed Central

    Gardner, Christopher L.; Pagliai, Fernando A.; Pan, Lei; Bojilova, Lora; Torino, Maria I.; Lorca, Graciela L.; Gonzalez, Claudio F.

    2016-01-01

    CLIBASIA_01510, PrbP, is a predicted RNA polymerase binding protein in Liberibacter asiaticus. PrbP was found to regulate expression of a small subset of ribosomal genes through interactions with the β-subunit of the RNA polymerase and a short, specific sequence on the promoter region. Molecular screening assays were performed to identify small molecules that interact with PrbP in vitro. Chemical hits were analyzed for therapeutic efficacy against L. asiaticus via an infected leaf assay, where the transcriptional activity of L. asiaticus was found to decrease significantly after exposure to tolfenamic acid. Similarly, tolfenamic acid was found to inhibit L. asiaticus infection in highly symptomatic citrus seedlings. Our results indicate that PrbP is an important transcriptional regulator for survival of L. asiaticus in planta, and the chemicals identified by molecular screening assays could be used as a therapeutic treatment for huanglongbing disease. PMID:27803694

  18. Accessory proteins assist exonuclease-deficient bacteriophage T4 DNA polymerase in replicating past an abasic site

    PubMed Central

    Blanca, Giuseppina; Delagoutte, Emmanuelle; Tanguy le gac, Nicolas; Johnson, Neil P.; Baldacci, Giuseppe; Villani, Giuseppe

    2006-01-01

    Replicative DNA polymerases, such as T4 polymerase, possess both elongation and 3′–5′ exonuclease proofreading catalytic activities. They arrest at the base preceding DNA damage on the coding DNA strand and specialized DNA polymerases have evolved to replicate across the lesion by a process known as TLS (translesion DNA synthesis). TLS is considered to take place in two steps that often require different enzymes, insertion of a nucleotide opposite the damaged template base followed by extension from the inserted nucleotide. We and others have observed that inactivation of the 3′–5′ exonuclease function of T4 polymerase enables TLS across a single site-specific abasic [AP (apurinic/apyrimidinic)] lesion. In the present study we report a role for auxiliary replicative factors in this reaction. When replication is performed with a large excess of DNA template over DNA polymerase in the absence of auxiliary factors, the exo− polymerase (T4 DNA polymerase deficient in the 3′–5′ exonuclease activity) inserts one nucleotide opposite the AP site but does not extend past the lesion. Addition of the clamp processivity factor and the clamp loader complex restores primer extension across an AP lesion on a circular AP-containing DNA substrate by the exo− polymerase, but has no effect on the wild-type enzyme. Hence T4 DNA polymerase exhibits a variety of responses to DNA damage. It can behave as a replicative polymerase or (in the absence of proofreading activity) as a specialized DNA polymerase and carry out TLS. As a specialized polymerase it can function either as an inserter or (with the help of accessory proteins) as an extender. The capacity to separate these distinct functions in a single DNA polymerase provides insight into the biochemical requirements for translesion DNA synthesis. PMID:17064253

  19. Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction.

    PubMed

    Wei, Dong; Li, Hui-Min; Tian, Chuan-Bei; Smagghe, Guy; Jia, Fu-Xian; Jiang, Hong-Bo; Dou, Wei; Wang, Jin-Jun

    2015-11-19

    In insects, the accessory gland proteins (Acps) secreted by male accessory glands (MAGs) account for the majority of seminal fluids proteins. Mixed with sperm, they are transferred to the female at mating and so impact reproduction. In this project, we identified 2,927 proteins in the MAG secretions of the oriental fruit fly Bactrocera dorsalis, an important agricultural pest worldwide, using LC-MS analysis, and all sequences containing open reading frames were analyzed using signalP. In total, 90 Acps were identified. About one third (26) of these 90 Acps had a specific functional description, while the other two thirds (64) had no functional description including dozens of new classes of proteins. Hence, several of these novel Acps were abundant in the MAG secretions, and we confirmed their MAG-specific expression by qPCR. Finally and interestingly, one of these novel proteins was functionally predicted as juvenile hormone-binding protein, suggesting the impact of Acps with reproductive events in the female. Our results will aid in the development of an experimental method to identify Acps in insects, and in turn this information with new Acps in B. dorsalis will pave the way of further exploration their function in reproduction and potential development as new insecticide targets.

  20. Proteome analysis of male accessory gland secretions in oriental fruit flies reveals juvenile hormone-binding protein, suggesting impact on female reproduction

    PubMed Central

    Wei, Dong; Li, Hui-Min; Tian, Chuan-Bei; Smagghe, Guy; Jia, Fu-Xian; Jiang, Hong-Bo; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    In insects, the accessory gland proteins (Acps) secreted by male accessory glands (MAGs) account for the majority of seminal fluids proteins. Mixed with sperm, they are transferred to the female at mating and so impact reproduction. In this project, we identified 2,927 proteins in the MAG secretions of the oriental fruit fly Bactrocera dorsalis, an important agricultural pest worldwide, using LC-MS analysis, and all sequences containing open reading frames were analyzed using signalP. In total, 90 Acps were identified. About one third (26) of these 90 Acps had a specific functional description, while the other two thirds (64) had no functional description including dozens of new classes of proteins. Hence, several of these novel Acps were abundant in the MAG secretions, and we confirmed their MAG-specific expression by qPCR. Finally and interestingly, one of these novel proteins was functionally predicted as juvenile hormone-binding protein, suggesting the impact of Acps with reproductive events in the female. Our results will aid in the development of an experimental method to identify Acps in insects, and in turn this information with new Acps in B. dorsalis will pave the way of further exploration their function in reproduction and potential development as new insecticide targets. PMID:26582577

  1. IL1 receptor accessory protein like, a protein involved in X-linked mental retardation, interacts with Neuronal Calcium Sensor-1 and regulates exocytosis.

    PubMed

    Bahi, Nadia; Friocourt, Gaelle; Carrié, Alain; Graham, Margaret E; Weiss, Jamie L; Chafey, Philippe; Fauchereau, Fabien; Burgoyne, Robert D; Chelly, Jamel

    2003-06-15

    Previously, human genetics-based approaches allowed us to show that mutations in the IL-1 receptor accessory protein-like gene (IL1RAPL) are responsible for a non-specific form of X-linked mental retardation. This gene encodes a predicted protein of 696 amino acids that belongs to a novel class of the IL-1/Toll receptor family. In addition to the extracellular portion consisting of three Ig-like domains and the intracellular TIR domain characteristic of the IL-1/Toll receptor family, IL1RAPL contains a specific 150 amino acid carboxy terminus that has no significant homology with any protein of known function. In order to begin to elucidate the function of this IL-1/Toll receptor-like protein, we have assessed the effect of recombinant IL1RAPL on the binding affinity of type I IL-1R for its ligands IL-1alpha and beta and searched for proteins interacting with the specific carboxy terminus domain of IL1RAPL. Our results show that IL1RAPL is not a protein receptor for IL-1. In addition we present here the identification of Neuronal Calcium Sensor-1 (NCS-1) as an IL1RAPL interactor. Remarkably, although NCS-1 and its non-mammalian homologue, frequenin, are members of a highly conserved EF-hand Ca(2+) binding protein family, our data show that IL1RAPL interacts only with NCS-1 through its specific C-terminal domain. The functional relevance of IL1RAPL activity was further supported by the inhibitory effect on exocytosis in PC12 cells overexpressing IL1RAPL. Taken together, our data suggest that IL1RAPL may regulate calcium-dependent exocytosis and provide insight into the understanding of physiopathological mechanisms underlying cognitive impairment resulting from IL1RAPL dysfunction.

  2. Differential expression of secretion machinery during bacterial growth: SecY and SecF decrease while SecA increases during transition from exponential phase to stationary phase.

    PubMed

    Yang, Chun-Kai; Lu, Chung-Dar; Tai, Phang C

    2013-12-01

    Transcription of many house-keeping genes, including secY and some other sec genes, decreases in the transition from the exponential phase to the stationary phase (feast to famine) in Bacillus subtilis. Unexpectedly and in contradiction to earlier reports, enhanced transcription was observed for another group of sec genes, including secA which codes for an essential ATPase for protein secretion. Consistent with the transcription data, the SecA protein of B. subtilis increases significantly in the stationary phase. Immunoblot analyses of Sec proteins during the transition in Escherichia coli also revealed the pronounced decreases of SecY and SecF and the increase of SecA, resulting in drastic increases of SecA/SecY and SecA/SecF ratios from exponential to stationary phases. The differential expression of Sec proteins in the stationary phase suggests the possibility of specific physiological functions.

  3. Structure-function analysis of small G proteins from Volvox and Chlamydomonas by complementation of Saccharomyces cerevisiae YPT/SEC mutations.

    PubMed

    Fabry, S; Steigerwald, R; Bernklau, C; Dietmaier, W; Schmitt, R

    1995-05-10

    cDNAs representing nine small G protein genes encoding Ypt proteins from the green algae Volvox carteri (YptV) and Chlamydomonas reinhardtii (YptC) were tested for their ability to complement mutations in the YPT1, SEC4, and YPT7 genes of Saccharomyces cerevisiae strains defective in different steps of intracellular vesicle transport. None of the heterologously expressed algal genes was able to complement mutations in SEC4 or YPT7, but three of them, yptV1, yptC1, and yptV2, restored a YPT1 null mutation. On the amino acid sequence level, and particularly with respect to known small G protein specificity domains, YptV1p and YptC1p are the closest algal analogs of yeast Ypt1p, with 70% overall identity and identical effector regions, but YptV2p is only 55% identical to Ypt1p, and its effector domain resembles that of Sec4p. To define more precisely the regions that supply Ypt1p function, six chimeras were constructed by reciprocal exchange of 68/72-, 122/123-, and 162/163-amino acid segments of the C-terminal regions between YptV1p (complementing) and YptV3p (non-complementing). Segments containing 68 amino acids of the hypervariable C-terminal, and 41 residues of the N-terminal region including the effector region, of YptV1p could be replaced by the corresponding parts of YptV3p without loss of function in yeast, but exchanges within the central core destroyed the ability to rescue the YPT1 mutation. Sequence analysis of ypt1-complementing and -noncomplementing Ypt types suggests that surface loop3 represents a novel specificity domain of small G proteins.

  4. IL1-receptor accessory protein-like 1 (IL1RAPL1), a protein involved in cognitive functions, regulates N-type Ca2+-channel and neurite elongation.

    PubMed

    Gambino, Frédéric; Pavlowsky, Alice; Béglé, Aurélie; Dupont, Jean-Luc; Bahi, Nadia; Courjaret, Raphael; Gardette, Robert; Hadjkacem, Hassen; Skala, Henriette; Poulain, Bernard; Chelly, Jamel; Vitale, Nicolas; Humeau, Yann

    2007-05-22

    Null mutations in the IL1-receptor accessory protein-like 1 gene (IL1RAPL1) are responsible for an inherited X-linked form of cognitive impairment. IL1RAPL1 protein physically interacts with neuronal calcium sensor-1 (NCS-1), but the functional impact of the IL1RAPL1/NCS-1 interaction remains unknown. Here, we demonstrate that stable expression of IL1RAPL1 in PC12 cells induces a specific silencing of N-type voltage-gated calcium channels (N-VGCC) activity that explains a secretion deficit observed in these IL1RAPL1 cells. Importantly, this modulation of VGCC activity is mediated by NCS-1. Indeed, a specific loss-of-function of N-VGCC was observed in PC12 cells overexpressing NCS-1, and a total recovery of N-VGCC activity was obtained by a down-regulation of NCS-1 in IL1RAPL1 cells. The functional relevance of the interaction between IL1RAPL1 and NCS-1 was also suggested by the reduction of neurite elongation observed in nerve growth factor (NGF)-treated IL1RAPL1 cells, a phenotype rescued by NCS-1 inactivation. Because both proteins are highly expressed in neurons, these results suggest that IL1RAPL1-related mental retardation could result from a disruption of N-VGCC and/or NCS-1-dependent synaptic and neuronal activities.

  5. Label-free Quantitative Proteomics Reveals a Role for the Mycobacterium tuberculosis SecA2 Pathway in Exporting Solute Binding Proteins and Mce Transporters to the Cell Wall.

    PubMed

    Feltcher, Meghan E; Gunawardena, Harsha P; Zulauf, Katelyn E; Malik, Seidu; Griffin, Jennifer E; Sassetti, Christopher M; Chen, Xian; Braunstein, Miriam

    2015-06-01

    Mycobacterium tuberculosis is an example of a bacterial pathogen with a specialized SecA2-dependent protein export system that contributes to its virulence. Our understanding of the mechanistic basis of SecA2-dependent export and the role(s) of the SecA2 pathway in M. tuberculosis pathogenesis has been hindered by our limited knowledge of the proteins exported by the pathway. Here, we set out to identify M. tuberculosis proteins that use the SecA2 pathway for their export from the bacterial cytoplasm to the cell wall. Using label-free quantitative proteomics involving spectral counting, we compared the cell wall and cytoplasmic proteomes of wild type M. tuberculosis to that of a ΔsecA2 mutant. This work revealed a role for the M. tuberculosis SecA2 pathway in the cell wall localization of solute binding proteins that work with ABC transporters to import solutes. Another discovery was a profound effect of SecA2 on the cell wall localization of the Mce1 and Mce4 lipid transporters, which contribute to M. tuberculosis virulence. In addition to the effects on solute binding proteins and Mce transporter export, our label-free quantitative analysis revealed an unexpected relationship between SecA2 and the hypoxia-induced DosR regulon, which is associated with M. tuberculosis latency. Nearly half of the transcriptionally controlled DosR regulon of cytoplasmic proteins were detected at higher levels in the ΔsecA2 mutant versus wild type M. tuberculosis. By increasing the list of M. tuberculosis proteins known to be affected by the SecA2 pathway, this study expands our appreciation of the types of proteins exported by this pathway and guides our understanding of the mechanism of SecA2-dependent protein export in mycobacteria. At the same time, the newly identified SecA2-dependent proteins are helpful for understanding the significance of this pathway to M. tuberculosis virulence and physiology.

  6. The need to accessorize: molecular roles of HTLV-1 p30 and HTLV-2 p28 accessory proteins in the viral life cycle.

    PubMed

    Anupam, Rajaneesh; Doueiri, Rami; Green, Patrick L

    2013-09-17

    Extensive studies of human T-cell leukemia virus (HTLV)-1 and HTLV-2 over the last three decades have provided detailed knowledge on viral transformation, host-viral interactions and pathogenesis. HTLV-1 is the etiological agent of adult T cell leukemia and multiple neurodegenerative and inflammatory diseases while HTLV-2 disease association remains elusive, with few infected individuals displaying neurodegenerative diseases similar to HTLV-1. The HTLV group of oncoretroviruses has a genome that encodes structural and enzymatic proteins Gag, Pro, and Env, regulatory proteins Tax and Rex, and several accessory proteins from the pX region. Of these proteins, HTLV-1 p30 and HTLV-2 p28 are encoded by the open reading frame II of the pX region. Like most other accessory proteins, p30 and p28 are dispensable for in vitro viral replication and transformation but are required for efficient viral replication and persistence in vivo. Both p30 and p28 regulate viral gene expression at the post-transcriptional level whereas p30 can also function at the transcriptional level. Recently, several reports have implicated p30 and p28 in multiple cellular processes, which provide novel insight into HTLV spread and survival and ultimately pathogenesis. In this review we summarize and compare what is known about p30 and p28, highlighting their roles in viral replication and viral pathogenesis.

  7. Melanocortin receptor accessory protein 2 (MRAP2) interplays with the zebrafish melanocortin 1 receptor (MC1R) but has no effect on its pharmacological profile.

    PubMed

    Cortés, Raúl; Agulleiro, Maria Josep; Navarro, Sandra; Guillot, Raúl; Sánchez, Elisa; Cerdá-Reverter, José Miguel

    2014-05-15

    The melanocortin system is probably one of the most complex hormonal systems since it integrates agonist, encoded in the proopiomelanocortin precursor, endogenous antagonist, agouti signaling protein and agouti-related protein, five different G-protein coupled receptors and two accessory proteins. These accessory proteins interact with melanocortin receptors to allow traffic to the plasma membrane or to regulate the pharmacological profile. The MC1R fill the extension locus, which is primarily responsible for the regulation of pigmentation. In zebrafish, both MC1R and MRAP2 system are expressed in the skin. We demonstrate that zebrafish MC1R physically, or closely, interacts with the MRAP2 system, although this interaction did not result in modification of the studied pharmacological profile. However, progressive fasting induced skin darkening but also an upregulation of the MRAP2 expression in the skin, suggesting an unknown role for MRAP2a that could involve receptor desensitization processes. We also demonstrate that crowding stress induces skin darkening and a downregulation of MC1R expression in the skin.

  8. Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia

    PubMed Central

    1988-01-01

    Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75

  9. The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

    PubMed

    Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

    2015-11-01

    Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry.

  10. The ns12.9 Accessory Protein of Human Coronavirus OC43 Is a Viroporin Involved in Virion Morphogenesis and Pathogenesis

    PubMed Central

    Zhang, Ronghua; Wang, Kai; Ping, Xianqiang; Yu, Wenjing

    2015-01-01

    ABSTRACT An accessory gene between the S and E gene loci is contained in all coronaviruses (CoVs), and its function has been studied in some coronaviruses. This gene locus in human coronavirus OC43 (HCoV-OC43) encodes the ns12.9 accessory protein; however, its function during viral infection remains unknown. Here, we engineered a recombinant mutant virus lacking the ns12.9 protein (HCoV-OC43-Δns12.9) to characterize the contributions of ns12.9 in HCoV-OC43 replication. The ns12.9 accessory protein is a transmembrane protein and forms ion channels in both Xenopus oocytes and yeast through homo-oligomerization, suggesting that ns12.9 is a newly recognized viroporin. HCoV-OC43-Δns12.9 presented at least 10-fold reduction of viral titer in vitro and in vivo. Intriguingly, exogenous ns12.9 and heterologous viroporins with ion channel activity could compensate for the production of HCoV-OC43-Δns12.9, indicating that the ion channel activity of ns12.9 plays a significant role in the production of infectious virions. Systematic dissection of single-cycle replication revealed that ns12.9 protein had no measurable effect on virus entry, subgenomic mRNA (sgmRNA) synthesis, and protein expression. Further characterization revealed that HCoV-OC43-Δns12.9 was less efficient in virion morphogenesis than recombinant wild-type virus (HCoV-OC43-WT). Moreover, reduced viral replication, inflammatory response, and virulence in HCoV-OC43-Δns12.9-infected mice were observed compared to the levels for HCoV-OC43-WT-infected mice. Taken together, our results demonstrated that the ns12.9 accessory protein functions as a viroporin and is involved in virion morphogenesis and the pathogenesis of HCoV-OC43 infection. IMPORTANCE HCoV-OC43 was isolated in the 1960s and is a major agent of the common cold. The functions of HCoV-OC43 structural proteins have been well studied, but few studies have focused on its accessory proteins. In the present study, we demonstrated that the ns12.9 protein

  11. IL-1 receptor accessory protein-like 1 associated with mental retardation and autism mediates synapse formation by trans-synaptic interaction with protein tyrosine phosphatase δ.

    PubMed

    Yoshida, Tomoyuki; Yasumura, Misato; Uemura, Takeshi; Lee, Sung-Jin; Ra, Moonjin; Taguchi, Ryo; Iwakura, Yoichiro; Mishina, Masayoshi

    2011-09-21

    Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

  12. Sec6, Sec8, and Sec15 are components of a multisubunit complex which localizes to small bud tips in Saccharomyces cerevisiae

    PubMed Central

    1995-01-01

    In the yeast Saccharomyces cerevisiae, the products of at least 14 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Two of these genes, SEC8 and SEC15, encode components of a 1-2-million D multi-subunit complex that is found in the cytoplasm and associated with the plasma membrane. In this study, oligonucleotide-directed mutagenesis is used to alter the COOH- terminal portion of Sec8 with a 6-histidine tag, a 9E10 c-myc epitope, or both, to allow the isolation of the Sec8/15 complex from yeast lysates either by immobilized metal affinity chromatography or by immunoprecipitation. Sec6 cofractionates with Sec8/15 by immobilized metal affinity chromatography, gel filtration chromatography, and by sucrose velocity centrifugation. Sec6 and Sec15 coimmunoprecipitate from lysates with c-myc-tagged Sec8. These data indicate that the Sec8/15 complex contains Sec6 as a stable component. Additional proteins associated with Sec6/8/15 were identified by immunoprecipitations from radiolabeled lysates. The entire Sec6/8/15 complex contains at least eight polypeptides which range in molecular mass from 70 to 144 kD. Yeast strains containing temperature sensitive mutations in the SEC genes were also transformed with the SEC8-c-myc-6- histidine construct and analyzed by immunoprecipitation. The composition of the Sec6/8/15 complex is disrupted specifically in the sec3-2, sec5-24, and sec10-2 strain backgrounds. The c-myc-Sec8 protein is localized by immunofluorescence to small bud tips indicating that the Sec6/8/15 complex may function at sites of exocytosis. PMID:7615633

  13. The interleukin 1 (IL-1) receptor accessory protein Toll/IL-1 receptor domain: analysis of putative interaction sites in vitro mutagenesis and molecular modeling.

    PubMed

    Radons, Jurgen; Dove, Stefan; Neumann, Detlef; Altmann, Reinhold; Botzki, Alexander; Martin, Michael U; Falk, Werner

    2003-12-05

    The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal alpha-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants delta528-534 and delta527-533, whereas the C-terminal beta-strand and/or alpha-helix is displaced in the nonresponsive mutant delta527-534.

  14. Synthesis, depletion and cell-type expression of a protein from the male accessory glands of the dengue vector mosquito Aedes aegypti

    PubMed Central

    Alfonso-Parra, Catalina; Avila, Frank W.; Deewatthanawong, Prasit; Sirot, Laura K.; Wolfner, Mariana F.; Harrington, Laura C.

    2014-01-01

    Aedes aegypti males transfer sperm and seminal fluid proteins (Sfps), primarily produced by male accessory glands (AGs), to females during mating. When collectively injected or transplanted into females, AG tissues and/or seminal fluid homogenates have profound effects on Aedes female physiology and behavior. To identify targets and design new strategies for vector control, it is important to understand the biology of the AGs. Thus, we examined characteristics of AG secretion and development in Ae. aegypti, using the AG-specific seminal fluid protein, AAEL010824, as a marker. We showed that AAEL010824 is first detectable by 12h post-eclosion, and increases in amount over the first 3 days of adult life. We then showed that the amount of AAEL0010824 in the AG decreases after mating, with each successive mating depleting it further; by 5 successive matings with no time for recovery, its levels are very low. AAEL010824 levels in a depleted male are replenished by 48hr post-mating. In addition to examining the level of AAEL010824 protein, we also characterized the expression of its gene. We did this by making a transgenic mosquito line that carries an Enhanced Green Fluorescence Protein (EGFP) fused to the AAEL0010824 promoter that we defined here. We showed that AAEL010824 is expressed in the anterior cells of the accessory glands, and that its RNA levels also respond to mating. In addition to further characterizing AAEL010824 expression, our results with the EGFP fusion provide a promoter for driving AG expression. By providing this information on the biology of an important male reproductive tissue and the production of one of its seminal proteins, our results lay the foundation for future work aimed at identifying novel targets for mosquito population control. PMID:25107876

  15. Synthesis, depletion and cell-type expression of a protein from the male accessory glands of the dengue vector mosquito Aedes aegypti.

    PubMed

    Alfonso-Parra, Catalina; Avila, Frank W; Deewatthanawong, Prasit; Sirot, Laura K; Wolfner, Mariana F; Harrington, Laura C

    2014-11-01

    Aedes aegypti males transfer sperm and seminal fluid proteins (Sfps), primarily produced by male accessory glands (AGs), to females during mating. When collectively injected or transplanted into females, AG tissues and/or seminal fluid homogenates have profound effects on Aedes female physiology and behavior. To identify targets and design new strategies for vector control, it is important to understand the biology of the AGs. Thus, we examined characteristics of AG secretion and development in A. aegypti, using the AG-specific seminal fluid protein, AAEL010824, as a marker. We showed that AAEL010824 is first detectable by 12h post-eclosion, and increases in amount over the first 3 days of adult life. We then showed that the amount of AAEL0010824 in the AG decreases after mating, with each successive mating depleting it further; by 5 successive matings with no time for recovery, its levels are very low. AAEL010824 levels in a depleted male are replenished by 48 h post-mating. In addition to examining the level of AAEL010824 protein, we also characterized the expression of its gene. We did this by making a transgenic mosquito line that carries an Enhanced Green Fluorescence Protein (EGFP) fused to the AAEL0010824 promoter that we defined here. We showed that AAEL010824 is expressed in the anterior cells of the accessory glands, and that its RNA levels also respond to mating. In addition to further characterizing AAEL010824 expression, our results with the EGFP fusion provide a promoter for driving AG expression. By providing this information on the biology of an important male reproductive tissue and the production of one of its seminal proteins, our results lay the foundation for future work aimed at identifying novel targets for mosquito population control.

  16. Adrenocorticotropic Hormone (ACTH) Responses Require Actions of the Melanocortin-2 Receptor Accessory Protein on the Extracellular Surface of the Plasma Membrane.

    PubMed

    Malik, Sundeep; Dolan, Terrance M; Maben, Zachary J; Hinkle, Patricia M

    2015-11-13

    The melanocortin-2 (MC2) receptor is a G protein-coupled receptor that mediates responses to ACTH. The MC2 receptor acts in concert with the MC2 receptor accessory protein (MRAP) that is absolutely required for ACTH binding and signaling. MRAP has a single transmembrane domain and forms a highly unusual antiparallel homodimer that is stably associated with MC2 receptors at the plasma membrane. Despite the physiological importance of the interaction between the MC2 receptor and MRAP, there is little understanding of how the accessory protein works. The dual topology of MRAP has made it impossible to determine whether highly conserved and necessary regions of MRAP are required on the intracellular or extracellular face of the plasma membrane. The strategy used here was to fix the orientation of two antiparallel MRAP molecules and then introduce inactivating mutations on one side of the membrane or the other. This was achieved by engineering proteins containing tandem copies of MRAP fused to the amino terminus of the MC2 receptor. The data firmly establish that only the extracellular amino terminus (Nout) copy of MRAP, oriented with critical segments on the extracellular side of the membrane, is essential. The transmembrane domain of MRAP is also required in only the Nout orientation. Finally, activity of MRAP-MRAP-MC2-receptor fusion proteins with inactivating mutations in either MRAP or the receptor was rescued by co-expression of free wild-type MRAP or free wild-type receptor. These results show that the basic MRAP-MRAP-receptor signaling unit forms higher order complexes and that these multimers signal.

  17. Crystallization and preliminary X-ray diffraction analysis of phospholipid-bound Sfh1p, a member of the Saccharomyces cerevisiae Sec14p-like phosphatidylinositol transfer protein family

    SciTech Connect

    Schaaf, Gabriel; Betts, Laurie; Garrett, Teresa A.; Raetz, Christian R. H.; Bankaitis, Vytas A.

    2006-11-01

    Yeast Sfh1p, a close homolog of the Sec14p phosphatidylinositol transfer protein, was crystallized in the absence of detergent. X-ray data have been collected to 2.5 Å. Sec14p is the major phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein in the budding yeast Saccharomyces cerevisiae and is the founding member of a large eukaryotic protein superfamily. This protein catalyzes the exchange of either PtdIns or PtdCho between membrane bilayers in vitro and this exchange reaction requires no external input of energy or of other protein cofactors. Despite the previous elucidation of the crystal structure of a detergent-bound form of Sec14p, the conformational changes that accompany the phospholipid-exchange reaction remain undefined. Moreover, a structural appreciation of how Sec14p or its homologs bind their various phospholipid substrates remains elusive. Here, the purification and crystallization of yeast Sfh1p, the protein most closely related to Sec14p, are reported. A combination of electrospray ionization mass-spectrometry and collision-induced decomposition mass-spectrometry methods indicate that recombinant Sfh1p loads predominantly with phosphatidylethanolamine. Unlike phospholipid-bound forms of Sec14p, this form of Sfh1p crystallizes readily in the absence of detergent. Sfh1p crystals diffract to 2.5 Å and belong to the orthorhombic primitive space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 49.40, b = 71.55, c = 98.21 Å, α = β = γ = 90°. One Sfh1p molecule is present in the asymmetric unit (V{sub M} = 2.5 Å{sup 3} Da{sup −1}; V{sub s} = 50%). Crystallization of a phospholipid-bound Sec14p-like protein is a critical first step in obtaining the first high-resolution picture of how proteins of the Sec14p superfamily bind their phospholipid ligands. This information will significantly extend our current understanding of how Sec14p-like proteins catalyze phospholipid exchange.

  18. ORF7-encoded accessory protein 7a of feline infectious peritonitis virus as a counteragent against IFN-α-induced antiviral response.

    PubMed

    Dedeurwaerder, Annelike; Olyslaegers, Dominique A J; Desmarets, Lowiese M B; Roukaerts, Inge D M; Theuns, Sebastiaan; Nauwynck, Hans J

    2014-02-01

    The type I IFN-mediated immune response is the first line of antiviral defence. Coronaviruses, like many other viruses, have evolved mechanisms to evade this innate response, ensuring their survival. Several coronavirus accessory genes play a central role in these pathways, but for feline coronaviruses this has never to our knowledge been studied. As it has been demonstrated previously that ORF7 is essential for efficient replication in vitro and virulence in vivo of feline infectious peritonitis virus (FIPV), the role of this ORF in the evasion of the IFN-α antiviral response was investigated. Deletion of ORF7 from FIPV strain 79-1146 (FIPV-Δ7) rendered the virus more susceptible to IFN-α treatment. Given that ORF7 encodes two proteins, 7a and 7b, it was further explored which of these proteins is active in this mechanism. Providing 7a protein in trans rescued the mutant FIPV-Δ7 from IFN sensitivity, which was not achieved by addition of 7b protein. Nevertheless, addition of protein 7a to FIPV-Δ3Δ7, a FIPV mutant deleted in both ORF3 and ORF7, could no longer increase the replication capacity of this mutant in the presence of IFN. These results indicate that FIPV 7a protein is a type I IFN antagonist and protects the virus from the antiviral state induced by IFN, but it needs the presence of ORF3-encoded proteins to exert its antagonistic function.

  19. IQ Motif and SEC7 Domain-containing Protein 3 (IQSEC3) Interacts with Gephyrin to Promote Inhibitory Synapse Formation.

    PubMed

    Um, Ji Won; Choii, Gayoung; Park, Dongseok; Kim, Dongwook; Jeon, Sangmin; Kang, Hyeyeon; Mori, Takuma; Papadopoulos, Theofilos; Yoo, Taesun; Lee, Yeunkum; Kim, Eunjoon; Tabuchi, Katsuhiko; Ko, Jaewon

    2016-05-06

    Gephyrin is a central scaffold protein that mediates development, function, and plasticity of mammalian inhibitory synapses by interacting with various inhibitory synaptic proteins. Here, we show that IQSEC3, a guanine nucleotide exchange factor for ARF6, directly interacts with gephyrin, an interaction that is critical for the inhibitory synapse localization of IQSEC3. Overexpression of IQSEC3 increases inhibitory, but not excitatory, synapse density in a guanine nucleotide exchange factor activity-dependent manner. Conversely, knockdown of IQSEC3 decreases size of gephyrin cluster without altering gephyrin puncta density. Collectively, these data reveal that IQSEC3 acts together with gephyrin to regulate inhibitory synapse development.

  20. Isolation and characterization of cDNA encoding the antigenic protein of the human tRNP(Ser)Sec complex recognized by autoantibodies from patients with type-1 autoimmune hepatitis

    PubMed Central

    Costa, M; Rodríguez-Sánchez, J L; Czaja, A J; Gelpí, C

    2000-01-01

    We previously described autoantibodies against a UGA serine tRNA–protein complex (tRNP(Ser)Sec) in patients with type-1 autoimmune hepatitis [1] and now define the specificity and frequency of this autoantibody and the DNA sequence encoding the tRNA(Ser)Sec-associated antigenic protein. The presence of anti‐tRNP(Ser)Sec antibodies was highly specific for type-1 autoimmune hepatitis, as 47·5% of patients were positive compared with none of the control subjects. To characterize the antigenic protein(s), we immunoscreened a human cDNA library with anti-tRNP(Ser)Sec-positive sera. Two clones (19 and 13) were isolated. Clone 19 encodes a protein with a predicted molecular mass of 48·8 kD. Clone 13 is a shorter cDNA, almost identical to clone 19, which encodes a 35·9-kD protein. Expression of both cDNAs was accomplished in Escherichia coli as His-tagged recombinant proteins. Antibodies eluted from both purified recombinant proteins were able to immunoprecipitate the tRNA(Ser)Sec from a HeLa S3 cell extract, demonstrating their cross-reactivity with the mammalian antigenic complex. Recent cloning data relating to the target antigen(s) of autoantibodies in autoimmune hepatitis patients that react with a soluble liver antigen (SLA) and a liver-pancreas antigen (LP) have revealed that these two autoantibodies are identical and that the cloned antigen shows 99% amino acid sequence homology with tRNP(Ser)Sec. PMID:10931155

  1. Loss of the Sec1/Munc18-family proteins VPS-33.2 and VPS-33.1 bypasses a block in endosome maturation in Caenorhabditis elegans

    PubMed Central

    Solinger, Jachen A.; Spang, Anne

    2014-01-01

    The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, whereas in HOPS knockdowns, RAB-5 was found also on membrane structures close to the cell center, indicating the RAB conversion might be impaired in the absence of these tethering complexes. Unlike in yeast, metazoans have two VPS33 homologues, which are Sec1/Munc18 (SM)-family proteins involved in the regulation of membrane fusion. We assume that in wild type, each tethering complex contains a specific SM protein but that they may be able to substitute for each other in case of absence of the other. Of importance, knockdown of both SM proteins allowed bypass of the endosome maturation block in sand-1 mutants. We propose a model in which the SM proteins in tethering complexes are required for coordinated flux of material through the endosomal system. PMID:25273556

  2. The ORF4b-encoded accessory proteins of Middle East respiratory syndrome coronavirus and two related bat coronaviruses localize to the nucleus and inhibit innate immune signalling.

    PubMed

    Matthews, Krystal L; Coleman, Christopher M; van der Meer, Yvonne; Snijder, Eric J; Frieman, Matthew B

    2014-04-01

    The recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV), a betacoronavirus, is associated with severe pneumonia and renal failure. The environmental origin of MERS-CoV is as yet unknown; however, its genome sequence is closely related to those of two bat coronaviruses, named BtCoV-HKU4 and BtCoV-HKU5, which were derived from Chinese bat samples. A hallmark of highly pathogenic respiratory viruses is their ability to evade the innate immune response of the host. CoV accessory proteins, for example those from severe acute respiratory syndrome CoV (SARS-CoV), have been shown to block innate antiviral signalling pathways. MERS-CoV, similar to SARS-CoV, has been shown to inhibit type I IFN induction in a variety of cell types in vitro. We therefore hypothesized that MERS-CoV and the phylogenetically related BtCoV-HKU4 and BtCoV-HKU5 may encode proteins with similar capabilities. In this study, we have demonstrated that the ORF4b-encoded accessory protein (p4b) of MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 may indeed facilitate innate immune evasion by inhibiting the type I IFN and NF-κB signalling pathways. We also analysed the subcellular localization of p4b from MERS-CoV, BtCoV-HKU4 and BtCoV-HKU5 and demonstrated that all are localized to the nucleus.

  3. Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland.

    PubMed

    Radha, Arumugam; Sree, Sreesha; Faisal, Kunnathodi; Kumar, G Pradeep; Oommen, Oommen V; Akbarsha, Mohammad A

    2014-10-01

    Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.

  4. Multiscale Dynamics in Soft-Matter Systems: Enzyme Catalysis, Sec-Facilitated Protein Translocation, and Ion-Conduction in Polymers

    NASA Astrophysics Data System (ADS)

    Miller, Thomas

    Nature exhibits dynamics that span extraordinary ranges of space and time. In some cases, these dynamical hierarchies are well separated, simplifying their understanding and description. But chemistry and biology are replete with examples of dynamically coupled scales. In this talk, we will discuss the use of high-performance computing and new simulation methods that enable the inclusion of nuclear quantum effects, such as zero point energy and tunneling, in the reaction dynamics of enzymes, as well as coarse-graining strategies to enable minute-timescale simulations of protein targeting to cell membranes and ion-conduction in polymer electrolytes for lithium-ion battery applications.

  5. Accessory mental foramen

    PubMed Central

    Balcioglu, Huseyin Avni; Kocaelli, Humeyra

    2009-01-01

    Context: Accessory mental foramen is a rare anatomical variation. Even so, in order to avoid neurovascular complications, particular attention should be paid to the possible occurrence of one or more accessory mental foramen during surgical procedures involving the mandible. Case report: A 3-dimensional computed tomography (3D-CT) scan of a female patient revealed an accessory mental foramen on the right side of her mandible. Conclusion: A 3D-CT scan should be obtained prior to mandibular surgeries so that the presence of accessory mental foramen can be detected, and so that the occurrence of a neurosensory disturbance or hemorrhage can be avoided. Although this anatomical variation is rare, it should be kept in mind that an accessory mental foramen may exist. PMID:22666714

  6. Studying the distribution pattern of selenium in nut proteins with information obtained from SEC-UV-ICP-MS and CE-ICP-MS.

    PubMed

    Kannamkumarath, Sasi S; Wrobel, Katarzyna; Wuilloud, Rodolfo G

    2005-03-31

    In this work, size exclusion chromatography (SEC) with UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used to study the association of selenium to proteins present in Brazil nuts (Bertholletia excelsa) under five different extraction conditions. As expected, better solubilization of proteins was observed using 0.05molL(-1) sodium hydroxide and 1% sodium dodecylsulfate (SDS) in Tris/HCl buffer (0.05molL(-1), pH 8) as compared to 0.05molL(-1) HCl, 0.05molL(-1) Tris/HCl or hot water (60 degrees C). Due to non-destructive character of Tris-SDS treatment, this was applied for studying molecular weight (MW) distribution patterns of selenium-containing nut proteins. Three different SEC columns were used for obtaining complete MW distribution of selenium: Superdex 75, Superdex Peptide, and Superdex 200 were tested with 50mmolL(-1) Tris buffer (pH 8), 150mmolL(-1) ammonium bicarbonate buffer (pH 7.8), phosphate (pH 7.5), and CAPS (pH 10.0) mobile phases. Using Superdex 200 column, the elution of at least three MW fractions was observed with UV detection (200-10kDa) and ICP-MS chromatogram showed the co-elution of selenium with the two earlier fractions. The apparent MWs of these selenium-containing fractions were respectively about 107 and 50kDa, as evaluated from the column calibration. For further characterization of individual selenium species, the defatted nuts were hydrolyzed with proteinase K and analyzed by capillary electrophoresis (CE) with ICP-MS detection. The suitability of CE for the separation of selenite, selenate, selenocystine and selenomethionine in the presence of the nut sample matrix is demonstrated. Complete separation of the above mentioned selenium species was obtained within a migration time of 7min. In the analysis of nut extracts with CE-ICP-MS, selenium was found to be present mainly as selenomethionine.

  7. Sorting of GLUT4 into its insulin-sensitive store requires the Sec1/Munc18 protein mVps45.

    PubMed

    Roccisana, Jennifer; Sadler, Jessica B A; Bryant, Nia J; Gould, Gwyn W

    2013-08-01

    Insulin stimulates glucose transport in fat and muscle cells by regulating delivery of the facilitative glucose transporter, glucose transporter isoform 4 (GLUT4), to the plasma membrane. In the absence of insulin, GLUT4 is sequestered away from the general recycling endosomal pathway into specialized vesicles, referred to as GLUT4-storage vesicles. Understanding the sorting of GLUT4 into this store is a major challenge. Here we examine the role of the Sec1/Munc18 protein mVps45 in GLUT4 trafficking. We show that mVps45 is up-regulated upon differentiation of 3T3-L1 fibroblasts into adipocytes and is expressed at stoichiometric levels with its cognate target-soluble N-ethylmaleimide-sensitive factor attachment protein receptor, syntaxin 16. Depletion of mVps45 in 3T3-L1 adipocytes results in decreased GLUT4 levels and impaired insulin-stimulated glucose transport. Using sub-cellular fractionation and an in vitro assay for GLUT4-storage vesicle formation, we show that mVps45 is required to correctly traffic GLUT4 into this compartment. Collectively our data reveal a crucial role for mVps45 in the delivery of GLUT4 into its specialized, insulin-regulated compartment.

  8. Analysis of a Soluble (UreD:UreF:UreG)2 Accessory Protein Complex and Its Interactions with Klebsiella aerogenes Urease by Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Farrugia, Mark A.; Han, Linjie; Zhong, Yueyang; Boer, Jodi L.; Ruotolo, Brandon T.; Hausinger, Robert P.

    2013-09-01

    Maturation of the nickel-containing urease of Klebsiella aerogenes is facilitated by the UreD, UreF, and UreG accessory proteins along with the UreE metallo-chaperone. A fusion of the maltose binding protein and UreD (MBP-UreD) was co-isolated with UreF and UreG in a soluble complex possessing a (MBP-UreD:UreF:UreG)2 quaternary structure. Within this complex a UreF:UreF interaction was identified by chemical cross-linking of the amino termini of its two UreF protomers, as shown by mass spectrometry of tryptic peptides. A pre-activation complex was formed by the interaction of (MBP-UreD:UreF:UreG)2 and urease. Mass spectrometry of intact protein species revealed a pathway for synthesis of the urease pre-activation complex in which individual hetero-trimer units of the (MBP-UreD:UreF:UreG)2 complex bind to urease. Together, these data provide important new insights into the structures of protein complexes associated with urease activation.

  9. Transcriptome analysis to identify genes for peptides and proteins involved in immunity and reproduction from male accessory glands and ejaculatory duct of Bactrocera dorsalis.

    PubMed

    Wei, Dong; Tian, Chuan-Bei; Liu, Shi-Huo; Wang, Tao; Smagghe, Guy; Jia, Fu-Xian; Dou, Wei; Wang, Jin-Jun

    2016-06-01

    In the male reproductive system of insects, the male accessory glands and ejaculatory duct (MAG/ED) are important organs and their primary function is to enhance the fertility of spermatozoa. Proteins secreted by the MAG/ED are also known to induce post-mating changes and immunity responses in the female insect. To understand the gene expression profile in the MAG/ED of the oriental fruit fly Bactrocera dorsalis (Hendel), that is an important pest in fruits, we performed an Illumina-based deep sequencing of mRNA. This yielded 54,577,630 clean reads corresponding to 4.91Gb total nucleotides that were assembled and clustered to 30,669 unigenes (average 645bp). Among them, 20,419 unigenes were functionally annotated to known proteins/peptides in Gene Orthology, Clusters of Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes pathway databases. Typically, many genes were involved in immunity and these included microbial recognition proteins and antimicrobial peptides. Subsequently, the inducible expression of these immunity-related genes was confirmed by qRT-PCR analysis when insects were challenged with immunity-inducible factors, suggesting their function in guaranteeing fertilization success. Besides, we identified some important reproductive genes such as juvenile hormone- and ecdysteroid-related genes in this de novo assembly. In conclusion, this transcriptomic sequencing of B. dorsalis MAG/ED provides insights to facilitate further functional research of reproduction, immunity and molecular evolution of reproductive proteins in this important agricultural pest.

  10. Dynamic Organization of SecA and SecY Secretion Complexes in the B. subtilis Membrane

    PubMed Central

    Dajkovic, Alex; Hinde, Elizabeth; MacKichan, Calum; Carballido-Lopez, Rut

    2016-01-01

    In prokaryotes, about one third of cellular proteins are translocated across the plasma membrane or inserted into it by concerted action of the cytoplasmic ATPase SecA and the universally conserved SecYEG heterotrimeric polypeptide-translocating pore. Secretion complexes have been reported to localize in specific subcellular sites in Bacillus subtilis. In this work, we used a combination of total internal reflection microscopy, scanning fluorescence correlation spectroscopy, and pair correlation function to study the localization and dynamics of SecA and SecY in growing Bacillus subtilis cells. Both SecA and SecY localized in transient and dynamic foci in the cytoplasmic membrane, which displayed no higher-level organization in helices. Foci of SecA and SecY were in constant flux with freely diffusing SecA and SecY molecules. Scanning FCS confirmed the existence of populations of cellular SecA and SecY molecules with a wide range of diffusion coefficients. Diffusion of SecY as an uncomplexed molecular species was short-lived and only local while SecY complexed with its protein partners traversed distances of over half a micrometer in the cell. PMID:27336478

  11. Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

    PubMed Central

    Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

    2015-01-01

    The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits Pc

  12. Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation

    PubMed Central

    Jadhav, Bhalchandra; McKenna, Michael; Johnson, Nicholas; High, Stephen; Sinning, Irmgard; Pool, Martin R.

    2015-01-01

    Two distinct pathways deliver secretory proteins to the Sec61 protein translocase in the endoplasmic reticulum membrane. The canonical pathway requires the signal recognition particle (SRP) and its cognate receptor (SR), and targets ribosome-associated proteins to the Sec translocase. The SRP-independent pathway requires the Sec translocase-associated ER membrane protein Sec62 and can be uncoupled from translation. Here we show that SR switches translocons to SRP-dependent translocation by displacing Sec62. This activity localizes to the charged linker region between the longin and GTPase domains of SRα. Using truncation variants, crosslinking and translocation assays reveals two elements with distinct functions as follows: one rearranges the translocon, displacing Sec62 from Sec61. A second promotes ribosome binding and is conserved between all eukaryotes. These specific regions in SRα reprogramme the Sec translocon and facilitate recruitment of ribosome-nascent chain complexes. Overall, our study identifies an important function of SR, which mechanistically links two seemingly independent modes of translocation. PMID:26634806

  13. Accessory Breast Carcinoma

    PubMed Central

    Youn, Hyun Jo; Jung, Sung Hoo

    2009-01-01

    Summary Background Ectopic breast tissue usually develops along the mammary ridges, and the incidence has been reported to be 2–6% of the general population. Occurrence of primary carcinoma in ectopic breast tissue is rare. Case Report We report the case of 59-year-old woman with accessory breast carcinoma in her left axilla. Conclusion Because an accessory areola or nipple is often missing and awareness of physicians and patients about these unsuspicious masses is lacking, clinical diagnosis of accessory breast carcinoma is frequently delayed. Therefore, a mass along the ‘milk line’ should be examined carefully, and any suspicious lesions should be evaluated. PMID:20847887

  14. Interleukin (IL)-1 in rat parturition: IL-1 receptors 1 and 2 and accessory proteins abundance in pregnant rat uterus at term - regulation by progesterone.

    PubMed

    Ishiguro, Tomohito; Takeda, Jun; Fang, Xin; Bronson, Heather; Olson, David M

    2016-07-01

    The role of interleukin-1 (IL-1), a pro-inflammatory cytokine, in parturition is typically noted by changes in its concentrations. Studying the expression of its receptor family, IL-1 receptor (IL-1R) 1, IL-1R2, IL-1R accessory protein (IL-1RAcP), and its predominantly brain isoform, IL-1RAcPb, during late gestation in the uterus in the Long-Evans rat is another. We assessed changes in their mRNA and protein relative abundance in the uterus and compared IL-1RAcP and IL-1RAcPb mRNA abundance in uterus, cervix, ovaries, placenta, and whole blood of Long-Evans rats during late gestation or in RU486 and progesterone-treated dams using quantitative real-time PCR and western immunoblotting. IL-1R1, IL-1RAcP, and IL-1RAcPb mRNA abundance significantly increased in the uterus at delivery whereas IL-1R2 mRNA abundance significantly decreased. IL-1R1 protein increased at term and IL-1R2 protein decreased at term compared to nonpregnant uteri. IL1-RAcPb mRNA abundance was less than IL-1RAcP, but in the lower uterine segment it was the highest of all tissues examined. RU486 stimulated preterm delivery and an increase in IL-1R1 mRNA abundance whereas progesterone administration extended pregnancy and suppressed the increase in IL-1R1. These data suggest that changes in uterine sensitivity to IL-1 occur during late gestation and suggest another level of regulation for the control of delivery. The roles for IL-1RAcP and IL-1RAcPb need to be determined, but may relate to different intracellular signaling pathways.

  15. Helping Hands for Budding Prospects: ENTH/ANTH/VHS Accessory Proteins in Endocytosis, Vacuolar Transport, and Secretion[W

    PubMed Central

    Zouhar, Jan; Sauer, Michael

    2014-01-01

    Coated vesicles provide a major mechanism for the transport of proteins through the endomembrane system of plants. Transport between the endoplasmic reticulum and the Golgi involves vesicles with COPI and COPII coats, whereas clathrin is the predominant coat in endocytosis and post-Golgi trafficking. Sorting of cargo, coat assembly, budding, and fission are all complex and tightly regulated processes that involve many proteins. The mechanisms and responsible factors are largely conserved in eukaryotes, and increasing organismal complexity tends to be associated with a greater numbers of individual family members. Among the key factors is the class of ENTH/ANTH/VHS domain-containing proteins, which link membrane subdomains, clathrin, and other adapter proteins involved in early steps of clathrin coated vesicle formation. More than 30 Arabidopsis thaliana proteins contain this domain, but their generally low sequence conservation has made functional classification difficult. Reports from the last two years have greatly expanded our knowledge of these proteins and suggest that ENTH/ANTH/VHS domain proteins are involved in various instances of clathrin-related endomembrane trafficking in plants. This review aims to summarize these new findings and discuss the broader context of clathrin-dependent plant vesicular transport. PMID:25415979

  16. Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator.

    PubMed

    Settlage, S B; Miller, A B; Gruissem, W; Hanley-Bowdoin, L

    2001-01-20

    Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.

  17. Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

    PubMed

    Dores, Robert M; Liang, Liang; Hollmann, Rebecca E; Sandhu, Navdeep; Vijayan, Mathilakath M

    2016-08-01

    The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs.

  18. Hypothesis and Theory: Revisiting Views on the Co-evolution of the Melanocortin Receptors and the Accessory Proteins, MRAP1 and MRAP2

    PubMed Central

    Dores, Robert M.

    2016-01-01

    The evolution of the melanocortin receptors (MCRs) is closely associated with the evolution of the melanocortin-2 receptor accessory proteins (MRAPs). Recent annotation of the elephant shark genome project revealed the sequence of a putative MRAP1 ortholog. The presence of this sequence in the genome of a cartilaginous fish raises the possibility that the mrap1 and mrap2 genes in the genomes of gnathostome vertebrates were the result of the chordate 2R genome duplication event. The presence of a putative MRAP1 ortholog in a cartilaginous fish genome is perplexing. Recent studies on melanocortin-2 receptor (MC2R) in the genomes of the elephant shark and the Japanese stingray indicate that these MC2R orthologs can be functionally expressed in CHO cells without co-expression of an exogenous mrap1 cDNA. The novel ligand selectivity of these cartilaginous fish MC2R orthologs is discussed. Finally, the origin of the mc2r and mc5r genes is reevaluated. The distinctive primary sequence conservation of MC2R and MC5R is discussed in light of the physiological roles of these two MCR paralogs. PMID:27445982

  19. The early origin of melanocortin receptors, agouti-related peptide, agouti signalling peptide, and melanocortin receptor-accessory proteins, with emphasis on pufferfishes, elephant shark, lampreys, and amphioxus.

    PubMed

    Västermark, Ake; Schiöth, Helgi B

    2011-06-11

    There are conflicting theories about the evolution of melanocortin MC receptors while only few studies have addressed the evolution of agouti-related peptide (AgRP) and agouti signalling peptide (ASIP), which are antagonists at the melanocortin receptors (MCRs), or the melanocortin MC(2) receptor accessory proteins (MRAP1 and MRAP2). Previously we have cloned melanocortin MC receptors (MC(a) and MC(b)) genes in river lamprey and here we identify orthologues to these melanocortin MC receptor sequences in the sea lamprey. We investigate the putative presence of the melanocortin MC receptor genes in lancelet (amphioxus; Branchiostoma floridae) but we find it unlikely that such gene exists, due to a sharp drop in sequence similarity beyond sequence clusters of known receptors. We show the presence of AgRP and ASIP in elephant shark, a cartilaginous fish belonging to the subclass of Elasmobranchii. However, we do not find any of these genes in lamprey or lancelet after detailed analysis of both targeted and whole proteome regular expression scans. We found MRAP2, but not MRAP1, to be present in elephant shark and sea lamprey while Fugu (T. rubripes) has both genes. This study shows that the most ancient presence of these melanocortin-related sequences is found in elephant shark and lampreys considering the current available sequence data.

  20. DNA polymerase III accessory proteins. I. holA and holB encoding delta and delta'.

    PubMed

    Dong, Z; Onrust, R; Skangalis, M; O'Donnell, M

    1993-06-05

    The genes encoding the delta and delta' subunits of the 10-subunit Escherichia coli replicase, DNA polymerase III holoenzyme, have been identified and sequenced. The holA gene encoding delta is located downstream of rlpB at 15.2 min and predicts a 38.7 kda protein. The holB gene encoding delta' is located at 24.3 min and predicts a 36.9-kDa protein. Hence the delta and delta' subunits are unrelated proteins encoded by separate genes. The genes have been used to express and purify delta and delta' in quantity. The predicted amino acid sequence of delta' is homologous to the sequences of the tau and gamma subunits revealing a large amount of structural redundancy within the holoenzyme.

  1. Human ARF4 expression rescues sec7 mutant yeast cells.

    PubMed Central

    Deitz, S B; Wu, C; Silve, S; Howell, K E; Melançon, P; Kahn, R A; Franzusoff, A

    1996-01-01

    Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics. PMID:8668142

  2. A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

    PubMed Central

    Durack, Juliana; Burke, Thomas P.

    2014-01-01

    The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis. PMID:25535272

  3. Lipopolysaccharide (LPS)-binding protein and soluble CD14 function as accessory molecules for LPS-induced changes in endothelial barrier function, in vitro.

    PubMed Central

    Goldblum, S E; Brann, T W; Ding, X; Pugin, J; Tobias, P S

    1994-01-01

    Bacterial LPS induces endothelial cell (EC) injury both in vivo and in vitro. We studied the effect of Escherichia coli 0111:B4 LPS on movement of 14C-BSA across bovine pulmonary artery EC monolayers. In the presence of serum, a 6-h LPS exposure augmented (P < 0.001) transendothelial 14C-BSA flux compared with the media control at concentrations > or = 0.5 ng/ml, and LPS (10 ng/ml) exposures of > or = 2-h increased (P < 0.005) the flux. In the absence of serum, LPS concentrations of up to 10 micrograms/ml failed to increase 14C-BSA flux at 6 h. The addition of 10% serum increased EC sensitivity to the LPS stimulus by > 10,000-fold. LPS (10 ng/ml, 6 h) failed to increase 14C-BSA flux at serum concentrations < 0.5%, and maximum LPS-induced increments could be generated in the presence of > or = 2.5%. LPS-binding protein (LBP) and soluble CD14 (sCD14) could each satisfy this serum requirement; either anti-LBP or anti-CD14 antibody each totally blocked (P < 0.00005) the LPS-induced changes in endothelial barrier function. LPS-LBP had a more rapid onset than did LPS-sCD14. The LPS effect in the presence of both LBP and sCD14 exceeded the effect in the presence of either protein alone. These data suggest that LBP and sCD14 each independently functions as an accessory molecule for LPS presentation to the non-CD14-bearing endothelial surface. However, in the presence of serum both molecules are required. Images PMID:7509346

  4. Phylogenetic Insights into the Functional Relationship between Primate Lentiviral Reverse Transcriptase and Accessory Proteins Vpx/Vpr

    PubMed Central

    Sakai, Yosuke; Doi, Naoya; Miyazaki, Yasuyuki; Adachi, Akio; Nomaguchi, Masako

    2016-01-01

    The efficiency of reverse transcription to synthesize viral DNA in infected cells greatly influences replication kinetics of retroviruses. However, viral replication in non-dividing cells such as resting T cells and terminally differentiated macrophages is potently and kinetically restricted by a host antiviral factor designated SAMHD1 (sterile alpha motif and HD-domain containing protein 1). SAMHD1 reduces cellular deoxynucleoside triphosphate (dNTP) pools and affects viral reverse transcription step. Human immunodeficiency virus type 2 (HIV-2) and some simian immunodeficiency viruses (SIVs) have Vpx or Vpr to efficiently degrade SAMHD1. Interestingly, the reverse transcriptase (RT) derived from HIV-1 that encodes no anti-SAMHD1 proteins has been previously demonstrated to uniquely exhibit a high enzymatic activity. It is thus not irrational to assume that some viruses may have acquired or lost the specific RT property to better adapt themselves to the low dNTP environments confronted in non-dividing cells. This adaptation process may probably be correlated with the SAMHD1-antagonizing ability by viruses. In this report, we asked whether such adaptive events can be inferable from Vpx/Vpr and RT phylogenetic trees overlaid with SAMHD1-degrading capacity of Vpx/Vpr and with kinetic characteristics of RT. Resultant two trees showed substantially similar clustering patterns, and therefore suggested that the properties of RT and Vpx/Vpr can be linked. In other words, HIV/SIVs may possess their own RT proteins to adequately react to various dNTP circumstances in target cells. PMID:27803699

  5. Core Binding Factor β Protects HIV, Type 1 Accessory Protein Viral Infectivity Factor from MDM2-mediated Degradation.

    PubMed

    Matsui, Yusuke; Shindo, Keisuke; Nagata, Kayoko; Yoshinaga, Noriyoshi; Shirakawa, Kotaro; Kobayashi, Masayuki; Takaori-Kondo, Akifumi

    2016-11-25

    HIV, type 1 overcomes host restriction factor apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins by organizing an E3 ubiquitin ligase complex together with viral infectivity factor (Vif) and a host transcription cofactor core binding factor β (CBFβ). CBFβ is essential for Vif to counteract APOBEC3 by enabling the recruitment of cullin 5 to the complex and increasing the steady-state level of Vif protein; however, the mechanisms by which CBFβ up-regulates Vif protein remains unclear. Because we have reported previously that mouse double minute 2 homolog (MDM2) is an E3 ligase for Vif, we hypothesized that CBFβ might protect Vif from MDM2-mediated degradation. Co-immunoprecipitation analyses showed that Vif mutants that do not bind to CBFβ preferentially interact with MDM2 and that overexpression of CBFβ disrupts the interaction between MDM2 and Vif. Knockdown of CBFβ reduced the steady-state level of Vif in MDM2-proficient cells but not in MDM2-null cells. Cycloheximide chase analyses revealed that Vif E88A/W89A, which does not interact with CBFβ, degraded faster than wild-type Vif in MDM2-proficient cells but not in MDM2-null cells, suggesting that Vif stabilization by CBFβ is mainly caused by impairing MDM2-mediated degradation. We identified Vif R93E as a Vif variant that does not bind to MDM2, and the virus with this substitution mutation was more resistant to APOBEC3G than the parental virus. Combinatory substitution of Vif residues required for CBFβ binding and MDM2 binding showed full recovery of Vif steady-state levels, supporting our hypothesis. Our data provide new insights into the mechanism of Vif augmentation by CBFβ.

  6. An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accessory proteins.

    PubMed

    Pacchia, A L; Adelson, M E; Kaul, M; Ron, Y; Dougherty, J P

    2001-03-30

    Lentiviral vectors based on human immunodeficiency virus type 1 (HIV-1) possess the ability to deliver exogenous genes to both dividing and nondividing cells and to subsequently establish a stable provirus in these target cells, which can allow long-term expression of the transferred gene. Herein we describe a stable packaging cell line that is devoid of HIV-1 tat, vif, vpr, vpu, and nef. In order to avoid any risk of cytotoxicity associated with constitutive expression of HIV-1 protease or the VSV-G envelope protein, transcription of the packaging and envelope constructs was tightly controlled by employing the ecdysone-inducible system. Using this cell line, we have been able to consistently generate concentrated pseudotyped vector virus stocks with titers in the range of 10(8) IU/ml, which can efficiently transduce actively dividing and growth-arrested cells in vitro. This novel packaging cell line for lentiviral vectors facilitates the production of high-titer virus stocks in the absence of replication-competent virus and provides us with an important tool for use in future gene transfer studies.

  7. The neuron-specific interleukin-1 receptor accessory protein is required for homeostatic sleep and sleep responses to influenza viral challenge in mice.

    PubMed

    Davis, Christopher J; Dunbrasky, Danielle; Oonk, Marcella; Taishi, Ping; Opp, Mark R; Krueger, James M

    2015-07-01

    Interleukin-1β (IL1) is involved in sleep regulation and sleep responses induced by influenza virus. The IL1 receptor accessory protein (AcP) and an alternatively spliced isoform of AcP found primarily in neurons, AcPb, form part of the IL1 signaling complex. IL1-induced sleep responses depend on injection time. In rat cortex, both IL1 mRNA and AcPb mRNA peak at Zeitgeber Time (ZT) 0 then decline over the daylight hours. Sleep deprivation enhances cortical IL1 mRNA and AcPb mRNA levels, but not AcP mRNA. We used wild type (WT) and AcPb knockout (KO) mice and performed sleep deprivation between ZT10 and 20 or between ZT22 and 8 based on the time of day expression profiles of AcPb and IL1. We hypothesized that the magnitude of the responses to sleep loss would be strain- and time of day-dependent. In WT mice, NREMS and REMS rebounds occurred regardless of when they were deprived of sleep. In contrast, when AcPbKO mice were sleep deprived from ZT10 to 20 NREMS and REMS rebounds were absent. The AcPbKO mice expressed sleep rebound if sleep loss occurred from ZT22 to 8 although the NREMS responses were not as robust as those that occurred in WT mice. We also challenged mice with intranasal H1N1 influenza virus. WT mice exhibited the expected enhanced sleep responses. In contrast, the AcPbKO mice had less sleep after influenza challenge compared to their own baseline values and compared to WT mice. Body temperature and locomotor activity responses after viral challenge were lower and mortality was higher in AcPbKO than in WT mice. We conclude that neuron-specific AcPb plays a critical role in host defenses and sleep homeostasis.

  8. The Rcs stress response and accessory envelope proteins are required for de novo generation of cell shape in Escherichia coli.

    PubMed

    Ranjit, Dev K; Young, Kevin D

    2013-06-01

    Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.

  9. Kv4 Accessory Protein DPPX (DPP6) is a Critical Regulator of Membrane Excitability in Hippocampal CA1 Pyramidal Neurons

    PubMed Central

    Kim, Jinhyun; Nadal, Marcela S.; Clemens, Ann M.; Baron, Matthew; Jung, Sung-Cherl; Misumi, Yoshio; Rudy, Bernardo; Hoffman, Dax A.

    2008-01-01

    A-type K+ currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic integration, action potential (AP) shape and firing patterns. In hippocampal CA1 pyramidal neurons, Kv4 channels make up the majority of the somatodendritic A-type current. Studies in heterologous expression systems have shown that Kv4 channels interact with transmembrane dipeptidyl-peptidase-like proteins (DPPLs) to regulate the surface trafficking and biophysical properties of Kv4 channels. To investigate the influence of DPPLs in a native system, we conducted voltage-clamp experiments in patches from CA1 pyramidal neurons expressing short-interfering RNA (siRNA) targeting the DPPL variant known to be expressed in hippocampal pyramidal neurons, DPPX (siDPPX). In accordance with heterologous studies, we found that DPPX downregulation in neurons resulted in depolarizing shifts of the steady-state inactivation and activation curves, a shallower conductance-voltage slope, slowed inactivation, and a delayed recovery from inactivation for A-type currents. We carried out current-clamp experiments to determine the physiological effect of the A-type current modifications by DPPX. Neurons expressing siDPPX exhibited a surprisingly large reduction in subthreshold excitability as measured by a decrease in input resistance, delayed time to AP onset, and an increased AP threshold. Suprathreshold DPPX downregulation resulted in slower AP rise and weaker repolarization. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitability. The Kv4 auxiliary subunit DPPX thus acts to increase neuronal responsiveness and enhance signal precision by advancing AP initiation and accelerating both the rise and repolarization of APs. PMID:18667548

  10. Analysis of SecA Dimerization in Solution

    PubMed Central

    2015-01-01

    The Sec pathway mediates translocation of protein across the inner membrane of bacteria. SecA is a motor protein that drives translocation of preprotein through the SecYEG channel. SecA reversibly dimerizes under physiological conditions, but different dimer interfaces have been observed in SecA crystal structures. Here, we have used biophysical approaches to address the nature of the SecA dimer that exists in solution. We have taken advantage of the extreme salt sensitivity of SecA dimerization to compare the rates of hydrogen–deuterium exchange of the monomer and dimer and have analyzed the effects of single-alanine substitutions on dimerization affinity. Our results support the antiparallel dimer arrangement observed in one of the crystal structures of Bacillus subtilis SecA. Additional residues lying within the preprotein binding domain and the C-terminus are also protected from exchange upon dimerization, indicating linkage to a conformational transition of the preprotein binding domain from an open to a closed state. In agreement with this interpretation, normal mode analysis demonstrates that the SecA dimer interface influences the global dynamics of SecA such that dimerization stabilizes the closed conformation. PMID:24786965

  11. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 3 Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority,...

  12. 46 CFR Sec. 3 - Premiums.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Premiums. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 3 Premiums. The bonds provided for shall be furnished without cost to the National Shipping Authority,...

  13. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  14. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  15. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  16. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  17. 46 CFR Sec. 1 - Purpose.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Purpose. Sec. 1 Section 1 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS OPERATING CONTRACT Sec. 1 Purpose. This part prescribes the standard form of marine terminal contract to be entered into by the...

  18. 46 CFR Sec. 9 - Communications.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Communications. Sec. 9 Section 9 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS RESTRICTIONS UPON THE TRANSFER OR CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 9 Communications....

  19. 46 CFR Sec. 9 - Communications.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Communications. Sec. 9 Section 9 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS RESTRICTIONS UPON THE TRANSFER OR CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 9 Communications....

  20. 46 CFR Sec. 12 - Audit.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance...

  1. 46 CFR Sec. 12 - Audit.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance...

  2. 46 CFR Sec. 12 - Audit.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance...

  3. 46 CFR Sec. 12 - Audit.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance...

  4. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Insurance. Sec. 13 Section 13 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance... respect to awarded work. Said Article 9 requires that the Contractor shall maintain insurance to...

  5. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Insurance. Sec. 13 Section 13 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance... respect to awarded work. Said Article 9 requires that the Contractor shall maintain insurance to...

  6. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Insurance. Sec. 13 Section 13 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance... respect to awarded work. Said Article 9 requires that the Contractor shall maintain insurance to...

  7. 46 CFR Sec. 5 - Accounting.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Accounting. Sec. 5 Section 5 Shipping MARITIME... Sec. 5 Accounting. The General Agent shall record the amounts of compensation paid from the NSA... Accounting Office, at which time the Maritime Administration will take custody of the records....

  8. 46 CFR Sec. 12 - Audit.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Audit. Sec. 12 Section 12 Shipping MARITIME... TRANSACTIONS UNDER AGENCY AGREEMENTS Reports and Audit Sec. 12 Audit. (a) The owner will audit as currently as possible subsequent to audit by the agent, all documents relating to the activities, maintenance...

  9. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Insurance. Sec. 13 Section 13 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance... respect to awarded work. Said Article 9 requires that the Contractor shall maintain insurance to...

  10. 46 CFR Sec. 13 - Insurance.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Insurance. Sec. 13 Section 13 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 13 Insurance... respect to awarded work. Said Article 9 requires that the Contractor shall maintain insurance to...

  11. 46 CFR Sec. 9 - Communications.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Communications. Sec. 9 Section 9 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION B-CONTROL AND UTILIZATION OF PORTS RESTRICTIONS UPON THE TRANSFER OR CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 9 Communications....

  12. 46 CFR Sec. 3 - Specifications.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Specifications. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec....

  13. Accessory nerve palsy.

    PubMed

    Olarte, M; Adams, D

    1977-11-01

    After apparently uncomplicated excision of benign lesions in the posterior cervical triangle, two patients had shoulder pain. In one, neck pain and trapezius weakness were not prominent until one month after surgery. Inability to elevate the arm above the horizontal without externally rotating it, and prominent scapular displacement on arm abduction, but not on forward pushing movements, highlighted the trapezius dysfunction and differentiated it from serratus anterior weakness. Spinal accessory nerve lesions should be considered when minor surgical procedures, lymphadenitis, minor trauma, or tumours involved the posterior triangle of the neck.

  14. Structural Studies of Apo Nosl, an Accessory Protein of the Nitrous Oxide Reductase System: Insights from Structural Homology with MerB, a Mercury Resistance Protein

    SciTech Connect

    Taubner, Lara M.; McGuirl, Michele A.; Dooley, David M.; Copie, Valerie

    2006-09-19

    The formation of the unique catalytic tetranuclear copper cluster (CuZ) of nitrous oxide reductase, N2OR, requires the coexpression of a multiprotein assembly apparatus encoded by the nosDFYL operon. NosL, one of the proteins encoded by this transcript, is a 20 kDa lipoprotein of the periplasm that has been shown to bind copper(I), although its function has yet to be detemined. Cu(I) EXAFS data collected on the holo protein demonstrated that features of the copper binding site are consistent with a role for this protein as a metallochaperone, a class of metal ion transporters involved in metal resistance, homeostasis, and metallocluster biosynthesis. To test this hypothesis and to gain insight into other potential functional roles for this protein in the N2OR system, the three-dimensional solution structure of apo NosL has been solved by solution NMR methods. The structure of apo NosL consists of two relatively independent homologous domains that adopt an unusual topology.

  15. Hph1 and Hph2 are novel components of the Sec63/Sec62 posttranslational translocation complex that aid in vacuolar proton ATPase biogenesis.

    PubMed

    Piña, Francisco J; O'Donnell, Allyson F; Pagant, Silvere; Piao, Hai Lan; Miller, John P; Fields, Stanley; Miller, Elizabeth A; Cyert, Martha S

    2011-01-01

    Hph1 and Hph2 are homologous integral endoplasmic reticulum (ER) membrane proteins required for Saccharomyces cerevisiae survival under environmental stress conditions. To investigate the molecular functions of Hph1 and Hph2, we carried out a split-ubiquitin-membrane-based yeast two-hybrid screen and identified their interactions with Sec71, a subunit of the Sec63/Sec62 complex, which mediates posttranslational translocation of proteins into the ER. Hph1 and Hph2 likely function in posttranslational translocation, as they interact with other Sec63/Sec62 complex subunits, i.e., Sec72, Sec62, and Sec63. hph1Δ hph2Δ cells display reduced vacuole acidification; increased instability of Vph1, a subunit of vacuolar proton ATPase (V-ATPase); and growth defects similar to those of mutants lacking V-ATPase activity. sec71Δ cells exhibit similar phenotypes, indicating that Hph1/Hph2 and the Sec63/Sec62 complex function during V-ATPase biogenesis. Hph1/Hph2 and the Sec63/Sec62 complex may act together in this process, as vacuolar acidification and Vph1 stability are compromised to the same extent in hph1Δ hph2Δ and hph1Δ hph2Δ sec71Δ cells. In contrast, loss of Pkr1, an ER protein that promotes posttranslocation assembly of Vph1 with V-ATPase subunits, further exacerbates hph1Δ hph2Δ phenotypes, suggesting that Hph1 and Hph2 function independently of Pkr1-mediated V-ATPase assembly. We propose that Hph1 and Hph2 aid Sec63/Sec62-mediated translocation of specific proteins, including factors that promote efficient biogenesis of V-ATPase, to support yeast cell survival during environmental stress.

  16. Torsion of Accessory Hepatic Lobe

    PubMed Central

    Natarajan, Saravanan; Jayasudha; Periasamy, Manikandhan; Rangasamy, Saminathan

    2017-01-01

    An accessory hepatic lobe is a rare congenital anomaly that can undergo torsion and present as an acute surgical emergency. A 5-year-old child admitted as acute abdomen, on laparotomy found to have torsion of accessory lobe of liver, is being reported. PMID:28082782

  17. Phylogenetic analysis and delineation of phytoplasmas based on the secY gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The secY gene, located in the operator-distal part of the spc ribosomal protein operon, codes for a protein translocase subunit secY. The secY gene sequence is more variable than that of the 16S rRNA gene. Comparative phylogenetic analyses with 16S rRNA and secY gene sequences from 80 and 83 phytop...

  18. Mycolactone subverts immunity by selectively blocking the Sec61 translocon.

    PubMed

    Baron, Ludivine; Paatero, Anja Onerva; Morel, Jean-David; Impens, Francis; Guenin-Macé, Laure; Saint-Auret, Sarah; Blanchard, Nicolas; Dillmann, Rabea; Niang, Fatoumata; Pellegrini, Sandra; Taunton, Jack; Paavilainen, Ville O; Demangel, Caroline

    2016-12-12

    Mycolactone, an immunosuppressive macrolide released by the human pathogen Mycobacterium ulcerans, was previously shown to impair Sec61-dependent protein translocation, but the underlying molecular mechanism was not identified. In this study, we show that mycolactone directly targets the α subunit of the Sec61 translocon to block the production of secreted and integral membrane proteins with high potency. We identify a single-amino acid mutation conferring resistance to mycolactone, which localizes its interaction site near the lumenal plug of Sec61α. Quantitative proteomics reveals that during T cell activation, mycolactone-mediated Sec61 blockade affects a selective subset of secretory proteins including key signal-transmitting receptors and adhesion molecules. Expression of mutant Sec61α in mycolactone-treated T cells rescued their homing potential and effector functions. Furthermore, when expressed in macrophages, the mycolactone-resistant mutant restored IFN-γ receptor-mediated antimicrobial responses. Thus, our data provide definitive genetic evidence that Sec61 is the host receptor mediating the diverse immunomodulatory effects of mycolactone and identify Sec61 as a novel regulator of immune cell functions.

  19. Determination of second virial coefficient of proteins using a dual-detector cell for simultaneous measurement of scattered light intensity and concentration in SEC-HPLC.

    PubMed

    Bajaj, Harminder; Sharma, Vikas K; Kalonia, Devendra S

    2004-12-01

    A method is proposed for the measurement of the B22 value of proteins in aqueous solutions in flow-mode that utilizes a novel fabricated dual-detector cell, which simultaneously measures protein concentration and the corresponding scattered light intensity at 90 degrees , after the protein elutes from a size-exclusion column. Each data point on the chromatograms obtained from the light scattering detector and the concentration (ultraviolet) detector is converted to Rayleigh's ratio, Rtheta, and concentration, c, respectively. The B22 value is calculated from the slope of the Debye plot (Kc/Rtheta versus c) generated from a range of concentrations obtained from these chromatograms for a single protein injection. It is shown that this method provides reliable determination of the B22 values for such proteins as lysozyme, chymotrypsinogen, and chymotrypsin in various solution conditions that agree well with those reported in literature.

  20. Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein.

    PubMed

    Brown, Laura T; Sutera, Vincent A; Zhou, Shen; Weitzel, Christopher S; Cheng, Yisha; Lovett, Susan T

    2015-11-01

    Elongating DNA polymerases frequently encounter lesions or structures that impede progress and require repair before DNA replication can be completed. Therefore, directing repair factors to a blocked fork, without interfering with normal replication, is important for proper cell function, and it is a process that is not well understood. To study this process, we have employed the chain-terminating nucleoside analog, 3' azidothymidine (AZT) and the E. coli genetic system, for which replication and repair factors have been well-defined. By using high-expression suppressor screens, we identified yoaA, encoding a putative helicase, and holC, encoding the Chi component of the replication clamp loader, as genes that promoted tolerance to AZT. YoaA is a putative Fe-S helicase in the XPD/RAD3 family for which orthologs can be found in most bacterial genomes; E. coli has a paralog to YoaA, DinG, which possesses 5' to 3' helicase activity and an Fe-S cluster essential to its activity. Mutants in yoaA are sensitive to AZT exposure; dinG mutations cause mild sensitivity to AZT and exacerbate the sensitivity of yoaA mutant strains. Suppression of AZT sensitivity by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Interactions of Chi with single-strand DNA binding protein (SSB) and with Psi were required to aid AZT tolerance, as was the proofreading 3' exonuclease, DnaQ. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to replication gaps and that unwinding of the nascent strand promotes repair and AZT excision. This recruitment prevents the toxicity of helicase activity and aids the handoff of repair with replication factors, ensuring timely repair and resumption of replication.

  1. Connecting Replication and Repair: YoaA, a Helicase-Related Protein, Promotes Azidothymidine Tolerance through Association with Chi, an Accessory Clamp Loader Protein

    PubMed Central

    Brown, Laura T.; Sutera, Vincent A.; Zhou, Shen; Weitzel, Christopher S.; Cheng, Yisha; Lovett, Susan T.

    2015-01-01

    Elongating DNA polymerases frequently encounter lesions or structures that impede progress and require repair before DNA replication can be completed. Therefore, directing repair factors to a blocked fork, without interfering with normal replication, is important for proper cell function, and it is a process that is not well understood. To study this process, we have employed the chain-terminating nucleoside analog, 3’ azidothymidine (AZT) and the E. coli genetic system, for which replication and repair factors have been well-defined. By using high-expression suppressor screens, we identified yoaA, encoding a putative helicase, and holC, encoding the Chi component of the replication clamp loader, as genes that promoted tolerance to AZT. YoaA is a putative Fe-S helicase in the XPD/RAD3 family for which orthologs can be found in most bacterial genomes; E. coli has a paralog to YoaA, DinG, which possesses 5’ to 3’ helicase activity and an Fe-S cluster essential to its activity. Mutants in yoaA are sensitive to AZT exposure; dinG mutations cause mild sensitivity to AZT and exacerbate the sensitivity of yoaA mutant strains. Suppression of AZT sensitivity by holC or yoaA was mutually codependent and we provide evidence here that YoaA and Chi physically interact. Interactions of Chi with single-strand DNA binding protein (SSB) and with Psi were required to aid AZT tolerance, as was the proofreading 3’ exonuclease, DnaQ. Our studies suggest that repair is coupled to blocked replication through these interactions. We hypothesize that SSB, through Chi, recruits the YoaA helicase to replication gaps and that unwinding of the nascent strand promotes repair and AZT excision. This recruitment prevents the toxicity of helicase activity and aids the handoff of repair with replication factors, ensuring timely repair and resumption of replication. PMID:26544712

  2. Sec14 Like PITPs Couple Lipid Metabolism with Phosphoinositide Synthesis to Regulate Golgi Functionality

    PubMed Central

    Davison, James M.; Bankaitis, Vytas A.

    2017-01-01

    An interface coordinating lipid metabolism with proteins that regulate membrane trafficking is necessary to regulate Golgi morphology and dynamics. Such an interface facilitates the membrane deformations required for vesicularization, forms platforms for protein recruitment and assembly on appropriate sites on a membrane surface and provides lipid co-factors for optimal protein activity in the proper spatio-temporally regulated manner. Importantly, Sec14 and Sec14-like proteins are a unique superfamily of proteins that sense specific aspects of lipid metabolism, employing this information to potentiate phosphoinositide production. Therefore, Sec14 and Sec14 like proteins form central conduits to integrate multiple aspects of lipid metabolism with productive phosphoinositide signaling. PMID:22374094

  3. 14 CFR 33.25 - Accessory attachments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Accessory attachments. 33.25 Section 33.25... STANDARDS: AIRCRAFT ENGINES Design and Construction; General § 33.25 Accessory attachments. The engine must operate properly with the accessory drive and mounting attachments loaded. Each engine accessory drive...

  4. Structural elements that govern Sec14-like PITP sensitivities to potent small molecule inhibitors.

    PubMed

    Khan, Danish; McGrath, Kaitlyn R; Dorosheva, Oleksandra; Bankaitis, Vytas A; Tripathi, Ashutosh

    2016-04-01

    Sec14-like phosphatidylinositol transfer proteins (PITPs) play important biological functions in integrating multiple aspects of intracellular lipid metabolism with phosphatidylinositol-4-phosphate signaling. As such, these proteins offer new opportunities for highly selective chemical interference with specific phosphoinositide pathways in cells. The first and best characterized small molecule inhibitors of the yeast PITP, Sec14, are nitrophenyl(4-(2-methoxyphenyl)piperazin-1-yl)methanones (NPPMs), and a hallmark feature of NPPMs is their exquisite targeting specificities for Sec14 relative to other closely related Sec14-like PITPs. Our present understanding of Sec14::NPPM binding interactions is based on computational docking and rational loss-of-function approaches. While those approaches have been informative, we still lack an adequate understanding of the basis for the high selectivity of NPPMs among closely related Sec14-like PITPs. Herein, we describe a Sec14 motif, which we term the VV signature, that contributes significantly to the NPPM sensitivity/resistance of Sec14-like phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer proteins. The data not only reveal previously unappreciated determinants that govern Sec14-like PITP sensitivities to NPPMs, but enable predictions of which Sec14-like PtdIns/PtdCho transfer proteins are likely to be NPPM resistant or sensitive based on primary sequence considerations. Finally, the data provide independent evidence in support of previous studies highlighting the importance of Sec14 residue Ser173 in the mechanism by which NPPMs engage and inhibit Sec14-like PITPs.

  5. Endoscopic Accessory Navicular Synchondrosis Fusion.

    PubMed

    Lui, Tun Hing

    2016-12-01

    The accessory navicular bone is one of the most common accessory ossicles of the foot. Fewer than 1% of accessory navicular bones are symptomatic, and most of these are type II accessory navicular bones. A separation of the synchondrosis is considered one of the main causes of pain. After an injury to the synchondrosis has resulted in a chondro-osseous disruption, the combined forces of tension and shear from the posterior tibial tendon and the foot aggravate the injury and prevent it from healing. Fusion of the synchondrosis is a logical surgical treatment option if the pain is recalcitrant to conservative measures. The purpose of this technical note is to report an endoscopic approach to achieve fusion. It has the advantages of better cosmesis, less scar pain, less risk of nonunion, and potential to examine the tibialis posterior tendon and the talonavicular joint.

  6. Crystal structure of Sec10, a subunit of the exocyst complex

    PubMed Central

    Chen, Jianxing; Yamagata, Atsushi; Kubota, Keiko; Sato, Yusuke; Goto-Ito, Sakurako; Fukai, Shuya

    2017-01-01

    The exocyst complex is a heterooctameric protein complex composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84. This complex plays an essential role in trafficking secretory vesicles to the plasma membrane through its interaction with phosphatidylinositol 4,5-bisphosphate and small GTPases. To date, the near-full-length structural information of each subunit has been limited to Exo70, although the C-terminal half structures of Sec6, Sec15 and Exo84 and the structures of the small GTPase-binding domains of Sec3, Sec5 and Exo84 have been reported. Here, we report the crystal structure of the near-full-length zebrafish Sec10 (zSec10) at 2.73 Å resolution. The structure of zSec10 consists of tandem antiparallel helix bundles that form a straight rod, like helical core regions of other exocyst subunits. This structure provides the first atomic details of Sec10, which may be useful for future functional and structural studies of this subunit and the exocyst complex. PMID:28098232

  7. Accessory drive for a turbine engine

    SciTech Connect

    Brogdon, J.W.; Allen, K.D.; Barton, J.S.; Hicks, R.J.

    1987-02-03

    This patent describes, in combination: a radial flow turbine engine having a main shaft and a casing with air inlets open radially at one end, and an accessory drive comprising: an accessory housing positioned axially adjacent the one end of the turbine engine casing, a gear ring rotatably mounted within the accessory housing, means for mechanically drivingly connecting the gear ring to the turbine main shaft, the connecting means comprising a planetary gear arrangement contained in the accessory housing, the accessory housing having apertures open to the gear ring and circumferentially spaced from each other, at least one accessory having a driven gear, and means for mounting the at least one accessory to the accessory housing so that the accessory registers with one of the plurality of apertures and so that the gear ring meshes with the driven gear, wherein each aperture is adapted for connection with a separate accessory.

  8. A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach

    PubMed Central

    Poussu, Eini; Jäntti, Jussi; Savilahti, Harri

    2005-01-01

    Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector. PMID:16006618

  9. Sec31 encodes an essential component of the COPII coat required for transport vesicle budding from the endoplasmic reticulum.

    PubMed Central

    Salama, N R; Chuang, J S; Schekman, R W

    1997-01-01

    The COPII vesicle coat protein promotes the formation of endoplasmic reticulum- (ER) derived transport vesicles that carry secretory proteins to the Golgi complex in Saccharomyces cerevisiae. This coat protein consists of Sar1p, the Sec23p protein complex containing Sec23p and Sec24p, and the Sec13p protein complex containing Sec13p and a novel 150-kDa protein, p150. Here, we report the cloning and characterization of the p150 gene. p150 is encoded by an essential gene. Depletion of this protein in vivo blocks the exit of secretory proteins from the ER and causes an elaboration of ER membranes, indicating that p150 is encoded by a SEC gene. Additionally, overproduction of the p150 gene product compromises the growth of two ER to Golgi sec mutants: sec16-2 and sec23-1. p150 is encoded by SEC31, a gene isolated in a genetic screen for mutations that accumulate unprocessed forms of the secretory protein alpha-factor. The sec31-1 mutation was mapped by gap repair, and sequence analysis revealed an alanine to valine change at position 1239, near the carboxyl terminus. Sec31p is a phosphoprotein and treatment of the Sec31p-containing fraction with alkaline phosphatase results in a 50-75% inhibition of transport vesicle formation activity in an ER membrane budding assay. Images PMID:9190202

  10. 46 CFR Sec. 9 - Payment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 9 Payment... Authority: (1) Repair Contractors will submit invoices for repair costs covered by job orders under Master... will submit invoices for repair costs covered by job orders under Master Repair Contracts or...

  11. Structure of the Munc18c/Syntaxin4 N-peptide complex defines universal features of the N-peptide binding mode of Sec1/Munc18 proteins

    PubMed Central

    Hu, Shu-Hong; Latham, Catherine F.; Gee, Christine L.; James, David E.; Martin, Jennifer L.

    2007-01-01

    Sec1/Munc18 proteins (SM proteins) bind to soluble NSF attachment protein receptors (SNAREs) and play an essential role in membrane fusion. Divergent modes of regulation have been proposed for different SM proteins indicating that they can either promote or inhibit SNARE assembly. This is in part because of discrete modes of binding that have been described for various SM/SNARE complexes. One mode suggests that SM proteins bind only to Syntaxins (Stx) preventing SNARE assembly, whereas in another they facilitate SNARE assembly and bind to SNARE complexes. The mammalian cell surface SM protein Munc18c binds to an N-peptide in Stx4, and this is compatible with its interaction with SNARE complexes. Here we describe the crystal structure of Munc18c in complex with the Stx4 N-peptide. This structure shows remarkable similarity with a yeast complex indicating that the mode of binding, which can accommodate SNARE complexes, is highly conserved throughout evolution. Modeling reveals the presence of the N-peptide binding mode in most but not all yeast and mammalian SM/Stx pairs, suggesting that it has coevolved to fulfill a specific regulatory function. It is unlikely that the N-peptide interaction alone accounts for the specificity in SM/SNARE binding, implicating other contact surfaces in this function. Together with other data, our results support a sequential two-state model for SM/SNARE binding involving an initial interaction via the Stx N-peptide, which somehow facilitates a second, more comprehensive interaction comprising other contact surfaces in both proteins. PMID:17517664

  12. Early stages of retinal development depend on Sec13 function

    PubMed Central

    Schmidt, Katy; Cavodeassi, Florencia; Feng, Yi; Stephens, David J.

    2013-01-01

    Summary ER-to-Golgi transport of proteins destined for the extracellular space or intracellular compartments depends on the COPII vesicle coat and is constitutive in all translationally active cells. Nevertheless, there is emerging evidence that this process is regulated on a cell- and tissue-specific basis, which means that components of the COPII coat will be of differential importance to certain cell types. The COPII coat consists of an inner layer, Sec23/24 and an outer shell, Sec13/31. We have shown previously that knock-down of Sec13 results in concomitant loss of Sec31. In zebrafish and cultured human cells this leads to impaired trafficking of large cargo, namely procollagens, and is causative for defects in craniofacial and gut development. It is now widely accepted that the outer COPII coat is key to the architecture and stability of ER export vesicles containing large, unusual cargo proteins. Here, we investigate zebrafish eye development following Sec13 depletion. We find that photoreceptors degenerate or fail to develop from the onset. Impaired collagen trafficking from the retinal pigment epithelium and defects in overall retinal lamination also seen in Sec13-depleted zebrafish might have been caused by increased apoptosis and reduced topical proliferation in the retina. Our data show that the outer layer of the COPII coat is also necessary for the transport of large amounts of cargo proteins, in this case rhodopsin, rather than just large cargo as previously thought. PMID:23519012

  13. Structure of Monomeric Yeast and Mammalian Sec61 Complexes Interacting with the Translating Ribosome

    PubMed Central

    Becker, Thomas; Bhushan, Shashi; Jarasch, Alexander; Armache, Jean-Paul; Funes, Soledad; Jossinet, Fabrice; Gumbart, James; Mielke, Thorsten; Berninghausen, Otto; Schulten, Klaus; Westhof, Eric; Gilmore, Reid; Mandon, Elisabet; Beckmann, Roland

    2010-01-01

    The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined sub-nanometer resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation. PMID:19933108

  14. 'Candidatus Phytoplasma pruni', a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes.

    PubMed

    Davis, Robert E; Zhao, Yan; Dally, Ellen L; Lee, Ing-Ming; Jomantiene, Rasa; Douglas, Sharon M

    2013-02-01

    X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Candidatus Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1(R) as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Ca. Phytoplasma pruni' lineage. We propose that the term 'Ca. Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and--within the tolerance of a single base difference in one unique sequence--peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' taxa.

  15. The Basis of Asymmetry in the SecA-SecB Complex

    PubMed Central

    Suo, Yuying; Hardy, Simon J. S.; Randall, Linda L.

    2015-01-01

    During export in Escherichia coli, SecB, a homotetramer, structurally organized as a dimer of dimers, forms a complex with two protomers of SecA, which is the ATPase that provides energy to transfer a precursor polypeptide through the membrane via the SecYEG translocon. There are two areas of contact on SecB that stabilize the SecA:SecB complex: one, the flat sides of the SecB tetramer and the second, the C-terminal thirteen residues of SecB. These contacts within the complex are distributed asymmetrically. Breaking contact between SecA and the sides of SecB results in release of only one protomer of SecA yielding a complex of stoichiometry SecA1:SecB4. This complex mediates export; however, the coupling of ATP hydrolysis to movements of the precursor through the translocon is much less efficient than the coupling by the SecA2:SecB4 complex. Here we used heterotetrameric species of SecB to understand the source of the asymmetry in the contacts and its role in the functioning of the complex. The model of interactions presented suggests a way that binding between SecA and SecB might decrease the affinity of precursor polypeptides for SecB and facilitate the transfer to SecA. PMID:25534082

  16. Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

    PubMed Central

    Ishii, Eiji; Chiba, Shinobu; Hashimoto, Narimasa; Kojima, Seiji; Homma, Michio; Ito, Koreaki; Akiyama, Yoshinori; Mori, Hiroyuki

    2015-01-01

    SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine–Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles. PMID:26392525

  17. Cloning and characterization of Kluyveromyces lactis SEC14, a gene whose product stimulates Golgi secretory function in Saccharomyces cerevisiae.

    PubMed Central

    Salama, S R; Cleves, A E; Malehorn, D E; Whitters, E A; Bankaitis, V A

    1990-01-01

    The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for secretory protein movement from the Golgi complex. That some conservation of SEC14p function may exist was initially suggested by experiments that revealed immunoreactive polypeptides in cell extracts of the divergent yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. We have cloned and characterized the K. lactis SEC14 gene (SEC14KL). Immunoprecipitation experiments indicated that SEC14KL encoded the K. lactis structural homolog of SEC14p. In agreement with those results, nucleotide sequence analysis of SEC14KL revealed a gene product of 301 residues (Mr, 34,615) and 77% identity to SEC14p. Moreover, a single ectopic copy of SEC14KL was sufficient to render S. cerevisiae sec14-1(Ts) mutants, or otherwise inviable sec14-129::HIS3 mutant strains, completely proficient for secretory pathway function by the criteria of growth, invertase secretion, and kinetics of vacuolar protein localization. This efficient complementation of sec14-129::HIS3 was observed to occur when the rates of SEC14pKL and SEC14p synthesis were reduced by a factor of 7 to 10 with respect to the wild-type rate of SEC14p synthesis. Taken together, these data provide evidence that the high level of structural conservation between SEC14p and SEC14pKL reflects a functional identity between these polypeptides as well. On the basis of the SEC14p and SEC14pKL primary sequence homology to the human retinaldehyde-binding protein, we suggest that the general function of these SEC14p species may be to regulate the delivery of a hydrophobic ligand to Golgi membranes so that biosynthetic secretory traffic can be supported. Images PMID:2198263

  18. Automobile accessories: Assessment and improvement

    SciTech Connect

    Jackson, M.

    1995-11-01

    With mandates and regulatory policies to meet both the California Air Resources Board (CARB) and the Partnership for a New Generation of Vehicles (PNGV), designing vehicles of the future will become a difficult task. As we look into the use of electric and hybrid vehicles, reduction of the required power demand by influential automobile components is necessary in order to obtain performance and range goals. Among those automobile components are accessories. Accessories have a profound impact on the range and mileage of future vehicles with limited amounts of energy or without power generating capabilities such as conventional vehicles. Careful assessment of major power consuming accessories helps us focus on those that need improvement and contributes to attainment of mileage and range goals for electric and hybrid vehicles.

  19. Teaching Techniques for Accessory Percussion

    ERIC Educational Resources Information Center

    Micallef, Ken

    2007-01-01

    Everyone is familiar with the main percussion instruments of the contemporary orchestra: bass drum, snare drum, suspended cymbal, vibraphone, and timpani. But as source material broadens, so do the demands placed on the percussion section. Accessory, or auxiliary percussion, can make the difference between a typical rendition of a well-known piece…

  20. Intrahepatic accessory spleen: imaging features.

    PubMed

    Izzo, Luciano; Caputo, Maria; Galati, Gaspare

    2004-06-01

    The authors present a case report of a 60-year-old man with a hepatic unknown mass. For diagnosis, they used ECO, CT (with and without contrast), MR (with and without contrast) and an ultrasound-assisted percutaneous lesion biopsy. Thus the mass-lesion in the liver appeared to be an intrahepatic accessory spleen in a patient afflicted with chronic hepatitis.

  1. 14 CFR 25.1163 - Powerplant accessories.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Powerplant accessories. (a) Each engine mounted accessory must— (1) Be approved for mounting on the engine involved; (2) Use the provisions on the engine for mounting; and (3) Be sealed to prevent contamination of the engine oil system and the accessory system. (b) Electrical equipment subject to arcing or...

  2. Genetic organization and molecular characterization of secA2 locus in Listeria species.

    PubMed

    Mishra, Krishna K; Mendonca, Marcelo; Aroonnual, Amornrat; Burkholder, Kristin M; Bhunia, Arun K

    2011-12-10

    The translocation of proteins across the bacterial cell wall is carried out by the general secretory (Sec) system. Most bacteria have a single copy of the secA gene, with the exception of a few Gram-positive bacteria, which have an additional copy of secA, designated secA2. secA2 is present in Listeria monocytogenes and is responsible for secretion and translocation of several proteins including virulence factors; however, little is known about the secA2 gene and its genetic organization in nonpathogenic members of the genus Listeria. The goal of this study was to determine the presence of secA2 locus and analyze the genetic relatedness among pathogenic and nonpathogenic Listeria species. Cloning experiments revealed that secA2 is present in all analyzed pathogenic (L. monocytogenes and L. ivanovii) and nonpathogenic (L. welshimeri, L. innocua, L. seeligeri, L. grayi and L. marthii) Listeria species except L. rocourtiae. Likewise, SecA2 transcripts were also detected in all species. Sequence analysis further revealed that 2331 nucleotides (776 amino acids) are conserved in L. monocytogenes, L. welshimeri, L. innocua and L. marthii. Three nucleotides are deleted in L. ivanovii and L. seeligeri and six in L. grayi, resulting in amino acid counts of 775, 775 and 774, respectively. secA2 is flanked upstream by iap (encoding p60) and downstream by a putative membrane protein (lmo0583, lmo f2365_0613) in all analyzed Listeria species, demonstrating conserved genetic organization of the secA2 locus in pathogenic and nonpathogenic species. Deletion of secA2 in L. innocua impaired accumulation of SecA2 substrate, N-acetyl muramidase (NamA) in the cell wall, providing evidence for the presence of functional SecA2 in nonpathogenic Listeria.

  3. Caesium accumulation in yeast and plants is selectively repressed by loss of the SNARE Sec22p/SEC22.

    PubMed

    Dräxl, Stephan; Müller, Johannes; Li, Wei B; Michalke, Bernhard; Scherb, Hagen; Hense, Burkhard A; Tschiersch, Jochen; Kanter, Ulrike; Schäffner, Anton R

    2013-01-01

    The non-essential cation caesium (Cs(+)) is assimilated by all organisms. Thus, anthropogenically released radiocaesium is of concern to agriculture. Cs(+) accumulates owing to its chemical similarity to the potassium ion (K(+)). The apparent lack of a Cs(+)-specific uptake mechanism has obstructed attempts to manipulate Cs(+) accumulation without causing pleiotropic effects. Here we show that the SNARE protein Sec22p/SEC22 specifically impacts Cs(+) accumulation in yeast and in plants. Loss of Saccharomyces cerevisiae Sec22p does not affect K(+) homeostasis, yet halves Cs(+) concentration compared with the wild type. Mathematical modelling of the uptake time course predicts a compromised vacuolar Cs(+) deposition in sec22Δ. Biochemical fractionation confirms this and indicates a new feature of Sec22p in enhancing non-selective cation deposition. A developmentally controlled loss-of-function mutant of the orthologous Arabidopsis thaliana SEC22 phenocopies the reduced Cs(+) uptake without affecting plant growth. This finding provides a new strategy to reduce radiocaesium entry into the food chain.

  4. Sec1p Binds to Snare Complexes and Concentrates at Sites of Secretion

    PubMed Central

    Carr, Chavela M.; Grote, Eric; Munson, Mary; Hughson, Frederick M.; Novick, Peter J.

    1999-01-01

    Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion. PMID:10427089

  5. The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli

    SciTech Connect

    Kuhn, Misty L.; Martinez, Salette; Gumataotao, Natalie; Bornscheuer, Uwe; Liu, Dali; Holz, Richard C.

    2012-10-10

    We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the {alpha}- and {beta}-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase{sup +Act}) and without (ReNHase{sup -Act}) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4 {angstrom} resolution revealing an {alpha}{beta} heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.

  6. The Fe-type nitrile hydratase from Comamonas testosteroni Ni1 does not require an activator accessory protein for expression in Escherichia coli.

    PubMed

    Kuhn, Misty L; Martinez, Salette; Gumataotao, Natalie; Bornscheuer, Uwe; Liu, Dali; Holz, Richard C

    2012-08-03

    We report herein the functional expression of an Fe-type nitrile hydratase (NHase) without the co-expression of an activator protein or the Escherichia coli chaperone proteins GroES/EL. Soluble protein was obtained when the α- and β-subunit genes of the Fe-type NHase Comamonas testosteroni Ni1 (CtNHase) were synthesized with optimized E. coli codon usage and co-expressed. As a control, the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase) was expressed with (ReNHase(+Act)) and without (ReNHase(-Act)) its activator protein, establishing that expression of a fully functional, metallated ReNHase enzyme requires the co-expression of its activator protein, similar to all other Fe-type NHase enzymes reported to date, whereas the CtNHase does not. The X-ray crystal structure of CtNHase was determined to 2.4Å resolution revealing an αβ heterodimer, similar to other Fe-type NHase enzymes, except for two important differences. First, two His residues reside in the CtNHase active site that are not observed in other Fe-type NHase enzymes and second, the active site Fe(III) ion resides at the bottom of a wide solvent exposed channel. The solvent exposed active site, along with the two active site histidine residues, are hypothesized to play a role in iron incorporation in the absence of an activator protein.

  7. A central cavity within the holo-translocon suggests a mechanism for membrane protein insertion

    PubMed Central

    Botte, Mathieu; Zaccai, Nathan R.; Nijeholt, Jelger Lycklama à.; Martin, Remy; Knoops, Kèvin; Papai, Gabor; Zou, Juan; Deniaud, Aurélien; Karuppasamy, Manikandan; Jiang, Qiyang; Roy, Abhishek Singha; Schulten, Klaus; Schultz, Patrick; Rappsilber, Juri; Zaccai, Giuseppe; Berger, Imre; Collinson, Ian; Schaffitzel, Christiane

    2016-01-01

    The conserved SecYEG protein-conducting channel and the accessory proteins SecDF-YajC and YidC constitute the bacterial holo-translocon (HTL), capable of protein-secretion and membrane-protein insertion. By employing an integrative approach combining small-angle neutron scattering (SANS), low-resolution electron microscopy and biophysical analyses we determined the arrangement of the proteins and lipids within the super-complex. The results guided the placement of X-ray structures of individual HTL components and allowed the proposal of a model of the functional translocon. Their arrangement around a central lipid-containing pool conveys an unexpected, but compelling mechanism for membrane-protein insertion. The periplasmic domains of YidC and SecD are poised at the protein-channel exit-site of SecY, presumably to aid the emergence of translocating polypeptides. The SecY lateral gate for membrane-insertion is adjacent to the membrane ‘insertase’ YidC. Absolute-scale SANS employing a novel contrast-match-point analysis revealed a dynamic complex adopting open and compact configurations around an adaptable central lipid-filled chamber, wherein polytopic membrane-proteins could fold, sheltered from aggregation and proteolysis. PMID:27924919

  8. The specificity of association of the IgD molecule with the accessory proteins BAP31/BAP29 lies in the IgD transmembrane sequence.

    PubMed

    Adachi, T; Schamel, W W; Kim, K M; Watanabe, T; Becker, B; Nielsen, P J; Reth, M

    1996-04-01

    Mature B cells co-express on their cell surface two classes of antigen receptor, the IgM and IgD immunoglobulins. The structural and functional differences between the two receptor classes are poorly understood. Recently two proteins of 29 and 31 kDa (BAP29 and BAP31) have been described that are preferentially associated with membrane IgD but only weakly with membrane IgM. We describe here the cloning of full-length murine and human BAP31 cDNAs encoding proteins of 245 and 246 amino acids respectively. The two BAP31 proteins are 95% identical. The BAP31 gene is ubiquitously expressed in murine tissues and is located on the X chromosome in both mouse and man. The murine BAP31 protein has 43% sequence identity to murine BAP29. Both proteins have a hydrophobic N-terminus and an alpha-helical C-terminus which ends with a KKXX motif implicated in vesicular transport. By a mutational analysis we have identified amino acids in the transmembrane sequence of the delta m chain that are critical for binding to BAP31/BAP29. A structural model of the BAPs and their potential functions are discussed.

  9. The gene encoding the VP16-accessory protein HCF (HCFC1) resides in human Xq28 and is highly expressed in fetal tissues and the adult kidney

    SciTech Connect

    Wilson, A.C.; Herr, W.; Parrish, J.E.; Massa, H.F.

    1995-01-20

    After herpes simplex virus (HSV) infection, the viral regulatory protein VP16 activates transcription of the HSV immediate-early promoters by directing complex formation with two cellular proteins, the POU-homeodomain transcription factor Oct-1 and the host cell factor HCF. The function of HCF in uninfected cells is unknown. Here we show by fluorescence in situ hybridization and somatic cell hybrid analysis that the gene encoding human HCF, HCFC1, maps to the q28 region of the X chromosome. Yeast artificial chromosome and cosmid mapping localizes the HCFC1 gene within 100 kb distal of the renal vasopressin type-2 receptor (V2R) gene and adjacent to the renin-binding protein gene (RENBP). The HCFC1 gene is apparently unique. HCF transcripts and protein are most abundant in fetal and placental tissues and cell lines, suggesting a role in cell proliferation. In adults, HCF protein is abundant in the kidney, but not in the brain, a site of latent HSV infection and where HCF levels may influence progression of HSV infection. 42 refs., 3 figs.

  10. Locally vascularized pelvic accessory spleen.

    PubMed

    Iorio, F; Frantellizzi, V; Drudi, Francesco M; Maghella, F; Liberatore, M

    2016-01-01

    Polysplenism and accessory spleen are congenital, usually asymptomatic anomalies. A rare case of polysplenism with ectopic spleen in pelvis of a 67-year-old, Caucasian female is reported here. A transvaginal ultrasound found a soft well-defined homogeneous and vascularized mass in the left pelvis. Patient underwent MRI evaluation and contrast-CT abdominal scan: images with parenchymal aspect, similar to spleen were obtained. Abdominal scintigraphy with 99mTc-albumin nanocolloid was performed and pelvic region was studied with planar scans and SPECT. The results showed the presence of an uptake area of the radiopharmaceutical in the pelvis, while the spleen was normally visualized. These findings confirmed the presence of an accessory spleen with an artery originated from the aorta and a vein that joined with the superior mesenteric vein. To our knowledge, in the literature, there is just only one case of a true ectopic, locally vascularized spleen in the pelvis.

  11. Molecular genetic manipulation of Pichia pastoris SEC4 governs cell growth and glucoamylase secretion

    SciTech Connect

    Liu, S.-H.; Chou, W.-I; Lin, S.-C.; Sheu, C.-C.; Chang, Margaret Dah-Tsyr . E-mail: dtchang@life.nthu.edu.tw

    2005-11-04

    We have previously engineered a recombinant Pichia pastoris GS115 transformant, MSPGA-7, harboring seven copies of glucoamylase (GA) fused with modified signal peptide. High yield secretion of GA was achieved as an extra copy of SEC4 was integrated to the transformant. To elucidate the physiological role of SEC4, a dominant-negative mutant of SEC4, SEC4 {sub S28N}, was overexpressed under the control of alchohol oxidase 1 (AOX1) promoter in P. pastoris strain MSPGA-7 as well as a set of host cells harboring multi-copy of wild type SEC4. We found that SEC4 {sub S28N} mutation in the key guanine nucleotide binding domain reduced guanine nucleotide binding affinity, hence it blocked the transport of vesicles required for targeting and fusion to the plasma membrane. The inhibitory levels of cell growth and GA secretion were correlated with the dosage of SEC4 {sub S28N} gene. In addition, overexpression of SEC4 driven by AOX1 promoter in MSPGA-7 improved the secretory production of GA, but demonstrated the delay of cell growth by increased gene dosage of SEC4. Interestingly, a limited level of Sec4p did not disturb the cell growth. It was because expression of only one copy of SEC4 resulted in delay of cell growth at an early stage while still maintaining high level Sec4p at long-term incubation. Accordingly, as glyceraldehyde-3-phosphate dehydrogenase promoter was used to substitute AOX1 promoter to drive the SEC4 expression, enhanced GA secretion but not inhibition of cell growth was achieved. Taken together, our results demonstrate that SEC4 is essential for P. pastoris in regulating cell growth and heterologous protein secretion in a dosage-dependent manner.

  12. Quaternary dynamics of the SecA motor drive translocase catalysis.

    PubMed

    Gouridis, Giorgos; Karamanou, Spyridoula; Sardis, Marios Frantzeskos; Schärer, Martin Alexander; Capitani, Guido; Economou, Anastassios

    2013-12-12

    Most secretory preproteins exit bacterial cells through the protein translocase, comprising the SecYEG channel and the dimeric peripheral ATPase motor SecA. Energetic coupling to work remains elusive. We now demonstrate that translocation is driven by unusually dynamic quaternary changes in SecA. The dimer occupies several successive states with distinct protomer arrangements. SecA docks on SecYEG as a dimer and becomes functionally asymmetric. Docking occurs via only one protomer. The second protomer allosterically regulates downstream steps. Binding of one preprotein signal peptide to the SecYEG-docked SecA protomer elongates the SecA dimer and triggers the translocase holoenzyme to obtain a lower activation energy conformation. ATP hydrolysis monomerizes the triggered SecA dimer, causing mature chain trapping and processive translocation. This is a unique example of one protein exploiting quaternary dynamics to become a substrate receptor, a "loading clamp," and a "processive motor." This mechanism has widespread implications on protein translocases, chaperones, and motors.

  13. Anomalous behavior of water inside the SecY translocon.

    PubMed

    Capponi, Sara; Heyden, Matthias; Bondar, Ana-Nicoleta; Tobias, Douglas J; White, Stephen H

    2015-07-21

    The heterotrimeric SecY translocon complex is required for the cotranslational assembly of membrane proteins in bacteria and archaea. The insertion of transmembrane (TM) segments during nascent-chain passage through the translocon is generally viewed as a simple partitioning process between the water-filled translocon and membrane lipid bilayer, suggesting that partitioning is driven by the hydrophobic effect. Indeed, the apparent free energy of partitioning of unnatural aliphatic amino acids on TM segments is proportional to accessible surface area, which is a hallmark of the hydrophobic effect [Öjemalm K, et al. (2011) Proc Natl Acad Sci USA 108(31):E359-E364]. However, the apparent partitioning solvation parameter is less than one-half the value expected for simple bulk partitioning, suggesting that the water in the translocon departs from bulk behavior. To examine the state of water in a SecY translocon complex embedded in a lipid bilayer, we carried out all-atom molecular-dynamics simulations of the Pyrococcus furiosus SecYE, which was determined to be in a "primed" open state [Egea PF, Stroud RM (2010) Proc Natl Acad Sci USA 107(40):17182-17187]. Remarkably, SecYE remained in this state throughout our 450-ns simulation. Water molecules within SecY exhibited anomalous diffusion, had highly retarded rotational dynamics, and aligned their dipoles along the SecY transmembrane axis. The translocon is therefore not a simple water-filled pore, which raises the question of how anomalous water behavior affects the mechanism of translocon function and, more generally, the partitioning of hydrophobic molecules. Because large water-filled cavities are found in many membrane proteins, our findings may have broader implications.

  14. Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28.

    PubMed

    Smargon, Aaron A; Cox, David B T; Pyzocha, Neena K; Zheng, Kaijie; Slaymaker, Ian M; Gootenberg, Jonathan S; Abudayyeh, Omar A; Essletzbichler, Patrick; Shmakov, Sergey; Makarova, Kira S; Koonin, Eugene V; Zhang, Feng

    2017-02-16

    CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

  15. Sec13 Regulates Expression of Specific Immune Factors Involved in Inflammation In Vivo

    PubMed Central

    Moreira, Thais G.; Zhang, Liang; Shaulov, Lihi; Harel, Amnon; Kuss, Sharon K.; Williams, Jessica; Shelton, John; Somatilaka, Bandarigoda; Seemann, Joachim; Yang, Jue; Sakthivel, Ramanavelan; Nussenzveig, Daniel R.; Faria, Ana M. C.; Fontoura, Beatriz M. A.

    2015-01-01

    The Sec13 protein functions in various intracellular compartments including the nuclear pore complex, COPII-coated vesicles, and inside the nucleus as a transcription regulator. Here we developed a mouse model that expresses low levels of Sec13 (Sec13H/−) to assess its functions in vivo, as Sec13 knockout is lethal. These Sec13 mutant mice did not present gross defects in anatomy and physiology. However, the reduced levels of Sec13 in vivo yielded specific immunological defects. In particular, these Sec13 mutant mice showed low levels of MHC I and II expressed by macrophages, low levels of INF-γ and IL-6 expressed by stimulated T cells, and low frequencies of splenic IFN-γ+CD8+ T cells. In contrast, the levels of soluble and membrane-bound TGF-β as well as serum immunoglobulin production are high in these mice. Furthermore, frequencies of CD19+CD5-CD95+ and CD19+CD5-IL-4+ B cells were diminished in Sec13H/− mice. Upon stimulation or immunization, some of the defects observed in the naïve mutant mice were compensated. However, TGF-β expression remained high suggesting that Sec13 is a negative modulator of TGF-β expression and of its immunosuppressive functions on certain immune cells. In sum, Sec13 regulates specific expression of immune factors with key functions in inflammation. PMID:26631972

  16. Conformational Flexibility and Peptide Interaction of the Translocation ATPase SecA

    SciTech Connect

    Zimmer, Jochen; Rapoport, Tom A.; Harvard-Med

    2010-09-21

    The SecA ATPase forms a functional complex with the protein-conducting SecY channel to translocate polypeptides across the bacterial cell membrane. SecA recognizes the translocation substrate and catalyzes its unidirectional movement through the SecY channel. The recent crystal structure of the Thermotoga maritima SecA-SecYEG complex shows the ATPase in a conformation where the nucleotide-binding domains (NBDs) have closed around a bound ADP-BeFx complex and SecA's polypeptide-binding clamp is shut. Here, we present the crystal structure of T. maritima SecA in isolation, determined in its ADP-bound form at 3.1 {angstrom} resolution. SecA alone has a drastically different conformation in which the nucleotide-binding pocket between NBD1 and NBD2 is open and the preprotein cross-linking domain has rotated away from both NBDs, thereby opening the polypeptide-binding clamp. To investigate how this clamp binds polypeptide substrates, we also determined a structure of Bacillus subtilis SecA in complex with a peptide at 2.5 {angstrom} resolution. This structure shows that the peptide augments the highly conserved {beta}-sheet at the back of the clamp. Taken together, these structures suggest a mechanism by which ATP hydrolysis can lead to polypeptide translocation.

  17. Engine starter and accessory drive system

    SciTech Connect

    Stockton, T.R.

    1986-10-07

    An engine starter and accessory drive system is described which consists of: an accessory drive means; a planetary gearset having a sun gear driveably connected to the accessory drive means, a ring gear, a carrier and planet pinions rotatably mounted on the carrier, fixed to the engine crankshaft, meshing with the sun gear and with the ring gear; means for holding the ring gear against rotation; and a starter motor and first clutch means for providing a one-way driving connection between the motor and the accessory drive means.

  18. FRACTIONATION OF PLASMA GLOBULIN FOR PROTHROMBIN, THROMBOKINASE, AND ACCESSORY THROMBOPLASTIN

    PubMed Central

    Milstone, J. H.

    1951-01-01

    1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner. PMID:14873922

  19. Advanced Accessory Power Supply Topologies

    SciTech Connect

    Marlino, L.D.

    2010-06-15

    This Cooperative Research and Development Agreement (CRADA) began December 8, 2000 and ended September 30, 2009. The total funding provided by the Participant (General Motors Advanced Technology Vehicles [GM]) during the course of the CRADA totaled $1.2M enabling the Contractor (UT-Battelle, LLC [Oak Ridge National Laboratory, a.k.a. ORNL]) to contribute significantly to the joint project. The initial task was to work with GM on the feasibility of developing their conceptual approach of modifying major components of the existing traction inverter/drive to develop low cost, robust, accessory power. Two alternate methods for implementation were suggested by ORNL and both were proven successful through simulations and then extensive testing of prototypes designed and fabricated during the project. This validated the GM overall concept. Moreover, three joint U.S. patents were issued and subsequently licensed by GM. After successfully fulfilling the initial objective, the direction and duration of the CRADA was modified and GM provided funding for two additional tasks. The first new task was to provide the basic development for implementing a cascaded inverter technology into hybrid vehicles (including plug-in hybrid, fuel cell, and electric). The second new task was to continue the basic development for implementing inverter and converter topologies and new technology assessments for hybrid vehicle applications. Additionally, this task was to address the use of high temperature components in drive systems. Under this CRADA, ORNL conducted further research based on GM’s idea of using the motor magnetic core and windings to produce bidirectional accessory power supply that is nongalvanically coupled to the terminals of the high voltage dc-link battery of hybrid vehicles. In order not to interfere with the motor’s torque, ORNL suggested to use the zero-sequence, highfrequency harmonics carried by the main fundamental motor current for producing the accessory power

  20. 46 CFR Sec. 4 - Funding of operations.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Funding of operations. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Funding of Operations Sec. 4 Funding of operations....

  1. 46 CFR Sec. 4 - Funding of operations.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Funding of operations. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Funding of Operations Sec. 4 Funding of operations....

  2. 46 CFR Sec. 4 - Funding of operations.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Funding of operations. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL RULES FOR FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Funding of Operations Sec. 4 Funding of operations....

  3. 46 CFR Sec. 4 - Posting of bond.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Posting of bond. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 4 Posting of bond. The General Agent shall retain an executed copy of each such bond in its principal...

  4. 46 CFR Sec. 2 - Amount of bond.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Amount of bond. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 2 Amount of bond. The amount of the bond must be governed by the amount of monies advanced or value of...

  5. 46 CFR Sec. 2 - Amount of bond.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Amount of bond. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 2 Amount of bond. The amount of the bond must be governed by the amount of monies advanced or value of...

  6. 46 CFR Sec. 4 - Posting of bond.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Posting of bond. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 4 Posting of bond. The General Agent shall retain an executed copy of each such bond in its principal...

  7. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental...

  8. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental...

  9. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental...

  10. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental...

  11. 46 CFR Sec. 7 - Job order numbering.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Job order numbering. Sec. 7 Section 7 Shipping MARITIME... REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 7 Job order numbering. (a) The NSA-LUMPSUMREP Contract number shall be inserted in every job order and supplemental...

  12. 14 CFR Sec. 1-5 - Records.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Records. Sec. 1-5 Section 1-5 Aeronautics... Provisions Sec. 1-5 Records. (a) The general books of account and all books, records, and memoranda which... Accounts. Registers, or other appropriate records, shall be maintained of the history and nature of...

  13. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Preparation of invoices. Sec. 3 Section 3 Shipping... FOLLOWED BY GENERAL AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service...

  14. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Preparation of invoices. Sec. 3 Section 3 Shipping... FOLLOWED BY GENERAL AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service...

  15. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Preparation of invoices. Sec. 3 Section 3 Shipping... FOLLOWED BY GENERAL AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service...

  16. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Preparation of invoices. Sec. 3 Section 3 Shipping... FOLLOWED BY GENERAL AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service...

  17. 46 CFR Sec. 3 - Preparation of invoices.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Preparation of invoices. Sec. 3 Section 3 Shipping... FOLLOWED BY GENERAL AGENTS IN PREPARATION OF INVOICES AND PAYMENT OF COMPENSATION PURSUANT TO PROVISIONS OF NSA ORDER NO. 47 Sec. 3 Preparation of invoices. (a) Pursuant to Article 4 of the Service...

  18. 46 CFR Sec. 3 - Accounting for revenues.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Accounting for revenues. Sec. 3 Section 3 Shipping... FINANCIAL TRANSACTIONS UNDER AGENCY AGREEMENTS Accounting for Revenues Sec. 3 Accounting for revenues. (a... shipper, consignee, weight or measurement, freight rate and basis (whether the freight rate applies...

  19. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 8 2012-10-01 2012-10-01 false Ship Repair Summaries. Sec. 19 Section 19 Shipping... Sec. 19 Ship Repair Summaries. (a) Ship Repair Summaries shall be prepared on Form MA-159 by the... jurisdiction and submitted to the District Ship Repair and Maintenance office involved. The summaries must...

  20. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Ship Repair Summaries. Sec. 19 Section 19 Shipping... Sec. 19 Ship Repair Summaries. (a) Ship Repair Summaries shall be prepared on Form MA-159 by the... jurisdiction and submitted to the District Ship Repair and Maintenance office involved. The summaries must...

  1. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Ship Repair Summaries. Sec. 19 Section 19 Shipping... Sec. 19 Ship Repair Summaries. (a) Ship Repair Summaries shall be prepared on Form MA-159 by the... jurisdiction and submitted to the District Ship Repair and Maintenance office involved. The summaries must...

  2. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Ship Repair Summaries. Sec. 19 Section 19 Shipping... Sec. 19 Ship Repair Summaries. (a) Ship Repair Summaries shall be prepared on Form MA-159 by the... jurisdiction and submitted to the District Ship Repair and Maintenance office involved. The summaries must...

  3. 46 CFR Sec. 19 - Ship Repair Summaries.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Ship Repair Summaries. Sec. 19 Section 19 Shipping... Sec. 19 Ship Repair Summaries. (a) Ship Repair Summaries shall be prepared on Form MA-159 by the... jurisdiction and submitted to the District Ship Repair and Maintenance office involved. The summaries must...

  4. 46 CFR Sec. 4 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false General provisions. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY SLOP CHESTS Sec. 4 General provisions. (a) All slop chest items, damaged or otherwise, shall be removed or transferred only in...

  5. 46 CFR Sec. 6 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false General provisions. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY REPATRIATION OF SEAMEN Sec. 6 General provisions. (a) In case of repatriation of any seaman as a passenger aboard a vessel operated for account...

  6. 46 CFR Sec. 4 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false General provisions. Sec. 4 Section 4 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY AUTHORITY AND RESPONSIBILITY OF GENERAL AGENTS TO UNDERTAKE EMERGENCY REPAIRS IN FOREIGN PORTS Sec. 4 General provisions. The...

  7. 46 CFR Sec. 6 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false General provisions. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY VOYAGE DATA Sec. 6 General provisions. (a) In cases of overlapping activities and all other questions arising in respect to...

  8. 46 CFR Sec. 6 - Awarding of work.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Awarding of work. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURE FOR ACCOMPLISHMENT OF VESSEL REPAIRS UNDER NATIONAL SHIPPING AUTHORITY MASTER LUMP SUM REPAIR CONTRACT-NSA-LUMPSUMREP Sec. 6...

  9. Kv4.2 and accessory dipeptidyl peptidase-like protein 10 (DPP10) subunit preferentially form a 4:2 (Kv4.2:DPP10) channel complex.

    PubMed

    Kitazawa, Masahiro; Kubo, Yoshihiro; Nakajo, Koichi

    2015-09-11

    Kv4 is a member of the voltage-gated K(+) channel family and forms a complex with various accessory subunits. Dipeptidyl aminopeptidase-like protein (DPP) is one of the auxiliary subunits for the Kv4 channel. Although DPP has been well characterized and is known to increase the current amplitude and accelerate the inactivation and recovery from inactivation of Kv4 current, it remains to be determined how many DPPs bind to one Kv4 channel. To examine whether the expression level of DPP changes the biophysical properties of Kv4, we expressed Kv4.2 and DPP10 in different ratios in Xenopus oocytes and analyzed the currents under two-electrode voltage clamp. The current amplitude and the speed of recovery from inactivation of Kv4.2 changed depending on the co-expression level of DPP10. This raised the possibility that the stoichiometry of the Kv4.2-DPP10 complex is variable and affects the biophysical properties of Kv4.2. We next determined the stoichiometry of DPP10 alone by subunit counting using single-molecule imaging. Approximately 70% of the DPP10 formed dimers in the plasma membrane, and the rest existed as monomers in the absence of Kv4.2. We next determined the stoichiometry of the Kv4.2-DPP10 complex; Kv4.2-mCherry and mEGFP-DPP10 were co-expressed in different ratios and the stoichiometries of Kv4.2-DPP10 complexes were evaluated by the subunit counting method. The stoichiometry of the Kv4.2-DPP10 complex was variable depending on the relative expression level of each subunit, with a preference for 4:2 stoichiometry. This preference may come from the bulky dimeric structure of the extracellular domain of DPP10.

  10. Extracranial spinal accessory nerve injury.

    PubMed

    Donner, T R; Kline, D G

    1993-06-01

    Eighty-three consecutive patients with extracranial accessory nerve injury seen over a 12-year period are reviewed. The most common etiology was iatrogenic injury to the nerve at the time of previous surgery. Such operations were usually minor in nature and often related to lymph node or benign tumor removal. Examination usually distinguished winging due to trapezius weakness from that of serratus anterior palsy. Trapezius weakness was seen in all cases. Sternocleidomastoid weakness was unusual. Patients with accessory palsy were evaluated by both clinical and electromyographic studies. Patients who exhibited no clinical or electrical evidence of regeneration were operated on (44 cases). Based on intraoperative nerve action potential studies, 8 lesions in continuity had neurolysis alone. Resection with repair either by end-to-end suture or by grafts was necessary in 31 cases. One case had suture removed from nerve, two had nerve placed into target muscle, and two had more proximal neurotization. Function was usually improved in both operative and nonoperative patients. Related anatomy is discussed.

  11. A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin.

    PubMed Central

    Garcia, E P; Gatti, E; Butler, M; Burton, J; De Camilli, P

    1994-01-01

    Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE. Images PMID:8134339

  12. Sec62 bridges the gap from 3q amplification to molecular cell biology in non-small cell lung cancer.

    PubMed

    Linxweiler, Maximilian; Linxweiler, Johannes; Barth, Monika; Benedix, Julia; Jung, Volker; Kim, Yoo-Jin; Bohle, Rainer M; Zimmermann, Richard; Greiner, Markus

    2012-02-01

    The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

  13. 21 CFR 890.5925 - Traction accessory.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Traction accessory. 890.5925 Section 890.5925 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Therapeutic Devices § 890.5925 Traction accessory....

  14. 21 CFR 890.5925 - Traction accessory.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Traction accessory. 890.5925 Section 890.5925 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Therapeutic Devices § 890.5925 Traction accessory....

  15. 21 CFR 890.5925 - Traction accessory.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Traction accessory. 890.5925 Section 890.5925 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Therapeutic Devices § 890.5925 Traction accessory....

  16. 21 CFR 890.5925 - Traction accessory.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Traction accessory. 890.5925 Section 890.5925 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Therapeutic Devices § 890.5925 Traction accessory....

  17. 21 CFR 890.5925 - Traction accessory.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Traction accessory. 890.5925 Section 890.5925 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Therapeutic Devices § 890.5925 Traction accessory....

  18. The cytohesin paralog Sec7 of Dictyostelium discoideum is required for phagocytosis and cell motility

    PubMed Central

    2013-01-01

    Background Dictyostelium harbors several paralogous Sec7 genes that encode members of three subfamilies of the Sec7 superfamily of guanine nucleotide exchange factors. One of them is the cytohesin family represented by three members in D. discoideum, SecG, Sec7 and a further protein distinguished by several transmembrane domains. Cytohesins are characterized by a Sec7-PH tandem domain and have roles in cell adhesion and migration. Results We study here Sec7. In vitro its PH domain bound preferentially to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). When following the distribution of GFP-Sec7 in vivo we observed the protein in the cytosol and at the plasma membrane. Strikingly, when cells formed pseudopods, macropinosomes or phagosomes, GFP-Sec7 was conspicuously absent from areas of the plasma membrane which were involved in these processes. Mutant cells lacking Sec7 exhibited an impaired phagocytosis and showed significantly reduced speed and less persistence during migration. Cellular properties associated with mammalian cytohesins like cell-cell and cell-substratum adhesion were not altered. Proteins with roles in membrane trafficking and signal transduction have been identified as putative interaction partners consistent with the data obtained from mutant analysis. Conclusions Sec7 is a cytosolic component and is associated with the plasma membrane in a pattern distinctly different from the accumulation of PI(3,4,5)P3. Mutant analysis reveals that loss of the protein affects cellular processes that involve membrane flow and the actin cytoskeleton. PMID:23915312

  19. SEC14 is a specific requirement for secretion of phospholipase B1 and pathogenicity of Cryptococcus neoformans

    PubMed Central

    Chayakulkeeree, Methee; Johnston, Simon Andrew; Oei, Johanes Bijosono; Lev, Sophie; Williamson, Peter Richard; Wilson, Christabel Frewen; Zuo, Xiaoming; Leal, Ana Lusia; Vainstein, Marilene Henning; Meyer, Wieland; Sorrell, Tania Christine; May, Robin Charles; Djordjevic, Julianne Teresa

    2011-01-01

    Summary Secreted phospholipase B1 (CnPlb1) is essential for dissemination of Cryptococcus neoformans to the central nervous system (CNS) yet essential components of its secretion machinery remain to be elucidated. Using gene deletion analysis we demonstrate that CnPlb1 secretion is dependent on the CnSEC14 product, CnSec14-1p. CnSec14-1p is a homologue of the phosphatidylinositol transfer protein (PITP) ScSec14p, which is essential for secretion and viability in Saccharomyces cerevisiae. In contrast to CnPlb1, neither laccase 1 (Lac1)-induced melanization within the cell wall nor capsule induction were negatively impacted in CnSEC14-1 deletion mutants (CnΔsec14-1 and CnΔsec14-1CnΔsfh5). Similar to the CnPLB1 deletion mutant (CnΔplb1), CnΔsec14-1 was hypo-virulent in mice and did not disseminate to the CNS by day 14 post infection. Furthermore, macrophage expulsion of live CnΔsec14-1 and CnΔplb1 (vomocytosis) was reduced. Individual deletion of CnSEC14-2, a closely-related CnSEC14-1 homologue, and CnSFH5, a distantly-related SEC fourteen-like homologue, did not abrogate CnPlb1 secretion or virulence. However, reconstitution of CnΔsec14-1 with CnSEC14-1 or CnSEC14-2 restored both phenotypes, consistent with functional genetic redundancy. We conclude that CnPlb1 secretion is SEC14-dependent and that C. neoformans preferentially exports virulence determinants to the cell periphery via distinct pathways. We also demonstrate that CnPlb1 secretion is essential for vomocytosis. PMID:21453402

  20. Exocyst SEC3 and Phosphoinositides Define Sites of Exocytosis in Pollen Tube Initiation and Growth1[OPEN

    PubMed Central

    Bloch, Daria; Pleskot, Roman; Vukašinović, Nemanja

    2016-01-01

    Polarized exocytosis is critical for pollen tube growth, but its localization and function are still under debate. The exocyst vesicle-tethering complex functions in polarized exocytosis. Here, we show that a sec3a exocyst subunit null mutant cannot be transmitted through the male gametophyte due to a defect in pollen tube growth. The green fluorescent protein (GFP)-SEC3a fusion protein is functional and accumulates at or proximal to the pollen tube tip plasma membrane. Partial complementation of sec3a resulted in the development of pollen with multiple tips, indicating that SEC3 is required to determine the site of pollen germination pore formation. Time-lapse imaging demonstrated that SEC3a and SEC8 were highly dynamic and that SEC3a localization on the apical plasma membrane predicts the direction of growth. At the tip, polar SEC3a domains coincided with cell wall deposition. Labeling of GFP-SEC3a-expressing pollen with the endocytic marker FM4-64 revealed the presence of subdomains on the apical membrane characterized by extensive exocytosis. In steady-state growing tobacco (Nicotiana tabacum) pollen tubes, SEC3a displayed amino-terminal Pleckstrin homology-like domain (SEC3a-N)-dependent subapical membrane localization. In agreement, SEC3a-N interacted with phosphoinositides in vitro and colocalized with a phosphatidylinositol 4,5-bisphosphate (PIP2) marker in pollen tubes. Correspondingly, molecular dynamics simulations indicated that SEC3a-N associates with the membrane by interacting with PIP2. However, the interaction with PIP2 is not required for polar localization and the function of SEC3a in Arabidopsis (Arabidopsis thaliana). Taken together, our findings indicate that SEC3a is a critical determinant of polar exocytosis during tip growth and suggest differential regulation of the exocytotic machinery depending on pollen tube growth modes. PMID:27516531

  1. Sec16 alternative splicing dynamically controls COPII transport efficiency

    PubMed Central

    Wilhelmi, Ilka; Kanski, Regina; Neumann, Alexander; Herdt, Olga; Hoff, Florian; Jacob, Ralf; Preußner, Marco; Heyd, Florian

    2016-01-01

    The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. Here we show that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, we show that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, we suggest the C-terminal Sec16 domain to be a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. Our work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments. PMID:27492621

  2. Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization

    PubMed Central

    Johansen, Jesper; Alfaro, Gabriel; Beh, Christopher T.

    2016-01-01

    Polarized growth is maintained by both polarized exocytosis, which transports membrane components to specific locations on the cell cortex, and endocytosis, which retrieves these components before they can diffuse away. Despite functional links between these two transport pathways, they are generally considered to be separate events. Using live cell imaging, in vivo and in vitro protein binding assays, and in vitro pyrene-actin polymerization assays, we show that the yeast Rab GTPase Sec4p couples polarized exocytosis with cortical actin polymerization, which induces endocytosis. After polarized exocytosis to the plasma membrane, Sec4p binds Las17/Bee1p (yeast Wiskott—Aldrich Syndrome protein [WASp]) in a complex with Sla1p and Sla2p during actin patch assembly. Mutations that inactivate Sec4p, or its guanine nucleotide exchange factor (GEF) Sec2p, inhibit actin patch formation, whereas the activating sec4-Q79L mutation accelerates patch assembly. In vitro assays of Arp2/3-dependent actin polymerization established that GTPγS-Sec4p overrides Sla1p inhibition of Las17p-dependent actin nucleation. These results support a model in which Sec4p relocates along the plasma membrane from polarized sites of exocytic vesicle fusion to nascent sites of endocytosis. Activated Sec4p then promotes actin polymerization and triggers compensatory endocytosis, which controls surface expansion and kinetically refines cell polarization. PMID:27526190

  3. Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules.

    PubMed

    Bonardi, Francesco; Halza, Erik; Walko, Martin; Du Plessis, François; Nouwen, Nico; Feringa, Ben L; Driessen, Arnold J M

    2011-05-10

    Protein translocation in Escherichia coli is mediated by the translocase that in its minimal form consists of the protein-conducting channel SecYEG, and the motor protein, SecA. SecYEG forms a narrow pore in the membrane that allows passage of unfolded proteins only. Molecular dynamics simulations suggest that the maximal width of the central pore of SecYEG is limited to . To access the functional size of the SecYEG pore, the precursor of outer membrane protein A was modified with rigid spherical tetraarylmethane derivatives of different diameters at a unique cysteine residue. SecYEG allowed the unrestricted passage of the precursor of outer membrane protein A conjugates carrying tetraarylmethanes with diameters up to , whereas a sized molecule blocked the translocation pore. Translocation of the protein-organic molecule hybrids was strictly proton motive force-dependent and occurred at a single pore. With an average diameter of an unfolded polypeptide chain of , the pore accommodates structures of at least , which is vastly larger than the predicted maximal width of a single pore by molecular dynamics simulations.

  4. SEC sensor parametric test and evaluation system

    NASA Technical Reports Server (NTRS)

    1978-01-01

    This system provides the necessary automated hardware required to carry out, in conjunction with the existing 70 mm SEC television camera, the sensor evaluation tests which are described in detail. The Parametric Test Set (PTS) was completed and is used in a semiautomatic data acquisition and control mode to test the development of the 70 mm SEC sensor, WX 32193. Data analysis of raw data is performed on the Princeton IBM 360-91 computer.

  5. Exocyst subunit SEC3A marks the germination site and is essential for pollen germination in Arabidopsis thaliana

    PubMed Central

    Li, Yan; Tan, Xiaoyun; Wang, Mengru; Li, Bingxuan; Zhao, Yanxue; Wu, Chengyun; Rui, Qingchen; Wang, Junxia; Liu, Zhongyuan; Bao, Yiqun

    2017-01-01

    Arabidopsis exocyst subunit SEC3A has been reported to participate in embryo development. Here we report that SEC3A is involved during pollen germination. A T-DNA insertion in SEC3A leads to an absolute, male-specific transmission defect that can be complemented by the expression of SEC3A coding sequence from the LAT52 promoter or SEC3A genomic DNA. No obvious abnormalities in the microgametogenesis are observed in the sec3a/SEC3A mutant, however, in vitro and in vivo pollen germination are defective. Further studies reveal that the callose, pectin, and cellulose are apparently not deposited at the germination site during pollen germination. SEC3A is expressed ubiquitously, including in pollen grains and pollen tubes. Notably, SEC3A-GFP fusion proteins are specifically recruited to the future pollen germination site. This particular localization pattern is independent of phosphatidylinositol 4,5-bisphosphate (PI-4,5P2), although SEC3-HIS fusion proteins are able to bind to several phosphoinositols in vitro. These results suggest that SEC3A plays an important role in the establishment of the polar site for pollen germination. PMID:28074928

  6. Coevolution of the ATPase ClpV, the sheath proteins TssB and TssC, and the accessory protein TagJ/HsiE1 distinguishes type VI secretion classes.

    PubMed

    Förster, Andreas; Planamente, Sara; Manoli, Eleni; Lossi, Nadine S; Freemont, Paul S; Filloux, Alain

    2014-11-21

    The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA(+) ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one of the classes. Using bacterial two-hybrid approaches, we showed that the P. aeruginosa homolog HsiE1 interacts strongly with ClpV1. We then resolved the crystal structure of HsiE1 in complex with the N terminus of HsiB1, a TssB homolog and component of the contractile sheath. Phylogenetic analysis confirmed that these differences distinguish T6SS classes that resulted from a functional co-evolution between TssB, TssC, TagJ/HsiE, and ClpV. The interaction of TagJ/HsiE with the sheath as well as with ClpV suggests an alternative mode of disassembly in which HsiE recruits the ATPase to the sheath.

  7. Coevolution of the ATPase ClpV, the Sheath Proteins TssB and TssC, and the Accessory Protein TagJ/HsiE1 Distinguishes Type VI Secretion Classes*

    PubMed Central

    Förster, Andreas; Planamente, Sara; Manoli, Eleni; Lossi, Nadine S.; Freemont, Paul S.; Filloux, Alain

    2014-01-01

    The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA+ ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one of the classes. Using bacterial two-hybrid approaches, we showed that the P. aeruginosa homolog HsiE1 interacts strongly with ClpV1. We then resolved the crystal structure of HsiE1 in complex with the N terminus of HsiB1, a TssB homolog and component of the contractile sheath. Phylogenetic analysis confirmed that these differences distinguish T6SS classes that resulted from a functional co-evolution between TssB, TssC, TagJ/HsiE, and ClpV. The interaction of TagJ/HsiE with the sheath as well as with ClpV suggests an alternative mode of disassembly in which HsiE recruits the ATPase to the sheath. PMID:25305017

  8. Normal Variants: Accessory Muscles About the Ankle.

    PubMed

    Cheung, Yvonne

    2017-02-01

    Accessory muscles around the ankle are commonly encountered as incidental findings on cross-sectional imaging. Mostly asymptomatic, accessory muscles sometimes mimic mass lesions. They have been implicated as the cause of tarsal tunnel syndrome, impingement of surrounding structures, and chronic pain. Distinguishing these muscles can be challenging, because some travel along a similar path. This article describes these accessory muscles in detail, including their relationships to the aponeurosis of the lower leg. An imaging algorithm is proposed to aid in identification of these muscles, providing a valuable tool in diagnostic accuracy and subsequent patient management.

  9. Manipulation of immunometabolism by HIV-accessories to the crime?

    PubMed

    Matheson, Nicholas J; Greenwood, Edward Jd; Lehner, Paul J

    2016-08-01

    Evolutionary pressure has produced an 'arms race' between cellular restriction factors (limiting viral replication) and viral proteins (overcoming host restriction). The host factors SAMHD1 and SLFN1 patrol metabolic bottlenecks required for HIV replication. Conversely, the HIV accessory proteins Vpx, Vpu and Nef manipulate cellular metabolism to enable viral replication. Recent work identifying Vpu-mediated downregulation of the alanine transporter SNAT1 and Nef-mediated downregulation of the serine carriers SERINC3/5 has uncovered the importance of HIV manipulation of the amino acid supply. Interference with CD4(+) T-cell amino acid metabolism suggests a novel paradigm of viral immunomodulation, and signposts fundamental aspects of lymphocyte biology.

  10. Development of a Microemulsion Formulation for Antimicrobial SecA Inhibitors

    PubMed Central

    Wang, Nian

    2016-01-01

    In our previous study, we have identified five antimicrobial small molecules via structure based design, which inhibit SecA of Candidatus Liberibacter asiaticus (Las). SecA is a critical protein translocase ATPase subunit and is involved in pre-protein translocation across and integration into the cellular membrane in bacteria. In this study, eleven compounds were identified using similarity search method based on the five lead SecA inhibitors identified previously. The identified SecA inhibitors have poor aqueous solubility. Thus a microemulsion master mix (MMX) was developed to address the solubility issue and for application of the antimicrobials. MMX consists of N-methyl-2-pyrrolidone and dimethyl sulfoxide as solvent and co-solvent, as well as polyoxyethylated castor oil, polyalkylene glycol, and polyoxyethylene tridecyl ether phosphate as surfactants. MMX has significantly improved the solubility of SecA inhibitors and has no or little phytotoxic effects at concentrations less than 5.0% (v/v). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the SecA inhibitors and streptomycin against eight bacteria including Agrobacterium tumefaciens, Liberibacter crescens, Rhizobium etli, Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti phylogenetically related to Las were determined using the broth microdilution method. MIC and MBC results showed that the 16 SecA inhibitors have antibacterial activities comparable to that of streptomycin. Overall, we have identified 11 potent SecA inhibitors using similarity search method. We have developed a microemulsion formulation for SecA inhibitors which improved the antimicrobial activities of SecA inhibitors. PMID:26963811

  11. 19 CFR 10.456 - Accessories, spare parts or tools.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Accessories, spare parts or tools. 10.456 Section... Trade Agreement Rules of Origin § 10.456 Accessories, spare parts or tools. Accessories, spare parts or tools that form part of the good's standard accessories, spare parts or tools and are delivered with...

  12. The Accessory Genome of Pseudomonas aeruginosa

    PubMed Central

    Kung, Vanderlene L.; Ozer, Egon A.; Hauser, Alan R.

    2010-01-01

    Summary: Pseudomonas aeruginosa strains exhibit significant variability in pathogenicity and ecological flexibility. Such interstrain differences reflect the dynamic nature of the P. aeruginosa genome, which is composed of a relatively invariable “core genome” and a highly variable “accessory genome.” Here we review the major classes of genetic elements comprising the P. aeruginosa accessory genome and highlight emerging themes in the acquisition and functional importance of these elements. Although the precise phenotypes endowed by the majority of the P. aeruginosa accessory genome have yet to be determined, rapid progress is being made, and a clearer understanding of the role of the P. aeruginosa accessory genome in ecology and infection is emerging. PMID:21119020

  13. Allosteric regulation of SecA: magnesium-mediated control of conformation and activity.

    PubMed

    Gold, Vicki A M; Robson, Alice; Clarke, Anthony R; Collinson, Ian

    2007-06-15

    In bacteria, the SecA protein associates with a ubiquitous protein channel SecYEG where it drives the post-translational secretion of pre-proteins across the plasma membrane. The high-resolution structures of both proteins have been determined in their resting states; however, the mechanism that couples ATP hydrolysis to active transport of substrate proteins through the membrane is not well understood. An analysis of the steady-state ATPase activity of the enzyme reveals that there is an allosteric binding site for magnesium distinct from that associated with hydrolysis of ATP. We have demonstrated that this regulation involves a large conformational change to the SecA dimer, which exerts a strong influence on the turnover and affinity for ATP, as well as the affinity for ADP. The strong inhibitory influence of magnesium on the ATPase activity can be countered by cardiolipin and conditions that promote protein translocation.

  14. Variation in the ribosome interacting loop of the Sec61α from Giardia lamblia.

    PubMed

    Sinha, Abhishek; Ray, Atrayee; Ganguly, Sandipan; Ghosh Dastidar, Shubhra; Sarkar, Srimonti

    2015-09-30

    The interaction between the ribosome and the endoplasmic reticulum-located Sec61 protein translocon is mediated through an arginine residue of Sec61α, which is conserved in all prokaryotic and eukaryotic orthologues characterized to date. Using in silico approaches we report that instead of arginine, this ribosome-interaction function is most likely discharged by a lysine residue in the protist Giardia lamblia. This functional substitution of the R with a K in GlSec61α may have taken place to accommodate a G-rich rRNA.

  15. 21 CFR 878.4950 - Manual operating table and accessories and manual operating chair and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Manual operating table and accessories and manual operating chair and accessories. 878.4950 Section 878.4950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY...

  16. 21 CFR 878.4960 - Operating tables and accessories and operating chairs and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Operating tables and accessories and operating chairs and accessories. 878.4960 Section 878.4960 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES...

  17. 21 CFR 878.4960 - Operating tables and accessories and operating chairs and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Operating tables and accessories and operating chairs and accessories. 878.4960 Section 878.4960 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES...

  18. 21 CFR 878.4950 - Manual operating table and accessories and manual operating chair and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Manual operating table and accessories and manual operating chair and accessories. 878.4950 Section 878.4950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY...

  19. 21 CFR 878.4960 - Operating tables and accessories and operating chairs and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Operating tables and accessories and operating chairs and accessories. 878.4960 Section 878.4960 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES...

  20. 21 CFR 878.4950 - Manual operating table and accessories and manual operating chair and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Manual operating table and accessories and manual operating chair and accessories. 878.4950 Section 878.4950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY...

  1. 21 CFR 878.4950 - Manual operating table and accessories and manual operating chair and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Manual operating table and accessories and manual operating chair and accessories. 878.4950 Section 878.4950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY...

  2. 21 CFR 878.4950 - Manual operating table and accessories and manual operating chair and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Manual operating table and accessories and manual operating chair and accessories. 878.4950 Section 878.4950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY...

  3. 21 CFR 878.4960 - Operating tables and accessories and operating chairs and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Operating tables and accessories and operating chairs and accessories. 878.4960 Section 878.4960 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES...

  4. 21 CFR 878.4960 - Operating tables and accessories and operating chairs and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Operating tables and accessories and operating chairs and accessories. 878.4960 Section 878.4960 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES...

  5. 46 CFR Sec. 4 - Evidence required.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONNECTION WITH FOREIGN REPAIR CUSTOM'S ENTRIES Sec. 4 Evidence required. When relief is claimed on the grounds of stress of weather or other casualty, there must be submitted to the Collector the following: (a) An affidavit of the Master which shall set out fully the nature of the casualty and/or stress...

  6. 46 CFR Sec. 4 - Evidence required.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONNECTION WITH FOREIGN REPAIR CUSTOM'S ENTRIES Sec. 4 Evidence required. When relief is claimed on the grounds of stress of weather or other casualty, there must be submitted to the Collector the following: (a) An affidavit of the Master which shall set out fully the nature of the casualty and/or stress...

  7. 46 CFR Sec. 4 - Evidence required.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONNECTION WITH FOREIGN REPAIR CUSTOM'S ENTRIES Sec. 4 Evidence required. When relief is claimed on the grounds of stress of weather or other casualty, there must be submitted to the Collector the following: (a) An affidavit of the Master which shall set out fully the nature of the casualty and/or stress...

  8. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Master's requirements. Sec. 3 Section 3 Shipping... Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items... the voyage. (e) Cause entry to be made in the ship's log authenticated by the person designated by...

  9. 46 CFR Sec. 3 - General provisions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false General provisions. Sec. 3 Section 3 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY AUTHORITY AND RESPONSIBILITY OF... General provisions. (a) The voyage repairs, as covered by section 2(a), may be awarded by the...

  10. 14 CFR Sec. 1-5 - Records.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-5 Records. (a) The general books of account and all books, records, and memoranda which... accounting records in a limited technical sense, but all other records such as organization tables and...

  11. 46 CFR Sec. 3 - Master's requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Master's requirements. Sec. 3 Section 3 Shipping... Master's requirements. The Master shall: (a) Receive and receipt for the quantities of slop chest items... therefor, at the unit price, specified by the Slop Chest Statement furnished the Master by the...

  12. The Structure of Sec12 Implicates Potassium Ion Coordination in Sar1 Activation*

    PubMed Central

    McMahon, Conor; Studer, Sean M.; Clendinen, Chaevia; Dann, Geoffrey P.; Jeffrey, Philip D.; Hughson, Frederick M.

    2012-01-01

    Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a β propeller fold. Conserved residues cluster around a loop we term the “K loop,” which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange. PMID:23109340

  13. Smallpox vaccination techniques. 2. Accessories and aftercare.

    PubMed

    Baxby, Derrick

    2003-03-28

    The various accessories used for smallpox vaccination are surveyed. These included modified vaccination instruments and various other items which facilitated the procedure, containers for preservation and transport of vaccine, sterilising equipment, aids to interpretation and recording, and a variety of skin preparations and dressings. Three phases can be discerned in the development and use of such items and procedures. Initially, in the pre-bacteriological era, there was little need for accessory equipment apart from the means of preserving and transporting vaccine. Later, particularly by the end of the 19th century, the importance of aseptic and antiseptic procedures was realised, use was made of more traumatic vaccination techniques and glass capillaries became the standard method for preservation and transport. All this led to the increasing availability of a wide range of accessories, particularly of skin preparations and dressings. Finally, from about 1930, it was appreciated that skin preparation and dressings were often unnecessary, and could be counter-productive. So, although accessories for this were still available their use was very much reduced. In some respects the use of accessories during this last phase, based on scientific analysis was a return to the earliest, 'pre-scientific', era.

  14. SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes

    PubMed Central

    Bruno, Joanne; Chaudhary, Natasha; Iaea, David

    2016-01-01

    RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10–SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4. PMID:27354378

  15. SEC16A is a RAB10 effector required for insulin-stimulated GLUT4 trafficking in adipocytes.

    PubMed

    Bruno, Joanne; Brumfield, Alexandria; Chaudhary, Natasha; Iaea, David; McGraw, Timothy E

    2016-07-04

    RAB10 is a regulator of insulin-stimulated translocation of the GLUT4 glucose transporter to the plasma membrane (PM) of adipocytes, which is essential for whole-body glucose homeostasis. We establish SEC16A as a novel RAB10 effector in this process. Colocalization of SEC16A with RAB10 is augmented by insulin stimulation, and SEC16A knockdown attenuates insulin-induced GLUT4 translocation, phenocopying RAB10 knockdown. We show that SEC16A and RAB10 promote insulin-stimulated mobilization of GLUT4 from a perinuclear recycling endosome/TGN compartment. We propose RAB10-SEC16A functions to accelerate formation of the vesicles that ferry GLUT4 to the PM during insulin stimulation. Because GLUT4 continually cycles between the PM and intracellular compartments, the maintenance of elevated cell-surface GLUT4 in the presence of insulin requires accelerated biogenesis of the specialized GLUT4 transport vesicles. The function of SEC16A in GLUT4 trafficking is independent of its previously characterized activity in ER exit site formation and therefore independent of canonical COPII-coated vesicle function. However, our data support a role for SEC23A, but not the other COPII components SEC13, SEC23B, and SEC31, in the insulin stimulation of GLUT4 trafficking, suggesting that vesicles derived from subcomplexes of COPII coat proteins have a role in the specialized trafficking of GLUT4.

  16. Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase1[OPEN

    PubMed Central

    2017-01-01

    Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40. PMID:28213560

  17. The Balance between Recombination Enzymes and Accessory Replicative Helicases in Facilitating Genome Duplication

    PubMed Central

    Syeda, Aisha H.; Atkinson, John; Lloyd, Robert G.; McGlynn, Peter

    2016-01-01

    Accessory replicative helicases aid the primary replicative helicase in duplicating protein-bound DNA, especially transcribed DNA. Recombination enzymes also aid genome duplication by facilitating the repair of DNA lesions via strand exchange and also processing of blocked fork DNA to generate structures onto which the replisome can be reloaded. There is significant interplay between accessory helicases and recombination enzymes in both bacteria and lower eukaryotes but how these replication repair systems interact to ensure efficient genome duplication remains unclear. Here, we demonstrate that the DNA content defects of Escherichia coli cells lacking the strand exchange protein RecA are driven primarily by conflicts between replication and transcription, as is the case in cells lacking the accessory helicase Rep. However, in contrast to Rep, neither RecA nor RecBCD, the helicase/exonuclease that loads RecA onto dsDNA ends, is important for maintaining rapid chromosome duplication. Furthermore, RecA and RecBCD together can sustain viability in the absence of accessory replicative helicases but only when transcriptional barriers to replication are suppressed by an RNA polymerase mutation. Our data indicate that the minimisation of replisome pausing by accessory helicases has a more significant impact on successful completion of chromosome duplication than recombination-directed fork repair. PMID:27483323

  18. Controlled Speed Accessory Drive demonstration program

    NASA Technical Reports Server (NTRS)

    Hoehn, F. W.

    1981-01-01

    A Controlled Speed Accessory Drive System was examined in an effort to improve the fuel economy of passenger cars. Concept feasibility and the performance of a typical system during actual road driving conditions were demonstrated. The CSAD system is described as a mechanical device which limits engine accessory speeds, thereby reducing parasitic horsepower losses and improving overall vehicle fuel economy. Fuel consumption data were compiled for fleets of GSA vehicles. Various motor pool locations were selected, each representing different climatic conditions. On the basis of a total accumulated fleet usage of nearly three million miles, an overall fuel economy improvement of 6 percent to 7 percent was demonstrated. Coincident chassis dynamometer tests were accomplished on selected vehicles to establish the effect of different accessory drive systems on exhaust emissions, and to evaluate the magnitude of the mileage benefits which could be derived.

  19. Structure of the Sec7 domain of the Arf exchange factor ARNO.

    PubMed

    Cherfils, J; Ménétrey, J; Mathieu, M; Le Bras, G; Robineau, S; Béraud-Dufour, S; Antonny, B; Chardin, P

    1998-03-05

    Small G proteins switch from a resting, GDP-bound state to an active, GTP-bound state. As spontaneous GDP release is slow, guanine-nucleotide-exchange factors (GEFs) are required to promote fast activation of small G proteins through replacement of GDP with GTP in vivo. Families of GEFs with no sequence similarity to other GEF families have now been assigned to most families of small G proteins. In the case of the small G protein Arf1, the exchange of bound GDP for GTP promotes the coating of secretory vesicles in Golgi traffic. An exchange factor for human Arf1, ARNO, and two closely related proteins, named cytohesin 1 and GPS1, have been identified. These three proteins are modular proteins with an amino-terminal coiled-coil, a central Sec7-like domain and a carboxy-terminal pleckstrin homology domain. The Sec7 domain contains the exchange-factor activity. It was first found in Sec7, a yeast protein involved in secretion, and is present in several other proteins, including the yeast exchange factors for Arf, Geal and Gea2. Here we report the crystal structure of the Sec7 domain of human ARNO at 2 A resolution and the identification of the site of interaction of ARNO with Arf.

  20. What's driving Gigabit/sec channels

    SciTech Connect

    Rupert, P.R.

    1991-09-01

    This paper summarizes the rationale for the drive towards Gigabit/sec communications for individual users. A description is given of a resulting prototype LAN which will deliver this capability to each user. The prototype is being built for the Lawrence Livermore National Laboratory. It will be delivered this winter and should be available for users by the end of the first quarter of next year. 19 refs., 8 figs.

  1. Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion

    PubMed Central

    Yue, Peng; Zhang, Yubo; Mei, Kunrong; Wang, Shaoxiao; Lesigang, Johannes; Zhu, Yueyao; Dong, Gang; Guo, Wei

    2017-01-01

    The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) constitute the core machinery for membrane fusion during eukaryotic cell vesicular trafficking. However, how the assembly of the SNARE complex is initiated is unknown. Here we report that Sec3, a component of the exocyst complex that mediates vesicle tethering during exocytosis, directly interacts with the t-SNARE protein Sso2. This interaction promotes the formation of an Sso2-Sec9 ‘binary' t-SNARE complex, the early rate-limiting step in SNARE complex assembly, and stimulates membrane fusion. The crystal structure of the Sec3-Sso2 complex suggests that Sec3 binding induces conformational changes of Sso2 that are crucial for the relief of its auto-inhibition. Interestingly, specific disruption of the Sec3–Sso2 interaction in cells blocks exocytosis without affecting the function of Sec3 in vesicle tethering. Our study reveals an activation mechanism for SNARE complex assembly, and uncovers a role of the exocyst in promoting membrane fusion in addition to vesicle tethering. PMID:28112172

  2. Segregated pathways to the vomeronasal amygdala: differential projections from the anterior and posterior divisions of the accessory olfactory bulb.

    PubMed

    Mohedano-Moriano, Alicia; Pro-Sistiaga, Palma; Ubeda-Bañón, Isabel; Crespo, Carlos; Insausti, Ricardo; Martinez-Marcos, Alino

    2007-04-01

    Apically and basally located receptor neurons in the vomeronasal sensory epithelium express G(i2 alpha)- and G(o alpha)-proteins, V1R and V2R vomeronasal receptors, project to the anterior and posterior accessory olfactory bulb and respond to different stimuli, respectively. The extent to which secondary projections from the two portions of the accessory olfactory bulb are convergent in the vomeronasal amygdala is controversial. This issue is addressed by using anterograde and retrograde tract-tracing methods in rats including electron microscopy. Injections of dextran-amines, Fluoro Gold, cholera toxin-B subunit and Fast Blue were delivered to the anterior and posterior accessory olfactory bulb, bed nucleus of the stria terminalis, dorsal anterior amygdala and bed nucleus of the accessory olfactory tract/anteroventral medial amygdaloid nucleus. We have demonstrated that, apart from common vomeronasal-recipient areas, only the anterior accessory olfactory bulb projects to the bed nucleus of the stria terminalis, medial division, posteromedial part, and only the posterior accessory olfactory bulb projects to the dorsal anterior amygdala and deep cell layers of the bed nucleus of the accessory olfactory tract and the anteroventral medial amygdaloid nucleus. These results provide evidence that, excluding areas of convergence, the V1R and V2R vomeronasal pathways project to specific areas of the amygdala. These two vomeronasal subsystems are therefore anatomically and functionally separated in the telencephalon.

  3. Cell cycle arrest of a Caulobacter crescentus secA mutant.

    PubMed Central

    Kang, P J; Shapiro, L

    1994-01-01

    Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function. Images PMID:8051008

  4. 46 CFR 169.671 - Accessories.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Accessories. 169.671 Section 169.671 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Electrical Installations Operating at Potentials of Less Than 50 Volts on Vessels of Less Than...

  5. 46 CFR 169.671 - Accessories.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Accessories. 169.671 Section 169.671 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Electrical Installations Operating at Potentials of Less Than 50 Volts on Vessels of Less Than...

  6. 46 CFR 169.671 - Accessories.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Accessories. 169.671 Section 169.671 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Electrical Installations Operating at Potentials of Less Than 50 Volts on Vessels of Less Than...

  7. 46 CFR 169.671 - Accessories.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Accessories. 169.671 Section 169.671 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Electrical Installations Operating at Potentials of Less Than 50 Volts on Vessels of Less Than...

  8. 14 CFR 23.1163 - Powerplant accessories.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...) Have torque limiting means on all accessory drives in order to prevent the torque limits established... approved as part of the powerplant driving the gearbox must— (1) Have torque limiting means to prevent the torque limits established for the affected drive from being exceeded; (2) Use the provisions on...

  9. 14 CFR 23.1163 - Powerplant accessories.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...) Have torque limiting means on all accessory drives in order to prevent the torque limits established... approved as part of the powerplant driving the gearbox must— (1) Have torque limiting means to prevent the torque limits established for the affected drive from being exceeded; (2) Use the provisions on...

  10. 14 CFR 23.1163 - Powerplant accessories.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...) Have torque limiting means on all accessory drives in order to prevent the torque limits established... approved as part of the powerplant driving the gearbox must— (1) Have torque limiting means to prevent the torque limits established for the affected drive from being exceeded; (2) Use the provisions on...

  11. 14 CFR 23.1163 - Powerplant accessories.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...) Have torque limiting means on all accessory drives in order to prevent the torque limits established... approved as part of the powerplant driving the gearbox must— (1) Have torque limiting means to prevent the torque limits established for the affected drive from being exceeded; (2) Use the provisions on...

  12. 14 CFR 23.1163 - Powerplant accessories.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...) Have torque limiting means on all accessory drives in order to prevent the torque limits established... approved as part of the powerplant driving the gearbox must— (1) Have torque limiting means to prevent the torque limits established for the affected drive from being exceeded; (2) Use the provisions on...

  13. 21 CFR 890.3910 - Wheelchair accessory.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Wheelchair accessory. 890.3910 Section 890.3910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Prosthetic Devices § 890.3910...

  14. 21 CFR 890.3910 - Wheelchair accessory.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Wheelchair accessory. 890.3910 Section 890.3910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Prosthetic Devices § 890.3910...

  15. 21 CFR 890.3910 - Wheelchair accessory.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Wheelchair accessory. 890.3910 Section 890.3910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Prosthetic Devices § 890.3910...

  16. 21 CFR 890.3910 - Wheelchair accessory.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Wheelchair accessory. 890.3910 Section 890.3910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Prosthetic Devices § 890.3910...

  17. 21 CFR 890.3910 - Wheelchair accessory.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Wheelchair accessory. 890.3910 Section 890.3910 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES PHYSICAL MEDICINE DEVICES Physical Medicine Prosthetic Devices § 890.3910...

  18. Electronic Position Sensor for Power Operated Accessory

    DOEpatents

    Haag, Ronald H.; Chia, Michael I.

    2005-05-31

    An electronic position sensor for use with a power operated vehicle accessory, such as a power liftgate. The position sensor includes an elongated resistive circuit that is mounted such that it is stationary and extends along the path of a track portion of the power operated accessory. The position sensor further includes a contact nub mounted to a link member that moves within the track portion such that the contact nub is slidingly biased against the elongated circuit. As the link member moves under the force of a motor-driven output gear, the contact nub slides along the surface of the resistive circuit, thereby affecting the overall resistance of the circuit. The position sensor uses the overall resistance to provide an electronic position signal to an ECU, wherein the signal is indicative of the absolute position of the power operated accessory. Accordingly, the electronic position sensor is capable of providing an electronic signal that enables the ECU to track the absolute position of the power operated accessory.

  19. Home Economics Careers in Apparel and Accessories.

    ERIC Educational Resources Information Center

    Texas Education Agency, Austin. Dept. of Occupational Education and Technology.

    This course of study on careers in apparel and accessories is one of a series on home economics careers designed to assist teacher-coordinators in Texas in promotion and/or teaching home economics cooperative education programs. The course of study consists of (1) an overview and job description, (2) a job analysis, (3) a course outline, (4)…

  20. 46 CFR 169.671 - Accessories.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 7 2010-10-01 2010-10-01 false Accessories. 169.671 Section 169.671 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) NAUTICAL SCHOOLS SAILING SCHOOL VESSELS Machinery and Electrical Electrical Installations Operating at Potentials of Less Than 50 Volts on Vessels of Less Than...

  1. 21 CFR 878.4700 - Surgical microscope and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Surgical microscope and accessories. 878.4700 Section 878.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... microscope and accessories. (a) Identification. A surgical microscope and accessories is an AC-powered...

  2. 21 CFR 878.4700 - Surgical microscope and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Surgical microscope and accessories. 878.4700 Section 878.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... microscope and accessories. (a) Identification. A surgical microscope and accessories is an AC-powered...

  3. 21 CFR 878.4700 - Surgical microscope and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Surgical microscope and accessories. 878.4700 Section 878.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... microscope and accessories. (a) Identification. A surgical microscope and accessories is an AC-powered...

  4. 21 CFR 878.4700 - Surgical microscope and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Surgical microscope and accessories. 878.4700 Section 878.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... microscope and accessories. (a) Identification. A surgical microscope and accessories is an AC-powered...

  5. 21 CFR 876.5540 - Blood access device and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Blood access device and accessories. 876.5540... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5540 Blood access device and accessories. (a) Identification. A blood access device and accessories is a device intended...

  6. 21 CFR 876.5540 - Blood access device and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Blood access device and accessories. 876.5540... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5540 Blood access device and accessories. (a) Identification. A blood access device and accessories is a device intended...

  7. 21 CFR 876.5540 - Blood access device and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Blood access device and accessories. 876.5540... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5540 Blood access device and accessories. (a) Identification. A blood access device and accessories is a device intended...

  8. 21 CFR 876.5540 - Blood access device and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Blood access device and accessories. 876.5540... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5540 Blood access device and accessories. (a) Identification. A blood access device and accessories is a device intended...

  9. 14 CFR 25.1192 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Engine accessory section diaphragm. 25.1192....1192 Engine accessory section diaphragm. For reciprocating engines, the engine power section and all portions of the exhaust system must be isolated from the engine accessory compartment by a diaphragm...

  10. 19 CFR 10.600 - Accessories, spare parts, or tools.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Accessories, spare parts, or tools. 10.600 Section... tools. (a) General. Accessories, spare parts, or tools that are delivered with a good and that form part of the good's standard accessories, spare parts, or tools will be treated as originating goods if...

  11. 19 CFR 10.537 - Accessories, spare parts, or tools.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Accessories, spare parts, or tools. 10.537 Section... Free Trade Agreement Rules of Origin § 10.537 Accessories, spare parts, or tools. Accessories, spare parts, or tools that are delivered with a good and that form part of the good's standard...

  12. 21 CFR 872.4120 - Bone cutting instrument and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Bone cutting instrument and accessories. 872.4120... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Surgical Devices § 872.4120 Bone cutting instrument and accessories. (a) Identification. A bone cutting instrument and accessories is a metal device intended for...

  13. 21 CFR 872.4120 - Bone cutting instrument and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Bone cutting instrument and accessories. 872.4120... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Surgical Devices § 872.4120 Bone cutting instrument and accessories. (a) Identification. A bone cutting instrument and accessories is a metal device intended for...

  14. 21 CFR 872.4120 - Bone cutting instrument and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Bone cutting instrument and accessories. 872.4120... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Surgical Devices § 872.4120 Bone cutting instrument and accessories. (a) Identification. A bone cutting instrument and accessories is a metal device intended for...

  15. 21 CFR 872.4120 - Bone cutting instrument and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Bone cutting instrument and accessories. 872.4120... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Surgical Devices § 872.4120 Bone cutting instrument and accessories. (a) Identification. A bone cutting instrument and accessories is a metal device intended for...

  16. 21 CFR 872.4120 - Bone cutting instrument and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Bone cutting instrument and accessories. 872.4120... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Surgical Devices § 872.4120 Bone cutting instrument and accessories. (a) Identification. A bone cutting instrument and accessories is a metal device intended for...

  17. 21 CFR 878.4700 - Surgical microscope and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Surgical microscope and accessories. 878.4700 Section 878.4700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES... microscope and accessories. (a) Identification. A surgical microscope and accessories is an AC-powered...

  18. 14 CFR 25.1192 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Engine accessory section diaphragm. 25.1192....1192 Engine accessory section diaphragm. For reciprocating engines, the engine power section and all portions of the exhaust system must be isolated from the engine accessory compartment by a diaphragm...

  19. 14 CFR 25.1192 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Engine accessory section diaphragm. 25.1192....1192 Engine accessory section diaphragm. For reciprocating engines, the engine power section and all portions of the exhaust system must be isolated from the engine accessory compartment by a diaphragm...

  20. 21 CFR 868.5860 - Pressure tubing and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Pressure tubing and accessories. 868.5860 Section... (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Therapeutic Devices § 868.5860 Pressure tubing and accessories. (a) Identification. Pressure tubing and accessories are flexible or rigid devices intended...

  1. 21 CFR 868.5860 - Pressure tubing and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Pressure tubing and accessories. 868.5860 Section... (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Therapeutic Devices § 868.5860 Pressure tubing and accessories. (a) Identification. Pressure tubing and accessories are flexible or rigid devices intended...

  2. 21 CFR 868.5860 - Pressure tubing and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pressure tubing and accessories. 868.5860 Section... (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Therapeutic Devices § 868.5860 Pressure tubing and accessories. (a) Identification. Pressure tubing and accessories are flexible or rigid devices intended...

  3. 21 CFR 868.5860 - Pressure tubing and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Pressure tubing and accessories. 868.5860 Section... (CONTINUED) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Therapeutic Devices § 868.5860 Pressure tubing and accessories. (a) Identification. Pressure tubing and accessories are flexible or rigid devices intended...

  4. 21 CFR 876.5540 - Blood access device and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Blood access device and accessories. 876.5540... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5540 Blood access device and accessories. (a) Identification. A blood access device and accessories is a device intended...

  5. 21 CFR 872.6300 - Rubber dam and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Rubber dam and accessories. 872.6300 Section 872...) MEDICAL DEVICES DENTAL DEVICES Miscellaneous Devices § 872.6300 Rubber dam and accessories. (a) Identification. A rubber dam and accessories is a device composed of a thin sheet of latex with a hole in...

  6. 21 CFR 872.6300 - Rubber dam and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Rubber dam and accessories. 872.6300 Section 872...) MEDICAL DEVICES DENTAL DEVICES Miscellaneous Devices § 872.6300 Rubber dam and accessories. (a) Identification. A rubber dam and accessories is a device composed of a thin sheet of latex with a hole in...

  7. 21 CFR 872.6300 - Rubber dam and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rubber dam and accessories. 872.6300 Section 872...) MEDICAL DEVICES DENTAL DEVICES Miscellaneous Devices § 872.6300 Rubber dam and accessories. (a) Identification. A rubber dam and accessories is a device composed of a thin sheet of latex with a hole in...

  8. 21 CFR 872.6300 - Rubber dam and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Rubber dam and accessories. 872.6300 Section 872...) MEDICAL DEVICES DENTAL DEVICES Miscellaneous Devices § 872.6300 Rubber dam and accessories. (a) Identification. A rubber dam and accessories is a device composed of a thin sheet of latex with a hole in...

  9. 21 CFR 872.6300 - Rubber dam and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Rubber dam and accessories. 872.6300 Section 872...) MEDICAL DEVICES DENTAL DEVICES Miscellaneous Devices § 872.6300 Rubber dam and accessories. (a) Identification. A rubber dam and accessories is a device composed of a thin sheet of latex with a hole in...

  10. 21 CFR 872.3980 - Endosseous dental implant accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Endosseous dental implant accessories. 872.3980... (CONTINUED) MEDICAL DEVICES DENTAL DEVICES Prosthetic Devices § 872.3980 Endosseous dental implant accessories. (a) Identification. Endosseous dental implant accessories are manually powered devices...

  11. 21 CFR 878.3925 - Plastic surgery kit and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Plastic surgery kit and accessories. 878.3925... (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Prosthetic Devices § 878.3925 Plastic surgery kit and accessories. (a) Identification. A plastic surgery kit and accessories is a device intended...

  12. 21 CFR 878.3925 - Plastic surgery kit and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Plastic surgery kit and accessories. 878.3925... (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Prosthetic Devices § 878.3925 Plastic surgery kit and accessories. (a) Identification. A plastic surgery kit and accessories is a device intended...

  13. 21 CFR 878.3925 - Plastic surgery kit and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Plastic surgery kit and accessories. 878.3925... (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Prosthetic Devices § 878.3925 Plastic surgery kit and accessories. (a) Identification. A plastic surgery kit and accessories is a device intended...

  14. 21 CFR 878.3925 - Plastic surgery kit and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Plastic surgery kit and accessories. 878.3925... (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Prosthetic Devices § 878.3925 Plastic surgery kit and accessories. (a) Identification. A plastic surgery kit and accessories is a device intended...

  15. 21 CFR 878.3925 - Plastic surgery kit and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Plastic surgery kit and accessories. 878.3925... (CONTINUED) MEDICAL DEVICES GENERAL AND PLASTIC SURGERY DEVICES Prosthetic Devices § 878.3925 Plastic surgery kit and accessories. (a) Identification. A plastic surgery kit and accessories is a device intended...

  16. 21 CFR 876.5250 - Urine collector and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Urine collector and accessories. 876.5250 Section... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5250 Urine collector and accessories. (a) Identification. A urine collector and accessories is a device intended to...

  17. 21 CFR 876.5250 - Urine collector and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Urine collector and accessories. 876.5250 Section... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5250 Urine collector and accessories. (a) Identification. A urine collector and accessories is a device intended to...

  18. 21 CFR 876.5250 - Urine collector and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Urine collector and accessories. 876.5250 Section... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5250 Urine collector and accessories. (a) Identification. A urine collector and accessories is a device intended to...

  19. 21 CFR 876.5250 - Urine collector and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Urine collector and accessories. 876.5250 Section... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5250 Urine collector and accessories. (a) Identification. A urine collector and accessories is a device intended to...

  20. 21 CFR 876.5250 - Urine collector and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Urine collector and accessories. 876.5250 Section... (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5250 Urine collector and accessories. (a) Identification. A urine collector and accessories is a device intended to...

  1. 21 CFR 884.6120 - Assisted reproduction accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Assisted reproduction accessories. 884.6120... (CONTINUED) MEDICAL DEVICES OBSTETRICAL AND GYNECOLOGICAL DEVICES Assisted Reproduction Devices § 884.6120 Assisted reproduction accessories. (a) Identification. Assisted reproduction accessories are a group...

  2. 21 CFR 884.6120 - Assisted reproduction accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Assisted reproduction accessories. 884.6120... (CONTINUED) MEDICAL DEVICES OBSTETRICAL AND GYNECOLOGICAL DEVICES Assisted Reproduction Devices § 884.6120 Assisted reproduction accessories. (a) Identification. Assisted reproduction accessories are a group...

  3. 21 CFR 884.6120 - Assisted reproduction accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Assisted reproduction accessories. 884.6120... (CONTINUED) MEDICAL DEVICES OBSTETRICAL AND GYNECOLOGICAL DEVICES Assisted Reproduction Devices § 884.6120 Assisted reproduction accessories. (a) Identification. Assisted reproduction accessories are a group...

  4. 21 CFR 884.6120 - Assisted reproduction accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Assisted reproduction accessories. 884.6120... (CONTINUED) MEDICAL DEVICES OBSTETRICAL AND GYNECOLOGICAL DEVICES Assisted Reproduction Devices § 884.6120 Assisted reproduction accessories. (a) Identification. Assisted reproduction accessories are a group...

  5. 21 CFR 884.6120 - Assisted reproduction accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Assisted reproduction accessories. 884.6120... (CONTINUED) MEDICAL DEVICES OBSTETRICAL AND GYNECOLOGICAL DEVICES Assisted Reproduction Devices § 884.6120 Assisted reproduction accessories. (a) Identification. Assisted reproduction accessories are a group...

  6. 21 CFR 876.5980 - Gastrointestinal tube and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Gastrointestinal tube and accessories. 876.5980... tube and accessories. (a) Identification. A gastrointestinal tube and accessories is a device that..., gastrointestinal string and tubes to locate internal bleeding, double lumen tube for intestinal decompression...

  7. 21 CFR 876.5980 - Gastrointestinal tube and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Gastrointestinal tube and accessories. 876.5980... tube and accessories. (a) Identification. A gastrointestinal tube and accessories is a device that..., gastrointestinal string and tubes to locate internal bleeding, double lumen tube for intestinal decompression...

  8. 21 CFR 876.5980 - Gastrointestinal tube and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Gastrointestinal tube and accessories. 876.5980... tube and accessories. (a) Identification. A gastrointestinal tube and accessories is a device that..., gastrointestinal string and tubes to locate internal bleeding, double lumen tube for intestinal decompression...

  9. 21 CFR 876.5980 - Gastrointestinal tube and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Gastrointestinal tube and accessories. 876.5980... tube and accessories. (a) Identification. A gastrointestinal tube and accessories is a device that..., gastrointestinal string and tubes to locate internal bleeding, double lumen tube for intestinal decompression...

  10. 21 CFR 876.5980 - Gastrointestinal tube and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Gastrointestinal tube and accessories. 876.5980... tube and accessories. (a) Identification. A gastrointestinal tube and accessories is a device that..., gastrointestinal string and tubes to locate internal bleeding, double lumen tube for intestinal decompression...

  11. Detection of accessory spleens with indium 111-labeled autologous platelets

    SciTech Connect

    Davis, H.H., II; Varki, A.; Heaton, W.A.; Siegel, B.A.

    1980-01-01

    In two patients with recurrent immune thrombocytopenia, accessory splenic tissue was demonstrated by radionuclide imaging following administration of indium 111-labeled autologous platelets. In one of these patients, no accessory splenic tissue was seen on images obtained with technetium 99m sulfur colloid. This new technique provides a simple means for demonstrating accessory spleens and simultaneously evaluating the life-span of autologous platelets.

  12. SecA—a New Twist in the Tale

    PubMed Central

    2016-01-01

    ABSTRACT A paper published in this issue of the Journal of Bacteriology (D. Huber, M. Jamshad, R. Hanmer, D. Schibich, K. Döring, I. Marcomini, G. Kramer, and B. Bukau, J Bacteriol 199:e0622-16, 2017, https://doi.org/10.1128/JB.00622-16) provides us with a timely reminder that all is not as clear as we had previously thought in the general bacterial secretion system. The paper describes a new mode of secretion through the Sec system—“uncoupled cotranslocation”—for the passage of proteins across the bacterial inner membrane and suggests that we might rethink the nature and mechanism of the targeting and transport steps toward protein export. PMID:27799326

  13. Sequential Events Control System (SECS) Overview

    NASA Technical Reports Server (NTRS)

    Interbartolo, Michael

    2009-01-01

    This slide presentation will cover the Sequential Events Control System (SECS), which is the Apollo spacecraft subsystem that controls the automatically sequenced functions during the mission and during any a borts that could be performed. Included in this presentation are its general architecture, its integration into and use of the spacecraft' s other systems, and details on the functions it is responsible for c ontrolling during the mission. The objectives are to describe the system's architecture, the major components in the system, and the major system functions.

  14. Differential regulation of Saccharomyces cerevisiae phospholipase D in sporulation and Sec14-independent secretion.

    PubMed Central

    Rudge, Simon A; Zhou, Chun; Engebrecht, JoAnne

    2002-01-01

    Saccharomyces cerevisiae Spo14, a phosphatidylcholine-specific, phosphatidylinositol (4,5) bisphosphate-activated phospholipase D (PLD), is essential for meiosis and spore formation. Spo14 is also required for secretion in the absence of the phosphatidylinositol/phosphatidylcholine transfer protein Sec14 (i.e., Sec14-independent secretion). In sporulating cells Spo14 is phosphorylated and relocalized within the cell. In contrast, Spo14 does not relocalize and is not phosphorylated in Sec14-independent secretion. Analysis of a partially phosphatidylinositol (4,5) bisphosphate-activated Spo14 mutant, spo14(R894G), revealed that Spo14 function in Sec14-independent secretion, unlike the situation in meiosis, requires fully stimulated PLD activity. Consistent with the differential regulation of Spo14 function during sporulation and secretion, we isolated a mutant allele, spo14-S251P, the product of which is improperly phosphorylated and fails to relocalize and rescue the sporulation phenotype of homozygous spo14 diploids, but supports Sec14-independent secretion. Furthermore, we show that the N-terminal domain of Spo14 is both phosphorylated and sufficient for prospore membrane localization during sporulation. These data indicate that Spo14 phosphorylation and relocalization are essential for the process of sporulation, but dispensable for Sec14-independent secretion. Finally, we demonstrate that Spo14 phosphorylation and relocalization are initiated by nitrogen and glucose limitation and occur independently of the process of meiosis. PMID:11973292

  15. In vitro refolding with simultaneous purification of recombinant human parathyroid hormone (rhPTH 1-34) from Escherichia coli directed by protein folding size exclusion chromatography (PF-SEC): implication of solution additives and their role on aggregates and renaturation.

    PubMed

    Vemula, Sandeep; Vemula, Sushma; Dedaniya, Akshay; Ronda, Srinivasa Reddy

    2016-01-01

    Recombinant proteins are frequently hampered by aggregation during the refolding and purification process. A simple and rapid method for in vitro refolding and purification of recombinant human parathyroid hormone (rhPTH 1-34) expressed in Escherichia coli with protein folding size exclusion chromatography (PF-SEC) was developed in the present work. Discrete effects of potential solution additives such as urea, polypolyethylene glycol, proline, and maltose on the refolding with simultaneous purification of rhPTH were investigated. The results of individual additives indicated that both maltose and proline had remarkable influences on the efficiency of refolding with a recovery yield of 65 and 66% respectively. Further, the synergistic effect of these additives on refolding was also explored. These results demonstrate that the additive combinations are more effective for inhibiting protein aggregation during purification of rhPTH in terms of recovery yield, purity, and specific activity. The maltose and proline combination system achieved the highest renatured rhPTH having a recovery yield of 78%, a purity of ≥99%, and a specific activity of 3.31 × 10(3) cAMP pM/cell respectively, when compared to the classical dilution method yield (41%) and purity (97%). In addition, the role of maltose and proline in a combined system on protein aggregation and refolding has been explained. The molecular docking (in silico) scores of maltose (-10.91) and proline (-9.0) support the in vitro results.

  16. Sec24 is a coincidence detector that simultaneously binds two signals to drive ER export

    PubMed Central

    Pagant, Silvere; Wu, Alexander; Edwards, Samuel; Diehl, Frances; Miller, Elizabeth A.

    2014-01-01

    Summary Background Incorporation of secretory proteins into ER-derived vesicles involves recognition of cytosolic signals by the COPII coat protein, Sec24. Additional cargo diversity is achieved through cargo receptors, which include the Erv14/Cornichon family that mediate export of transmembrane proteins despite the potential for such clients to directly interact with Sec24. The molecular function of Erv14 thus remains unclear, with possible roles in COPII-binding, membrane domain chaperoning and lipid organization. Results Using a targeted mutagenesis approach to define the mechanism of Erv14 function, we identify conserved residues in the second transmembrane domain of Erv14 that mediate interaction with a subset of Erv14 clients. We further show that interaction of Erv14 with a novel cargo-binding surface on Sec24 is necessary for efficient trafficking of all of its clients. However, we also determine that some Erv14 clients also engage directly an adjacent cargo-binding domain of Sec24, suggesting a novel mode of dual interaction between cargo and coat. Conclusions We conclude that Erv14 functions as a canonical cargo receptor that couples membrane proteins to the COPII coat, but that maximal export requires a bivalent signal that derives from motifs on both the cargo protein and Erv14. Sec24 can thus be considered a coincidence detector that binds simultaneously to multiple signals to drive packaging of polytopic membrane proteins. This mode of dual signal binding to a single coat protein might serve as a general mechanism to trigger efficient capture, or may be specifically employed in ER export to control deployment of nascent proteins. PMID:25619760

  17. Phosphorylation Provides a Negative Mode of Regulation for the Yeast Rab GTPase Sec4p

    PubMed Central

    Heger, Christopher D.; Wrann, Christiane D.; Collins, Ruth N.

    2011-01-01

    The Rab family of Ras-related GTPases are part of a complex signaling circuitry in eukaryotic cells, yet we understand little about the mechanisms that underlie Rab protein participation in such signal transduction networks, or how these networks are integrated at the physiological level. Reversible protein phosphorylation is widely used by cells as a signaling mechanism. Several phospho-Rabs have been identified, however the functional consequences of the modification appear to be diverse and need to be evaluated on an individual basis. In this study we demonstrate a role for phosphorylation as a negative regulatory event for the action of the yeast Rab GTPase Sec4p in regulating polarized growth. Our data suggest that the phosphorylation of the Rab Sec4p prevents interactions with its effector, the exocyst component Sec15p, and that the inhibition may be relieved by a PP2A phosphatase complex containing the regulatory subunit Cdc55p. PMID:21931684

  18. Accessory cells for β-cell transplantation.

    PubMed

    Staels, W; De Groef, S; Heremans, Y; Coppens, V; Van Gassen, N; Leuckx, G; Van de Casteele, M; Van Riet, I; Luttun, A; Heimberg, H; De Leu, N

    2016-02-01

    Despite recent advances, insulin therapy remains a treatment, not a cure, for diabetes mellitus with persistent risk of glycaemic alterations and life-threatening complications. Restoration of the endogenous β-cell mass through regeneration or transplantation offers an attractive alternative. Unfortunately, signals that drive β-cell regeneration remain enigmatic and β-cell replacement therapy still faces major hurdles that prevent its widespread application. Co-transplantation of accessory non-islet cells with islet cells has been shown to improve the outcome of experimental islet transplantation. This review will highlight current travails in β-cell therapy and focuses on the potential benefits of accessory cells for islet transplantation in diabetes.

  19. Sec16A is critical for both conventional and unconventional secretion of CFTR

    PubMed Central

    Piao, He; Kim, Jiyoon; Noh, Shin Hye; Kweon, Hee-Seok; Kim, Joo Young; Lee, Min Goo

    2017-01-01

    CFTR is a transmembrane protein that reaches the cell surface via the conventional Golgi mediated secretion pathway. Interestingly, ER-to-Golgi blockade or ER stress induces alternative GRASP-mediated, Golgi-bypassing unconventional trafficking of wild-type CFTR and the disease-causing ΔF508-CFTR, which has folding and trafficking defects. Here, we show that Sec16A, the key regulator of conventional ER-to-Golgi transport, plays a critical role in the ER exit of protein cargos during unconventional secretion. In an initial gene silencing screen, Sec16A knockdown abolished the unconventional secretion of wild-type and ΔF508-CFTR induced by ER-to-Golgi blockade, whereas the knockdown of other COPII-related components did not. Notably, during unconventional secretion, Sec16A was redistributed to cell periphery and associated with GRASP55 in mammalian cells. Molecular and morphological analyses revealed that IRE1α-mediated signaling is an upstream regulator of Sec16A during ER-to-Golgi blockade or ER stress associated unconventional secretion. These findings highlight a novel function of Sec16A as an essential mediator of ER stress-associated unconventional secretion. PMID:28067262

  20. Plasmodium falciparum Sec24 marks transitional ER that exports a model cargo via a diacidic motif.

    PubMed

    Lee, Marcus C S; Moura, Pedro A; Miller, Elizabeth A; Fidock, David A

    2008-06-01

    Exit from the endoplasmic reticulum (ER) often occurs at distinct sites of vesicle formation known as transitional ER (tER) that are enriched for COPII vesicle coat proteins. We have characterized the organization of ER export in the malaria parasite, Plasmodium falciparum, by examining the localization of two components of the COPII machinery, PfSec12 and PfSec24a. PfSec12 was found throughout the ER, whereas the COPII cargo adaptor, PfSec24a, was concentrated at distinct foci that likely correspond to tER sites. These foci were closely apposed to cis-Golgi sites marked by PfGRASP-GFP, and upon treatment with brefeldin A they accumulated a model cargo protein via a process dependent on the presence of an intact diacidic export motif. Our data suggest that the cargo-binding function of PfSec24a is conserved and that accumulation of cargo in discrete tER sites depends upon positive sorting signals. Furthermore, the number and position of tER sites with respect to the cis-Golgi suggests a co-ordinated biogenesis of these domains.

  1. Exertional responses to sprint interval training: a comparison of 30-sec. and 60-sec. conditions.

    PubMed

    Kilpatrick, Marcus W; Greeley, Samuel J

    2014-06-01

    The purpose of this study was to assess the effect of sprint interval training on rating of perceived exertion. 20 healthy participants (11 men, 9 women; M age = 23 yr.) completed a maximal cycle ergometer test and two high-intensity interval training cycling sessions. Each session utilized the same work-to-rest ratio (1:1), work intensity (90% max), recovery intensity (10% work intensity), and session duration (16 min.). Trials differed on duration of the interval segment, with a 30-sec. trial and a 60-sec. trial. Sessions required the same amount of total work over the duration of the trial. Rating of perceived exertion assessed before, during, and after exercise were higher for the 60-sec. trial than the 30-sec. trial despite no difference in total work. High intensity interval training trials utilizing the same total external work but differing in interval length produced different ratings of perceived exertion. Perceived exertion is significantly higher for sessions of exercise that utilize longer work intervals. These findings suggest that shorter intervals may produce more favorable exertional responses that could positively affect future behavior.

  2. Sec61β Controls Sensitivity to Platinum-Containing Chemotherapeutic Agents through Modulation of the Copper-Transporting ATPase ATP7A

    PubMed Central

    Larson, Christopher A.; Manorek, Gerald; Adams, Preston; Howell, Stephen B.

    2012-01-01

    The Sec61 protein translocon is a multimeric complex that transports proteins across lipid bilayers. We discovered that the Sec61β subunit modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. To investigate the mechanism, expression of Sec61β was constitutively knocked down in 2008 ovarian cancer cells. Sec61β knockdown (KD) resulted in 8-, 16.8-, and 9-fold resistance to cisplatin (cDDP), carboplatin, and oxaliplatin, respectively. Sec61β KD reduced the cellular accumulation of cDDP to 67% of that in parental cells. Baseline copper levels, copper uptake, and copper cytotoxicity were also reduced. Because copper transporters and chaperones regulate platinum drug accumulation and efflux, their expression in 2008 Sec61β-KD cells was analyzed; ATP7A was found to be 2- to 3-fold overexpressed, whereas there was no change in ATP7B, ATOX1, CTR1, or CTR2 levels. Cells lacking ATP7A did not exhibit increased cDDP resistance upon knockdown of Sec61β. Sec61β-KD cells also exhibited altered ATP7A cellular distribution. We conclude that Sec61β modulates the cytotoxicity of many chemotherapeutic agents, with the largest effect being on the platinum drugs. This modulation occurs through effects of Sec61β on the expression and distribution of ATP7A, which was shown previously to control platinum drug sequestration and cytotoxicity. PMID:22710939

  3. Sec61β controls sensitivity to platinum-containing chemotherapeutic agents through modulation of the copper-transporting ATPase ATP7A.

    PubMed

    Abada, Paolo B; Larson, Christopher A; Manorek, Gerald; Adams, Preston; Howell, Stephen B

    2012-09-01

    The Sec61 protein translocon is a multimeric complex that transports proteins across lipid bilayers. We discovered that the Sec61β subunit modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum drugs. To investigate the mechanism, expression of Sec61β was constitutively knocked down in 2008 ovarian cancer cells. Sec61β knockdown (KD) resulted in 8-, 16.8-, and 9-fold resistance to cisplatin (cDDP), carboplatin, and oxaliplatin, respectively. Sec61β KD reduced the cellular accumulation of cDDP to 67% of that in parental cells. Baseline copper levels, copper uptake, and copper cytotoxicity were also reduced. Because copper transporters and chaperones regulate platinum drug accumulation and efflux, their expression in 2008 Sec61β-KD cells was analyzed; ATP7A was found to be 2- to 3-fold overexpressed, whereas there was no change in ATP7B, ATOX1, CTR1, or CTR2 levels. Cells lacking ATP7A did not exhibit increased cDDP resistance upon knockdown of Sec61β. Sec61β-KD cells also exhibited altered ATP7A cellular distribution. We conclude that Sec61β modulates the cytotoxicity of many chemotherapeutic agents, with the largest effect being on the platinum drugs. This modulation occurs through effects of Sec61β on the expression and distribution of ATP7A, which was shown previously to control platinum drug sequestration and cytotoxicity.

  4. The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans.

    PubMed

    Zhao, Yani; Holmgren, Benjamin T; Hinas, Andrea

    2017-03-01

    Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22 SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing.

  5. The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans

    PubMed Central

    Zhao, Yani; Holmgren, Benjamin T.

    2017-01-01

    Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(−) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22. SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing. PMID:27974622

  6. Somatic overgrowth associated with homozygous mutations in both MAN1B1 and SEC23A

    PubMed Central

    Gupta, Swati; Fahiminiya, Somayyeh; Wang, Tracy; Dempsey Nunez, Laura; Rosenblatt, David S.; Gibson, William T.; Gilfix, Brian; Bergeron, John J. M.; Jerome-Majewska, Loydie A.

    2016-01-01

    Using whole-exome sequencing, we identified homozygous mutations in two unlinked genes, SEC23A c.1200G>C (p.M400I) and MAN1B1 c.1000C>T (p.R334C), associated with congenital birth defects in two patients from a consanguineous family. Patients presented with carbohydrate-deficient transferrin, tall stature, obesity, macrocephaly, and maloccluded teeth. The parents were healthy heterozygous carriers for both mutations and an unaffected sibling with tall stature carried the heterozygous mutation in SEC23A only. Mutations in SEC23A are responsible for craniolenticosultura dysplasia (CLSD). CLSD patients are short, have late-closing fontanels, and have reduced procollagen (pro-COL1A1) secretion because of abnormal pro-COL1A1 retention in the endoplasmic reticulum (ER). The mutation we identified in MAN1B1 was previously associated with reduced MAN1B1 protein and congenital disorders of glycosylation (CDG). CDG patients are also short, are obese, and have abnormal glycan remodeling. Molecular analysis of fibroblasts from the family revealed normal levels of SEC23A in all cells and reduced levels of MAN1B1 in cells with heterozygous or homozygous mutations in SEC23A and MAN1B1. Secretion of pro-COL1A1 was increased in fibroblasts from the siblings and patients, and pro-COL1A1 was retained in Golgi of heterozygous and homozygous mutant cells, although intracellular pro-COL1A1 was increased in patient fibroblasts only. We postulate that increased pro-COL1A1 secretion is responsible for tall stature in these patients and an unaffected sibling, and not previously discovered in patients with mutations in either SEC23A or MAN1B1. The patients in this study share biochemical and cellular characteristics consistent with mutations in MAN1B1 and SEC23A, indicating a digenic disease. PMID:27148587

  7. Cloning of SEC61 homologues from Schizosaccharomyces pombe and Yarrowia lipolytica reveals the extent of functional conservation within this core component of the ER translocation machinery.

    PubMed

    Broughton, J; Swennen, D; Wilkinson, B M; Joyet, P; Gaillardin, C; Stirling, C J

    1997-11-01

    The Sec61 protein is required for protein translocation across the ER membrane in both yeast and mammals and is found in close association with polypeptides during their membrane transit. In Saccharomyces cerevisiae Sec61p is essential for viability and the extent of sequence similarity between the yeast and mammalian proteins (55% sequence identity) suggests that the role of Sec61p in the translocation mechanism is likely to be conserved. In order to further our understanding of the structure and function of Sec61p we have cloned homologues from both Schizosaccharomyces pombe and Yarrowia lipolytica. The S. pombe gene comprises six exons encoding a 479 residue protein which we have immunolocalised to the endoplasmic reticulum. Sequence comparisons reveal that S. pombe Sec61p is 58.6% identical to that of S. cerevisiae. The deduced amino acid sequence of the Y. lipolytica protein shares 68.8% sequence identity with S. cerevisiae Sec61p. Gene disruption studies have shown that the SEC61 is required for viability in both S. pombe and Y. lipolytica demonstrating that the essential nature of this protein is not unique to S. cerevisiae. Moreover, heterologous complementation studies indicate that the Y. lipolytica SEC61 gene can complement a null mutation in S. cerevisiae. Sequence comparisons between the various eukaryotic Sec61p homologues reveal a number of highly conserved domains, including several transmembrane sequences and the majority of cytosolic loops. These comparisons will provide an important framework for the detailed analysis of interactions between Sec61p and other components of the translocation machinery and between Sec61p and translocating polypeptide chains.

  8. Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

    PubMed

    Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

    2013-07-01

    Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

  9. Dermal fibroblasts contribute to multiple tissues in the accessory limb model.

    PubMed

    Hirata, Ayako; Gardiner, David M; Satoh, Akira

    2010-05-01

    The accessory limb model has become an alternative model for performing investigations of limb regeneration in an amputated limb. In the accessory limb model, a complete patterned limb can be induced as a result of an interaction between the wound epithelium, a nerve and dermal fibroblasts in the skin. Studies should therefore focus on examining these tissues. To date, however, a study of cellular contributions in the accessory limb model has not been reported. By using green fluorescent protein (GFP) transgenic axolotl tissues, we can trace cell fate at the tissue level. Therefore, in the present study, we transgrafted GFP skin onto the limb of a non-GFP host and induced an accessory limb to investigate cellular contributions. Previous studies of cell contribution to amputation-induced blastemas have demonstrated that dermal cells are the progenitors of many of the early blastema cells, and that these cells contribute to regeneration of the connective tissues, including cartilage. In the present study, we have determined that this same population of progenitor cells responds to signaling from the nerve and wound epithelium in the absence of limb amputation to form an ectopic blastema and regenerate the connective tissues of an ectopic limb. Blastema cells from dermal fibroblasts, however, did not differentiate into either muscle or neural cells, and we conclude that dermal fibroblasts are dedifferentiated along its developmental lineage.

  10. Accessory slips of the extensor digiti minimi.

    PubMed

    Li, Jing; Mao, Qing Hua

    2014-01-01

    During the educational dissection of a 69-year-old Chinese male cadaver, an extensor digiti minimi (EDM) with five slips on the right hand was discovered. Except for the two slips of the little finger, the two radial slips were inserted into the dorsal aponeurosis of the middle finger and the ring finger, respectively. The middle slip was connected to the junctura tendinum in the fourth intermetacarpal spaces. Variations in this region are of paramount importance for the reconstructive surgeons, who may utilize the accessory slips to restore functional capacity of the fingers.

  11. Optimization of diffuse reflectance infrared spectroscopy accessories

    SciTech Connect

    Hirschfeld, T.

    1986-11-01

    The value of diffuse reflectance as an infrared or near-infrared spectroscopic sampling procedure has been limited by the low efficiency of accessories designed for it. In terms of signal-to-noise ratio, these average 2-6% for integrating spheres and 10-12% for various ellipsoidal mirror arrangements. Much better performances, up to 37% efficiency, can be obtained by optimizing a concentric confocal ellipsoidal mirror arrangement by using a very large central opening in the amular collector mirror, and adapting the throughput of the detector to the geometry of the collected beam.

  12. Reviewing prescription spending and accessory usage.

    PubMed

    Oxenham, Julie

    This article aims to explore the role of the stoma nurse specialist in the community and how recent initiatives within the NHS have impacted on the roles in stoma care to react to the rising prescription costs in the specialty. The article will explore how the stoma care nurse conducted her prescription reviews within her own clinical commissioning group (CCG). The findings of the reviews will be highlighted by a small case history and a mini audit that reveals that some stoma patients may be using their stoma care accessories inappropriately, which may contribute to the rise in stoma prescription spending. To prevent the incorrect use of stoma appliances it may necessitate an annual review of ostomates (individuals who have a stoma), as the author's reviews revealed that inappropriate usage was particularly commonplace when a patient may have not been reviewed by a stoma care specialist for some considerable amount of time. Initial education of the ostomate and ongoing education of how stoma products work is essential to prevent the misuse of stoma appliances, particularly accessories, as the reviews revealed that often patients were not always aware of how their products worked in practice.

  13. Enhanced oral bioavailability of insulin using PLGA nanoparticles co-modified with cell-penetrating peptides and Engrailed secretion peptide (Sec).

    PubMed

    Zhu, Siqi; Chen, Shuangxi; Gao, Yuan; Guo, Feng; Li, Fengying; Xie, Baogang; Zhou, Jianliang; Zhong, Haijun

    2016-07-01

    Biodegradable polymer nanoparticle drug carriers are an attractive strategy for oral delivery of peptide and protein drugs. However, their ability to cross the intestinal epithelium membrane is largely limited. Therefore, in the present study, cell-penetrating peptides (R8, Tat, penetratin) and a secretion peptide (Sec) with N-terminal stearylation were introduced to modify nanoparticles (NPs) on the surface to improve oral bioavailability of peptide and protein drugs. In vitro studies conducted in Caco-2 cells showed the value of the apparent permeability coefficient (Papp) of the nanoparticles co-modified with Sec and penetratin (Sec-Pen-NPs) was about two-times greater than that of the nanoparticles modified with only penetratin (Pen-NPs), while the increase of transcellular transport of nanoparticles modified together with Sec and R8 (Sec-R8-NPs), or Sec and Tat (Sec-Tat-NPs), was not significant compared with nanoparticles modified with only R8 (R8-NPs) or Tat (Tat-NPs). Using insulin as the model drug, in vivo studies performed on rats indicated that compared to Pen-NPs, the relative bioavailability of insulin for Sec-Pen-NPs was 1.71-times increased after ileal segments administration, and stronger hypoglycemic effects was also observed. Therefore, the nanoparticles co-modified with penetratin and Sec could act as attractive carriers for oral delivery of insulin.

  14. Potential role of Arabidopsis PHP as an accessory subunit of the PAF1 transcriptional cofactor.

    PubMed

    Park, Sunchung; Ek-Ramos, Maria Julissa; Oh, Sookyung; van Nocker, Steven

    2011-08-01

    Paf1C is a transcriptional cofactor that has been implicated in various transcription-associated mechanisms spanning initiation, elongation and RNA processing, and is important for multiple aspects of development in Arabidopsis. Our recent studies suggest Arabidopsis Paf1C is crucial for proper regulation of genes within H3K27me3-enriched chromatin, and that a protein named PHP may act as an accessory subunit of Paf1C that promotes this function.

  15. 46 CFR Sec. 8 - Extra work and changes.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Sec. 8 Extra work and changes. (a) At any time after the award of an original job order and during the time the work thereunder is being performed, additional or extra work or changes in the work covered by... 46 Shipping 8 2011-10-01 2011-10-01 false Extra work and changes. Sec. 8 Section 8...

  16. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by...

  17. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required...

  18. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required...

  19. 46 CFR Sec. 6 - Surety and form of bond.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Surety and form of bond. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY BONDING OF SHIP'S PERSONNEL Sec. 6 Surety and form of bond. Each bond provided for by this order shall be duly executed by...

  20. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  1. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  2. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  3. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 4 2014-01-01 2014-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  4. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 4 2013-01-01 2013-01-01 false Accounting entities. Sec. 1-6 Section 1-6... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each...

  5. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 4 2011-01-01 2011-01-01 false Accounting period. Sec. 2-4 Section 2-4... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this...

  6. 46 CFR Sec. 4 - Port facilities predesignated for emergency use.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Port facilities predesignated for emergency use. Sec. 4... FACILITIES Sec. 4 Port facilities predesignated for emergency use. (a) Certain port facilities selected for...) Facilities which are not required by the United States immediately on the effective date of this part will...

  7. 46 CFR Sec. 3 - Federal control of port facilities.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Federal control of port facilities. Sec. 3 Section 3... RESTRICTIONS UPON THE TRANSFER OR CHANGE IN USE OR IN TERMS GOVERNING UTILIZATION OF PORT FACILITIES Sec. 3 Federal control of port facilities. During any period when the provisions of this part are in effect...

  8. 46 CFR Sec. 12 - Disposition of removed equipment and scrap.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Disposition of removed equipment and scrap. Sec. 12... CONTRACT-NSA-LUMPSUMREP Sec. 12 Disposition of removed equipment and scrap. (a) Article 8 of the NSA-LUMPSUMREP Contract provides that any ship equipment, fuel, lube oil, supplies, stores, furniture,...

  9. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 8 2014-10-01 2014-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's...

  10. 46 CFR Sec. 5 - Measures to protect ship's payrolls.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 8 2013-10-01 2013-10-01 false Measures to protect ship's payrolls. Sec. 5 Section 5... SHIP'S PERSONNEL Sec. 5 Measures to protect ship's payrolls. (a) General Agents are not required to... paying off the crew should be either the Master, or purser, or some other member of the ship's...

  11. Financial Journalism under Fire: The SEC and Newsroom Ethics.

    ERIC Educational Resources Information Center

    Spellman, Robert L.

    Although noting that the Securities and Exchange Commission (SEC) has been a valuable ally of journalists, this paper suggests that recent efforts of the SEC in prosecuting the case of R. Foster Winans, Jr., a former writer for the "Wall Street Journal," may be unconstitutional. Following an introduction to the First Amendment issues…

  12. 46 CFR Sec. 2 - Authority for launch hire.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Authority for launch hire. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY LAUNCH SERVICES Sec. 2 Authority for launch hire. Launch hire in foreign and domestic ports will be accepted by National...

  13. 46 CFR Sec. 2 - Authority for launch hire.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Authority for launch hire. Sec. 2 Section 2 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY LAUNCH SERVICES Sec. 2 Authority for launch hire. Launch hire in foreign and domestic ports will be accepted by National...

  14. 14 CFR Sec. 19-2 - Maintenance of data.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Maintenance of data. Sec. 19-2 Section 19-2 Aeronautics and Space OFFICE OF THE SECRETARY, DEPARTMENT OF TRANSPORTATION (AVIATION PROCEEDINGS) ECONOMIC... Classifications Sec. 19-2 Maintenance of data. (a) Each air carrier required to file Form 41 Schedule T-100...

  15. 14 CFR Sec. 1-6 - Accounting entities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 1-6 Accounting entities. (a) Separate accounting records shall be maintained for each air... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accounting entities. Sec. 1-6 Section...

  16. 14 CFR Sec. 2-5 - Revenue and accounting practices.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... General Accounting Provisions Sec. 2-5 Revenue and accounting practices. (a) Revenue accounting practices shall conform to the provisions of account 2160, Air Traffic Liability. (b) Each route air carrier shall... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Revenue and accounting practices. Sec....

  17. 14 CFR Sec. 2-4 - Accounting period.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... REGULATIONS UNIFORM SYSTEM OF ACCOUNTS AND REPORTS FOR LARGE CERTIFICATED AIR CARRIERS General Accounting Provisions Sec. 2-4 Accounting period. (a) The accounting year of each air carrier subject to this Uniform... 14 Aeronautics and Space 4 2010-01-01 2010-01-01 false Accounting period. Sec. 2-4 Section...

  18. Lst1p and Sec24p Cooperate in Sorting of the Plasma Membrane Atpase into Copii Vesicles in Saccharomyces cerevisiae

    PubMed Central

    Shimoni, Yuval; Kurihara, Tatsuo; Ravazzola, Mariella; Amherdt, Mylène; Orci, Lelio; Schekman, Randy

    2000-01-01

    Formation of ER-derived protein transport vesicles requires three cytosolic components, a small GTPase, Sar1p, and two heterodimeric complexes, Sec23/24p and Sec13/31p, which comprise the COPII coat. We investigated the role of Lst1p, a Sec24p homologue, in cargo recruitment into COPII vesicles in Saccharomyces cerevisiae. A tagged version of Lst1p was purified and eluted as a heterodimer complexed with Sec23p comparable to the Sec23/24p heterodimer. We found that cytosol from an lst1-null strain supported the packaging of α-factor precursor into COPII vesicles but was deficient in the packaging of Pma1p, the essential plasma membrane ATPase. Supplementation of mutant cytosol with purified Sec23/Lst1p restored Pma1p packaging into the vesicles. When purified COPII components were used in the vesicle budding reaction, Pma1p packaging was optimal with a mixture of Sec23/24p and Sec23/Lst1p; Sec23/Lst1p did not replace Sec23/24p. Furthermore, Pma1p coimmunoprecipitated with Lst1p and Sec24p from vesicles. Vesicles formed with a mixture of Sec23/Lst1p and Sec23/24p were similar morphologically and in their buoyant density, but larger than normal COPII vesicles (87-nm vs. 75-nm diameter). Immunoelectronmicroscopic and biochemical studies revealed both Sec23/Lst1p and Sec23/24p on the membranes of the same vesicles. These results suggest that Lst1p and Sec24p cooperate in the packaging of Pma1p and support the view that biosynthetic precursors of plasma membrane proteins must be sorted into ER-derived transport vesicles. Sec24p homologues may comprise a more complex coat whose combinatorial subunit composition serves to expand the range of cargo to be packaged into COPII vesicles. By changing the geometry of COPII coat polymerization, Lst1p may allow the transport of bulky cargo molecules, polymers, or particles. PMID:11086000

  19. Pmt-mediated O mannosylation stabilizes an essential component of the secretory apparatus, Sec20p, in Candida albicans.

    PubMed

    Weber, Yvonne; Prill, Stephan K-H; Ernst, Joachim F

    2004-10-01

    Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.

  20. 21 CFR 876.5820 - Hemodialysis system and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... conditions and that consists of an extracorporeal blood system, a conventional dialyzer, a dialysate delivery system, and accessories. Blood from a patient flows through the tubing of the extracorporeal blood system and accessories to the blood compartment of the dialyzer, then returns through further tubing of...

  1. 21 CFR 876.5820 - Hemodialysis system and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... conditions and that consists of an extracorporeal blood system, a conventional dialyzer, a dialysate delivery system, and accessories. Blood from a patient flows through the tubing of the extracorporeal blood system and accessories to the blood compartment of the dialyzer, then returns through further tubing of...

  2. 21 CFR 876.5820 - Hemodialysis system and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... conditions and that consists of an extracorporeal blood system, a conventional dialyzer, a dialysate delivery system, and accessories. Blood from a patient flows through the tubing of the extracorporeal blood system and accessories to the blood compartment of the dialyzer, then returns through further tubing of...

  3. 21 CFR 876.5820 - Hemodialysis system and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... conditions and that consists of an extracorporeal blood system, a conventional dialyzer, a dialysate delivery system, and accessories. Blood from a patient flows through the tubing of the extracorporeal blood system and accessories to the blood compartment of the dialyzer, then returns through further tubing of...

  4. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  5. 14 CFR 125.149 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 3 2012-01-01 2012-01-01 false Engine accessory section diaphragm. 125.149... Requirements § 125.149 Engine accessory section diaphragm. Unless equivalent protection can be shown by other means, a diaphragm that complies with § 125.145 must be provided on air-cooled engines to isolate...

  6. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  7. 14 CFR 125.149 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 3 2013-01-01 2013-01-01 false Engine accessory section diaphragm. 125.149... Requirements § 125.149 Engine accessory section diaphragm. Unless equivalent protection can be shown by other means, a diaphragm that complies with § 125.145 must be provided on air-cooled engines to isolate...

  8. 14 CFR 23.1437 - Accessories for multiengine airplanes.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Accessories for multiengine airplanes. 23... TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Equipment Miscellaneous Equipment § 23.1437 Accessories for multiengine airplanes. For multiengine...

  9. 14 CFR 23.1437 - Accessories for multiengine airplanes.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Accessories for multiengine airplanes. 23... TRANSPORTATION AIRCRAFT AIRWORTHINESS STANDARDS: NORMAL, UTILITY, ACROBATIC, AND COMMUTER CATEGORY AIRPLANES Equipment Miscellaneous Equipment § 23.1437 Accessories for multiengine airplanes. For multiengine...

  10. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  11. 14 CFR 125.149 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Engine accessory section diaphragm. 125.149... Requirements § 125.149 Engine accessory section diaphragm. Unless equivalent protection can be shown by other means, a diaphragm that complies with § 125.145 must be provided on air-cooled engines to isolate...

  12. 14 CFR 23.1192 - Engine accessory compartment diaphragm.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Engine accessory compartment diaphragm. 23... Powerplant Powerplant Fire Protection § 23.1192 Engine accessory compartment diaphragm. For aircooled radial engines, the engine power section and all portions of the exhaust sytem must be isolated from the...

  13. 14 CFR 125.149 - Engine accessory section diaphragm.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 3 2011-01-01 2011-01-01 false Engine accessory section diaphragm. 125.149... Requirements § 125.149 Engine accessory section diaphragm. Unless equivalent protection can be shown by other means, a diaphragm that complies with § 125.145 must be provided on air-cooled engines to isolate...

  14. 21 CFR 864.3600 - Microscopes and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Microscopes and accessories. 864.3600 Section 864.3600 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Pathology Instrumentation and Accessories §...

  15. 21 CFR 884.2700 - Intrauterine pressure monitor and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Intrauterine pressure monitor and accessories. 884... Monitoring Devices § 884.2700 Intrauterine pressure monitor and accessories. (a) Identification. An intrauterine pressure monitor is a device designed to detect and measure intrauterine and amniotic...

  16. 21 CFR 884.2660 - Fetal ultrasonic monitor and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Fetal ultrasonic monitor and accessories. 884.2660... Devices § 884.2660 Fetal ultrasonic monitor and accessories. (a) Identification. A fetal ultrasonic monitor is a device designed to transmit and receive ultrasonic energy into and from the pregnant...

  17. 21 CFR 884.2660 - Fetal ultrasonic monitor and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Fetal ultrasonic monitor and accessories. 884.2660... Devices § 884.2660 Fetal ultrasonic monitor and accessories. (a) Identification. A fetal ultrasonic monitor is a device designed to transmit and receive ultrasonic energy into and from the pregnant...

  18. 49 CFR 192.147 - Flanges and flange accessories.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ....147 Flanges and flange accessories. (a) Each flange or flange accessory (other than cast iron) must... be subjected in service. (c) Each flange on a flanged joint in cast iron pipe must conform in dimensions, drilling, face and gasket design to ASME/ANSI B16.1 and be cast integrally with the pipe,...

  19. 46 CFR 98.25-40 - Valves, fittings, and accessories.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... Anhydrous Ammonia in Bulk § 98.25-40 Valves, fittings, and accessories. (a) All valves, flanges, fittings and accessory equipment shall be of a type suitable for use with anhydrous ammonia and shall be made... Engineering) of this chapter. Valves shall be fitted with noncorrosive material suitable for ammonia...

  20. 46 CFR 98.25-40 - Valves, fittings, and accessories.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... Anhydrous Ammonia in Bulk § 98.25-40 Valves, fittings, and accessories. (a) All valves, flanges, fittings and accessory equipment shall be of a type suitable for use with anhydrous ammonia and shall be made... Engineering) of this chapter. Valves shall be fitted with noncorrosive material suitable for ammonia...

  1. 46 CFR 98.25-40 - Valves, fittings, and accessories.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Anhydrous Ammonia in Bulk § 98.25-40 Valves, fittings, and accessories. (a) All valves, flanges, fittings and accessory equipment shall be of a type suitable for use with anhydrous ammonia and shall be made... Engineering) of this chapter. Valves shall be fitted with noncorrosive material suitable for ammonia...

  2. 46 CFR 98.25-40 - Valves, fittings, and accessories.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Anhydrous Ammonia in Bulk § 98.25-40 Valves, fittings, and accessories. (a) All valves, flanges, fittings and accessory equipment shall be of a type suitable for use with anhydrous ammonia and shall be made... Engineering) of this chapter. Valves shall be fitted with noncorrosive material suitable for ammonia...

  3. 46 CFR 98.25-40 - Valves, fittings, and accessories.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... Anhydrous Ammonia in Bulk § 98.25-40 Valves, fittings, and accessories. (a) All valves, flanges, fittings and accessory equipment shall be of a type suitable for use with anhydrous ammonia and shall be made... Engineering) of this chapter. Valves shall be fitted with noncorrosive material suitable for ammonia...

  4. A human exchange factor for ARF contains Sec7- and pleckstrin-homology domains.

    PubMed

    Chardin, P; Paris, S; Antonny, B; Robineau, S; Béraud-Dufour, S; Jackson, C L; Chabre, M

    1996-12-05

    The small G protein ARF1 is involved in the coating of vesicles that bud from the Golgi compartments. Its activation is controlled by as-yet unidentified guanine-nucleotide exchange factors. Gea1, the first ARF exchange factor to be discovered in yeast, is a large protein containing a domain of homology with Sec7, another yeast protein that is also involved in secretion. Here we characterized a smaller human protein (relative molecular mass 47K) named ARNO, which contains a central Sec7 domain that promotes guanine-nucleotide exchange on ARF1. ARNO also contains an amino-terminal coiled-coil motif and a carboxy-terminal pleckstrin-homology (PH) domain. The PH domain mediates an enhancement of ARNO exchange activity by negatively charged phospholipid vesicles supplemented with phosphatidylinositol bisphosphate. The exchange activity of ARNO is not inhibited by brefeldin A, an agent known to block vesicular transport and inhibit the exchange activity on ARF1 in cell extracts. This suggests that a regulatory component which is sensitive to brefeldin A associates with ARNO in vivo, possibly through the amino-terminal coiled-coil. We propose that other proteins with a Sec7 domain regulate different members of the ARF family.

  5. From the Sec complex to the membrane insertase YidC.

    PubMed

    Kuhn, Andreas

    2009-08-01

    The key enzymes that catalyze the insertion of proteins into membranes are the Sec translocase and the YidC membrane insertase. Recent insights into the structure and functional intermediates of these enzymes have provided a first molecular glimpse of how they help the newly synthesized proteins to enter the membrane bilayer. In this process, the new proteins undergo a number of specific interactions in the cytoplasm and at the membrane surface before they insert into the bilayer and translocate their external domains across the membrane. The components involved in this pathway recognize each other at the molecular level, forming a route the membrane protein can move along.

  6. Identification of small-molecule inhibitors against SecA by structure-based virtual ligand screening.

    PubMed

    De Waelheyns, Evelien; Segers, Kenneth; Sardis, Marios Frantzeskos; Anné, Jozef; Nicolaes, Gerry A F; Economou, Anastassios

    2015-11-01

    The rapid rise of antibiotic-resistant bacteria is one of the major concerns in modern medicine. Therefore, to treat bacterial infections, there is an urgent need for new antibacterials-preferably directed against alternative bacterial targets. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec secretion pathway, the major route for bacterial protein export across or into the cytoplasmic membrane. As SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target. Using an in silico approach, we have defined several potentially druggable and conserved pockets on the surface of SecA. We show that three of these potentially druggable sites are important for SecA function. A starting collection of ~500 000 commercially available small-molecules was virtually screened against a predicted druggable pocket in the preprotein-binding domain of Escherichia coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of E. coli SecA. Five inhibitors of the translocation ATPase, and not of basal or membrane ATPase, were identified with IC50 values <65 μm. The most potent inhibitor showed an IC50 of 24 μm. The antimicrobial activity was determined for the five most potent SecA inhibitors. Two compounds were found to possess weak antibacterial activity (IC50 ~198 μm) against E. coli, whereas some compounds showed moderate antibacterial activity (IC50 ~100 μm) against Staphylococcus aureus.

  7. Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity1[OPEN

    PubMed Central

    Du, Yu; Mpina, Mohamed H.; Birch, Paul R.J.; Bouwmeester, Klaas; Govers, Francine

    2015-01-01

    Phytophthora infestans secretes numerous RXLR effectors that modulate host defense and thereby pave the way for successful invasion. Here, we show that the RXLR effector AVR1 is a virulence factor that promotes colonization and suppresses callose deposition, a hallmark of basal defense. To identify host targets of AVR1, we performed yeast two-hybrid screens and selected Sec5 as a candidate. Sec5 is a subunit of the exocyst, a protein complex that is involved in vesicle trafficking. AVR1-like (A-L), a close homolog of AVR1, also acts as a virulence factor, but unlike AVR1, A-L does not suppress CRINKLER2 (CRN2)-induced cell death or interact with Sec5. Compared with AVR1, A-L is shorter and lacks the carboxyl-terminal tail, the T-region that is crucial for CRN2-induced cell death suppression and Sec5 interaction. In planta analyses revealed that AVR1 and Sec5 are in close proximity, and coimmunoprecipitation confirmed the interaction. Sec5 is required for secretion of the pathogenesis-related protein PR-1 and callose deposition and also plays a role in CRN2-induced cell death. Our findings show that P. infestans manipulates an exocyst subunit and thereby potentially disturbs vesicle trafficking, a cellular process that is important for basal defense. This is a novel strategy that oomycete pathogens exploit to modulate host defense. PMID:26336092

  8. Hunting for eruption ages in accessory minerals

    NASA Astrophysics Data System (ADS)

    Vazquez, J. A.

    2012-12-01

    A primary goal in geochronology is to provide precise and accurate ages for tephras that serve as chronostratigraphic markers for constraining the timing and rates of volcanism, sedimentation, climate change, and catastrophic events in Earth history. Zircon remains the most versatile accessory mineral for dating silicic tephras due to its common preservation in distal pyroclastic deposits, as well as the robustness of its U-Pb and U-series systems even after host materials have been hydrothermally altered or weathered. Countless studies document that zircon may be complexly zoned in age due to inheritance, contamination, recycling of antecrysts, protracted crystallization in long-lived magma reservoirs, or any combination of these. Other accessory minerals such as allanite or chevkinite can retain similar records of protracted crystallization. If the goal is to date the durations of magmatic crystallization, differentiation, and/or magma residence, then these protracted chronologies within and between accessory minerals are a blessing. However, if the goal is to date the timing of eruption with high precision, i.e., absolute ages with millennial-scale uncertainties, then this age zoning is a curse. Observations from ion microprobe 238U-230Th dating of Pleistocene zircon and allanite provide insight into the record of near-eruption crystallization in accessory minerals and serve as a guide for high-precision whole-crystal dating. Although imprecise relative to conventional techniques, ion probe analysis allows high-spatial resolution 238U-230Th dating that can document multi-millennial age distributions at the crystal scale. Analysis of unpolished rims and continuous depth profiling of zircon from small and large volume eruptions (e.g., Coso, Mono Craters, Yellowstone) reveals that the final several micrometers of crystallization often yield ages that are indistinguishable from associated eruption ages from the 40Ar/39Ar or (U-Th)/He methods. Using this approach, we

  9. Intrapancreatic accessory spleen diagnosed on radionuclide imaging.

    PubMed

    Belkhir, Sara Melboucy; Archambaud, Frédérique; Prigent, Alain; Chaumet-Riffaud, Philippe

    2009-09-01

    Intrapancreatic accessory spleen (IPAS) is ectopic splenic tissue distinct from the main spleen. A 46-year-old man with chronic hepatitis C, presented in 2006 with low right chest pain which led to a diagnosis of tuberculosis pleurisy. CT scan and MRI showed a round, homogenous, well limited mass of 3cm in the pancreas tail. Tc-99m heat-damaged red blood cell scintigraphy with SPECT-CT was performed to confirm the diagnosis of IPAS. Most cases of IPAS described in the literature were diagnosed by pathologists after distal pancreatectomy and splenectomy performed for a suspicion of pancreatic tumor. However, heat-damaged red blood cell scintigraphy remains the most commonly used diagnostic procedure for IPAS, even if superparamagnetic iron oxide MRI contrast agent may be used in the future.

  10. Fluid assisted installation of electrical cable accessories

    DOEpatents

    Mayer, Robert W.; Silva, Frank A.

    1977-01-01

    An electrical cable accessory includes a generally tubular member of elastomeric material which is to be installed by placement over a cylindrical surface to grip the cylindrical surface, when in appropriate assembled relation therewith, with a predetermined gripping force established by dilation of the tubular member, the installation being facilitated by introducing fluid under pressure, through means provided in the tubular member, between the tubular member and the cylindrical surface, and simultaneously impeding the escape of the fluid under pressure from between the tubular member and the cylindrical surface by means adjacent one of the ends of the tubular member to cause dilation of the tubular member and establish a fluid layer between the tubular member and the cylindrical surface, thereby reducing the gripping force during installation.

  11. Germline Heterozygous Variants in SEC23B Are Associated with Cowden Syndrome and Enriched in Apparently Sporadic Thyroid Cancer

    PubMed Central

    Yehia, Lamis; Niazi, Farshad; Ni, Ying; Ngeow, Joanne; Sankunny, Madhav; Liu, Zhigang; Wei, Wei; Mester, Jessica L.; Keri, Ruth A.; Zhang, Bin; Eng, Charis

    2015-01-01

    Cancer-predisposing genes associated with inherited cancer syndromes help explain mechanisms of sporadic carcinogenesis and often inform normal development. Cowden syndrome (CS) is an autosomal-dominant disorder characterized by high lifetime risks of epithelial cancers, such that ∼50% of affected individuals are wild-type for known cancer-predisposing genes. Using whole-exome and Sanger sequencing of a multi-generation CS family affected by thyroid and other cancers, we identified a pathogenic missense heterozygous SEC23B variant (c.1781T>G [p.Val594Gly]) that segregates with the phenotype. We also found germline heterozygous SEC23B variants in 3/96 (3%) unrelated mutation-negative CS probands with thyroid cancer and in The Cancer Genome Atlas (TCGA), representing apparently sporadic cancers. We note that the TCGA thyroid cancer dataset is enriched with unique germline deleterious SEC23B variants associated with a significantly younger age of onset. SEC23B encodes Sec23 homolog B (S. cerevisiae), a component of coat protein complex II (COPII), which transports proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. Interestingly, germline homozygous or compound-heterozygous SEC23B mutations cause an unrelated disorder, congenital dyserythropoietic anemia type II, and SEC23B-deficient mice suffer from secretory organ degeneration due to ER-stress-associated apoptosis. By characterizing the p.Val594Gly variant in a normal thyroid cell line, we show that it is a functional alteration that results in ER-stress-mediated cell-colony formation and survival, growth, and invasion, which reflect aspects of a cancer phenotype. Our findings suggest a different role for SEC23B, whereby germline heterozygous variants associate with cancer predisposition potentially mediated by ER stress “addiction.” PMID:26522472

  12. Resistance to a novel antichlamydial compound is mediated through mutations in Chlamydia trachomatis secY.

    PubMed

    Sandoz, Kelsi M; Eriksen, Steven G; Jeffrey, Brendan M; Suchland, Robert J; Putman, Timothy E; Hruby, Dennis E; Jordan, Robert; Rockey, Daniel D

    2012-08-01

    A novel and quantitative high-throughput screening approach was explored as a tool for the identification of novel compounds that inhibit chlamydial growth in mammalian cells. The assay is based on accumulation of a fluorescent marker by intracellular chlamydiae. Its utility was demonstrated by screening 42,000 chemically defined compounds against Chlamydia caviae GPIC. This analysis led to the identification of 40 primary-hit compounds. Five of these compounds were nontoxic to host cells and had similar activities against both C. caviae GPIC and Chlamydia trachomatis. The inhibitory activity of one of the compounds, (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA), was chlamydia specific and was selected for further study. Selection for resistance to MDQA led to the generation of three independent resistant clones of C. trachomatis. Amino acid changes in SecY, a protein involved in Sec-dependent secretion in Gram-negative bacteria, were associated with the resistance phenotype. The amino acids changed in each of the resistant mutants are located in the predicted central channel of a SecY crystal structure, based on the known structure of Thermus thermophilus SecY. These experiments model a process that can be used for the discovery of antichlamydial, anti-intracellular, or antibacterial compounds and has led to the identification of compounds that may have utility in both antibiotic discovery and furthering our understanding of chlamydial biology.

  13. Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

    SciTech Connect

    Meining, Winfried; Scheuring, Johannes; Fischer, Markus; Weinkauf, Sevil

    2006-06-01

    SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å, α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.

  14. Morphology of accessory genital glands of spotted paca (Agouti paca Linnaeus, 1766).

    PubMed

    Borges, Edson Moreira; Branco, Érika; de Lima, Ana Rita; Leal, Leonardo Martins; Martins, Leandro Luiz; Reis, Ana Carolina Gonçalves; Cruz, Claudinei; Machado, Márcia Rita Fernandes; Miglino, Maria Angelica

    2014-02-01

    The spotted paca is the second largest rodent in Brazil, where it is of great economic interest in impoverished regions in view of its prominence as a low-cost source of protein. Little is known about the morphology of the accessory genital glands of this species. Thus, we studied the position and morphology of the genitals in ten adult male spotted pacas. The animals were divided into two groups, five animals were used for fixing of samples in 10% aqueous formaldehyde for macroscopic studies and the other five animals were designated for microscopic analysis. These were arranged in pairs and had the vesicular, prostate, coagulating and bulbourethral glands identified, being structured as mucous glands, which lead into the pelvic urethra. It was concluded that the accessory genital glands found in the paca are the same as those found in most rodents, showing similar histological aspects.

  15. The scolopidial accessory organ in the Jerusalem cricket (Orthoptera: Stenopelmatidae).

    PubMed

    Strauß, Johannes

    2017-03-01

    Multiple mechanosensory organs form the subgenual organ complex in orthopteroid insects, located in the proximal tibia. In several Ensifera (Orthoptera), a small chordotonal organ, the so-called accessory organ, is the most posterior part of this sensory complex. In order to document the presence of this accessory organ among the Ensifera, the chordotonal sensilla and their innervation in the posterior tibia of two species of Jerusalem crickets (Stenopelmatidae: Stenopelmatus) is described. The sensory structures were stained by axonal tracing. Scolopidial sensilla occur in the posterior subgenual organ and the accessory organ in all leg pairs. The accessory organ contains 10-17 scolopidial sensilla. Both groups of sensilla are commonly spatially separated. However, in few cases neuronal fibres occurred between both organs. The two sensillum groups are considered as separate organs by the general spatial separation and innervation by different nerve branches. A functional role for mechanoreception is considered: since the accessory organ is located closely under the cuticle, sensilla may be suited to detect vibrations transferred over the leg's surface. This study extends the known taxa with an accessory organ, which occurs in several taxa of Ensifera. Comparative neuroanatomy thus suggests that the accessory organ may be conserved at least in Tettigoniidea.

  16. Successful treatment of accessory breast cancer with endocrine therapy#

    PubMed Central

    Wang, Chun-Xi; Guo, Shu-Li; Han, Li-Na

    2017-01-01

    Accessory breast cancers in males are extremely rare, and only a few cases have been reported in the literature. In this paper, an 87-year-old male patient was diagnosed with an accessory breast cancer by means of computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography-computed tomography (PET-CT), and immunohistochemistry based on needle biopsy, and has undergone successful resection and postoperative adjuvant endocrine therapy. He was the oldest male patient with an accessory breast cancer reported in the Chinese Hospital Knowledge Database and PubMed literature from 1975 to 2015. PMID:28070998

  17. 37. Webster Avenue Bridge. New Rochelle, Westchester Co., NY. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    37. Webster Avenue Bridge. New Rochelle, Westchester Co., NY. Sec. 4207, MP 19.12. - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  18. 36. Webster Avenue Bridge. New Rochelle, Westchester Co., NY. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    36. Webster Avenue Bridge. New Rochelle, Westchester Co., NY. Sec. 4207, MP 19.12. - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  19. 24. Pedestrian Overbridges. West Farms, Bronx Co., NY. Sec. 4207, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    24. Pedestrian Overbridges. West Farms, Bronx Co., NY. Sec. 4207, MP 12.50. - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  20. Loudon Park Substation. Violetville, Baltimore City, MD. Sec. 1201, MP ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Loudon Park Substation. Violetville, Baltimore City, MD. Sec. 1201, MP 100.50. - Northeast Railroad Corridor, Amtrak route between District of Columbia/Maryland state line & Maryland/Delaware state line, Baltimore, Independent City, MD

  1. 7. RARITAN AVENUE BRIDGE. NEW BRUNSWICK, MIDDLESEX CO., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    7. RARITAN AVENUE BRIDGE. NEW BRUNSWICK, MIDDLESEX CO., NJ. Sec. 1401, MP 30.92. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  2. 23. Haynes Avenue Bridge. Newark, Essex Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    23. Haynes Avenue Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 11.39. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  3. 25. Lehigh Valley Railroad Bridge. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    25. Lehigh Valley Railroad Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 10.56. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  4. 16. Rahway Avenue Bridge. Elizabeth, Union Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    16. Rahway Avenue Bridge. Elizabeth, Union Co., NJ. Sec. 1401, MP 14.49. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  5. 8. Raritan River Bridge. New Brunswick, Middlesex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    8. Raritan River Bridge. New Brunswick, Middlesex Co., NJ. Sec. 1401, MP 30.92. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  6. 32. Passaic River Bridge. Newark, Essex Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    32. Passaic River Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 8.50. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  7. 9. Raritan River Bridge. New Brunswick, Middlesex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    9. Raritan River Bridge. New Brunswick, Middlesex Co., NJ. Sec. 1401, MP 30.92. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  8. 6. RARITAN AVENUE BRIDGE. NEW BRUNSWICK, MIDDLESEX CO., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. RARITAN AVENUE BRIDGE. NEW BRUNSWICK, MIDDLESEX CO., NJ. Sec. 1401, MP 30.92 - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  9. 1. BLOOMSBURY STREET BRIDGE. TRENTON, MERCER CO., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. BLOOMSBURY STREET BRIDGE. TRENTON, MERCER CO., NJ. Sec. 1401, MP 57.61. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  10. 22. Haynes Avenue Bridge. Newark, Essex Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    22. Haynes Avenue Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 11.39. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  11. 34. Passaic River Bridge. Newark, Essex Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    34. Passaic River Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 8.50. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  12. 33. Passaic River Bridge. Newark, Essex Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    33. Passaic River Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 8.50. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  13. 10. Milton Avenue Bridge. Rahway, Union Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    10. Milton Avenue Bridge. Rahway, Union Co., NJ. Sec. 1401, MP 19.41. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  14. 14. Rahway River Bridge. Rahway, Union Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. Rahway River Bridge. Rahway, Union Co., NJ. Sec. 1401, MP 19.13. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  15. 27. Lehigh Valley Railroad Bridge. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    27. Lehigh Valley Railroad Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 10.56. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  16. 30. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    30. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. 1401, MP 8.60. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  17. 28. Lafayette Street Bridge. Newark, Essex Co., NJ. Sec. 1401, ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    28. Lafayette Street Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 8.95. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  18. 31. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    31. Pennsylvania Railroad: Newark Station. Newark, Essex Co., NJ. Sec. 1401, MP 8.60. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  19. 26. Lehigh Valley Railroad Bridge. Newark, Essex Co., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    26. Lehigh Valley Railroad Bridge. Newark, Essex Co., NJ. Sec. 1401, MP 10.56. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ

  20. 2. SOUTH CLINTON AVENUE BRIDGE. TRENTON, MERCER CO., NJ. Sec. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    2. SOUTH CLINTON AVENUE BRIDGE. TRENTON, MERCER CO., NJ. Sec. 1401, MP 56.77. - Northeast Railroad Corridor, Amtrak Route between Pennsylvania/New Jersey & New Jersey/New York State Lines, Newark, Essex County, NJ