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Sample records for gap envelope transmembrane

  1. Several Novel Nuclear Envelope Transmembrane Proteins Identified in Skeletal Muscle Have Cytoskeletal Associations*

    PubMed Central

    Wilkie, Gavin S.; Korfali, Nadia; Swanson, Selene K.; Malik, Poonam; Srsen, Vlastimil; Batrakou, Dzmitry G.; de las Heras, Jose; Zuleger, Nikolaj; Kerr, Alastair R. W.; Florens, Laurence; Schirmer, Eric C.

    2011-01-01

    Nuclear envelopes from liver and a neuroblastoma cell line have previously been analyzed by proteomics; however, most diseases associated with the nuclear envelope affect muscle. To determine whether muscle has unique nuclear envelope proteins, rat skeletal muscle nuclear envelopes were prepared and analyzed by multidimensional protein identification technology. Many novel muscle-specific proteins were identified that did not appear in previous nuclear envelope data sets. Nuclear envelope residence was confirmed for 11 of these by expression of fusion proteins and by antibody staining of muscle tissue cryosections. Moreover, transcript levels for several of the newly identified nuclear envelope transmembrane proteins increased during muscle differentiation using mouse and human in vitro model systems. Some of these proteins tracked with microtubules at the nuclear surface in interphase cells and accumulated at the base of the microtubule spindle in mitotic cells, suggesting they may associate with complexes that connect the nucleus to the cytoskeleton. The finding of tissue-specific proteins in the skeletal muscle nuclear envelope proteome argues the importance of analyzing nuclear envelopes from all tissues linked to disease and suggests that general investigation of tissue differences in organellar proteomes might yield critical insights. PMID:20876400

  2. TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation

    PubMed Central

    Batrakou, Dzmitry G.; de las Heras, Jose I.; Czapiewski, Rafal; Mouras, Rabah; Schirmer, Eric C.

    2015-01-01

    Recent work indicates that the nuclear envelope is a major signaling node for the cell that can influence tissue differentiation processes. Here we present two nuclear envelope trans-membrane proteins TMEM120A and TMEM120B that are paralogs encoded by the Tmem120A and Tmem120B genes. The TMEM120 proteins are expressed preferentially in fat and both are induced during 3T3-L1 adipocyte differentiation. Knockdown of one or the other protein altered expression of several genes required for adipocyte differentiation, Gata3, Fasn, Glut4, while knockdown of both together additionally affected Pparg and Adipoq. The double knockdown also increased the strength of effects, reducing for example Glut4 levels by 95% compared to control 3T3-L1 cells upon pharmacologically induced differentiation. Accordingly, TMEM120A and B knockdown individually and together impacted on adipocyte differentiation/metabolism as measured by lipid accumulation through binding of Oil Red O and coherent anti-Stokes Raman scattering microscopy (CARS). The nuclear envelope is linked to several lipodystrophies through mutations in lamin A; however, lamin A is widely expressed. Thus it is possible that the TMEM120A and B fat-specific nuclear envelope transmembrane proteins may play a contributory role in the tissue-specific pathology of this disorder or in the wider problem of obesity. PMID:26024229

  3. Immunising with the transmembrane envelope proteins of different retroviruses including HIV-1

    PubMed Central

    Denner, Joachim

    2013-01-01

    The induction of neutralizing antibodies is a promising way to prevent retrovirus infections. Neutralizing antibodies are mainly directed against the envelope proteins, which consist of two molecules, the surface envelope (SU) protein and the transmembrane envelope (TM) protein. Antibodies broadly neutralizing the human immunodeficiencvy virus-1 (HIV-1) and binding to the TM protein gp41 of the virus have been isolated from infected individuals. Their epitopes are located in the membrane proximal external region (MPER). Since there are difficulties to induce such neutralizing antibodies as basis for an effective AIDS vaccine, we performed a comparative analysis immunising with the TM proteins of different viruses from the family Retroviridae. Both subfamilies, the Orthoretrovirinae and the Spumaretrovirinae were included. In this study, the TM proteins of three gammaretroviruses including (1) the porcine endogenous retrovirus (PERV), (2) the Koala retrovirus (KoRV), (3) the feline leukemia virus (FeLV), of two lentiviruses, HIV-1, HIV-2, and of two spumaviruses, the feline foamy virus (FFV) and the primate foamy virus (PFV) were used for immunisation. Whereas in all immunisation studies binding antibodies were induced, neutralizing antibodies were only found in the case of the gammaretroviruses. The induced antibodies were directed against the MPER and the fusion peptide proximal region (FPPR) of their TM proteins; however only the antibodies against the MPER were neutralizing. Most importantly, the epitopes in the MPER were localized in the same position as the epitopes of the antibodies broadly neutralizing HIV-1 in the TM protein gp41 of HIV-1, indicating that the MPER is an effective target for the neutralization of retroviruses. PMID:23249763

  4. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1

    PubMed Central

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus—1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  5. Antigenic and immunosuppressive properties of a trimeric recombinant transmembrane envelope protein gp41 of HIV-1.

    PubMed

    Mühle, Michael; Lehmann, Melissa; Hoffmann, Kerstin; Stern, Daniel; Kroniger, Tobias; Luttmann, Werner; Denner, Joachim

    2017-01-01

    The transmembrane envelope (TM) protein gp41 of the human immunodeficiency virus-1 (HIV-1) plays an important role during virus infection inducing the fusion of the viral and cellular membranes. In addition, there are indications that the TM protein plays a role in the immunopathogenesis leading to the acquired immunodeficiency syndrome (AIDS). Inactivated virus particles and recombinant gp41 have been reported to inhibit lymphocyte proliferation, as well as to alter cytokine release and gene expression. The same was shown for a peptide corresponding to a highly conserved domain of all retroviral TM proteins, the immunosuppressive domain. Due to its propensity to aggregate and to be expressed at low levels, studies comprising authentic gp41 produced in eukaryotic cells are extremely rare. Here we describe the production of a secreted, soluble recombinant gp41 in 293 cells. The antigen was purified to homogeneity and characterised thoroughly by various biochemical and immunological methods. It was shown that the protein was glycosylated and assembled into trimers. Binding studies by ELISA and surface plasmon resonance using conformation-specific monoclonal antibodies implied a six-helix bundle conformation. The low binding of broadly neutralising antibodies (bnAb) directed against the membrane proximal external region (MPER) suggested that this gp41 is probably not suited as vaccine to induce such bnAb. Purified gp41 bound to monocytes and to a lesser extent to lymphocytes and triggered the production of specific cytokines when added to normal peripheral blood mononuclear cells. In addition, gp41 expressed on target cells inhibited the antigen-specific response of murine CD8+ T cells by drastically impairing their IFNγ production. To our knowledge, this is the first comprehensive analysis of a gp41 produced in eukaryotic cells including its immunosuppressive properties. Our data provide another line of evidence that gp41 might be directly involved in HIV-1

  6. Targeting of a polytopic membrane protein to the inner envelope membrane of chloroplasts in vivo involves multiple transmembrane segments

    PubMed Central

    Okawa, Kumiko; Inoue, Hitoshi; Adachi, Fumi; Nakayama, Katsuhiro; Ito-Inaba, Yasuko; Schnell, Danny J.; Uehara, Susumu; Inaba, Takehito

    2014-01-01

    The inner envelope membrane (IEM) of the chloroplast plays crucial roles in forming an osmotic barrier and controlling metabolite exchange between the organelle and the cytosol. The IEM therefore harbours a number of membrane proteins and requires the import and integration of these nuclear-encoded proteins for its biogenesis. Recent studies have demonstrated that the transmembrane segment of single-spanning IEM proteins plays key roles in determining their IEM localization. However, few studies have focused on the molecular mechanisms by which polytopic membrane proteins are targeted to the IEM. In this study, we investigated the targeting mechanism of polytopic IEM proteins using the protein Cor413im1 as a model substrate. Cor413im1 does not utilize a soluble intermediate for its targeting to the IEM. Furthermore, we show that the putative fifth transmembrane segment of Cor413im1 is necessary for its targeting to the IEM. The C-terminal portion containing this transmembrane segment is also able to deliver Cor413im1 protein to the IEM. However, the fifth transmembrane segment of Cor413im1 itself is insufficient to target a fusion protein to the IEM. These data suggest that the targeting of polytopic membrane proteins to the chloroplast IEM in vivo involves multiple transmembrane segments and that chloroplasts have evolved a unique mechanism for the integration of polytopic proteins to the IEM. PMID:25013120

  7. Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells.

    PubMed

    Christodoulou, Andri; Santarella-Mellwig, Rachel; Santama, Niovi; Mattaj, Iain W

    2016-04-15

    The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation.

  8. Transmembrane protein TMEM170A is a newly discovered regulator of ER and nuclear envelope morphogenesis in human cells

    PubMed Central

    Christodoulou, Andri; Santarella-Mellwig, Rachel; Santama, Niovi

    2016-01-01

    ABSTRACT The mechanism of endoplasmic reticulum (ER) morphogenesis is incompletely understood. ER tubules are shaped by the reticulons (RTNs) and DP1/Yop1p family members, but the mechanism of ER sheet formation is much less clear. Here, we characterize TMEM170A, a human transmembrane protein, which localizes in ER and nuclear envelope membranes. Silencing or overexpressing TMEM170A in HeLa K cells alters ER shape and morphology. Ultrastructural analysis reveals that downregulation of TMEM170A specifically induces tubular ER formation, whereas overexpression of TMEM170A induces ER sheet formation, indicating that TMEM170A is a newly discovered ER-sheet-promoting protein. Additionally, downregulation of TMEM170A alters nuclear shape and size, decreases the density of nuclear pore complexes (NPCs) in the nuclear envelope and causes either a reduction in inner nuclear membrane (INM) proteins or their relocalization to the ER. TMEM170A interacts with RTN4, a member of the reticulon family; simultaneous co-silencing of TMEM170A and RTN4 rescues ER, NPC and nuclear-envelope-related phenotypes, implying that the two proteins have antagonistic effects on ER membrane organization, and nuclear envelope and NPC formation. PMID:26906412

  9. Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening.

    PubMed

    Mühle, Michael; Bleiholder, Anne; Kolb, Susanne; Hübner, Janine; Löchelt, Martin; Denner, Joachim

    2011-04-10

    The transmembrane envelope (TM) proteins of retroviruses are used as antigen in diagnostic immunoassays and they represent a conserved target for neutralizing antibodies. To analyze the situation in infections with the feline foamy virus (FFV), its recombinant TM protein was produced and used for ELISA and Western blot analyses. Screening sera from 404 German cats showed that 39% reacted against the TM protein, the same infection rate was determined using the Gag protein. Epitope mapping showed antibodies against the membrane proximal external region (MPER) of the TM protein in the sera from infected cats, but attempts to induce neutralizing antibodies by immunization with the recombinant TM protein failed. This is the first report demonstrating that the TM protein of the FFV is highly immunogenic and valuable for serological screening. Similar to HIV-1, but in contrast to different gammaretroviruses, immunization with the TM protein of FFV did not induce neutralizing antibodies.

  10. The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

    PubMed

    Wozniak, R W; Blobel, G

    1992-12-01

    The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.

  11. The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope

    PubMed Central

    1992-01-01

    The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components. PMID:1281815

  12. The HIV-1 envelope transmembrane domain binds TLR2 through a distinct dimerization motif and inhibits TLR2-mediated responses.

    PubMed

    Reuven, Eliran Moshe; Ali, Mohammad; Rotem, Etai; Schwarzer, Roland; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H; Shai, Yechiel

    2014-08-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.

  13. The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

    PubMed Central

    Rotem, Etai; Schwarzter, Roland; Gramatica, Andrea; Futerman, Anthony H.; Shai, Yechiel

    2014-01-01

    HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation. PMID:25121610

  14. Gap and out-gap solitons in modulated systems of finite length: exact solutions in the slowly varying envelope limit

    NASA Astrophysics Data System (ADS)

    Johansson, M.; Kirr, K.; Kovalev, A. S.; Kroon, L.

    2011-06-01

    We discuss nonlinear excitations in finite-size one-dimensional modulated systems. Considering a binary modulated discrete nonlinear Schrödinger chain of large but finite length with periodic boundary conditions, we obtain exact elliptic-function solutions corresponding to stationary excitations in the slowly varying envelope limit. From these solutions, we analyze how the transformation between (localized) gap and (delocalized) out-gap solitons manifests itself in a system of finite length. The analogue of a localized gap soliton appears through a bifurcation at a critical point, so that gap soliton analogues exist only for chains longer than a critical value, which scales inversely proportional to the modulation depth. The total norm of these gap-out-gap states is found to be a monotonic function of the frequency, always inside a 'nonlinear gap' with edges defined by the main nonlinear modes which approach the linear spectrum gap boundaries in the small-amplitude limit. The transformation from a gap to an out-gap state is associated with a particular frequency, close to the lower boundary of the linear gap; at this point the elliptic functions become trigonometric, corresponding to a finite-size analogue of an algebraic soliton. We compare the scenario with earlier results obtained numerically for purely discrete chains with few degrees of freedom.

  15. Mutations within a putative cysteine loop of the transmembrane protein of an attenuated immunodeficiency-inducing feline leukemia virus variant inhibit envelope protein processing.

    PubMed Central

    Burns, C C; Poss, M L; Thomas, E; Overbaugh, J

    1995-01-01

    A replication-defective feline leukemia virus molecular clone, 61B, has been shown to cause immunodeficiency in cats and cytopathicity in T cells after a long latency period when coinfected with a minimally pathogenic helper virus (J. Overbaugh, E. A. Hoover, J. I. Mullins, D. P. W. Burns, L. Rudensey, S. L. Quackenbush, V. Stallard, and P. R. Donahue, Virology 188:558-569, 1992). The long-latency phenotype of 61B has been mapped to four mutations in the extracellular domain of the envelope transmembrane protein, and we report here that these mutations cause a defect in envelope protein processing. Immunoprecipitation analyses demonstrated that the 61B gp85 envelope precursor was produced but that further processing to generate the surface protein (SU/gp70) and the transmembrane protein (TM/p15E) did not occur. The 61B precursor was not expressed on the cell surface and appeared to be retained in the endoplasmic reticulum or Golgi apparatus. Two of the four 61B-specific amino acid changes are located within a putative cysteine loop in a region of TM that is conserved among retroviruses. Introduction of these two amino acid changes into a replication-competent highly cytopathic virus resulted in the production of noninfectious virus that exhibited an envelope-protein-processing defect. This analysis suggests that mutations in a conserved region within a putative cysteine loop affect retroviral envelope protein maturation and viral infectivity. PMID:7884859

  16. A flow cytometry-based screen of nuclear envelope transmembrane proteins identifies NET4/Tmem53 as involved in stress-dependent cell cycle withdrawal.

    PubMed

    Korfali, Nadia; Srsen, Vlastimil; Waterfall, Martin; Batrakou, Dzmitry G; Pekovic, Vanja; Hutchison, Christopher J; Schirmer, Eric C

    2011-04-14

    Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N:2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53(-/-) cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held.

  17. A Flow Cytometry-Based Screen of Nuclear Envelope Transmembrane Proteins Identifies NET4/Tmem53 as Involved in Stress-Dependent Cell Cycle Withdrawal

    PubMed Central

    Waterfall, Martin; Batrakou, Dzmitry G.; Pekovic, Vanja; Hutchison, Christopher J.; Schirmer, Eric C.

    2011-01-01

    Disruption of cell cycle regulation is one mechanism proposed for how nuclear envelope protein mutation can cause disease. Thus far only a few nuclear envelope proteins have been tested/found to affect cell cycle progression: to identify others, 39 novel nuclear envelope transmembrane proteins were screened for their ability to alter flow cytometry cell cycle/DNA content profiles when exogenously expressed. Eight had notable effects with seven increasing and one decreasing the 4N∶2N ratio. We subsequently focused on NET4/Tmem53 that lost its effects in p53−/− cells and retinoblastoma protein-deficient cells. NET4/TMEM53 knockdown by siRNA altered flow cytometry cell cycle/DNA content profiles in a similar way as overexpression. NET4/TMEM53 knockdown did not affect total retinoblastoma protein levels, unlike nuclear envelope-associated proteins Lamin A and LAP2α. However, a decrease in phosphorylated retinoblastoma protein was observed along with a doubling of p53 levels and a 7-fold increase in p21. Consequently cells withdrew from the cell cycle, which was confirmed in MRC5 cells by a drop in the percentage of cells expressing Ki-67 antigen and an increase in the number of cells stained for ß-galactosidase. The ß-galactosidase upregulation suggests that cells become prematurely senescent. Finally, the changes in retinoblastoma protein, p53, and p21 resulting from loss of NET4/Tmem53 were dependent upon active p38 MAP kinase. The finding that roughly a fifth of nuclear envelope transmembrane proteins screened yielded alterations in flow cytometry cell cycle/DNA content profiles suggests a much greater influence of the nuclear envelope on the cell cycle than is widely held. PMID:21533191

  18. Regulated Erlin-dependent release of the B12 transmembrane J-protein promotes ER membrane penetration of a non-enveloped virus

    PubMed Central

    2017-01-01

    The molecular mechanism by which non-enveloped viruses penetrate biological membranes remains enigmatic. The non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol and cause infection. We previously demonstrated that SV40 creates its own membrane penetration structure by mobilizing select transmembrane proteins to distinct puncta in the ER membrane called foci that likely function as the cytosol entry sites. How these ER membrane proteins reorganize into the foci is unknown. B12 is a transmembrane J-protein that mobilizes into the foci to promote cytosol entry of SV40. Here we identify two closely related ER membrane proteins Erlin1 and Erlin2 (Erlin1/2) as B12-interaction partners. Strikingly, SV40 recruits B12 to the foci by inducing release of this J-protein from Erlin1/2. Our data thus reveal how a non-enveloped virus promotes its own membrane translocation by triggering the release and recruitment of a critical transport factor to the membrane penetration site. PMID:28614383

  19. Activation of the erythropoietin receptor by the gp55-P viral envelope protein is determined by a single amino acid in its transmembrane domain.

    PubMed Central

    Constantinescu, S N; Liu, X; Beyer, W; Fallon, A; Shekar, S; Henis, Y I; Smith, S O; Lodish, H F

    1999-01-01

    The spleen focus forming virus (SFFV) gp55-P envelope glycoprotein specifically binds to and activates murine erythropoietin receptors (EpoRs) coexpressed in the same cell, triggering proliferation of erythroid progenitors and inducing erythroleukemia. Here we demonstrate specific interactions between the single transmembrane domains of the two proteins that are essential for receptor activation. The human EpoR is not activated by gp55-P but by mutation of a single amino acid, L238, in its transmembrane sequence to its murine counterpart serine, resulting in its ability to be activated. The converse mutation in the murine EpoR (S238L) abolishes activation by gp55-P. Computational searches of interactions between the membrane-spanning segments of murine EpoR and gp55-P provide a possible explanation: the face of the EpoR transmembrane domain containing S238 is predicted to interact specifically with gp55-P but not gp55-A, a variant which is much less effective in activating the murine EpoR. Mutational studies on gp55-P M390, which is predicted to interact with S238, provide additional support for this model. Mutation of M390 to isoleucine, the corresponding residue in gp55-A, abolishes activation, but the gp55-P M390L mutation is fully functional. gp55-P is thought to activate signaling by the EpoR by inducing receptor oligomerization through interactions involving specific transmembrane residues. PMID:10369674

  20. The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

    PubMed Central

    Inabe, Kazunori; Nishizawa, Masako; Tajima, Shigeru; Ikuta, Kazuyoshi; Aida, Yoko

    1999-01-01

    The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV. PMID:9882334

  1. Transmembrane Domains of Hepatitis C Virus Envelope Glycoproteins: Residues Involved in E1E2 Heterodimerization and Involvement of These Domains in Virus Entry▿

    PubMed Central

    Ciczora, Yann; Callens, Nathalie; Penin, François; Pécheur, Eve-Isabelle; Dubuisson, Jean

    2007-01-01

    The transmembrane (TM) domains of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 have been shown to play multiple roles during the biogenesis of the E1E2 heterodimer. By using alanine scanning insertion mutagenesis within the TM domains of HCV envelope glycoproteins, we have previously shown that the central regions of these domains as well as the N-terminal part of the TM domain of E1 are involved in heterodimerization. Here, we used a tryptophan replacement scan of these regions to identify individual residues that participate in those interactions. Our mutagenesis study identified at least four residues involved in heterodimerization: Gly 354, Gly 358, Lys 370, and Asp 728. Interestingly, Gly 354 and Gly 358 belong to a GXXXG oligomerization motif. Our tryptophan mutants were also used to generate retrovirus-based, HCV-pseudotyped particles (HCVpp) in order to analyze the effects of these mutations on virus entry. Surprisingly, two mutants consistently displayed higher infectivity compared to that of the wild type. In contrast, HCVpp infectivity was strongly affected for many mutants, despite normal E1E2 heterodimerization and normal levels of incorporation of HCV glycoproteins into HCVpp. The characterization of some of these HCVpp mutants in the recently developed in vitro fusion assay using fluorescent-labeled liposomes indicated that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affected the fusion properties of HCV envelope glycoproteins. In conclusion, this mutational analysis identified residues involved in E1E2 heterodimerization and revealed that the TM domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins. PMID:17166909

  2. Interactions of the Cytoplasmic Domains of Human and Simian Retroviral Transmembrane Proteins with Components of the Clathrin Adaptor Complexes Modulate Intracellular and Cell Surface Expression of Envelope Glycoproteins

    PubMed Central

    Berlioz-Torrent, Clarisse; Shacklett, Barbara L.; Erdtmann, Lars; Delamarre, Lelia; Bouchaert, Isabelle; Sonigo, Pierre; Dokhelar, Marie Christine; Benarous, Richard

    1999-01-01

    The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXØ, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXØ motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXØ motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXØ motifs interact with the μ1 and μ2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the β2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXØ-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved. PMID:9882340

  3. MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells.

    PubMed

    Jafferali, Mohammed Hakim; Vijayaraghavan, Balaje; Figueroa, Ricardo A; Crafoord, Ellinor; Gudise, Santhosh; Larsson, Veronica J; Hallberg, Einar

    2014-10-01

    Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle. Copyright © 2014. Published by Elsevier B.V.

  4. Nuclear translocation of RanGAP1 coincides with virtual nuclear envelope breakdown in fission yeast meiosis.

    PubMed

    Asakawa, Haruhiko; Hiraoka, Yasushi; Haraguchi, Tokuko

    2011-05-01

    In higher eukaryotes, mitosis proceeds with nuclear envelope breakdown (NEBD) and disassembly of the nuclear pore complex (NPC); this is designated "open" mitosis. On the other hand, in many fungi, mitosis and chromosome segregation takes place without NEBD; this is designated "closed" mitosis. In a recent study on Schizosaccharomyces pombe, a closed mitosis organism, we reported a novel phenomenon that is equivalent to NEBD: a mixing of nuclear proteins and cytoplasmic proteins occurred transiently for a few minutes in meiosis without physical breakdown of the nuclear envelope. We designated this event virtual nuclear envelope breakdown (V-NEBD). In S. pombe, nuclear translocation of Rna1, a RanGAP1 homolog in S. pombe, occurs during meiosis, and this translocation of Rna1 leads to collapse of the Ran-GTP gradient across the nuclear envelope and occurs coincidently with V-NEBD. Here, we describe possible roles of RanGAP1 in V-NEBD in S. pombe and provide insights into the roles V-NEBD may play in meiosis.

  5. Coreceptor Tropism Can Be Influenced by Amino Acid Substitutions in the gp41 Transmembrane Subunit of Human Immunodeficiency Virus Type 1 Envelope Protein▿

    PubMed Central

    Huang, Wei; Toma, Jonathan; Fransen, Signe; Stawiski, Eric; Reeves, Jacqueline D.; Whitcomb, Jeannette M.; Parkin, Neil; Petropoulos, Christos J.

    2008-01-01

    Many studies have demonstrated that the third variable region (V3) of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env) is a major determinant of coreceptor tropism. Other regions in the surface gp120 subunit of Env can modulate coreceptor tropism in a manner that is not fully understood. In this study, we evaluated the effect of env determinants outside of V3 on coreceptor usage through the analysis of (i) patient-derived env clones that differ in coreceptor tropism, (ii) chimeric env sequences, and (iii) site-directed mutants. The introduction of distinct V3 sequences from CXCR4-using clones into an R5-tropic env backbone conferred the inefficient use of CXCR4 in some but not all cases. Conversely, in many cases, X4- and dual-tropic env backbones containing the V3 sequences of R5-tropic clones retained the ability to use CXCR4, suggesting that sequences outside of the V3 regions of these CXCR4-using clones were responsible for CXCR4 use. The determinants of CXCR4 use in a set of dual-tropic env sequences with V3 sequences identical to those of R5-tropic clones mapped to the gp41 transmembrane (TM) subunit. In one case, a single-amino-acid substitution in the fusion peptide of TM was able to confer CXCR4 use; however, TM substitutions associated with CXCR4 use varied among different env sequences. These results demonstrate that sequences in TM can modulate coreceptor specificity and that env sequences other than that of V3 may facilitate efficient CXCR4-mediated entry. We hypothesize that the latter plays an important role in the transition from CCR5 to CXCR4 coreceptor use. PMID:18353956

  6. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions.

  7. Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

    PubMed Central

    Mahajan, Rohit; Gerace, Larry; Melchior, Frauke

    1998-01-01

    The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role. PMID:9442102

  8. VAP-B binds to Rab3GAP1 at the ER: its implication in nuclear envelope formation through the ER-Golgi intermediate compartment.

    PubMed

    Hantan, Degejirihu; Yamamoto, Yasunori; Sakisaka, Toshiaki

    2014-10-01

    The vesicle-associated membrane protein-associated protein B (VAP-B) is a tail-anchored protein in the endoplasmic reticulum (ER). VAP-B functions as an adaptor protein to recruit target proteins to the ER and execute various cellular functions, lipid transport, membrane traffic, ER stress etc. Recently, VAP-B has been shown to regulate the nuclear envelope protein transport through the ER-Golgi intermediate compartment (ERGIC). We showed here that VAP-B directly binds to Rab3 GTPase activating protein 1 (Rab3GAP1), the catalytic subunit of Rab3GAP, through the two phenylalanines (FF) in an acidic tract (FFAT)-like motif of Rab3GAP1. Rab3GAP consists of two subunits, the catalytic subunit Rab3GAP1 and the non-catalytic subunit Rab3GAP2. VAP-B binds to Rab3GAP1 even in the Rab3GAP1/2 heterodimer complex. A single amino acid substitution of the FFAT-like motif reduces the binding activity of Rab3GAP1 to VAP-B. On the other hand, the FFAT-like motif mutation increases the binding activity of Rab3GAP1 to ERGIC-53, the ERGIC marker protein. Overexpression of Rab3GAP1 affects nuclear envelope formation more potently than that of Rab3GAP1 FFAT-like motif mutant. These results suggest that the binding of VAP-B to Rab3GAP1 is implicated in the regulation of nuclear envelope formation through ERGIC.

  9. Gap Detection in School-Age Children and Adults: Effects of Inherent Envelope Modulation and the Availability of Cues across Frequency

    ERIC Educational Resources Information Center

    Buss, Emily; Hall, Joseph W., III; Porter, Heather; Grose, John H.

    2014-01-01

    Purpose: The present study evaluated the effects of inherent envelope modulation and the availability of cues across frequency on behavioral gap detection with noise-band stimuli in school-age children. Method: Listeners were 34 normal-hearing children (ages 5.2-15.6 years) and 12 normal-hearing adults (ages 18.5-28.8 years). Stimuli were…

  10. Gap Detection in School-Age Children and Adults: Effects of Inherent Envelope Modulation and the Availability of Cues across Frequency

    ERIC Educational Resources Information Center

    Buss, Emily; Hall, Joseph W., III; Porter, Heather; Grose, John H.

    2014-01-01

    Purpose: The present study evaluated the effects of inherent envelope modulation and the availability of cues across frequency on behavioral gap detection with noise-band stimuli in school-age children. Method: Listeners were 34 normal-hearing children (ages 5.2-15.6 years) and 12 normal-hearing adults (ages 18.5-28.8 years). Stimuli were…

  11. Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32.

    PubMed

    Brennan, Matthew J; Karcz, Jennifer; Vaughn, Nicholas R; Woolwine-Cunningham, Yvonne; DePriest, Adam D; Escalona, Yerko; Perez-Acle, Tomas; Skerrett, I Martha

    2015-07-10

    Tryptophan was substituted for residues in all four transmembrane domains of connexin32. Function was assayed using dual cell two-electrode voltage clamp after expression in Xenopus oocytes. Tryptophan substitution was poorly tolerated in all domains, with the greatest impact in TM1 and TM4. For instance, in TM1, 15 substitutions were made, six abolished coupling and five others significantly reduced function. Only TM2 and TM3 included a distinct helical face that lacked sensitivity to tryptophan substitution. Results were visualized on a comparative model of Cx32 hemichannel. In this model, a region midway through the membrane appears highly sensitive to tryptophan substitution and includes residues Arg-32, Ile-33, Met-34, and Val-35. In the modeled channel, pore-facing regions of TM1 and TM2 were highly sensitive to tryptophan substitution, whereas the lipid-facing regions of TM3 and TM4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also highly sensitive to tryptophan substitution. Although future studies will be required to separate trafficking-defective mutants from those that alter channel function, a subset of interactions important for voltage gating was identified. Interactions important for voltage gating occurred mainly in the mid-region of the channel and focused on TM1. To determine whether results could be extrapolated to other connexins, TM1 of Cx43 was scanned revealing similar but not identical sensitivity to TM1 of Cx32.

  12. Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

    PubMed

    Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  13. Analysis of Trafficking, Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

    PubMed Central

    Ambrosi, Cinzia; Walker, Amy E.; DePriest, Adam D.; Cone, Angela C.; Lu, Connie; Badger, John; Skerrett, I. Martha; Sosinsky, Gina E.

    2013-01-01

    Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased

  14. The C-terminal tail of the gp41 transmembrane envelope glycoprotein of HIV-1 clades A, B, C, and D may exist in two conformations: an analysis of sequence, structure, and function

    SciTech Connect

    Hollier, Mark J.; Dimmock, Nigel J. . E-mail: n.j.dimmock@warwick.ac.uk

    2005-07-05

    In addition to the major ectodomain, the gp41 transmembrane glycoprotein of HIV-1 is now known to have a minor ectodomain that is part of the long C-terminal tail. Both ectodomains are highly antigenic, carry neutralizing and non-neutralizing epitopes, and are involved in virus-mediated fusion activity. However, data have so far been biologically based, and derived solely from T cell line-adapted (TCLA), B clade viruses. Here we have carried out sequence and theoretically based structural analyses of 357 gp41 C-terminal sequences of mainly primary isolates of HIV-1 clades A, B, C, and D. Data show that all these viruses have the potential to form a tail loop structure (the minor ectodomain) supported by three, {beta}-sheet, membrane-spanning domains (MSDs). This means that the first (N-terminal) tyrosine-based sorting signal of the gp41 tail is situated outside the cell membrane and is non-functional, and that gp41 that reaches the cell surface may be recycled back into the cytoplasm through the activity of the second tyrosine-sorting signal. However, we suggest that only a minority of cell-associated gp41 molecules - those destined for incorporation into virions - has 3 MSDs and the minor ectodomain. Most intracellular gp41 has the conventional single MSD, no minor ectodomain, a functional first tyrosine-based sorting signal, and in line with current thinking is degraded intracellularly. The gp41 structural diversity suggested here can be viewed as an evolutionary strategy to minimize HIV-1 envelope glycoprotein expression on the cell surface, and hence possible cytotoxicity and immune attack on the infected cell.

  15. Effect of interfaces and the spin-orbit band on the band gaps of InAs/GaSb superlattices beyond the standard envelope-function approximation

    NASA Astrophysics Data System (ADS)

    Szmulowicz, Frank; Haugan, Heather; Brown, Gail; Mahalingam, Krishnamurthy

    2004-03-01

    We develop a modified 8x8 envelope-function approximation (EFA) formalism for the noncommon-atom (NCA) superlattices (SLs), incorporating the effect of anisotropic and other interface (IF) interactions that go beyond the standard EFA. The boundary conditions in the presence of IF interactions are used to set up a novel secular equation (including a transfer matrix derivation) whose physical transparency makes possible a number of valuable insights (possibility of IF bound states, analytic solutions, indirect gaps, etc.). We show that the heavy hole - spin-orbit IF coupling is very important due to the IF localization of the SO wave function components and the ability of the IF potential to potentially bind a hole at the IFs. With two adjustable parameter for the two possible IFs, we find a very good agreement between experiment and theory for the band gaps of several sets of very long- and mid-infrared InAs/GaSb SLs grown at several laboratories and by us. The band gaps as a function of GaSb and InAs widths are explained in terms of variations of the HH and conduction (C) band bandwidths. We show that the cut-off wavelengths can be reduced by increasing the GaSb layer width. Thus, a consistent application of the EFA method with the inclusion of well established IF effects can provide useful physical insights and possesses good predictive capacity in the design of NCA SLs.

  16. PTF1 J085713+331843, a new post-common-envelope binary in the orbital period gap of cataclysmic variables

    NASA Astrophysics Data System (ADS)

    van Roestel, J.; Groot, P. J.; Levitan, D.; Prince, T. A.; Bloemen, S.; Marsh, T. R.; Dhillon, V. S.; Shupe, D.; Laher, R.

    2017-07-01

    We report the discovery and analysis of PTF1 J085713+331843, a new eclipsing post-common-envelope detached white-dwarf-red-dwarf binary with a 2.5 h orbital period discovered by the Palomar Transient Factory. ULTRACAM multicolour photometry over multiple orbital periods reveals a light curve with a deep flat-bottomed primary eclipse and a strong reflection effect. Phase-resolved spectroscopy shows broad Balmer absorption lines from the DA white dwarf and phase-dependent Balmer emission lines originating on the irradiated side of the red dwarf. The temperature of the DA white dwarf is TWD = 25 700 ± 400 K and the spectral type of the red dwarf is M3-M5. A combined modelling of the light curve and the radial velocity variations results in a white dwarf mass of M_WD = 0.61^{+0.18}_{-0.17} M_{⊙} and radius of R_WD = 0.0175^{+0.0012}_{-0.0011} R_{⊙}, and a red dwarf mass and radius of M_RD = 0.19^{+0.10}_{-0.08} M_{⊙} and R_RD = 0.24^{+0.04}_{-0.04} R_{⊙}. The system is either a detached cataclysmic variable or has emerged like from the common-envelope phase at nearly its current orbital period. In {˜ }70 Myr, this system will become a cataclysmic variable in the period gap.

  17. Effect of interfaces and the spin-orbit band on the band gaps of InAs/GaSb superlattices beyond the standard envelope-function approximation

    NASA Astrophysics Data System (ADS)

    Szmulowicz, F.; Haugan, H.; Brown, G. J.

    2004-04-01

    We develop a modified 8×8 envelope-function approximation (EFA) formalism for the noncommon-atom (NCA) superlattices (SL’s), incorporating the effect of anisotropic and other interface (IF) interactions that go beyond the standard EFA. The boundary conditions in the presence of IF interactions are used to set up a secular equation (including a transfer matrix derivation) whose physical transparency makes possible a number of valuable insights (possibility of IF bound states, analytic solutions, indirect gaps, etc.). We show that the heavy-hole spin-orbit IF coupling is very important due to the IF localization of the SO wave function components and the ability of the IF potential to potentially bind a hole at the IF’s, all of which pose convergence problems for perturbative solutions. With two adjustable parameter for the two possible IF’s, we find a very good agreement between experiment and theory for the band gaps of several sets of very long-infrared and midinfrared InAs/GaSb SL’s grown at several laboratories and by us. The band gaps as a function of GaSb and InAs widths are explained in terms of variations of the HH and conduction (C) band bandwidths. We show that the cut-off wavelengths can be reduced by increasing the GaSb layer width. Thus, a consistent application of the EFA method with the inclusion of well established IF effects can provide useful physical insights and possesses good predictive capacity in the design of NCA SL’s.

  18. Functional expression of Ca²⁺ dependent mammalian transmembrane gap junction protein Cx43 in slime mold Dictyostelium discoideum.

    PubMed

    Kaufmann, Stefan; Weiss, Ingrid M; Eckstein, Volker; Tanaka, Motomu

    2012-03-09

    In this paper, we expressed murine gap junction protein Cx43 in Dictyostelium discoideum by introducing the specific vector pDXA. In the first step, the successful expression of Cx43 and Cx43-eGFP was verified by (a) Western blot (anti-Cx43, anti-GFP), (b) fluorescence microscopy (eGFP-Cx43 co-expression, Cx43 immunostaining), and (c) flow cytometry analysis (eGFP-Cx43 co-expression). Although the fluorescence signals from cells expressing Cx43-eGFP detected by fluorescence microscopy seem relatively low, analysis by flow cytometry demonstrated that more than 60% of cells expressed Cx43-eGFP. In order to evaluate the function of expressed Cx43 in D. discoideum, we examined the hemi-channel function of Cx43. In this series of experiments, the passive uptake of carboxyfluorescein was monitored using flow cytometric analysis. A significant number of the transfected cells showed a prominent dye uptake in the absence of Ca(2+). The dye uptake by transfected cells in the presence of Ca(2+) was even lower than the non-specific dye uptake by non-transformed Ax3 orf+ cells, confirming that Cx43 expressed in D. discoideum retains its Ca(2+)-dependent, specific gating function. The expression of gap junction proteins expressed in slime molds opens a possibility to the biological significance of intercellular communications in development and maintenance of multicellular organisms.

  19. Coupling between the bacteriorhodopsin photocycle and the protonmotive force in Halobacterium halobium cell envelope vesicles. III. Time-resolved increase in the transmembrane electric potential and modeling of the associated ion fluxes.

    PubMed Central

    Helgerson, S L; Mathew, M K; Bivin, D B; Wolber, P K; Heinz, E; Stoeckenius, W

    1985-01-01

    Bacteriorhodopsin functions as an electrogenic, light-driven proton pump in Halobacterium halobium. In cell envelope vesicles, its photocycle kinetics can be correlated with membrane potential. The initial decay rate of the M photocycle intermediate(s) decreases with increasing membrane potential, allowing the construction of a calibration curve. The laser (592.5 nm) was flashed at various time delays following the start of background illumination (592 +/- 25 nm) and transient absorbance changes at 418 nm monitored in cell envelope vesicles. The vesicles were loaded with and suspended in either 3 M NaCl or 3 M KCl buffered with 50 mM HEPES at pH 7.5 and the membrane permeability to protons modified by pretreatment with N,N'-dicyclohexylcarbodiimide. In each case the membrane potential rose with a halftime of approximately 75 ms. The steady-state potential achieved depends on the cation present and the proton permeability of the membrane, i.e., higher potentials are developed in dicyclohexylcarbodiimide treated vesicles or in NaCl media as compared with KCl media. The results are modeled using an irreversible thermodynamics formulation, which assumes a constant driving reaction affinity (Ach) and a variable reaction rate (Jr) for the proton-pumping cycle of bacteriorhodopsin. Additionally, the model includes a voltage-gated, electrogenic Na+/H+ antiporter that is active when vesicles are suspended in NaCl. Estimates for the linear phenomenological coefficients describing the overall proton-pumping cycle (Lr = 3.5 X 10(-11)/mol2/J X g X s), passive cation permeabilities (LHu = 2 X 10(-10), LKu = 2.2 X 10(-10), LNau = 1 X 10(-11)), and the Na+/H+ exchange via the antiporter (Lex = 5 X 10(-11)) have been obtained. PMID:4074833

  20. Transmembrane signaling proteoglycans.

    PubMed

    Couchman, John R

    2010-01-01

    Virtually all metazoan cells contain at least one and usually several types of transmembrane proteoglycans. These are varied in protein structure and type of polysaccharide, but the total number of vertebrate genes encoding transmembrane proteoglycan core proteins is less than 10. Some core proteins, including those of the syndecans, always possess covalently coupled glycosaminoglycans; others do not. Syndecan has a long evolutionary history, as it is present in invertebrates, but many other transmembrane proteoglycans are vertebrate inventions. The variety of proteins and their glycosaminoglycan chains is matched by diverse functions. However, all assume roles as coreceptors, often working alongside high-affinity growth factor receptors or adhesion receptors such as integrins. Other common themes are an ability to signal through their cytoplasmic domains, often to the actin cytoskeleton, and linkage to PDZ protein networks. Many transmembrane proteoglycans associate on the cell surface with metzincin proteases and can be shed by them. Work with model systems in vivo and in vitro reveals roles in growth, adhesion, migration, and metabolism. Furthermore, a wide range of phenotypes for the core proteins has been obtained in mouse knockout experiments. Here some of the latest developments in the field are examined in hopes of stimulating further interest in this fascinating group of molecules.

  1. SAFEGUARDS ENVELOPE

    SciTech Connect

    Duc Cao; Richard Metcalf

    2010-07-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z-testing. A brief analysis of the impact of the safeguards optimization on the rest of plant efficiency, criticality concerns, and overall requirements is presented.

  2. Cluster formation of transmembrane proteins due to hydrophobic mismatching.

    PubMed

    Schmidt, Ulrich; Guigas, Gernot; Weiss, Matthias

    2008-09-19

    Membranes are the defining envelopes of living cells. At this boundary a multitude of transmembrane proteins mediate signal and mass transfer between cells and their environment. Clustering of these proteins is a frequent and often vital phenomenon that relies at least in part on membrane-mediated interactions. Indeed, the mismatch between proteins' hydrophobic transmembrane domains and the surrounding lipid bilayer has been predicted to facilitate clustering, yet unequivocal quantitative data in support of these predictions have been lacking. Here, we have used coarse-grained membrane simulations to thoroughly address the clustering of transmembrane proteins in detail. Our results emphasize the universal nature of membrane-mediated attraction which relaxes the need for a plethora of fine-tuned interactions between membrane proteins.

  3. Elevated temperature envelope forming

    NASA Technical Reports Server (NTRS)

    Burg, Bruce M. (Inventor); Gane, David H. (Inventor); Starowski, Robert M. (Inventor)

    1992-01-01

    Elevated temperature envelope forming includes enclosing a part blank and form tool within an envelope sealed against the atmosphere, heat treating the combination while forming pressure holds the envelope and part against the form tool, and allowing part cool down to occur in an inert atmosphere with forming pressure removed. The forming pressure is provided by evacuating the envelope and may be aided by differential force applied between the envelope and the form tool.

  4. The stability of the three transmembrane and the four transmembrane human vitamin K epoxide reductase models

    NASA Astrophysics Data System (ADS)

    Wu, Sangwook

    2016-04-01

    The three transmembrane and the four transmembrane helix models are suggested for human vitamin K epoxide reductase (VKOR). In this study, we investigate the stability of the human three transmembrane/four transmembrane VKOR models by employing a coarse-grained normal mode analysis and molecular dynamics simulation. Based on the analysis of the mobility of each transmembrane domain, we suggest that the three transmembrane human VKOR model is more stable than the four transmembrane human VKOR model.

  5. The plant nuclear envelope.

    PubMed

    Rose, Annkatrin; Patel, Shalaka; Meier, Iris

    2004-01-01

    This review summarizes our present knowledge about the composition and function of the plant nuclear envelope. Compared with animals or yeast, our molecular understanding of the nuclear envelope in higher plants is in its infancy. However, fundamental differences in the structure and function of the plant and animal nuclear envelope have already been found. Here, we compare and contrast these differences with respect to nuclear pore complexes, targeting of Ran signaling to the nuclear envelope, inner nuclear envelope proteins, and the role and fate of the nuclear envelope during mitosis. Further investigation of the emerging fundamental differences as well as the similarities between kingdoms might illuminate why there appears to be more than one blueprint for building a nucleus.

  6. The transmembrane domains of the prM and E proteins of yellow fever virus are endoplasmic reticulum localization signals.

    PubMed

    Op De Beeck, Anne; Rouillé, Yves; Caron, Mélanie; Duvet, Sandrine; Dubuisson, Jean

    2004-11-01

    The immature flavivirus particle contains two envelope proteins, prM and E, that are associated as a heterodimer. Virion morphogenesis of the flaviviruses occurs in association with endoplasmic reticulum (ER) membranes, suggesting that there should be accumulation of the virion components in this compartment. This also implies that ER localization signals must be present in the flavivirus envelope proteins. In this work, we looked for potential subcellular localization signals in the yellow fever virus envelope proteins. Confocal immunofluorescence analysis of the subcellular localization of the E protein in yellow fever virus-infected cells indicated that this protein accumulates in the ER. Similar results were obtained with cells expressing only prM and E. Chimeric proteins containing the ectodomain of CD4 or CD8 fused to the transmembrane domains of prM or E were constructed, and their subcellular localization was studied by confocal immunofluorescence and by analyzing the maturation of their associated glycans. Although a small fraction was detected in the ER-to-Golgi intermediate and Golgi compartments, these chimeric proteins were located mainly in the ER. The C termini of prM and E form two antiparallel transmembrane alpha-helices. Interestingly, the first transmembrane passage contains enough information for ER localization. Taken altogether, these data indicate that, besides their role as membrane anchors, the transmembrane domains of yellow fever virus envelope proteins are ER retention signals. In addition, our data show that the mechanisms of ER retention of the flavivirus and hepacivirus envelope proteins are different.

  7. Nuclear envelope protein autoantibodies in primary biliary cirrhosis.

    PubMed

    Courvalin, J C; Worman, H J

    1997-02-01

    A subset of patients with primary biliary cirrhosis (PBC) have autoantibodies directed against nuclear envelope proteins. The major autoantigen is gp210, a 210 kilodalton (kD) transmembrane protein of the nuclear pore complex, that is recognized by antibodies in approximately 25% of patients. The predominant epitope in gp210 that is recognized by most of the autoantibodies is a 15 amino acid stretch in the cytoplasmic, carboxyl-terminal domain of the protein. Gp210 autoantibodies are specific for PBC, as are the less frequent autoantibodies directed against LBR, a transmembrane protein of the inner nuclear membrane. Although autoantibodies against nuclear lamins, abundant intermediate filament proteins associated with the inner nuclear membrane, may be found in PBC, they are not specific for this disease. Nuclear envelope protein autoantibodies are also present in some patients without detectable antimitochondrial antibodies and may be of particular utility in diagnosing individuals with atypical presentations of PBC.

  8. Nicotinic Receptor Fourth Transmembrane Domain

    PubMed Central

    Bouzat, Cecilia; Barrantes, Francisco; Sine, Steven

    2000-01-01

    The fourth transmembrane domain (M4) of the nicotinic acetylcholine receptor (AChR) contributes to the kinetics of activation, yet its close association with the lipid bilayer makes it the outermost of the transmembrane domains. To investigate mechanistic and structural contributions of M4 to AChR activation, we systematically mutated αT422, a conserved residue that has been labeled by hydrophobic probes, and evaluated changes in rate constants underlying ACh binding and channel gating steps. Aromatic and nonpolar mutations of αT422 selectively affect the channel gating step, slowing the rate of opening two- to sevenfold, and speeding the rate of closing four- to ninefold. Additionally, kinetic modeling shows a second doubly liganded open state for aromatic and nonpolar mutations. In contrast, serine and asparagine mutations of αT422 largely preserve the kinetics of the wild-type AChR. Thus, rapid and efficient gating of the AChR channel depends on a hydrogen bond involving the side chain at position 422 of the M4 transmembrane domain. PMID:10779322

  9. Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis.

    PubMed

    de Brogniez, Alix; Mast, Jan; Willems, Luc

    2016-03-24

    Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis.

  10. Circumplanetary disc or circumplanetary envelope?

    NASA Astrophysics Data System (ADS)

    Szulágyi, J.; Masset, F.; Lega, E.; Crida, A.; Morbidelli, A.; Guillot, T.

    2016-08-01

    We present three-dimensional simulations with nested meshes of the dynamics of the gas around a Jupiter mass planet with the JUPITER and FARGOCA codes. We implemented a radiative transfer module into the JUPITER code to account for realistic heating and cooling of the gas. We focus on the circumplanetary gas flow, determining its characteristics at very high resolution (80 per cent of Jupiter's diameter). In our nominal simulation where the temperature evolves freely by the radiative module and reaches 13000 K at the planet, a circumplanetary envelope was formed filling the entire Roche lobe. Because of our equation of state is simplified and probably overestimates the temperature, we also performed simulations with limited maximal temperatures in the planet region (1000, 1500, and 2000 K). In these fixed temperature cases circumplanetary discs (CPDs) were formed. This suggests that the capability to form a CPD is not simply linked to the mass of the planet and its ability to open a gap. Instead, the gas temperature at the planet's location, which depends on its accretion history, plays also fundamental role. The CPDs in the simulations are hot and cooling very slowly, they have very steep temperature and density profiles, and are strongly sub-Keplerian. Moreover, the CPDs are fed by a strong vertical influx, which shocks on the CPD surfaces creating a hot and luminous shock-front. In contrast, the pressure supported circumplanetary envelope is characterized by internal convection and almost stalled rotation.

  11. Opacities for Stellar Envelopes

    NASA Astrophysics Data System (ADS)

    Seaton, M. J.; Yan, Y.; Mihalas, D.; Pradhan, A. K.

    1994-02-01

    We define stellar envelopes to be those regions of stellar interiors in which atoms exist and are not markedly perturbed by the plasma environment. Availability of accurate and extensive atomic data is a prime requirement for the calculation of envelope opacities. For envelopes we adopt the criterion of mass density p < 0.01 ρ≥g cm-3. We present radiative Rosseland mean opacities for envelopes obtained using atomic data calculated in an international collaboration referred to as the Opacity Project, or OP. Equations of state are calculated using an occupation-probability formalism. To a good approximation, ionization equilibria and level populations in envelopes depend only on the temperature T and electron density Ne and are insensitive to chemical mixtures. Monochromatic opacities for all abundant chemical elements are therefore calculated on a grid of (T, Ne) values and are archived. Rosseland mean opacities are then readily calculated for any chemical mixture. Tables of Rosseland means, for any required mixtures and as functions of ρ and T, are available on request in computer-readable form. The present, op, results are compared with those from another recent study, referred to as OPAL, by C. A. Iglesias and F. A. Rogers at the Lawrence Livermore National Laboratory. The agreement between the OP and OPAL calculations is generally good, although there are some differences. Both calculations give results larger than those obtained in earlier work, by factors of up to 3 or more.

  12. LINCing complex functions at the nuclear envelope

    PubMed Central

    Rothballer, Andrea; Schwartz, Thomas U.; Kutay, Ulrike

    2013-01-01

    Linker of nucleoskeleton and cytoskeleton (LINC) complexes span the double membrane of the nuclear envelope (NE) and physically connect nuclear structures to cytoskeletal elements. LINC complexes are envisioned as force transducers in the NE, which facilitate processes like nuclear anchorage and migration, or chromosome movements. The complexes are built from members of two evolutionary conserved families of transmembrane (TM) proteins, the SUN (Sad1/UNC-84) domain proteins in the inner nuclear membrane (INM) and the KASH (Klarsicht/ANC-1/SYNE homology) domain proteins in the outer nuclear membrane (ONM). In the lumen of the NE, the SUN and KASH domains engage in an intimate assembly to jointly form a NE bridge. Detailed insights into the molecular architecture and atomic structure of LINC complexes have recently revealed the molecular basis of nucleo-cytoskeletal coupling. They bear important implications for LINC complex function and suggest new potential and as yet unexplored roles, which the complexes may play in the cell. PMID:23324460

  13. FRACTIONAL CRYSTALLIZATION FEED ENVELOPE

    SciTech Connect

    HERTING DL

    2008-03-19

    Laboratory work was completed on a set of evaporation tests designed to establish a feed envelope for the fractional crystallization process. The feed envelope defines chemical concentration limits within which the process can be operated successfully. All 38 runs in the half-factorial design matrix were completed successfully, based on the qualitative definition of success. There is no feed composition likely to be derived from saltcake dissolution that would cause the fractional crystallization process to not meet acceptable performance requirements. However, some compositions clearly would provide more successful operation than other compositions.

  14. Virtual breakdown of the nuclear envelope in fission yeast meiosis.

    PubMed

    Asakawa, Haruhiko; Kojidani, Tomoko; Mori, Chie; Osakada, Hiroko; Sato, Mamiko; Ding, Da-Qiao; Hiraoka, Yasushi; Haraguchi, Tokuko

    2010-11-09

    Asymmetric localization of Ran regulators (RanGAP1 and RanGEF/RCC1) produces a gradient of RanGTP across the nuclear envelope. In higher eukaryotes, the nuclear envelope breaks down as the cell enters mitosis (designated "open" mitosis). This nuclear envelope breakdown (NEBD) leads to collapse of the RanGTP gradient and the diffusion of nuclear and cytoplasmic macromolecules in the cell, resulting in irreversible progression of the cell cycle. On the other hand, in many fungi, chromosome segregation takes place without NEBD (designated "closed" mitosis). Here we report that in the fission yeast Schizosaccharomyces pombe, despite the nuclear envelope and the nuclear pore complex remaining intact throughout both the meiotic and mitotic cell cycles, nuclear proteins diffuse into the cytoplasm transiently for a few minutes at the onset of anaphase of meiosis II. We also found that nuclear protein diffusion into the cytoplasm occurred coincidently with nuclear localization of Rna1, an S. pombe RanGAP1 homolog that is usually localized in the cytoplasm. These results suggest that nuclear localization of RanGAP1 and depression of RanGTP activity in the nucleus may be mechanistically tied to meiosis-specific diffusion of nuclear proteins into the cytoplasm. This nucleocytoplasmic shuffling of RanGAP1 and nuclear proteins represents virtual breakdown of the nuclear envelope.

  15. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    PubMed

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  16. HIV-1 envelope glycoprotein

    DOEpatents

    Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

    2017-08-22

    The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

  17. Jacketed lamp bulb envelope

    DOEpatents

    MacLennan, Donald A.; Turner, Brian P.; Gitsevich, Aleksandr; Bass, Gary K.; Dolan, James T.; Kipling, Kent; Kirkpatrick, Douglas A.; Leng, Yongzhang; Levin, Izrail; Roy, Robert J.; Shanks, Bruce; Smith, Malcolm; Trimble, William C.; Tsai, Peter

    2001-01-01

    A jacketed lamp bulb envelope includes a ceramic cup having an open end and a partially closed end, the partially closed end defining an aperture, a lamp bulb positioned inside the ceramic cup abutting the aperture, and a reflective ceramic material at least partially covering a portion of the bulb not abutting the aperture. The reflective ceramic material may substantially fill an interior volume of the ceramic cup not occupied by the bulb. The ceramic cup may include a structural feature for aiding in alignment of the jacketed lamp bulb envelope in a lamp. The ceramic cup may include an external flange about a periphery thereof. One example of a jacketed lamp bulb envelope includes a ceramic cup having an open end and a closed end, a ceramic washer covering the open end of the ceramic cup, the washer defining an aperture therethrough, a lamp bulb positioned inside the ceramic cup abutting the aperture, and a reflective ceramic material filling an interior volume of the ceramic cup not occupied by the bulb. A method of packing a jacketed lamp bulb envelope of the type comprising a ceramic cup with a lamp bulb disposed therein includes the steps of filling the ceramic cup with a flowable slurry of reflective material, and applying centrifugal force to the cup to pack the reflective material therein.

  18. Pushing the endogenous envelope

    PubMed Central

    Henzy, Jamie E.; Johnson, Welkin E.

    2013-01-01

    The majority of retroviral envelope glycoproteins characterized to date are typical of type I viral fusion proteins, having a receptor binding subunit associated with a fusion subunit. The fusion subunits of lentiviruses and alpha-, beta-, delta- and gammaretroviruses have a very conserved domain organization and conserved features of secondary structure, making them suitable for phylogenetic analyses. Such analyses, along with sequence comparisons, reveal evidence of numerous recombination events in which retroviruses have acquired envelope glycoproteins from heterologous sequences. Thus, the envelope gene (env) can have a history separate from that of the polymerase gene (pol), which is the most commonly used gene in phylogenetic analyses of retroviruses. Focusing on the fusion subunits of the genera listed above, we describe three distinct types of retroviral envelope glycoproteins, which we refer to as gamma-type, avian gamma-type and beta-type. By tracing these types within the ‘fossil record’ provided by endogenous retroviruses, we show that they have surprisingly distinct evolutionary histories and dynamics, with important implications for cross-species transmissions and the generation of novel lineages. These findings validate the utility of env sequences in contributing phylogenetic signal that enlarges our understanding of retrovirus evolution. PMID:23938755

  19. An Autonomously Reciprocating Transmembrane Nanoactuator.

    PubMed

    Watson, Matthew A; Cockroft, Scott L

    2016-01-22

    Biological molecular machines operate far from equilibrium by coupling chemical potential to repeated cycles of dissipative nanomechanical motion. This principle has been exploited in supramolecular systems that exhibit true machine behavior in solution and on surfaces. However, designed membrane-spanning assemblies developed to date have been limited to simple switches or stochastic shuttles, and true machine behavior has remained elusive. Herein, we present a transmembrane nanoactuator that turns over chemical fuel to drive autonomous reciprocating (back-and-forth) nanomechanical motion. Ratcheted reciprocating motion of a DNA/PEG copolymer threaded through a single α-hemolysin pore was induced by a combination of DNA strand displacement processes and enzyme-catalyzed reactions. Ion-current recordings revealed saw-tooth patterns, indicating that the assemblies operated in autonomous, asymmetric cycles of conformational change at rates of up to one cycle per minute. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Model scattering envelopes of young stellar objects. II - Infalling envelopes

    NASA Technical Reports Server (NTRS)

    Whitney, Barbara A.; Hartmann, Lee

    1993-01-01

    We present scattered light images for models of young stellar objects surrounded by dusty envelopes. The envelopes are assumed to have finite angular momentum and are falling in steady flow onto a disk. The model envelopes include holes, such as might be created by energetic bipolar flows. We calculate images using the Monte Carlo method to follow the light scattered in the dusty envelope and circumstellar disk, assuming that the photons originate from the central source. Adopting typical interstellar medium dust opacities and expected mass infall rates for protostars of about 10 exp -6 solar mass/yr, we find that detectable amounts of optical radiation can escape from envelopes falling into a disk as small as about 10-100 AU, depending upon the viewing angle and the size of the bipolar flow cavity. We suggest that the extended optical and near-IR light observed around several young stars is scattered by dusty infalling envelopes rather than disks.

  1. Model scattering envelopes of young stellar objects. II - Infalling envelopes

    NASA Technical Reports Server (NTRS)

    Whitney, Barbara A.; Hartmann, Lee

    1993-01-01

    We present scattered light images for models of young stellar objects surrounded by dusty envelopes. The envelopes are assumed to have finite angular momentum and are falling in steady flow onto a disk. The model envelopes include holes, such as might be created by energetic bipolar flows. We calculate images using the Monte Carlo method to follow the light scattered in the dusty envelope and circumstellar disk, assuming that the photons originate from the central source. Adopting typical interstellar medium dust opacities and expected mass infall rates for protostars of about 10 exp -6 solar mass/yr, we find that detectable amounts of optical radiation can escape from envelopes falling into a disk as small as about 10-100 AU, depending upon the viewing angle and the size of the bipolar flow cavity. We suggest that the extended optical and near-IR light observed around several young stars is scattered by dusty infalling envelopes rather than disks.

  2. Structural Basis for Membrane Anchoring of HIV-1 Envelope Spike

    PubMed Central

    Fu, Qingshan; Chen, Jia; Ha, Heather Jiwon; Ghantous, Fadi; Herrmann, Tobias; Chang, Weiting; Liu, Zhijun; Frey, Gary; Seaman, Michael S.; Chen, Bing; Chou, James J.

    2016-01-01

    HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We use NMR to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An N-terminal coiled-coil and a C-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design. PMID:27338706

  3. Refrigerated cryogenic envelope

    DOEpatents

    Loudon, John D.

    1976-11-16

    An elongated cryogenic envelope including an outer tube and an inner tube coaxially spaced within said inner tube so that the space therebetween forms a vacuum chamber for holding a vacuum. The inner and outer tubes are provided with means for expanding or contracting during thermal changes. A shield is located in the vacuum chamber intermediate the inner and outer tubes; and, a refrigeration tube for directing refrigeration to the shield is coiled about at least a portion of the inner tube within the vacuum chamber to permit the refrigeration tube to expand or contract along its length during thermal changes within said vacuum chamber.

  4. Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth

    PubMed Central

    Watanabe, Keisuke; Bizen, Norihisa; Sato, Noboru; Takebayashi, Hirohide

    2016-01-01

    The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development. PMID:27959946

  5. Simulation of random envelope processes.

    NASA Technical Reports Server (NTRS)

    Yang, J.-N.

    1972-01-01

    Efficient and practical methods of simulating stationary and nonstationary random envelope processes are presented. The stationary envelope processes are simulated by using the fast Fourier transform while the nonstationary envelope processes are simulated as the square root of the sum of a series of cosine functions and a series of sine functions with random phase angles. Typical applications of the envelope simulation are the simulations of peaks and troughs which play an important role in the analyses of the first excursion probability, fatigue and crack propagation. In particular, applications to the crack propagation under random loadings are demonstrated in detail.

  6. Inducible expression of transmembrane proteins on bacterial magnetic particles in Magnetospirillum magneticum AMB-1.

    PubMed

    Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

    2010-02-01

    Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.

  7. Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1▿

    PubMed Central

    Yoshino, Tomoko; Shimojo, Akiko; Maeda, Yoshiaki; Matsunaga, Tadashi

    2010-01-01

    Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs. PMID:20038711

  8. The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis.

    PubMed

    Gomez-Cavazos, J Sebastian; Hetzer, Martin W

    2015-03-16

    Previously, we identified the nucleoporin gp210/Nup210 as a critical regulator of muscle and neuronal differentiation, but how this nucleoporin exerts its function and whether it modulates nuclear pore complex (NPC) activity remain unknown. Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space. Removal of the C-terminal domain, which partially mislocalizes gp210/Nup210 away from NPCs, efficiently rescues the differentiation defect caused by the knockdown of endogenous gp210/Nup210. Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation. We demonstrate that the endoplasmic reticulum (ER) stress-specific caspase cascade is exacerbated during Nup210 depletion and that blocking ER stress-mediated apoptosis rescues differentiation of Nup210-deficient cells. Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis. © 2015 Gomez-Cavazos and Hetzer.

  9. The nucleoporin gp210/Nup210 controls muscle differentiation by regulating nuclear envelope/ER homeostasis

    PubMed Central

    Gomez-Cavazos, J. Sebastian

    2015-01-01

    Previously, we identified the nucleoporin gp210/Nup210 as a critical regulator of muscle and neuronal differentiation, but how this nucleoporin exerts its function and whether it modulates nuclear pore complex (NPC) activity remain unknown. Here, we show that gp210/Nup210 mediates muscle cell differentiation in vitro via its conserved N-terminal domain that extends into the perinuclear space. Removal of the C-terminal domain, which partially mislocalizes gp210/Nup210 away from NPCs, efficiently rescues the differentiation defect caused by the knockdown of endogenous gp210/Nup210. Unexpectedly, a gp210/Nup210 mutant lacking the NPC-targeting transmembrane and C-terminal domains is sufficient for C2C12 myoblast differentiation. We demonstrate that the endoplasmic reticulum (ER) stress-specific caspase cascade is exacerbated during Nup210 depletion and that blocking ER stress-mediated apoptosis rescues differentiation of Nup210-deficient cells. Our results suggest that the role of gp210/Nup210 in cell differentiation is mediated by its large luminal domain, which can act independently of NPC association and appears to play a pivotal role in the maintenance of nuclear envelope/ER homeostasis. PMID:25778917

  10. A novel family of plant nuclear envelope-associated proteins.

    PubMed

    Pawar, Vidya; Poulet, Axel; Détourné, Gwénaëlle; Tatout, Christophe; Vanrobays, Emmanuel; Evans, David E; Graumann, Katja

    2016-10-01

    This paper describes the characterisation of a new family of higher plant nuclear envelope-associated proteins (NEAPs) that interact with other proteins of the nuclear envelope. In the model plant Arabidopsis thaliana, the family consists of three genes expressed ubiquitously (AtNEAP1-3) and a pseudogene (AtNEAP4). NEAPs consist of extensive coiled-coil domains, followed by a nuclear localisation signal and a C-terminal predicted transmembrane domain. Domain deletion mutants confirm the presence of a functional nuclear localisation signal and transmembrane domain. AtNEAP proteins localise to the nuclear periphery as part of stable protein complexes, are able to form homo- and heteromers, and interact with the SUN domain proteins AtSUN1 and AtSUN2, involved in the linker of nucleoskeleton and cytoskeleton (LINC) complex. An A. thaliana cDNA library screen identified a putative transcription factor called AtbZIP18 as a novel interactor of AtNEAP1, which suggest a connection between NEAP and chromatin. An Atneap1 Atneap3 double-knockout mutant showed reduced root growth, and altered nuclear morphology and chromatin structure. Thus AtNEAPs are suggested as inner nuclear membrane-anchored coiled-coil proteins with roles in maintaining nuclear morphology and chromatin structure.

  11. Fast Moreau envelope computation I

    NASA Astrophysics Data System (ADS)

    Lucet, Yves

    2006-11-01

    The present article summarizes the state of the art algorithms to compute the discrete Moreau envelope, and presents a new linear-time algorithm, named NEP for NonExpansive Proximal mapping. Numerical comparisons between the NEP and two existing algorithms: The Linear-time Legendre Transform (LLT) and the Parabolic Envelope (PE) algorithms are performed. Worst-case time complexity, convergence results, and examples are included. The fast Moreau envelope algorithms first factor the Moreau envelope as several one-dimensional transforms and then reduce the brute force quadratic worst-case time complexity to linear time by using either the equivalence with Fast Legendre Transform algorithms, the computation of a lower envelope of parabolas, or, in the convex case, the non expansiveness of the proximal mapping.

  12. Integral membrane proteins of the chloroplast envelope: Identification and subcellular localization of new transporters

    PubMed Central

    Ferro, Myriam; Salvi, Daniel; Rivière-Rolland, Hélène; Vermat, Thierry; Seigneurin-Berny, Daphné; Grunwald, Didier; Garin, Jérôme; Joyard, Jacques; Rolland, Norbert

    2002-01-01

    A two-membrane system, or envelope, surrounds plastids. Because of the integration of chloroplast metabolism within the plant cell, the envelope is the site of many specific transport activities. However, only a few proteins involved in the processes of transport across the chloroplast envelope have been identified already at the molecular level. To discover new envelope transporters, we developed a subcellular proteomic approach, which is aimed to identify the most hydrophobic envelope proteins. This strategy combined the use of highly purified and characterized membrane fractions, extraction of the hydrophobic proteins with organic solvents, SDS/PAGE separation, and tandem mass spectrometry analysis. To process the large amount of MS/MS data, a blast-based program was developed for searching in protein, expressed sequence tag, and genomic plant databases. Among the 54 identified proteins, 27 were new envelope proteins, with most of them bearing multiple α-helical transmembrane regions and being very likely envelope transporters. The present proteomic study also allowed us to identify common features among the known and newly identified putative envelope inner membrane transporters. These features were used to mine the complete Arabidopsis genome and allowed us to establish a virtual plastid envelope integral protein database. Altogether, both proteomic and in silico approaches identified more than 50 candidates for the as yet previously uncharacterized plastid envelope transporters. The predictable function of some of these proteins opens up areas of investigation that may lead to a better understanding of the chloroplast metabolism. The present subcellular proteomic approach is amenable to the analysis of the hydrophobic core of other intracellular membrane systems. PMID:12177442

  13. Structural analysis of the principal immunodominant domain of the feline immunodeficiency virus transmembrane glycoprotein.

    PubMed Central

    Pancino, G; Camoin, L; Sonigo, P

    1995-01-01

    In the transmembrane envelope glycoprotein (TM) of lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV), two cysteine residues, conserved in most retroviruses, are thought to form a loop containing five to seven amino acids. These elements make up a B-cell epitope recognized by nearly 100% of sera from infected patients or animals, designated the principal immunodominant domain (PID). The PID amino acid sequences are highly conserved between isolates of the same lentivirus but are unrelated, except for the two cysteines, when divergent lentiviruses are compared. The aim of this study was to analyze the relationship between amino acid sequence in the PID and envelope function. We introduced two kinds of mutations in the PID of FIV: mutations which impeded the formation of a loop and mutations which substituted the sequence of FIV with the corresponding sequences from other lentiviruses, HIV-1, visna virus, and equine infectious anemia virus. We analyzed antibody recognition, processing, and fusogenic properties of the modified envelopes, using two methods of Env expression: a cell-free expression system and transfection of a feline fibroblast cell line with gag-pol-deleted FIV proviruses. Most mutations in the PID of FIV severely affected envelope processing and abolished syncytium formation. Only the chimeric envelope containing the HIV-1 PID sequence was correctly processed and maintained the capacity to induce syncytium formation, although less efficiently than the wild-type envelope. We computed three-dimensional structural models of the PID, which were consistent with mutagenesis data and confirmed the similarity of FIV and HIV-1 PID structures, despite their divergence in amino acid sequence. Considering these results, we discussed the respective importance of selection exerted by functional requirements or host antibodies to explain the observed variations of the PIDs in lentiviruses. PMID:7884857

  14. Multifamily Envelope Leakage Model

    SciTech Connect

    Faakye, Omari; Griffiths, Dianne

    2015-05-08

    “The cost for blower testing is high, because it is labor intensive, and it may disrupt occupants in multiple units. This high cost and disruption deter program participants, and dissuade them from pursuing energy improvements that would trigger air leakage testing, such as improvements to the building envelope.” This statement found in a 2012 report by Heschong Mahone Group for several California interests emphasizes the importance of reducing the cost and complexity of blower testing in multifamily buildings. Energy efficiency opportunities are being bypassed. The cost of single blower testing is on the order of $300. The cost for guarded blower door testing—the more appropriate test for assessing energy savings opportunities—could easily be six times that, and that’s only if you have the equipment and simultaneous access to multiple apartments. Thus, the proper test is simply not performed. This research seeks to provide an algorithm for predicting the guarded blower door test result based upon a single, total blower door test.

  15. Masonry building envelope analysis

    NASA Astrophysics Data System (ADS)

    McMullan, Phillip C.

    1993-04-01

    Over the past five years, infrared thermography has proven an effective tool to assist in required inspections on new masonry construction. However, with more thermographers providing this inspection service, establishing a standard for conducting these inspections is imperative. To attempt to standardize these inspections, it is important to understand the nature of the inspection as well as the context in which the inspection is typically conducted. The inspection focuses on evaluating masonry construction for compliance with the design specifications with regard to structural components and thermal performance of the building envelope. The thermal performance of the building includes both the thermal resistance of the material as well as infiltration/exfiltration characteristics. Given that the inspections occur in the 'field' rather than the controlled environment of a laboratory, there are numerous variables to be considered when undertaking this type of inspection. Both weather and site conditions at the time of the inspection can vary greatly. In this paper we will look at the variables encountered during recent inspections. Additionally, the author will present the standard which was employed in collecting this field data. This method is being incorporated into a new standard to be included in the revised version of 'Guidelines for Specifying and Performing Infrared Inspections' developed by the Infraspection Institute.

  16. Envelope glycoprotein of arenaviruses.

    PubMed

    Burri, Dominique J; da Palma, Joel Ramos; Kunz, Stefan; Pasquato, Antonella

    2012-10-17

    Arenaviruses include lethal human pathogens which pose serious public health threats. So far, no FDA approved vaccines are available against arenavirus infections, and therapeutic options are limited, making the identification of novel drug targets for the development of efficacious therapeutics an urgent need. Arenaviruses are comprised of two RNA genome segments and four proteins, the polymerase L, the envelope glycoprotein GP, the matrix protein Z, and the nucleoprotein NP. A crucial step in the arenavirus life-cycle is the biosynthesis and maturation of the GP precursor (GPC) by cellular signal peptidases and the cellular enzyme Subtilisin Kexin Isozyme-1 (SKI-1)/Site-1 Protease (S1P) yielding a tripartite mature GP complex formed by GP1/GP2 and a stable signal peptide (SSP). GPC cleavage by SKI-1/S1P is crucial for fusion competence and incorporation of mature GP into nascent budding virion particles. In a first part of our review, we cover basic aspects and newer developments in the biosynthesis of arenavirus GP and its molecular interaction with SKI-1/S1P. A second part will then highlight the potential of SKI-1/S1P-mediated processing of arenavirus GPC as a novel target for therapeutic intervention to combat human pathogenic arenaviruses.

  17. Assembly of transmembrane proteins on oil-water interfaces

    NASA Astrophysics Data System (ADS)

    Yunker, Peter; Landry, Corey; Chong, Shaorong; Weitz, David

    2015-03-01

    Transmembrane proteins are difficult to handle by aqueous solution-based biochemical and biophysical approaches, due to the hydrophobicity of transmembrane helices. Detergents can solubilize transmembrane proteins; however, surfactant coated transmembrane proteins are not always functional, and purifying detergent coated proteins in a micellar solution can be difficult. Motivated by this problem, we study the self-assembly of transmembrane proteins on oil-water interfaces. We found that the large water-oil interface of oil drops prevents nascent transmembrane proteins from forming non-functional aggregates. The oil provides a hydrophobic environment for the transmembrane helix, allowing the ectodomain to fold into its natural structure and orientation. Further, modifying the strength or valency of hydrophobic interactions between transmembrane proteins results in the self-assembly of spatially clustered, active proteins on the oil-water interface. Thus, hydrophobic interactions can facilitate, rather than inhibit, the assembly of transmembrane proteins.

  18. Isolation of bacteria envelope proteins.

    PubMed

    Quan, Shu; Hiniker, Annie; Collet, Jean-François; Bardwell, James C A

    2013-01-01

    Proteomic analysis on cell envelope proteins from Gram-negative bacteria requires specific isolation techniques. We found that conventional extraction methods such as osmotic shock cause extracts to be heavily contaminated with soluble cytoplasmic proteins. These cytoplasmic protein contaminants constitute the major signal in proteomic analysis and can overwhelm the signals coming from genuine envelope components. After extensive testing of various protocols for the preparation of envelope contents, we found that a modified version of the method of Oliver and Beckwith consistently produces the cleanest extract of periplasmic and outer membrane proteins.We have designated this very simple method TSE extraction because it uses a Tris-sucrose solution supplemented with EDTA.Cytoplasmic and inner membrane protein contaminants are not evident on 1D SDS polyacrylamide gels and contribute to less than 6% of total signal in very sensitive mass spectrometry analysis. This straightforward method is therefore ideal for -analyzing specific proteomic changes in the cell envelope.

  19. Determinants of the Bovine Leukemia Virus Envelope Glycoproteins Involved in Infectivity, Replication and Pathogenesis

    PubMed Central

    de Brogniez, Alix; Mast, Jan; Willems, Luc

    2016-01-01

    Interaction of viral envelope proteins with host cell membranes has been extensively investigated in a number of systems. However, the biological relevance of these interactions in vivo has been hampered by the absence of adequate animal models. Reverse genetics using the bovine leukemia virus (BLV) genome highlighted important functional domains of the envelope protein involved in the viral life cycle. For example, immunoreceptor tyrosine-based activation motifs (ITAM) of the envelope transmembrane protein (TM) are essential determinants of infection. Although cell fusion directed by the aminoterminal end of TM is postulated to be essential, some proviruses expressing fusion-deficient envelope proteins unexpectedly replicate at wild-type levels. Surprisingly also, a conserved N-linked glycosylation site of the extracellular envelope protein (SU) inhibits cell-to-cell transmission suggesting that infectious potential has been limited during evolution. In this review, we summarize the knowledge pertaining to the BLV envelope protein in the context of viral infection, replication and pathogenesis. PMID:27023592

  20. Detection of an Immunogenic HERV-E Envelope with Selective Expression in Clear Cell Kidney Cancer.

    PubMed

    Cherkasova, Elena; Scrivani, Claire; Doh, Susan; Weisman, Quinn; Takahashi, Yoshiyuki; Harashima, Nanae; Yokoyama, Hisayuki; Srinivasan, Ramaprasad; Linehan, W Marston; Lerman, Michael I; Childs, Richard W

    2016-04-15

    VHL-deficient clear cell renal cell carcinomas (ccRCC), the most common form of kidney cancer, express transcripts derived from the novel human endogenous retrovirus HERV-E (named CT-RCC HERV-E). In this study, we define a transcript encoding the entire envelope gene of HERV-E as expressed selectively in ccRCC tumors, as distinct from normal kidney tissues or other tumor types. Sequence analysis of this envelope transcript revealed long open reading frames encoding putative surface and transmembrane envelope proteins. Retroviral envelopes are known to be capable of eliciting immunity in humans. Accordingly, we found that HLA-A*0201-restricted peptides predicted to be products of the CT-RCC HERV-E envelope transcript-stimulated CD8(+) T cells, which could recognize HLA-A*0201-positive HERV-E-expressing kidney tumor cells. Overall, our results offer evidence of unique HERV-E envelope peptides presented on the surface of ccRCC cells, offering potentially useful tumor-restricted targets for T-cell-based immunotherapy of kidney cancer. Cancer Res; 76(8); 2177-85. ©2016 AACR. ©2016 American Association for Cancer Research.

  1. Membrane shape modulates transmembrane protein distribution

    PubMed Central

    Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E. S.; Bassereau, Patricia

    2014-01-01

    Summary Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown due to the numerous sorting mechanisms taking place concurrently in cells. To isolate the effect of membrane shape, cellsized Giant Unilamellar Vesicles (GUVs) containing either the potassium channel, KvAP, or water channel, AQP0, were used to form membrane nanotubes with controlled radii. While the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. FRAP measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins, and provides a method for determining the effective shape and flexibility of membrane proteins. PMID:24480645

  2. TRAMPLE: the transmembrane protein labelling environment.

    PubMed

    Fariselli, Piero; Finelli, Michele; Rossi, Ivan; Amico, Mauro; Zauli, Andrea; Martelli, Pier Luigi; Casadio, Rita

    2005-07-01

    TRAMPLE (http://gpcr.biocomp.unibo.it/biodec/) is a web application server dedicated to the detection and the annotation of transmembrane protein sequences. TRAMPLE includes different state-of-the-art algorithms for the prediction of signal peptides, transmembrane segments (both beta-strands and alpha-helices), secondary structure and fast fold recognition. TRAMPLE also includes a complete content management system to manage the results of the predictions. Each user of the server has his/her own workplace, where the data can be stored, organized, accessed and annotated with documents through a simple web-based interface. In this manner, TRAMPLE significantly improves usability with respect to other more traditional web servers.

  3. Crystallizing Transmembrane Peptides in Lipidic Mesophases

    SciTech Connect

    Höfer, Nicole; Aragão, David; Caffrey, Martin

    2011-09-28

    Structure determination of membrane proteins by crystallographic means has been facilitated by crystallization in lipidic mesophases. It has been suggested, however, that this so-called in meso method, as originally implemented, would not apply to small protein targets having {le}4 transmembrane crossings. In our study, the hypothesis that the inherent flexibility of the mesophase would enable crystallogenesis of small proteins was tested using a transmembrane pentadecapeptide, linear gramicidin, which produced structure-grade crystals. This result suggests that the in meso method should be considered as a viable means for high-resolution structure determination of integral membrane peptides, many of which are predicted to be coded for in the human genome.

  4. Spatial changes in the transmembrane potential during extracellular electric stimulation.

    PubMed

    Zhou, X; Knisley, S B; Smith, W M; Rollins, D; Pollard, A E; Ideker, R E

    1998-11-16

    The purpose of this study was to determine the spatial changes in the transmembrane potential caused by extracellular electric field stimulation. The transmembrane potential was recorded in 10 guinea pig papillary muscles in a tissue bath using a double-barrel microelectrode. After 20 S1 stimuli, a 10-ms square wave S2 shock field with a 30-ms S1-S2 coupling interval was given via patch shock electrodes 1 cm on either side of the tissue during the action potential plateau. Two shock strengths (2.1+/-0.2 and 6.5+/-0.6 V/cm) were tested with both shock polarities. The recording site was moved across the tissue along fibers with either 200 micrometer (macroscopic group [n=5], 12 consecutive recording sites over a 2. 2-mm tissue length in each muscle) or 20 micrometer (microscopic group [n=5], 21 consecutive recording sites over a 0.4-mm tissue length in each muscle) between adjacent recording sites. In the macroscopic group, the portion of the tissue toward the anode was hyperpolarized, whereas the portion toward the cathode was depolarized, with 1 zero-potential crossing from hyperpolarization to depolarization present near the center of the tissue. In the microscopic group, only 1 zero-potential crossing was observed in the center region of the tissue, whereas, away from the center, only hyperpolarization was observed toward the anode and depolarization toward the cathode. Although these results are consistent with predictions from field stimulation of continuous representations of myocardial structure, ie, the bidomain and cable equation models, they are not consistent with the prediction of depolarization-hyperpolarization oscillation from representations based on cellular-level resistive discontinuities associated with gap junctions, ie, the sawtooth model.

  5. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

    PubMed Central

    Corradi, Valentina; Vergani, Paola; Tieleman, D. Peter

    2015-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette (ABC) transporter superfamily. CFTR controls the flow of anions through the apical membrane of epithelia. Dysfunctional CFTR causes the common lethal genetic disease cystic fibrosis. Transitions between open and closed states of CFTR are regulated by ATP binding and hydrolysis on the cytosolic nucleotide binding domains, which are coupled with the transmembrane (TM) domains forming the pathway for anion permeation. Lack of structural data hampers a global understanding of CFTR and thus the development of “rational” approaches directly targeting defective CFTR. In this work, we explored possible conformational states of the CFTR gating cycle by means of homology modeling. As templates, we used structures of homologous ABC transporters, namely TM(287–288), ABC-B10, McjD, and Sav1866. In the light of published experimental results, structural analysis of the transmembrane cavity suggests that the TM(287–288)-based CFTR model could correspond to a commonly occupied closed state, whereas the McjD-based model could represent an open state. The models capture the important role played by Phe-337 as a filter/gating residue and provide structural information on the conformational transition from closed to open channel. PMID:26229102

  6. Plant nuclear envelope proteins.

    PubMed

    Rose, Annkatrin; Patel, Shalaka; Meier, Iris

    2004-01-01

    Compared to research in the animal field, the plant NE has been clearly under-investigated. The available data so far indicate similarities as well as striking differences that raise interesting questions about the function and evolution of the NE in different kingdoms. Despite a seemingly similar structure and organization of the NE, many of the proteins that are integral components of the animal NE appear to lack homologues in plant cells. The sequencing of the Arabidopsis genome has not led to the identification of homologues of animal NE components, but has indicated that the plant NE must have a distinct protein composition different from that found in metazoan cells. Besides providing a selective barrier between the nucleoplasm and the cytoplasm, the plant NE functions as a scaffold for chromatin but the scaffolding components are not identical to those found in animal cells. The NE comprises an MTOC in higher plant cells, a striking difference to the organization of microtubule nucleation in other eukaryotic cells. Nuclear pores are present in the plant NE, but identifiable orthologues of most animal and yeast nucleoporins are presently lacking. The transport pathway through the nuclear pores via the action of karyopherins and the Ran cycle is conserved in plant cells. Interestingly, RanGAP is sequestered to the NE in plant cells and animal cells, yet the targeting domains and mechanisms of attachment are different between the two kingdoms. At present, only a few proteins localized at the plant NE have been identified molecularly. Future research will have to expand the list of known protein components involved in building a functional plant NE.

  7. Envelope and phase evolution of femtosecond pulses in hollow photonic-crystal fibres

    SciTech Connect

    Konorov, Stanislav O; Fedotov, Andrei B; Zheltikov, Aleksei M; Beloglazov, V I; Skibina, N B; Shcherbakov, Andrei V

    2004-01-31

    Changes in the envelope and the evolution of the spectral phase and chirp of femtosecond pulses propagating through hollow fibres with a photonic-crystal cladding are experimentally studied. Envelope and phase distortions of ultrashort pulses transmitted through such fibres are shown to be controlled by the detuning of the carrier frequency of laser pulses from the central frequency of the passband in the transmission of the fibre. Near the passband edges, which map the edges of photonic band gaps of the fibre cladding, ultrashort pulses transmitted through the fibre display considerable envelope distortions, as well as frequency- and time-dependent phase shifts. (optical fibres)

  8. Wind-Resistant Filler for Tile Gaps

    NASA Technical Reports Server (NTRS)

    Bellavia, J.; Quigley, I. A.; Callahan, T. S.

    1982-01-01

    Filler developed for gaps between insulating tiles on Space Shuttle finds application in industries that use tiles for thermal or environmental protection. Filler consists of tight-fitting ceramic tubes and fibrous alumina. Combination resists high wind loads while providing requisite heat protection. Quartz-thread stitching holds envelope together.

  9. Nuclear envelope and genome interactions in cell fate

    PubMed Central

    Talamas, Jessica A.; Capelson, Maya

    2015-01-01

    The eukaryotic cell nucleus houses an organism’s genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate. PMID:25852741

  10. Gap junctions.

    PubMed

    Goodenough, Daniel A; Paul, David L

    2009-07-01

    Gap junctions are aggregates of intercellular channels that permit direct cell-cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology.

  11. Gap Junctions

    PubMed Central

    Goodenough, Daniel A.; Paul, David L.

    2009-01-01

    Gap junctions are aggregates of intercellular channels that permit direct cell–cell transfer of ions and small molecules. Initially described as low-resistance ion pathways joining excitable cells (nerve and muscle), gap junctions are found joining virtually all cells in solid tissues. Their long evolutionary history has permitted adaptation of gap-junctional intercellular communication to a variety of functions, with multiple regulatory mechanisms. Gap-junctional channels are composed of hexamers of medium-sized families of integral proteins: connexins in chordates and innexins in precordates. The functions of gap junctions have been explored by studying mutations in flies, worms, and humans, and targeted gene disruption in mice. These studies have revealed a wide diversity of function in tissue and organ biology. PMID:20066080

  12. Envelope Inflation or Stellar Wind?

    NASA Astrophysics Data System (ADS)

    Ro, S.; Matzner, C. D.

    We an optically-thick, transonic, steady wind model for a H-free Wolf-Rayet star. A bifurcation is found across a critical mass loss rate Mb. Slower winds M < Mb extend by several hydrostatic stellar radii, reproduce features of envelope in ation from Petrovic et al. (2006) and Gräfener et al. (2012), and are energetically unbound. This work is of particular interest for extended envelopes and winds, radiative hydrodynamic instabilities (eg. wind stagnation, clumping, etc.), and NLTE atmospheric models.

  13. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, F. C.

    1986-01-01

    The use of nonmetallic or fabric structures for space application is considered. The following structures are suggested: (1) unpressurized space hangars; (2) extendable tunnels for soft docking; and (3) manned habitat for space stations, storage facilities, and work structures. The uses of the tunnel as a passageway: for personnel and equipment, eliminating extravehicular activity, for access to a control cabin on a space crane and between free flyers and the space station are outlined. The personnal occupied woven envelope robot (POWER) device is shown. The woven envelope (tunnel) acts as part of the boom of a crane. Potential applications of POWER are outlined. Several possible deflection mechanisms and design criteria are determined.

  14. Carbon chemistry of circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Bieging, John H.

    1990-01-01

    The chemical composition of envelopes surrounding cool evolved stars, as determined from microwave spectroscopic observations, is reviewed. Emphasis is placed on recent observations with the new large mm-wavelength telescopes and interferometer arrays, and on new theoretical work, especially concerning ion-molecule chemistry of carbon-bearing in these envelopes. Thermal (as opposed to maser) emission lines are discussed. Much progress has been made in the past few years in the theoretical understanding of these objects. It is already clear, however, that observations with the new generation of mm-telescopes will require substantial improvements in the theoretical models to achieve a thorough understanding of the data now becoming available.

  15. Identification of a Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Variant Resistant to Cold Inactivation▿ †

    PubMed Central

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R.; Smith, Amos B.; Sodroski, Joseph

    2009-01-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability. PMID:19211747

  16. Identification of a human immunodeficiency virus type 1 envelope glycoprotein variant resistant to cold inactivation.

    PubMed

    Kassa, Aemro; Finzi, Andrés; Pancera, Marie; Courter, Joel R; Smith, Amos B; Sodroski, Joseph

    2009-05-01

    The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein trimer consists of gp120 and gp41 subunits and undergoes a series of conformational changes upon binding to the receptors, CD4 and CCR5/CXCR4, that promote virus entry. Surprisingly, we found that the envelope glycoproteins of some HIV-1 strains are functionally inactivated by prolonged incubation on ice. Serial exposure of HIV-1 to extremes of temperature, followed by expansion of replication-competent viruses, allowed selection of a temperature-resistant virus. The envelope glycoproteins of this virus resisted cold inactivation due to a single passage-associated change, H66N, in the gp120 exterior envelope glycoprotein. Histidine 66 is located within the gp41-interactive inner domain of gp120 and, in other studies, has been shown to decrease the sampling of the CD4-bound conformation by unliganded gp120. Substituting asparagine or other amino acid residues for histidine 66 in cold-sensitive HIV-1 envelope glycoproteins resulted in cold-stable phenotypes. Cold inactivation of the HIV-1 envelope glycoproteins occurred even at high pH, indicating that protonation of histidine 66 is not necessary for this process. Increased exposure of epitopes in the ectodomain of the gp41 transmembrane envelope glycoprotein accompanied cold inactivation, but shedding of gp120 did not. An amino acid change in gp120 (S375W) that promotes the CD4-bound state or treatment with soluble CD4 or a small-molecule CD4 mimic resulted in increased cold sensitivity. These results indicate that the CD4-bound intermediate of the HIV-1 envelope glycoproteins is cold labile; avoiding the CD4-bound state increases temperature stability.

  17. Trimeric Transmembrane Domain Interactions in Paramyxovirus Fusion Proteins

    PubMed Central

    Smith, Everett Clinton; Smith, Stacy E.; Carter, James R.; Webb, Stacy R.; Gibson, Kathleen M.; Hellman, Lance M.; Fried, Michael G.; Dutch, Rebecca Ellis

    2013-01-01

    Paramyxovirus fusion (F) proteins promote membrane fusion between the viral envelope and host cell membranes, a critical early step in viral infection. Although mutational analyses have indicated that transmembrane (TM) domain residues can affect folding or function of viral fusion proteins, direct analysis of TM-TM interactions has proved challenging. To directly assess TM interactions, the oligomeric state of purified chimeric proteins containing the Staphylococcal nuclease (SN) protein linked to the TM segments from three paramyxovirus F proteins was analyzed by sedimentation equilibrium analysis in detergent and buffer conditions that allowed density matching. A monomer-trimer equilibrium best fit was found for all three SN-TM constructs tested, and similar fits were obtained with peptides corresponding to just the TM region of two different paramyxovirus F proteins. These findings demonstrate for the first time that class I viral fusion protein TM domains can self-associate as trimeric complexes in the absence of the rest of the protein. Glycine residues have been implicated in TM helix interactions, so the effect of mutations at Hendra F Gly-508 was assessed in the context of the whole F protein. Mutations G508I or G508L resulted in decreased cell surface expression of the fusogenic form, consistent with decreased stability of the prefusion form of the protein. Sedimentation equilibrium analysis of TM domains containing these mutations gave higher relative association constants, suggesting altered TM-TM interactions. Overall, these results suggest that trimeric TM interactions are important driving forces for protein folding, stability and membrane fusion promotion. PMID:24178297

  18. Gap junctions.

    PubMed

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2012-07-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1853-1872, 2012.

  19. Gap Junctions

    PubMed Central

    Nielsen, Morten Schak; Axelsen, Lene Nygaard; Sorgen, Paul L.; Verma, Vandana; Delmar, Mario; Holstein-Rathlou, Niels-Henrik

    2013-01-01

    Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease. © 2012 American Physiological Society. Compr Physiol 2:1981-2035, 2012. PMID:23723031

  20. Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

    PubMed Central

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-01-01

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

  1. Coat as a dagger: the use of capsid proteins to perforate membranes during non-enveloped DNA viruses trafficking.

    PubMed

    Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

    2014-07-23

    To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection.

  2. CERAMIDE-RICH PLATFORMS IN TRANSMEMBRANE SIGNALING

    PubMed Central

    Stancevic, Branka; Kolesnick, Richard

    2015-01-01

    Recent evidence suggests that ceramide regulates stress signaling via reorganization of the plasma membrane. The focus of this review will be to discuss the mechanism by which acid sphingomyelinase (ASMase)-generated ceramide initiates transmembrane signaling in the plasma membrane exoplasmic leaflet. In particular, we review the unique biophysical properties of ceramide that render it proficient in formation of signaling domains termed ceramide-rich platforms (CRPs), and the role of CRPs in the pathophysiology of various diseases. The biomedical significance of CRPs makes these structures an attractive therapeutic target. PMID:20178791

  3. Gap junctions.

    PubMed

    Shimizu, Kazumichi; Stopfer, Mark

    2013-12-02

    In vertebrates and invertebrates, signaling among neurons is most commonly mediated by chemical synapses. At these synapses neurotransmitter released by presynaptic neurons is detected by receptors on the postsynaptic neurons, leading to an influx of ions through the receptors themselves or through channels activated by intracellular signaling downstream of the receptors. But neurons can communicate with each other in a more direct way, by passing signals composed of small molecules and ions through pores called gap junctions. Gap junctions that transmit electrical signals are called electrical synapses. Unlike most chemical synapses, electrical synapses interact through axon-to-axon or dendrite-to-dendrite contacts. Found throughout the nervous system, they are probably best known for linking the relatively few inhibitory, GABAergic, neurons into large, effective networks within vertebrate brains. They are particularly important early in development before the formation of most chemical synapses, but recent work shows gap junctions play important roles in the adult nervous system, too. Gap junctions are sometimes thought to be mere passageways between cells. But, as recent work shows, their properties can be complex and surprising. Gap junctions help generate, propagate, and regulate neural oscillations, can filter electrical signals, and can be modulated in a variety of ways. Here we discuss recent work highlighting the diversity and importance of gap junctions throughout the nervous system. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. [Efficient extraction of transmembrane proteins using ProteoExtract Transmembrane Protein Extraction Kit].

    PubMed

    Błachnio, Karina

    2010-01-01

    Detergents commonly used for solubilization of membrane proteins may be ionic or non-ionic. Exposing membrane proteins to detergents, however, can adversely affect their native structure, which can be a major hindrance for functional studies. This is especially true for proteins with multiple transmembrane domains. The ProteoExtract Transmembrane Protein Extraction Kit (TM-PEK), offered by Merck, provides a detergent-free novel reagents to enable the mild and efficient extraction of proteins containing seven transmembrane domains, such as GPCRs (G-Protein Coupled Receptors) e.g.: Frizzled-4 and CELSR-3, from mammalian cells. The fraction enriched in transmembrane proteins using TM-PEK is directly compatible with enzyme assays, non-denaturing gel electrophoresis, 1- and 2-D SDS-PAGE, MS analysis, Western blotting, immunoprecipitation and ELISA. Unlike many alternatives, TM-PEK extraction procedure does not require sonication, extended rigorous vortexing, ultracentrifugation, or incubation of samples at elevated temperatures--thus minimizing the risk of post-extraction degradation or modifications.

  5. Characterization and interactome study of white spot syndrome virus envelope protein VP11.

    PubMed

    Liu, Wang-Jing; Shiung, Hui-Jui; Lo, Chu-Fang; Leu, Jiann-Horng; Lai, Ying-Jang; Lee, Tai-Lin; Huang, Wei-Tung; Kou, Guang-Hsiung; Chang, Yun-Shiang

    2014-01-01

    White spot syndrome virus (WSSV) is a large enveloped virus. The WSSV viral particle consists of three structural layers that surround its core DNA: an outer envelope, a tegument and a nucleocapsid. Here we characterize the WSSV structural protein VP11 (WSSV394, GenBank accession number AF440570), and use an interactome approach to analyze the possible associations between this protein and an array of other WSSV and host proteins. Temporal transcription analysis showed that vp11 is an early gene. Western blot hybridization of the intact viral particles and fractionation of the viral components, and immunoelectron microscopy showed that VP11 is an envelope protein. Membrane topology software predicted VP11 to be a type of transmembrane protein with a highly hydrophobic transmembrane domain at its N-terminal. Based on an immunofluorescence assay performed on VP11-transfected Sf9 cells and a trypsin digestion analysis of the virion, we conclude that, contrary to topology software prediction, the C-terminal of this protein is in fact inside the virion. Yeast two-hybrid screening combined with co-immunoprecipitation assays found that VP11 directly interacted with at least 12 other WSSV structural proteins as well as itself. An oligomerization assay further showed that VP11 could form dimers. VP11 is also the first reported WSSV structural protein to interact with the major nucleocapsid protein VP664.

  6. Spectropolarimetry of stripped-envelope supernovae: observations and modelling

    NASA Astrophysics Data System (ADS)

    Tanaka, Masaomi

    2017-09-01

    Spectropolarimetry is one of the most powerful methods to study the multi-dimensional geometry of supernovae (SNe). We present a brief summary of the spectropolarimetric observations of stripped-envelope core-collapse SNe. Observations indicate that stripped-envelope SNe generally have a non-axisymmetric ion distribution in the ejecta. Three-dimensional clumpy geometry nicely explains the observed properties. A typical size of the clumps deduced from observations is relatively large: 25% of the photosphere. Such a large-scale clumpy structure is similar to that observed in Cassiopeia A, and suggests that large-scale convection or standing accretion shock instability takes place at the onset of the explosion. This article is part of the themed issue 'Bridging the gap: from massive stars to supernovae'.

  7. Full-Envelope Launch Abort System Performance Analysis Methodology

    NASA Technical Reports Server (NTRS)

    Aubuchon, Vanessa V.

    2014-01-01

    The implementation of a new dispersion methodology is described, which dis-perses abort initiation altitude or time along with all other Launch Abort System (LAS) parameters during Monte Carlo simulations. In contrast, the standard methodology assumes that an abort initiation condition is held constant (e.g., aborts initiated at altitude for Mach 1, altitude for maximum dynamic pressure, etc.) while dispersing other LAS parameters. The standard method results in large gaps in performance information due to the discrete nature of initiation conditions, while the full-envelope dispersion method provides a significantly more comprehensive assessment of LAS abort performance for the full launch vehicle ascent flight envelope and identifies performance "pinch-points" that may occur at flight conditions outside of those contained in the discrete set. The new method has significantly increased the fidelity of LAS abort simulations and confidence in the results.

  8. Functional role of the zipper motif region of human immunodeficiency virus type 1 transmembrane protein gp41.

    PubMed Central

    Chen, S S

    1994-01-01

    To study the functional role of the zipper motif region, located in the N-terminal region of the envelope transmembrane protein of human immunodeficiency virus type 1, a series of vaccinia virus-expressed mutant proteins containing a proline substitution in this region were characterized. All of the mutant proteins showed partial or no inhibition in gp160 cleavage, demonstrated impaired ability of gp120 to associate with gp41, and were unable to mediate syncytium formation with CD4+ cells. Moreover, mutants 580 and 587 secreted excessive gp120 into the medium compared with the wild type. Mutations in this region affected the conformation of the local or proximal sequence but did not alter the conformation conferred by a distal site. These studies reveal the crucial role of the C-terminal segment of the zipper motif region in envelope heterodimeric association and suggest that this sequence forms a gp120 contact site. Images PMID:7509005

  9. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes

    PubMed Central

    Hernáez, Bruno; Guerra, Milagros; Salas, María L.

    2016-01-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs. PMID:27110717

  10. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    PubMed

    Hernáez, Bruno; Guerra, Milagros; Salas, María L; Andrés, Germán

    2016-04-01

    African swine fever virus (ASFV) is a nucleocytoplasmic large DNA virus (NCLDV) that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  11. Nuclear envelopathies: a complex LINC between nuclear envelope and pathology.

    PubMed

    Janin, Alexandre; Bauer, Delphine; Ratti, Francesca; Millat, Gilles; Méjat, Alexandre

    2017-08-30

    Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of "nuclear envelopathies" is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

  12. TSTMP: target selection for structural genomics of human transmembrane proteins.

    PubMed

    Varga, Julia; Dobson, László; Reményi, István; Tusnády, Gábor E

    2017-01-04

    The TSTMP database is designed to help the target selection of human transmembrane proteins for structural genomics projects and structure modeling studies. Currently, there are only 60 known 3D structures among the polytopic human transmembrane proteins and about a further 600 could be modeled using existing structures. Although there are a great number of human transmembrane protein structures left to be determined, surprisingly only a small fraction of these proteins have 'selected' (or above) status according to the current version the TargetDB/TargetTrack database. This figure is even worse regarding those transmembrane proteins that would contribute the most to the structural coverage of the human transmembrane proteome. The database was built by sorting out proteins from the human transmembrane proteome with known structure and searching for suitable model structures for the remaining proteins by combining the results of a state-of-the-art transmembrane specific fold recognition algorithm and a sequence similarity search algorithm. Proteins were searched for homologues among the human transmembrane proteins in order to select targets whose successful structure determination would lead to the best structural coverage of the human transmembrane proteome. The pipeline constructed for creating the TSTMP database guarantees to keep the database up-to-date. The database is available at http://tstmp.enzim.ttk.mta.hu. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. TSTMP: target selection for structural genomics of human transmembrane proteins

    PubMed Central

    Varga, Julia; Dobson, László; Reményi, István; Tusnády, Gábor E.

    2017-01-01

    The TSTMP database is designed to help the target selection of human transmembrane proteins for structural genomics projects and structure modeling studies. Currently, there are only 60 known 3D structures among the polytopic human transmembrane proteins and about a further 600 could be modeled using existing structures. Although there are a great number of human transmembrane protein structures left to be determined, surprisingly only a small fraction of these proteins have ‘selected’ (or above) status according to the current version the TargetDB/TargetTrack database. This figure is even worse regarding those transmembrane proteins that would contribute the most to the structural coverage of the human transmembrane proteome. The database was built by sorting out proteins from the human transmembrane proteome with known structure and searching for suitable model structures for the remaining proteins by combining the results of a state-of-the-art transmembrane specific fold recognition algorithm and a sequence similarity search algorithm. Proteins were searched for homologues among the human transmembrane proteins in order to select targets whose successful structure determination would lead to the best structural coverage of the human transmembrane proteome. The pipeline constructed for creating the TSTMP database guarantees to keep the database up-to-date. The database is available at http://tstmp.enzim.ttk.mta.hu. PMID:27924015

  14. Anchors aweigh: protein localization and transport mediated by transmembrane domains.

    PubMed

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S

    2013-10-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in cytosolic domains, TMD sorting determinants are not conserved amino acid sequences but physical properties such as the length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized, but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport.

  15. Anchors Aweigh: Protein Traffic Mediated by Transmembrane Domains

    PubMed Central

    Cosson, Pierre; Perrin, Jackie; Bonifacino, Juan S.

    2013-01-01

    The transmembrane domains (TMDs) of integral membrane proteins have emerged as major determinants of intracellular localization and transport in the secretory and endocytic pathways. Unlike sorting signals in the cytosolic domains, TMD sorting determinants are not conserved amino-acid sequences but physical properties such as length and hydrophilicity of the transmembrane span. The underlying sorting machinery is still poorly characterized but several mechanisms have been proposed, including TMD recognition by transmembrane sorting receptors and partitioning into membrane lipid domains. Here we review the nature of TMD sorting determinants and how they may dictate transmembrane protein localization and transport. PMID:23806646

  16. Safeguards Envelope Progress FY08

    SciTech Connect

    Robert Bean; Richard Metcalf; Aaron Bevill

    2008-09-01

    The Safeguards Envelope Project met its milestones by creating a rudimentary safeguards envelope, proving the value of the approach on a small scale, and determining the most appropriate path forward. The Idaho Chemical Processing Plant’s large cache of reprocessing process monitoring data, dubbed UBER Data, was recovered and used in the analysis. A probabilistic Z test was used on a Markov Monte Carlo simulation of expected diversion data when compared with normal operating data. The data regarding a fully transient event in a tank was used to create a simple requirement, representative of a safeguards envelope, whose impact was a decrease in operating efficiency by 1.3% but an increase in material balance period of 26%. This approach is operator, state, and international safeguards friendly and should be applied to future reprocessing plants. Future requirements include tank-to-tank correlations in reprocessing facilities, detailed operations impact studies, simulation inclusion, automated optimization, advanced statistics analysis, and multi-attribute utility analysis.

  17. Heat recovery in building envelopes

    SciTech Connect

    Walker, Iain S.; Sherman, Max H.

    2003-08-01

    Infiltration has traditionally been assumed to contribute to the energy load of a building by an amount equal to the product of the infiltration flow rate and the enthalpy difference between inside and outside. Some studies have indicated that application of such a simple formula may produce an unreasonably high contribution because of heat recovery within the building envelope. The major objective of this study was to provide an improved prediction of the energy load due to infiltration by introducing a correction factor that multiplies the expression for the conventional load. This paper discusses simplified analytical modeling and CFD simulations that examine infiltration heat recovery (IHR) in an attempt to quantify the magnitude of this effect for typical building envelopes. For comparison, we will also briefly examine the results of some full-scale field measurements of IHR based on infiltration rates and energy use in real buildings. The results of this work showed that for houses with insulated walls the heat recovery is negligible due to the small fraction of the envelope that participates in heat exchange with the infiltrating air. However; there is the potential for IHR to have a significant effect for higher participation dynamic walls/ceilings or uninsulated walls. This result implies that the existing methods for evaluating infiltration related building loads provide adequate results for typical buildings.

  18. Transmembrane protein sorting driven by membrane curvature

    NASA Astrophysics Data System (ADS)

    Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

    2015-11-01

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  19. Transmembrane protein sorting driven by membrane curvature.

    PubMed

    Strahl, H; Ronneau, S; González, B Solana; Klutsch, D; Schaffner-Barbero, C; Hamoen, L W

    2015-11-02

    The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

  20. A periodicity analysis of transmembrane helices.

    PubMed

    Leonov, Hadas; Arkin, Isaiah T

    2005-06-01

    Transmembrane helices and the helical bundles which they form are the major building blocks of membrane proteins. Since helices are characterized by a given periodicity, it is possible to search for patterns of traits which typify one side of the helix and not the other (e.g. amphipathic helices contain a polar and apolar sides). Using Fourier transformation we have analyzed solved membrane protein structures as well as sequences of membrane proteins from the Swiss-Prot database. The traits searched included aromaticity, volume and ionization. While a number of motifs were already recognized in the literature, many were not. One particular example involved helix VII of lactose permease which contains seven aromatic residues on six helical turns. Similarly six glycine residues in four consecutive helical turns were identified as forming a motif in the chloride channel. A tabulation of all the findings is presented as well as a possible rationalization of the function of the motif.

  1. Enteropeptidase, a type II transmembrane serine protease.

    PubMed

    Zheng, X Long; Kitamoto, Yasunori; Sadler, J Evan

    2009-06-01

    Enteropeptidase, a type II transmembrane serine protease, is localized to the brush border of the duodenal and jejunal mucosa. It is synthesized as a zymogen (proenteropeptidase) that requires activation by another protease, either trypsin or possibly duodenase. Active enteropeptidase then converts the pancreatic precursor, trypsinogen, to trypsin by cleavage of the specific trypsinogen activation peptide, Asp-Asp-Asp-Asp-Lys- Ile that is highly conserved in vertebrates. Trypsin, in turn, activates other digestive zymogens such as chymotrypsinogen, proelastase, procarboxypeptidase and prolipase in the lumen of the gut. The important biological function of enteropeptidase is highlighted by the manifestation of severe diarrhea, failure to thrive, hypoproteinemia and edema as a result of congenital deficiency of enteropeptidase activity in the gut. Conversely, duodenopancreatic reflux of proteolytically active enteropeptidase may cause acute and chronic pancreatitis.

  2. Molecular mechanisms for generating transmembrane proton gradients.

    PubMed

    Gunner, M R; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side.

  3. Molecular mechanisms for generating transmembrane proton gradients

    PubMed Central

    Gunner, M.R.; Amin, Muhamed; Zhu, Xuyu; Lu, Jianxun

    2013-01-01

    Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side. PMID:23507617

  4. The structure of common-envelope remnants

    NASA Astrophysics Data System (ADS)

    Hall, Philip D.

    2015-05-01

    We investigate the structure and evolution of the remnants of common-envelope evolution in binary star systems. In a common-envelope phase, two stars become engulfed in a gaseous envelope and, under the influence of drag forces, spiral to smaller separations. They may merge to form a single star or the envelope may be ejected to leave the stars in a shorter period orbit. This process explains the short orbital periods of many observed binary systems, such as cataclysmic variables and low-mass X-ray binary systems. Despite the importance of these systems, and of common-envelope evolution to their formation, it remains poorly understood. Specifically, we are unable to confidently predict the outcome of a common-envelope phase from the properties at its onset. After presenting a review of work on stellar evolution, binary systems, common-envelope evolution and the computer programs used, we describe the results of three computational projects on common-envelope evolution. Our work specifically relates to the methods and prescriptions which are used for predicting the outcome. We use the Cambridge stellar-evolution code STARS to produce detailed models of the structure and evolution of remnants of common-envelope evolution. We compare different assumptions about the uncertain end-of-common envelope structure and envelope mass of remnants which successfully eject their common envelopes. In the first project, we use detailed remnant models to investigate whether planetary nebulae are predicted after common-envelope phases initiated by low-mass red giants. We focus on the requirement that a remnant evolves rapidly enough to photoionize the nebula and compare the predictions for different ideas about the structure at the end of a common-envelope phase. We find that planetary nebulae are possible for some prescriptions for the end-of-common envelope structure. In our second contribution, we compute a large set of single-star models and fit new formulae to the core radii of

  5. Acoustic Band Gap Formation in Metamaterials

    NASA Astrophysics Data System (ADS)

    Elford, D. P.; Chalmers, L.; Kusmartsev, F.; Swallowe, G. M.

    We present several new classes of metamaterials and/or locally resonant sonic crystal that are comprised of complex resonators. The proposed systems consist of multiple resonating inclusion that correspond to different excitation frequencies. This causes the formation of multiple overlapped resonance band gaps. We demonstrate theoretically and experimentally that the individual band gaps achieved, span a far greater range (≈ 2kHz) than previously reported cases. The position and width of the band gap is independent of the crystal's lattice constant and forms in the low frequency regime significantly below the conventional Bragg band gap. The broad envelope of individual resonance band gaps is attractive for sound proofing applications and furthermore the devices can be tailored to attenuate lower or higher frequency ranges, i.e., from seismic to ultrasonic.

  6. Acoustic Band Gap Formation in Metamaterials

    NASA Astrophysics Data System (ADS)

    Elford, D. P.; Chalmers, L.; Kusmartsev, F.; Swallowe, G. M.

    2011-03-01

    We present several new classes of metamaterials and/or locally resonant sonic crystal that are comprised of complex resonators. The proposed systems consist of multiple resonating inclusion that correspond to different excitation frequencies. This causes the formation of multiple overlapped resonance band gaps. We demonstrate theoretically and experimentally that the individual band gaps achieved, span a far greater range (≈ 2kHz) than previously reported cases. The position and width of the band gap is independent of the crystal's lattice constant and forms in the low frequency regime significantly below the conventional Bragg band gap. The broad envelope of individual resonance band gaps is attractive for sound proofing applications and furthermore the devices can be tailored to attenuate lower or higher frequency ranges, i.e., from seismic to ultrasonic.

  7. A pump-pore model for transmembrane transport of hydrophilic solutes.

    PubMed Central

    Roberts, E

    1993-01-01

    Transmembrane transport of a hydrophilic solute is presumed to begin when hydrated ligand adheres in Velcro-like fashion to hydrated membrane surface. Asymmetric physical forces cause rolling movements of ligand over membrane surface until contact occurs with appropriate transport machinery, consisting of a pump (Pu) to which is tethered a ligand (Li)-specific perm-selective pore (Po). The Po is in the open form when the Li is attached to an external high-affinity allosteric site on it. The active form of the Pu is stabilized by attachment of the Li to high-affinity internal or low-affinity external allosteric sites. The active form of the Pu induces closure of the Po, even when ligand is bound to it; the inactive conformation of the Pu permits Po opening. Attachment of Li to either one of two binding sites on the active Pu and irreversible envelopment by it in Venus fly-trap fashion trigger transmembrane transport of Li. Multistep attachment of Li is rate-limiting in the transport process. Application of a simple equation derived from relevant kinetic considerations relating velocity of transport (V) to concentration of Li (L), V = k1(L)1/2, gives V-L curves approximating transport data obtained in a variety of biological systems. This model is congruent with the ability of cells to concentrate substances from extremely dilute solutions and with the adaptive informational value to cells of rates of transport. PMID:8102798

  8. Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission.

    PubMed

    Lorizate, Maier; Huarte, Nerea; Sáez-Cirión, Asier; Nieva, José L

    2008-01-01

    Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley-White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure-function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

  9. Investigation on perceptual influence of spatial index on enveloping sound

    NASA Astrophysics Data System (ADS)

    Oh, Jiseong

    This thesis is to discover the influence of Spatial Index on the region of listener envelopment. Audio engineers often realize the perceptual difference between convolution and conventional artificial reverberation techniques. The understanding of uncorrelatedness via investigating spatial index may provide one of clues on this difference. The primary task is to diverse the decorrelation degrees of the reverberation tail using various algorithms, which can be proved by psychoacoustic test. Some optimal results may lead us to perceive no difference between measured binaural impulse response and created reverberation tail. Through this discovery, the researchers acquire a hint to resolve the perceptual gap between the real world experience and virtual space.

  10. Structure of a Dengue Virus Envelope Protein Late-Stage Fusion Intermediate

    PubMed Central

    Klein, Daryl E.; Choi, Jason L.

    2013-01-01

    The final stages of dengue virus fusion are thought to occur when the membrane-proximal stem drives the transmembrane anchor of the viral envelope protein (E) toward the fusion loop, buried in the target cell membrane. Crystal structures of E have lacked this essential stem region. We expressed and crystallized soluble mutant forms of the dengue virus envelope protein (sE) that include portions of the juxtamembrane stem. Their structures represent late-stage fusion intermediates. The proximal part of the stem has both intra- and intermolecular interactions, so the chain “zips up” along the trimer seam. The penultimate interaction we detected involves the conserved residue F402, which has hydrophobic contacts with a conserved surface on domain II. These interactions do not require any larger-scale changes in trimer packing. The techniques for expression and crystallization of sE containing stem reported here may allow further characterization of the final stages of flavivirus fusion. PMID:23236058

  11. Targeting and biogenesis of transporters and channels in chloroplast envelope membranes: Unsolved questions.

    PubMed

    Oh, Young Jun; Hwang, Inhwan

    2015-07-01

    Chloroplasts produce carbohydrates, hormones, vitamins, amino acids, pigments, nucleotides, ATP, and secondary metabolites. Channels and transporters are required for the movement of molecules across the two chloroplast envelope membranes. These transporters and channel proteins are grouped into two different types, including β-barrel proteins and transmembrane-domain (TMD) containing proteins. Most β-barrel proteins are localized at the outer chloroplast membrane, and TMD-containing proteins are localized at the inner chloroplast membrane. Many of these transporters and channels are encoded by nuclear genes; therefore, they have to be imported into chloroplasts after translation on cytosolic ribosomes. These proteins should have specific targeting signals for their final destination in the chloroplast membrane and for assembly into specific complexes. In this review, we summarize recent progress in the identification, functional characterization, and biogenesis of transporters and channels at the chloroplast envelope membranes, and discuss outstanding questions regarding transporter and channel protein biogenesis.

  12. Synthesis, Assembly, and Processing of the Env ERVWE1/Syncytin Human Endogenous Retroviral Envelope

    PubMed Central

    Cheynet, V.; Ruggieri, A.; Oriol, G.; Blond, J.-L.; Boson, B.; Vachot, L.; Verrier, B.; Cosset, F.-L.; Mallet, F.

    2005-01-01

    Syncytin is a fusogenic protein involved in the formation of the placental syncytiotrophoblast layer. This protein is encoded by the envelope gene of the ERVWE1 proviral locus belonging to the human endogenous retrovirus W (HERV-W) family. The HERV-W infectious ancestor entered the primate lineage 25 to 40 million years ago. Although the syncytin fusion property has been clearly demonstrated, little is known about this cellular protein maturation process with respect to classical infectious retrovirus envelope proteins. Here we show that the cellular syncytin protein is synthesized as a glycosylated gPr73 precursor cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM). These SU and TM subunits are found associated as homotrimers. The intracytoplasmic tail is critical to the fusogenic phenotype, although its cleavage requirements seem to have diverged from those of classical retroviral maturation. PMID:15827173

  13. Synthesis, assembly, and processing of the Env ERVWE1/syncytin human endogenous retroviral envelope.

    PubMed

    Cheynet, V; Ruggieri, A; Oriol, G; Blond, J-L; Boson, B; Vachot, L; Verrier, B; Cosset, F-L; Mallet, F

    2005-05-01

    Syncytin is a fusogenic protein involved in the formation of the placental syncytiotrophoblast layer. This protein is encoded by the envelope gene of the ERVWE1 proviral locus belonging to the human endogenous retrovirus W (HERV-W) family. The HERV-W infectious ancestor entered the primate lineage 25 to 40 million years ago. Although the syncytin fusion property has been clearly demonstrated, little is known about this cellular protein maturation process with respect to classical infectious retrovirus envelope proteins. Here we show that the cellular syncytin protein is synthesized as a glycosylated gPr73 precursor cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM). These SU and TM subunits are found associated as homotrimers. The intracytoplasmic tail is critical to the fusogenic phenotype, although its cleavage requirements seem to have diverged from those of classical retroviral maturation.

  14. Cortical processing of dynamic sound envelope transitions.

    PubMed

    Zhou, Yi; Wang, Xiaoqin

    2010-12-08

    Slow envelope fluctuations in the range of 2-20 Hz provide important segmental cues for processing communication sounds. For a successful segmentation, a neural processor must capture envelope features associated with the rise and fall of signal energy, a process that is often challenged by the interference of background noise. This study investigated the neural representations of slowly varying envelopes in quiet and in background noise in the primary auditory cortex (A1) of awake marmoset monkeys. We characterized envelope features based on the local average and rate of change of sound level in envelope waveforms and identified envelope features to which neurons were selective by reverse correlation. Our results showed that envelope feature selectivity of A1 neurons was correlated with the degree of nonmonotonicity in their static rate-level functions. Nonmonotonic neurons exhibited greater feature selectivity than monotonic neurons in quiet and in background noise. The diverse envelope feature selectivity decreased spike-timing correlation among A1 neurons in response to the same envelope waveforms. As a result, the variability, but not the average, of the ensemble responses of A1 neurons represented more faithfully the dynamic transitions in low-frequency sound envelopes both in quiet and in background noise.

  15. Cell entry of enveloped viruses.

    PubMed

    Cosset, François-Loic; Lavillette, Dimitri

    2011-01-01

    Enveloped viruses penetrate their cell targets following the merging of their membrane with that of the cell. This fusion process is catalyzed by one or several viral glycoproteins incorporated on the membrane of the virus. These envelope glycoproteins (EnvGP) evolved in order to combine two features. First, they acquired a domain to bind to a specific cellular protein, named "receptor." Second, they developed, with the help of cellular proteins, a function of finely controlled fusion to optimize the replication and preserve the integrity of the cell, specific to the genus of the virus. Following the activation of the EnvGP either by binding to their receptors and/or sometimes the acid pH of the endosomes, many changes of conformation permit ultimately the action of a specific hydrophobic domain, the fusion peptide, which destabilizes the cell membrane and leads to the opening of the lipidic membrane. The comprehension of these mechanisms is essential to develop medicines of the therapeutic class of entry inhibitor like enfuvirtide (Fuzeon) against human immunodeficiency virus (HIV). In this chapter, we will summarize the different envelope glycoprotein structures that viruses develop to achieve membrane fusion and the entry of the virus. We will describe the different entry pathways and cellular proteins that viruses have subverted to allow infection of the cell and the receptors that are used. Finally, we will illustrate more precisely the recent discoveries that have been made within the field of the entry process, with a focus on the use of pseudoparticles. These pseudoparticles are suitable for high-throughput screenings that help in the development of natural or artificial inhibitors as new therapeutics of the class of entry inhibitors. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Isolating The Building Thermal Envelope

    NASA Astrophysics Data System (ADS)

    Harrje, D. T.; Dutt, G. S.; Gadsby, K. J.

    1981-01-01

    The evaluation of the thermal integrity of building envelopes by infrared scanning tech-niques is often hampered in mild weather because temperature differentials across the envelope are small. Combining the infrared scanning with positive or negative building pressures, induced by a "blower door" or the building ventilation system, considerably extends the periods during which meaningful diagnostics can be conducted. Although missing or poorly installed insulation may lead to a substantial energy penalty, it is the search for air leakage sites that often has the largest potential for energy savings. Infrared inspection of the attic floor with air forced from the occupied space through ceiling by-passes, and inspecting the interior of the building when outside air is being sucked through the envelope reveals unexpected leakage sites. Portability of the diagnostic equipment is essential in these surveys which may include access into some tight spaces. A catalog of bypass heat losses that have been detected in residential housing using the combined infrared pressure differential technique is included to point out the wide variety of leakage sites which may compromise the benefits of thermal insulation and allow excessive air infiltration. Detection and suppression of such leaks should be key items in any building energy audit program. Where a calibrated blower door is used to pressurize or evacuate the house, the leakage rate can be quantified and an excessively tight house recognized. Houses that are too tight may be improved with a minimal energy penalty by forced ventilation,preferably with a heat recuperator and/or by providing combustion air directly to the furnace.

  17. Aircraft maneuver envelope warning system

    NASA Technical Reports Server (NTRS)

    Bivens, Courtland C. (Inventor); Rosado, Joel M. (Inventor); Lee, Burnett (Inventor)

    1994-01-01

    A maneuver envelope warning system for an aircraft having operating limits, operating condition sensors and an indicator driver. The indicator driver has a plurality of visual indicators. The indicator driver determines a relationship between sensed operating conditions and the operating limits; such as, a ratio therebetween. The indicator driver illuminates a number of the indicators in proportion to the determined relationship. The position of the indicators illuminated represents to a pilot in an easily ascertainable manner whether the operational conditions are approaching operational limits of the aircraft, and the degree to which operational conditions lie within or exceed operational limits.

  18. Flexible Envelope Request Notation (FERN)

    NASA Technical Reports Server (NTRS)

    Zoch, David R.; Lavallee, David; Weinstein, Stuart

    1991-01-01

    The following topics are presented in view graph form and include the following: scheduling application; the motivation for the Flexible Envelope Request Notation (FERN); characteristics of FERN; types of information needed in requests; where information is stored in requests; FERN structures; generic requests; resource availability for pooled resources; expressive notation; temporal constraints; time formats; changes to FERN; sample FERN requests; the temporal relationship between two steps; maximum activity length to limit step delays; alternative requests; the temporal relationship between two activities; and idle resource usage between steps.

  19. Synthesis and assembly of Hepatitis B virus envelope protein-derived particles in Escherichia coli.

    PubMed

    Li, Hao; Onbe, Keisuke; Liu, Qiushi; Iijima, Masumi; Tatematsu, Kenji; Seno, Masaharu; Tada, Hiroko; Kuroda, Shun' Ichi

    2017-08-19

    Hepatitis B virus (HBV) envelope particles have been synthesized in eukaryotic cells (e.g., mammalian cells, insect cells, and yeast cells) as an HB vaccine immunogen and drug delivery system (DDS) nanocarrier. Many researchers had made attempts to synthesize the particles in Escherichia coli for minimize the cost and time for producing HBV envelope particles, but the protein was too deleterious to be synthesized in E. coli. In this study, we generated deletion mutants of HBV envelope L protein (389 amino acid residues (aa)) containing three transmembrane domains (TM1, TM2, TM3). The ΔNC mutant spanning from TM2 to N-terminal half of TM3 (from 237 aa to 335 aa) was found as a shortest form showing spontaneous particle formation. After the N-terminal end of ΔNC mutant was optimized by the N-end rule for E. coli expression, the modified ΔNC mutant (mΔNC) was efficiently expressed as particles in E. coli. The molecular mass of mΔNC particle was approx. 670 kDa, and the diameter was 28.5 ± 6.2 nm (mean ± SD, N = 61). The particle could react with anti-HBV envelope S protein antibody, indicating the particles exhibited S antigenic domain outside as well as HBV envelope particles. Taken together, the E. coli-derived mΔNC particles could be used as a substitute of eukaryotic cell-derived HBV envelope particles for versatile applications. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Safeguards Envelope Progress FY10

    SciTech Connect

    Richard Metcalf

    2010-10-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters within which nuclear facilities may operate to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details the additions to the advanced operating techniques that will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). Research this year focused on combining disparate pieces of data together to maximize operating time with minimal downtime due to safeguards. A Chi-Square and Croiser's cumulative sum were both included as part of the new analysis. Because of a major issue with the original data, the implementation of the two new tests did not add to the existing set of tests, though limited one-variable optimization made a small increase in detection probability. Additional analysis was performed to determine if prior analysis would have caused a major security or safety operating envelope issue. It was determined that a safety issue would have resulted from the prior research, but that the security may have been increased under certain conditions.

  1. The dynamic nature of the nuclear envelope

    PubMed Central

    Arnone, James T; Walters, Alison D; Cohen-Fix, Orna

    2013-01-01

    In eukaryotes, chromosomes are encased by a dynamic nuclear envelope. In contrast to metazoans, where the nuclear envelope disassembles during mitosis, many fungi including budding yeast undergo “closed mitosis,” where the nuclear envelope remains intact throughout the cell cycle. Consequently, during closed mitosis the nuclear envelope must expand to accommodate chromosome segregation to the two daughter cells. A recent study by Witkin et al. in budding yeast showed that if progression through mitosis is delayed, for example due to checkpoint activation, the nuclear envelope continues to expand despite the block to chromosome segregation. Moreover, this expansion occurs at a specific region of the nuclear envelope- adjacent to the nucleolus- forming an extension referred to as a “flare.” These observations raise questions regarding the regulation of nuclear envelope expansion both in budding yeast and in higher eukaryotes, the mechanisms confining mitotic nuclear envelope expansion to a particular region and the possible consequences of failing to regulate nuclear envelope expansion during the cell cycle. PMID:23873576

  2. Nonstationary envelope process and first excursion probability.

    NASA Technical Reports Server (NTRS)

    Yang, J.-N.

    1972-01-01

    The definition of stationary random envelope proposed by Cramer and Leadbetter, is extended to the envelope of nonstationary random process possessing evolutionary power spectral densities. The density function, the joint density function, the moment function, and the crossing rate of a level of the nonstationary envelope process are derived. Based on the envelope statistics, approximate solutions to the first excursion probability of nonstationary random processes are obtained. In particular, applications of the first excursion probability to the earthquake engineering problems are demonstrated in detail.

  3. Testing the Definition of the ESC Envelope

    NASA Technical Reports Server (NTRS)

    Vincent, Mark A.

    2017-01-01

    The previous effort, including a successful Change Control Request, addressed shrinking the size of the Earth Science Constellations' (ESC) Envelope by reducing the Margin. Fundamental to the purpose of the Envelope is the case where the argument of perigee of the secondary object circulates from 90 degrees to 270 degrees. This ("outside of the envelope, always outside the envelope") case was tested both numerically in a spreadsheet and analytically. Results showed how it is important to include the fact that a secondary with a different semi-major axis has a different frozen eccentricity value.

  4. The Origins of Transmembrane Ion Channels

    NASA Technical Reports Server (NTRS)

    Pohorille, Andrew; Wilson, Michael A.

    2012-01-01

    Even though membrane proteins that mediate transport of ions and small molecules across cell walls are among the largest and least understood biopolymers in contemporary cells, it is still possible to shed light on their origins and early evolution. The central observation is that transmembrane portions of most ion channels are simply bundles of -helices. By combining results of experimental and computer simulation studies on synthetic models and natural channels, mostly of non-genomic origin, we show that the emergence of -helical channels was protobiologically plausible, and did not require highly specific amino acid sequences. Despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly larger complexity. Specifically, we explain how the antiamoebin channels, which are made of identical helices, 16 amino acids in length, achieve efficiency comparable to that of highly evolved channels. We further show that antiamoebin channels are extremely flexible, compared to modern, genetically coded channels. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire stable rigid structures and mechanisms for precise regulation rather than improve efficiency. In general, even though architectures of membrane proteins are not nearly as diverse as those of water-soluble proteins, they are sufficiently flexible to adapt readily to the functional demands arising during evolution.

  5. Molecular archeological studies of transmembrane transport systems

    NASA Astrophysics Data System (ADS)

    Saier, Milton H.; Wang, Bin; Sun, Eric I.; Matias, Madeleine; Yen, Ming Ren

    We here review studies concerned with the evolutionary pathways taken for the appearance of complex transport systems. The transmembrane protein constituents of these systems generally arose by (1) intragenic duplications, (2) gene fusions, and (3) the superimposition of enzymes onto carriers. In a few instances, we have documented examples of “reverse” or “retrograde” evolution where complex carriers have apparently lost parts of their polypeptide chains to give rise to simpler channels. Some functional superfamilies of transporters that are energized by adenosine triphosphate (ATP) or phosphoenolpyruvate (PEP) include several independently evolving permease families. The ubiquitous ATP-binding cassette (ABC) superfamily couples transport to ATP hydrolysis where the ATPases are superimposed on at least three distinct, independently evolving families of permeases. The prokaryotic sugar transporting phosphotransferase system (PTS) uses homologous PEP-dependent general energy-coupling phosphoryl transfer enzymes superimposed on at least three independently arising families of permeases to give rise to complex group translocators that modify their sugar substrates during transport, releasing cytoplasmic sugar phosphates. We suggest that simple carriers evolved independently of the energizing enzymes, and that chemical energization of transport resulted from the physical and functional coupling of the enzymes to the carriers.

  6. Measuring Mitochondrial Transmembrane Potential by TMRE Staining.

    PubMed

    Crowley, Lisa C; Christensen, Melinda E; Waterhouse, Nigel J

    2016-12-01

    Adenosine triphosphate (ATP) is the main source of energy for metabolism. Mitochondria provide the majority of this ATP by a process known as oxidative phosphorylation. This process involves active transfer of positively charged protons across the mitochondrial inner membrane resulting in a net internal negative charge, known as the mitochondrial transmembrane potential (ΔΨm). The proton gradient is then used by ATP synthase to produce ATP by fusing adenosine diphosphate and free phosphate. The net negative charge across a healthy mitochondrion is maintained at approximately -180 mV, which can be detected by staining cells with positively charged dyes such as tetramethylrhodamine ethyl ester (TMRE). TMRE emits a red fluorescence that can be detected by flow cytometry or fluorescence microscopy and the level of TMRE fluorescence in stained cells can be used to determine whether mitochondria in a cell have high or low ΔΨm. Cytochrome c is essential for producing ΔΨm because it promotes the pumping the protons into the mitochondrial intermembrane space as it shuttles electrons from Complex III to Complex IV along the electron transport chain. Cytochrome c is released from the mitochondrial intermembrane space into the cytosol during apoptosis. This impairs its ability to shuttle electrons between Complex III and Complex IV and results in rapid dissipation of ΔΨm. Loss of ΔΨm is therefore closely associated with cytochrome c release during apoptosis and is often used as a surrogate marker for cytochrome c release in cells.

  7. The infrared dichroism of transmembrane helical polypeptides.

    PubMed Central

    Axelsen, P H; Kaufman, B K; McElhaney, R N; Lewis, R N

    1995-01-01

    Polarized attenuated total internal reflectance techniques were applied to study the infrared dichroism of the amide I transition moment in two membrane-bound peptides that are known to form oriented transmembrane helices: gramicidin A in a supported phospholipid monolayer and Ac-Lys2-Leu24-Lys2-amide (L24) in oriented multibilayers. These studies were performed to test the ability of these techniques to determine the orientation of these peptides, to verify the value of optical parameters used to calculate electric field strengths, to examine the common assumptions regarding the amide I transition moment orientation, and to ascertain the effect of surface imperfections on molecular disorder. The two peptides exhibit marked differences in the shape and frequency of their amide I absorption bands. Yet both peptides are highly ordered and oriented with their helical axes perpendicular to the membrane surface. In the alpha-helix formed by L24, there is evidence for a mode with type E1 symmetry contributing to amide I, and the amide I transition moment must be more closely aligned with the peptide C=O (< 34 degrees) than earlier studies have suggested. These results indicate that long-standing assumptions about the orientation of amide I in a peptide require some revision, but that in general, infrared spectroscopy yields reliable information about the orientation of membrane-bound helical peptides. Images FIGURE 1 PMID:8599683

  8. Transmembrane beta-barrel protein structure prediction

    NASA Astrophysics Data System (ADS)

    Randall, Arlo; Baldi, Pierre

    Transmembrane β-barrel (TMB) proteins are embedded in the outer membranes of mitochondria, Gram-negative bacteria, and chloroplasts. These proteins perform critical functions, including active ion-transport and passive nutrient intake. Therefore, there is a need for accurate prediction of secondary and tertiary structures of TMB proteins. A variety of methods have been developed for predicting the secondary structure and these predictions are very useful for constructing a coarse topology of TMB structure; however, they do not provide enough information to construct a low-resolution tertiary structure for a TMB protein. In addition, while the overall structural architecture is well conserved among TMB proteins, the amino acid sequences are highly divergent. Thus, traditional homology modeling methods cannot be applied to many putative TMB proteins. Here, we describe the TMBpro: a pipeline of methods for predicting TMB secondary structure, β-residue contacts, and finally tertiary structure. The tertiary prediction method relies on the specific construction rules that TMB proteins adhere to and on the predicted β-residue contacts to dramatically reduce the search space for the model building procedure.

  9. Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41.

    PubMed

    Gong, Zhen; Kessans, Sarah A; Song, Lusheng; Dörner, Katerina; Lee, Ho-Hsien; Meador, Lydia R; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2014-11-01

    The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼ 1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1.

  10. Envelope solitons of acoustic plate modes and surface waves.

    PubMed

    Mayer, Andreas P; Kovalev, Alexander S

    2003-06-01

    The problem of the existence of evelope solitons in elastic plates and at solid surfaces covered by an elastic film is revisited with special attention paid to nonlinear long-wave short-wave interactions. Using asymptotic expansions and multiple scales, conditions for the existence of envelope solitons are established and it is shown how their parameters can be expressed in terms of the elastic moduli and mass densities of the materials involved. In addition to homogeneous plates, weak periodic modulation of the plate's material parameters are also considered. In the case of wave propagation in an elastic plate, modulations of weakly nonlinear carrier waves are governed by a coupled system of partial differential equations consisting of evolution equations for the complex amplitude of the carrier wave (the nonlinear Schrödinger equation for envelope solitons and the Mills-Trullinger equations for gap solitons), and the wave equation for long-wavelength acoustic plate modes. In contrast to this situation, envelope solitons of surface acoustic waves in a layered structure are normally described by the nonlinear Schrödinger equation alone. However, at higher orders of the carrier wave amplitude, the envelope soliton is found to be accompanied by a quasistatic long-wavelength strain field, which may be localized at the surface with penetration depth into the substrate of the order of the inverse amplitude or which may radiate energy into the bulk. A new set of modulation equations is derived for the resonant case of the carrier wave's group velocity being equal to the phase velocity of long-wavelength Rayleigh waves of the uncoated substrate.

  11. Consonant confusions for temporally smeared envelope of syllables in noise

    NASA Astrophysics Data System (ADS)

    Yoon, Yang-Soo; Gooler, David; Allen, Jont

    2005-09-01

    The purpose of this study was to better understand the contribution of speech temporal envelope cues to consonant recognition in noise (-8, 0, 8, 16, 24, and quiet dB SNR). Four tasks were performed for hearing-impaired listeners: Speech-in-Noise test for the measure of sentence repeat ability in noise, gap and intensity detection for the measure of ability to detect changes in sound over time, and consonant identification for confusion matrices. For consonant identification, the temporal envelope of 16 consonants with a vowel /a/ (CV) was extracted from each of 26 critical bands. Each processed CV was temporally smeared by low-pass filtering (4, 8, and 16 Hz). The results show that the mean Pc increases gradually as SNR increases SNR < 16 dB for all conditions, but decreases at SNR > 16 dB for no-smearing and 8-Hz smearing conditions. Consonant confusions result from certain sets of consonants, but the degree of confusion is a function of SNR. The results also show that measures of Speech-in-Noise and gap detection are more related to pure-tone threshold, while intensity detection is less related to pure-tone threshold. However, the effect of interstimulus interval is consistent regardless of degree and configuration of hearing loss.

  12. Exogenous agents that target transmembrane domains of proteins.

    PubMed

    Yin, Hang

    2008-01-01

    Although membrane proteins account for approximately one third of all proteins encoded in the human genome, the functions and structures of their transmembrane domains are much less understood than the water-soluble regions. A major hurdle in studying these transmembrane domains is the lack of appropriate exogenous agents that can be used as specific probes. Despite the daunting challenges, major strides have recently been made in targeting the transmembrane domains of a variety of membrane proteins. High affinity and selectivity have been achieved in model biophysical systems, membranes of bacteria, and mammalian cells.

  13. NET23/STING Promotes Chromatin Compaction from the Nuclear Envelope

    PubMed Central

    de las Heras, Jose I.; Saiz-Ros, Natalia; Makarov, Alexandr A.; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A.; Schirmer, Eric C.

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture. PMID:25386906

  14. NET23/STING promotes chromatin compaction from the nuclear envelope.

    PubMed

    Malik, Poonam; Zuleger, Nikolaj; de las Heras, Jose I; Saiz-Ros, Natalia; Makarov, Alexandr A; Lazou, Vassiliki; Meinke, Peter; Waterfall, Martin; Kelly, David A; Schirmer, Eric C

    2014-01-01

    Changes in the peripheral distribution and amount of condensed chromatin are observed in a number of diseases linked to mutations in the lamin A protein of the nuclear envelope. We postulated that lamin A interactions with nuclear envelope transmembrane proteins (NETs) that affect chromatin structure might be altered in these diseases and so screened thirty-one NETs for those that promote chromatin compaction as determined by an increase in the number of chromatin clusters of high pixel intensity. One of these, NET23 (also called STING, MITA, MPYS, ERIS, Tmem173), strongly promoted chromatin compaction. A correlation between chromatin compaction and endogenous levels of NET23/STING was observed for a number of human cell lines, suggesting that NET23/STING may contribute generally to chromatin condensation. NET23/STING has separately been found to be involved in innate immune response signaling. Upon infection cells make a choice to either apoptose or to alter chromatin architecture to support focused expression of interferon genes and other response factors. We postulate that the chromatin compaction induced by NET23/STING may contribute to this choice because the cells expressing NET23/STING eventually apoptose, but the chromatin compaction effect is separate from this as the condensation was still observed when cells were treated with Z-VAD to block apoptosis. NET23/STING-induced compacted chromatin revealed changes in epigenetic marks including changes in histone methylation and acetylation. This indicates a previously uncharacterized nuclear role for NET23/STING potentially in both innate immune signaling and general chromatin architecture.

  15. Transmembrane Location of Retinal in Purple Membrane

    PubMed Central

    Kometani, Tadaatsu; Kinosita, Kazuhiko; Furuno, Taiji; Kouyama, Tsutomu; Ikegami, Akira

    1987-01-01

    Transmembrane location of the retinal chromophore in the purple membrane of Halobacterium halobium was investigated in three different systems in which excitation energy transfer between the chromophore and external dye molecules condensed on the membrane surfaces was observed. In system ii, the energy donor was the retinal chromophore converted in situ to a fluorescent derivative. The fluorescent membranes were embedded in solid cobalt-EDTA, which served as energy acceptors. System iii was similar to system ii, except that the acceptors were tris(2,2′-bipyridyl)ruthenium(II) complex in solid form. The positively charged ruthenium complex had a radius of 0.7 nm, whereas the cobalt complex in system ii was smaller (radius ∼0.4 nm) and negatively charged. System iv was stacked sheets of native purple membrane with interspersed ruthenium complex; energy transfer from the luminescent ruthenuim complex to the native retinal chromophore was observed. The energy transfer rates in these three systems, and in two additional systems already described (Kouyama, T., K. Kinosita, Jr., and A. Ikegami, 1983, J. Mol. Biol., 165:91-107), were all consistent with a location of the retinal chromophore at a depth of 1.0 ± 0.3 nm from a surface of the purple membrane. All the analyses in the present work involved an assumption that contacts between the external dye molecules and membrane surfaces were maximal; the depth values obtained cannot be underestimates. The chromophore therefore must be outside the middle one-third of the thickness, ∼4.5 nm, of the purple membrane. PMID:19431704

  16. Molecular mechanisms of intercellular communication: transmembrane signaling

    SciTech Connect

    Bitensky, M.W.; George, J.S.; Siegel, H.N.; McGregor, D.M.

    1982-01-01

    This short discussion of transmembrane signaling depicts a particular class of signaling devices whose functional characteristics may well be representative of broader classes of membrane switches. These multicomponent aggregates are characterized by tight organization of interacting components which function by conformational interactions to provide sensitive, amplified, rapid, and modulated responses. It is clear that the essential role of such switches in cell-cell interactions necessitated their appearance early in the history of the development of multicellular organisms. It also seems clear that once such devices made their appearance, the conformationally interactive moieties were firmly locked into a regulatory relationship. Since modification of interacting components could perturb or interfere with the functional integrity of the whole switch, genetic drift was only permitted at the input and outflow extremes. However, the GTP binding moiety and its interacting protein domains on contiguous portions of the receptor and readout components were highly conserved. The observed stringent evolutionary conservation of the molecular features of these membrane switches thus applies primarily to the central (GTP binding) elements. An extraordinary degree of variation was permitted within the domains of signal recognition and enzymatic output. Thus, time and evolution have adapted the central logic of the regulatory algorithm to serve a great variety of cellular purposes and to recognize a great variety of chemical and physical signals. This is exemplified by the richness of the hormonal and cellular dialogues found in primates such as man. Here the wealth of intercellular communiation can support the composition and performance of symphonies and the study of cellular immunology.

  17. Polar residue tagging of transmembrane peptides.

    PubMed

    Melnyk, Roman A; Partridge, Anthony W; Yip, Jeannie; Wu, Yanqiu; Goto, Natalie K; Deber, Charles M

    2003-01-01

    Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes. Copyright 2004 Wiley Periodicals, Inc.

  18. A folded protein can be transported across the chloroplast envelope and thylakoid membranes.

    PubMed Central

    Clark, S A; Theg, S M

    1997-01-01

    Many thylakoid lumenal proteins are nuclear encoded, cytosolically synthesized, and reach their functional location after posttranslational targeting across two chloroplast envelope membranes and the thylakoid membrane via proteinaceous transport systems. To study whether these transmembrane transport machineries can translocate folded structures, we overexpressed the 17-kDa subunit of the oxygen-evolving complex of photosystem II (prOE17) that had been modified to contain a unique C-terminal cysteine. This allowed us to chemically link a terminal 6.5-kDa bovine pancreatic trypsin inhibitor (BPTI) moiety to prOE17 to create the chimeric protein prOE17-BPTI. Redox reagents and an irreversible sulfhydryl-specific cross-linker, bis-maleimidohexane, were used to manipulate the structure of BPTI. Import of prOE17-BPTI into isolated chloroplasts and thylakoids demonstrates that the small tightly folded BPTI domain is carried across both the chloroplast envelopes and the delta pH-dependent transmembrane transporter of the thylakoid membrane when linked to the correctly targeted OE17 precursor. Transport proceeded even when the BPTI moiety was internally cross-linked into a protease-resistant form. These data indicate that unfolding is not a ubiquitous requirement for protein translocation and that at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts. Images PMID:9168475

  19. Plotting max/min data envelopes

    NASA Technical Reports Server (NTRS)

    Furuike, T.; Long, J. C.

    1979-01-01

    Study of maximum and minimum load distributions along structural section is aided by visual display of load distribution data. Maximum/minimum envelope plot program plots these envelopes of the stresses and shear loads at selected points in beam modeled by series of finite elements. Digital output for engineers and management is presented for quick analysis and understanding.

  20. The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins.

    PubMed

    Chum, Tomáš; Glatzová, Daniela; Kvíčalová, Zuzana; Malínský, Jan; Brdička, Tomáš; Cebecauer, Marek

    2016-01-01

    Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins. © 2016. Published by The Company of Biologists Ltd.

  1. TOPPER: topology prediction of transmembrane protein based on evidential reasoning.

    PubMed

    Deng, Xinyang; Liu, Qi; Hu, Yong; Deng, Yong

    2013-01-01

    The topology prediction of transmembrane protein is a hot research field in bioinformatics and molecular biology. It is a typical pattern recognition problem. Various prediction algorithms are developed to predict the transmembrane protein topology since the experimental techniques have been restricted by many stringent conditions. Usually, these individual prediction algorithms depend on various principles such as the hydrophobicity or charges of residues. In this paper, an evidential topology prediction method for transmembrane protein is proposed based on evidential reasoning, which is called TOPPER (topology prediction of transmembrane protein based on evidential reasoning). In the proposed method, the prediction results of multiple individual prediction algorithms can be transformed into BPAs (basic probability assignments) according to the confusion matrix. Then, the final prediction result can be obtained by the combination of each individual prediction base on Dempster's rule of combination. The experimental results show that the proposed method is superior to the individual prediction algorithms, which illustrates the effectiveness of the proposed method.

  2. A deterministic algorithm for constrained enumeration of transmembrane protein folds.

    SciTech Connect

    Brown, William Michael; Young, Malin M.; Sale, Kenneth L.; Faulon, Jean-Loup Michel; Schoeniger, Joseph S.

    2004-07-01

    A deterministic algorithm for enumeration of transmembrane protein folds is presented. Using a set of sparse pairwise atomic distance constraints (such as those obtained from chemical cross-linking, FRET, or dipolar EPR experiments), the algorithm performs an exhaustive search of secondary structure element packing conformations distributed throughout the entire conformational space. The end result is a set of distinct protein conformations, which can be scored and refined as part of a process designed for computational elucidation of transmembrane protein structures.

  3. Characterization of Disease-Associated Mutations in Human Transmembrane Proteins

    PubMed Central

    Molnár, János; Szakács, Gergely; Tusnády, Gábor E.

    2016-01-01

    Transmembrane protein coding genes are commonly associated with human diseases. We characterized disease causing mutations and natural polymorphisms in transmembrane proteins by mapping missense genetic variations from the UniProt database on the transmembrane protein topology listed in the Human Transmembrane Proteome database. We found characteristic differences in the spectrum of amino acid changes within transmembrane regions: in the case of disease associated mutations the non-polar to non-polar and non-polar to charged amino acid changes are equally frequent. In contrast, in the case of natural polymorphisms non-polar to charged amino acid changes are rare while non-polar to non-polar changes are common. The majority of disease associated mutations result in glycine to arginine and leucine to proline substitutions. Mutations to positively charged amino acids are more common in the center of the lipid bilayer, where they cause more severe structural and functional anomalies. Our analysis contributes to the better understanding of the effect of disease associated mutations in transmembrane proteins, which can help prioritize genetic variations in personal genomic investigations. PMID:26986070

  4. Simultaneous prediction of protein secondary structure and transmembrane spans.

    PubMed

    Leman, Julia Koehler; Mueller, Ralf; Karakas, Mert; Woetzel, Nils; Meiler, Jens

    2013-07-01

    Prediction of transmembrane spans and secondary structure from the protein sequence is generally the first step in the structural characterization of (membrane) proteins. Preference of a stretch of amino acids in a protein to form secondary structure and being placed in the membrane are correlated. Nevertheless, current methods predict either secondary structure or individual transmembrane states. We introduce a method that simultaneously predicts the secondary structure and transmembrane spans from the protein sequence. This approach not only eliminates the necessity to create a consensus prediction from possibly contradicting outputs of several predictors but bears the potential to predict conformational switches, i.e., sequence regions that have a high probability to change for example from a coil conformation in solution to an α-helical transmembrane state. An artificial neural network was trained on databases of 177 membrane proteins and 6048 soluble proteins. The output is a 3 × 3 dimensional probability matrix for each residue in the sequence that combines three secondary structure types (helix, strand, coil) and three environment types (membrane core, interface, solution). The prediction accuracies are 70.3% for nine possible states, 73.2% for three-state secondary structure prediction, and 94.8% for three-state transmembrane span prediction. These accuracies are comparable to state-of-the-art predictors of secondary structure (e.g., Psipred) or transmembrane placement (e.g., OCTOPUS). The method is available as web server and for download at www.meilerlab.org. Copyright © 2013 Wiley Periodicals, Inc.

  5. Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

    PubMed

    Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

    2010-01-01

    The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

  6. Fusogenic activity of reconstituted newcastle disease virus envelopes: a role for the hemagglutinin-neuraminidase protein in the fusion process.

    PubMed

    Cobaleda, C; Muñoz-Barroso, I; Sagrera, A; Villar, E

    2002-04-01

    Enveloped viruses, such as newcastle disease virus (NDV), make their entry into the host cell by membrane fusion. In the case of NDV, the fusion step requires both transmembrane hemagglutinin-neuraminidase (HN) and fusion (F) viral envelope glycoproteins. The HN protein should show fusion promotion activity. To date, the nature of HN-F interactions is a controversial issue. In this work, we aim to clarify the role of the HN glycoprotein in the membrane fusion step. Four types of reconstituted detergent-free NDV envelopes were used, on differing in their envelope protein contents. Fusion of the different virosomes and erythrocyte ghosts was monitored using the octadecyl rhodamine B chloride assay. Only the reconstituted envelopes having the F protein, even in the absence of HN protein, displayed residual fusion activity. Treatment of such virosomes with denaturing agents affecting the F protein abolished fusion, indicating that the fusion detected was viral protein-dependent. Interestingly, the rate of fusion in the reconstituted systems was similar to that of intact viruses in the presence of the inhibitor of HN sialidase activity 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. The results show that the residual fusion activity detected in the reconstituted systems was exclusively due to F protein activity, with no contribution from the fusion promotion activity of HN protein.

  7. Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

    PubMed Central

    Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.; Roston, Rebecca L.; Yamaguchi, Toshio; Kamikubo, Hironari

    2016-01-01

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope. PMID:27936133

  8. Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

    DOE PAGES

    Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.; ...

    2016-12-09

    PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

  9. The theoretical polarization of pure scattering axisymmetric circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Fox, G. K.

    1994-01-01

    The Sobolev approach to the scattering of starlight through a pure scattering circumstellar envelope is developed. The theoretical polarization due to electron scattering in Be star envelopes is calculated for two geometries (an equatorially enhanced envelope and a spheroidal envelope). Only the disk-type envelope is found to yield a maximum polarization consistent with the observed range for Be stars. A lower limit, analytical approximation to the theoretical polarization from a pure scattering envelope is obtained.

  10. Grazing envelope evolution towards Type IIb supernovae

    NASA Astrophysics Data System (ADS)

    Soker, Noam

    2017-09-01

    I propose a scenario where the majority of the progenitors of Type IIb supernovae (SNe IIb) lose most of their hydrogen-rich envelope during a grazing envelope evolution (GEE). In the GEE, the orbital radius of the binary system is about equal to the radius of the giant star, and the more compact companion accretes mass through an accretion disc. The accretion disc is assumed to launch two opposite jets that efficiently remove gas from the envelope along the orbit of the companion. The efficient envelope removal by jets prevents the binary system from entering a common envelope evolution, at least for part of the time. The GEE might be continuous or intermittent. I crudely estimate the total GEE time period to be in the range of about hundreds of years, for a continuous GEE, and up to few tens of thousands of years for intermittent GEE. The key new point is that the removal of envelope gas by jets during the GEE prevents the system from entering a common envelope evolution, and by that substantially increases the volume of the stellar binary parameter space that leads to SNe IIb, both to lower secondary masses and to closer orbital separations.

  11. Envelope theory in spectral geometry

    NASA Astrophysics Data System (ADS)

    Hall, Richard L.

    1993-07-01

    It is shown that the discrete spectrum of Schrödinger Hamiltonians of the form H=-Δ+vf may be represented by the semiclassical expression Enl=minr≳0 {K(f)nl(r) + vf(r)}. The K functions are found to be invariant with respect to coupling and shifts: K(Af+B)=K(f). For pure power laws, f(r)=sgn(q)rq, and the log potential, they are also invariant with respect to scale, and have the simple forms (Pnl(q)/r)2 and (Lnl/r)2, respectively. K functions are also derived for sech-squared and Hulthén potentials. If f=g(h), where g is a smooth transformation, then the envelope approximation is expressed in terms of K by the relation K(f)≂K(h). When the transformation g has definite convexity, then the approximation immediately yields eigenvalue bounds for all n and l. The theory is used to prove the log-power theorem Lnl = Pnl(0), which, in turn, generates a simple eigenvalue formula for the log potential.

  12. Safeguards Envelope Progress FY09

    SciTech Connect

    Richard Metcalf; Robert Bean

    2009-09-01

    The Safeguards Envelope is a strategy to determine a set of specific operating parameters which nuclear facilities may operate within to maximize safeguards effectiveness without sacrificing safety or plant efficiency. This paper details advanced statistical techniques will be applied to real plant process monitoring (PM) data from the Idaho Chemical Processing Plant (ICPP). As a result of the U.S. having no operating nuclear chemical reprocessing plants, there has been a strong interest in obtaining process monitoring data from the ICPP. The ICPP was shut down in 1996 and a recent effort has been made to retrieve the PM data from storage in a data mining effort. In a simulation based on this data, multi-tank and multi-attribute correlations were tested against synthetic diversion scenarios. Kernel regression smoothing was used to fit a curve to the historical data, and multivariable, residual analysis and cumulative sum techniques set parameters for operating conditions. Diversion scenarios were created and tested, showing improved results when compared with a previous study utilizing only one-variable Z- testing7.

  13. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, Francis; Teoh, William; Ziemke, M. Carl

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) provides an alternative to extravehicular activity (EVA) of space suited astronauts and/or use of long slender manipulator arms such as are used in the Shuttle Remote Manipulator System. POWER provides the capability for a shirt sleeved astronaut to perform such work by entering a control pod through air locks at both ends of an inflated flexible bellows (access tunnel). The exoskeleton of the tunnel is a series of six degrees of freedom (Six-DOF) articulated links compressible to 1/6 of their fully extended length. The operator can maneuver the control pod to almost any location within about 50 m of the base attachment to the space station. POWER can be envisioned as a series of hollow Six-DOF manipulator segments or arms wherein each arm grasps the shoulder of the next arm. Inside the hollow arms ia a bellow-type access tunnel. The control pod is the fist of the series of linked hollow arms. The fingers of the fist are conventional manipulator arms under direct visual control of the nearby operator in the pod. The applications and progress to date of the POWER system is given.

  14. Personnel occupied woven envelope robot

    NASA Technical Reports Server (NTRS)

    Wessling, Francis; Teoh, William; Ziemke, M. Carl

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) provides an alternative to extravehicular activity (EVA) of space suited astronauts and/or use of long slender manipulator arms such as are used in the Shuttle Remote Manipulator System. POWER provides the capability for a shirt sleeved astronaut to perform such work by entering a control pod through air locks at both ends of an inflated flexible bellows (access tunnel). The exoskeleton of the tunnel is a series of six degrees of freedom (Six-DOF) articulated links compressible to 1/6 of their fully extended length. The operator can maneuver the control pod to almost any location within about 50 m of the base attachment to the space station. POWER can be envisioned as a series of hollow Six-DOF manipulator segments or arms wherein each arm grasps the shoulder of the next arm. Inside the hollow arms ia a bellow-type access tunnel. The control pod is the fist of the series of linked hollow arms. The fingers of the fist are conventional manipulator arms under direct visual control of the nearby operator in the pod. The applications and progress to date of the POWER system is given.

  15. Palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions

    SciTech Connect

    Gonzalez, Silvia A.; Paladino, Monica G.; Affranchino, Jose L.

    2012-06-20

    The feline immunodeficiency virus (FIV) envelope glycoprotein (Env) possesses a short cytoplasmic domain of 53 amino acids containing four highly conserved cysteines at Env positions 804, 811, 815 and 848. Since palmitoylation of transmembrane proteins occurs at or near the membrane anchor, we investigated whether cysteines 804, 811 and 815 are acylated and analyzed the relevance of these residues for Env functions. Replacement of cysteines 804, 811 and 815 individually or in combination by serine residues resulted in Env glycoproteins that were efficiently expressed and processed. However, mutations C804S and C811S reduced Env fusogenicity by 93% and 84%, respectively, compared with wild-type Env. By contrast, mutant C815S exhibited a fusogenic capacity representing 50% of the wild-type value. Remarkably, the double mutation C804S/C811S abrogated both Env fusion activity and Env incorporation into virions. Finally, by means of Click chemistry assays we demonstrated that the four FIV Env cytoplasmic cysteines are palmitoylated.

  16. New pharmacological strategies to fight enveloped viruses.

    PubMed

    Wisskirchen, Karin; Lucifora, Julie; Michler, Thomas; Protzer, Ulrike

    2014-09-01

    Enveloped viruses pose an important health threat because most of the persistent and many emerging viruses are enveloped. In particular, newly emerging viruses create a need to develop broad-spectrum antivirals, which usually are obtained by targeting host cell factors. Persistent viruses have developed efficient strategies to escape host immune control, and treatment options are limited. Targeting host cell factors essential for virus persistence, or immune-based therapies provide alternative approaches. In this review, we therefore focus on recent developments to generate antivirals targeting host cell factors or immune-based therapeutic approaches to fight infections with enveloped viruses. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Resource envelope concepts for mission planning

    NASA Technical Reports Server (NTRS)

    Ibrahim, K. Y.; Weiler, J. D.; Tokaz, J. C.

    1991-01-01

    Seven proposed methods for creating resource envelopes for Space Station Freedom mission planning are detailed. Four reference science activity models are used to illustrate the effect of adding operational flexibility to mission timelines. For each method, a brief explanation is given along with graphs to illustrate the application of the envelopes to the power and crew resources. The benefits and costs of each method are analyzed in terms of resource utilization. In addition to the effect on individual activities, resource envelopes are analyzed at the experiment level.

  18. Region- and Cell-Specific Expression of Transmembrane Collagens in Mouse Brain.

    PubMed

    Monavarfeshani, Aboozar; Knill, Courtney N; Sabbagh, Ubadah; Su, Jianmin; Fox, Michael A

    2017-01-01

    Unconventional collagens are nonfribrillar proteins that not only contribute to the structure of extracellular matrices but exhibit unique bio-activities. Although roles for unconventional collagens have been well-established in the development and function of non-neural tissues, only recently have studies identified roles for these proteins in brain development, and more specifically, in the formation and refinement of synaptic connections between neurons. Still, our understanding of the full cohort of unconventional collagens that are generated in the mammalian brain remains unclear. Here, we sought to address this gap by assessing the expression of transmembrane collagens (i.e., collagens XIII, XVII, XXIII and XXV) in mouse brain. Using quantitative PCR and in situ hybridization (ISH), we demonstrate both region- and cell-specific expression of these unique collagens in the developing brain. For the two most highly expressed transmembrane collagens (i.e., collagen XXIII and XXV), we demonstrate that they are expressed by select subsets of neurons in different parts of the brain. For example, collagen XXIII is selectively expressed by excitatory neurons in the mitral/tufted cell layer of the accessory olfactory bulb (AOB) and by cells in the inner nuclear layer (INL) of the retina. On the other hand, collagen XXV, which is more broadly expressed, is generated by subsets of excitatory neurons in the dorsal thalamus and midbrain and by inhibitory neurons in the retina, ventral thalamus and telencephalon. Not only is col25a1 expression present in retina, it appears specifically enriched in retino-recipient nuclei within the brain (including the suprachiasmatic nucleus (SCN), lateral geniculate complex, olivary pretectal nucleus (OPN) and superior colliculus). Taken together, the distinct region- and cell-specific expression patterns of transmembrane collagens suggest that this family of unconventional collagens may play unique, yet-to-be identified roles in brain

  19. Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

    PubMed Central

    Kolesnitchenko, V; Wahl, L M; Tian, H; Sunila, I; Tani, Y; Hartmann, D P; Cossman, J; Raffeld, M; Orenstein, J; Samelson, L E

    1995-01-01

    Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8524869

  20. EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus

    PubMed Central

    Bagchi, Parikshit; Inoue, Takamasa; Tsai, Billy

    2016-01-01

    Destabilization of a non-enveloped virus generates a membrane transport-competent viral particle. Here we probe polyomavirus SV40 endoplasmic reticulum (ER)-to-cytosol membrane transport, a decisive infection step where destabilization initiates this non-enveloped virus for membrane penetration. We find that a member of the ER membrane protein complex (EMC) called EMC1 promotes SV40 ER membrane transport and infection. Surprisingly, EMC1 does so by using its predicted transmembrane residue D961 to bind to and stabilize the membrane-embedded partially destabilized SV40, thereby preventing premature viral disassembly. EMC1-dependent stabilization enables SV40 to engage a cytosolic extraction complex that ejects the virus into the cytosol. Thus EMC1 acts as a molecular chaperone, bracing the destabilized SV40 in a transport-competent state. Our findings reveal the novel principle that coordinated destabilization-stabilization drives membrane transport of a non-enveloped virus. DOI: http://dx.doi.org/10.7554/eLife.21470.001 PMID:28012275

  1. Radiative accelerations in stellar envelopes

    NASA Astrophysics Data System (ADS)

    Seaton, M. J.

    1997-08-01

    In stars which are sufficiently quiescent, changes in the relative abundances of the chemical elements can result from gravitational settling and from levitation produced by radiation pressure forces, usually expressed as radiative accelerations g_rad. Those changes can affect the structure of such stars, due to modifications in opacities, and can lead to marked peculiarities in observed atmospheric abundances. It is necessary to consider diffusive movements both in the atmospheres and in much deeper layers of the stellar envelopes. For the envelopes the equation of radiative transfer can be solved in a diffusion approximation and, for an element k in ionization stage j, one obtains expressions for g_rad(j, k) proportional to the total radiative flux, to the Rosseland-mean opacity kappa_R (which may depend on the abundance of k), and to a dimensionless quantity gamma(j, k) which, due to saturation effects, can be sensitive to the abundance of k. The radiative accelerations are required for each ionization stage, because the diffusion coefficients depend on j. Using atomic data obtained in the course of the work of the Opacity Project (OP), we calculate kappa_R and gamma(j, k) for the chemical elements C, N, O, Ne, Na, Mg, Al, Si, S, Ar, Ca, Cr, Mn, Fe and Ni. We start from standard Solar system abundances, and then vary the abundance of one element at a time (element k) by a factor chi. The following results are obtained and are available at the Centre de Donnees astronomiques de Strasbourg (CDS). (1) Files stages.zz (where zz specifies the nuclear charge of the selected element k) containing values of kappa_R and gamma(j, k) on a mesh of values of (T, N_e, chi), where T is temperature, and N_e is electron density. We include derivatives of kappa_R and gamma(j, k) with respect to chi, which are used for making interpolations. (2) A code add.f which reads a file stages.zz and writes a file acc.zz containing values of gamma(k) obtained on summing the gamma(j, k

  2. Transcriptional regulation at the yeast nuclear envelope

    PubMed Central

    Steglich, Babett; Sazer, Shelley; Ekwall, Karl

    2013-01-01

    The spatial organization of the genome inside the nucleus affects many nuclear processes, such as DNA replication, DNA repair, and gene transcription. In metazoans, the nuclear periphery harbors mainly repressed genes that associate with the nuclear lamina. This review discusses how peripheral positioning is connected to transcriptional regulation in yeasts. Tethering of reporter genes to the nuclear envelope was found to result in transcriptional silencing. Similarly, repression of the silent mating type loci and subtelomeric genes is influenced by their position close to the nuclear envelope. In contrast, active genes are bound by nucleoporins and inducible genes associate with the nuclear pore complex upon activation. Taken together, these results portray the nuclear envelope as a platform for transcriptional regulation, both through activation at nuclear pores and silencing at the nuclear envelope. PMID:24021962

  3. Personnel occupied woven envelope robot power

    NASA Technical Reports Server (NTRS)

    Wessling, F. C.

    1988-01-01

    The Personnel Occupied Woven Envelope Robot (POWER) concept has evolved over the course of the study. The goal of the project was the development of methods and algorithms for solid modeling for the flexible robot arm.

  4. Dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes.

    PubMed

    Li, Rui; Fan, Jianfen; Li, Hui; Yan, Xiliang; Yu, Yi

    2015-07-07

    Classical molecular dynamics simulations have been performed to investigate the dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes (CPNTs) with various radii, i.e., 8×(WL¯)n=3,4,5/POPE. The results show that ethanol molecules spontaneously fill the octa- and deca-CPNTs, but not the hexa-CPNT. In the octa-CPNT, ethanol molecules are trapped at individual gaps with their carbon skeletons perpendicular to the tube axis and hydroxyl groups towards the tube wall, forming a broken single-file chain. As the channel radius increases, ethanol molecules inside the deca-CPNT tend to form a tubular layer and the hydroxyl groups mainly stretch towards the tube axis. Computations of diffusion coefficients indicate that ethanol molecules in the octa-CPNT nearly lost their diffusion abilities, while those in the deca-CPNT diffuse as 4.5 times as in a (8, 8) carbon nanotube with a similar tube diameter. The osmotic and diffusion permeabilities (pf and pd, respectively) of the octa- and deca-CPNTs transporting ethanol were deduced for the first time. The distributions of the gauche and trans conformers of ethanol molecules in two CPNTs are quite similar, both with approximately 57% gauche conformers. The non-bonded interactions of channel ethanol with a CPNT wall and surrounding ethanol were explored. The potential of mean force elucidates the mechanism underlying the transporting characteristics of channel ethanol in a transmembrane CPNT.

  5. Dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes

    NASA Astrophysics Data System (ADS)

    Li, Rui; Fan, Jianfen; Li, Hui; Yan, Xiliang; Yu, Yi

    2015-07-01

    Classical molecular dynamics simulations have been performed to investigate the dynamic behaviors and transport properties of ethanol molecules in transmembrane cyclic peptide nanotubes (CPNTs) with various radii, i.e., 8 × ( W L ¯ ) n = 3 , 4 , 5 / POPE . The results show that ethanol molecules spontaneously fill the octa- and deca-CPNTs, but not the hexa-CPNT. In the octa-CPNT, ethanol molecules are trapped at individual gaps with their carbon skeletons perpendicular to the tube axis and hydroxyl groups towards the tube wall, forming a broken single-file chain. As the channel radius increases, ethanol molecules inside the deca-CPNT tend to form a tubular layer and the hydroxyl groups mainly stretch towards the tube axis. Computations of diffusion coefficients indicate that ethanol molecules in the octa-CPNT nearly lost their diffusion abilities, while those in the deca-CPNT diffuse as 4.5 times as in a (8, 8) carbon nanotube with a similar tube diameter. The osmotic and diffusion permeabilities (pf and pd, respectively) of the octa- and deca-CPNTs transporting ethanol were deduced for the first time. The distributions of the gauche and trans conformers of ethanol molecules in two CPNTs are quite similar, both with approximately 57% gauche conformers. The non-bonded interactions of channel ethanol with a CPNT wall and surrounding ethanol were explored. The potential of mean force elucidates the mechanism underlying the transporting characteristics of channel ethanol in a transmembrane CPNT.

  6. Transmembrane helix prediction using amino acid property features and latent semantic analysis.

    PubMed

    Ganapathiraju, Madhavi; Balakrishnan, N; Reddy, Raj; Klein-Seetharaman, Judith

    2008-01-01

    Prediction of transmembrane (TM) helices by statistical methods suffers from lack of sufficient training data. Current best methods use hundreds or even thousands of free parameters in their models which are tuned to fit the little data available for training. Further, they are often restricted to the generally accepted topology "cytoplasmic-transmembrane-extracellular" and cannot adapt to membrane proteins that do not conform to this topology. Recent crystal structures of channel proteins have revealed novel architectures showing that the above topology may not be as universal as previously believed. Thus, there is a need for methods that can better predict TM helices even in novel topologies and families. Here, we describe a new method "TMpro" to predict TM helices with high accuracy. To avoid overfitting to existing topologies, we have collapsed cytoplasmic and extracellular labels to a single state, non-TM. TMpro is a binary classifier which predicts TM or non-TM using multiple amino acid properties (charge, polarity, aromaticity, size and electronic properties) as features. The features are extracted from sequence information by applying the framework used for latent semantic analysis of text documents and are input to neural networks that learn the distinction between TM and non-TM segments. The model uses only 25 free parameters. In benchmark analysis TMpro achieves 95% segment F-score corresponding to 50% reduction in error rate compared to the best methods not requiring an evolutionary profile of a protein to be known. Performance is also improved when applied to more recent and larger high resolution datasets PDBTM and MPtopo. TMpro predictions in membrane proteins with unusual or disputed TM structure (K+ channel, aquaporin and HIV envelope glycoprotein) are discussed. TMpro uses very few free parameters in modeling TM segments as opposed to the very large number of free parameters used in state-of-the-art membrane prediction methods, yet achieves very

  7. Creating a Lunar EVA Work Envelope

    NASA Technical Reports Server (NTRS)

    Griffin, Brand N.; Howard, Robert; Rajulu, Sudhakar; Smitherman, David

    2009-01-01

    A work envelope has been defined for weightless Extravehicular Activity (EVA) based on the Space Shuttle Extravehicular Mobility Unit (EMU), but there is no equivalent for planetary operations. The weightless work envelope is essential for planning all EVA tasks because it determines the location of removable parts, making sure they are within reach and visibility of the suited crew member. In addition, using the envelope positions the structural hard points for foot restraints that allow placing both hands on the job and provides a load path for reacting forces. EVA operations are always constrained by time. Tasks are carefully planned to ensure the crew has enough breathing oxygen, cooling water, and battery power. Planning first involves computers using a virtual work envelope to model tasks, next suited crew members in a simulated environment refine the tasks. For weightless operations, this process is well developed, but planetary EVA is different and no work envelope has been defined. The primary difference between weightless and planetary work envelopes is gravity. It influences anthropometry, horizontal and vertical mobility, and reaction load paths and introduces effort into doing "overhead" work. Additionally, the use of spacesuits other than the EMU, and their impacts on range of motion, must be taken into account. This paper presents the analysis leading to a concept for a planetary EVA work envelope with emphasis on lunar operations. There is some urgency in creating this concept because NASA has begun building and testing development hardware for the lunar surface, including rovers, habitats and cargo off-loading equipment. Just as with microgravity operations, a lunar EVA work envelope is needed to guide designers in the formative stages of the program with the objective of avoiding difficult and costly rework.

  8. Control load envelope shaping by live twist

    NASA Technical Reports Server (NTRS)

    Tarzanin, F. J., Jr.; Mirick, P. H.

    1974-01-01

    Rotor control systems experience a rapid load growth resulting from retreating blade stall during flight conditions of high blade loading or airspeeds. An investigation was undertaken to determine the effect of changing blade torsional properties over the rotor flight envelope. The results of this study show that reducing the blade stiffness to introduce more blade live twist significantly reduces the large retreating blade control loads, while expanding the flight envelope and reducing retreating blade stall loads.

  9. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    PubMed

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca

    2017-04-07

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material, so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a metastable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements that control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith, E. C., Smith, S. E., Carter, J. R., Webb, S. R., Gibson, K. M., Hellman, L. M., Fried, M. G., and Dutch, R. E. (2013) J. Biol. Chem. 288, 35726-35735). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found, and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together, our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. The Unique Transmembrane Hairpin of Flavivirus Fusion Protein E Is Essential for Membrane Fusion▿

    PubMed Central

    Fritz, Richard; Blazevic, Janja; Taucher, Christian; Pangerl, Karen; Heinz, Franz X.; Stiasny, Karin

    2011-01-01

    The fusion of enveloped viruses with cellular membranes is mediated by proteins that are anchored in the lipid bilayer of the virus and capable of triggered conformational changes necessary for driving fusion. The flavivirus envelope protein E is the only known viral fusion protein with a double membrane anchor, consisting of two antiparallel transmembrane helices (TM1 and TM2). TM1 functions as a stop-transfer sequence and TM2 as an internal signal sequence for the first nonstructural protein during polyprotein processing. The possible role of this peculiar C-terminal helical hairpin in membrane fusion has not been investigated so far. We addressed this question by studying TM mutants of tick-borne encephalitis virus (TBEV) recombinant subviral particles (RSPs), an established model system for flavivirus membrane fusion. The engineered mutations included the deletion of TM2, the replacement of both TM domains (TMDs) by those of the related Japanese encephalitis virus (JEV), and the use of chimeric TBEV-JEV membrane anchors. Using these mutant RSPs, we provide evidence that TM2 is not just a remnant of polyprotein processing but, together with TM1, plays an active role in fusion. None of the TM mutations, including the deletion of TM2, affected early steps of the fusion process, but TM interactions apparently contribute to the stability of the postfusion E trimer and the completion of the merger of the membranes. Our data provide evidence for both intratrimer and intertrimer interactions mediated by the TMDs of E and thus extend the existing models of flavivirus membrane fusion. PMID:21325407

  11. Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions

    PubMed Central

    Seitz, Stefan; Urban, Stephan; Antoni, Christoph; Böttcher, Bettina

    2007-01-01

    Hepatitis B virus (HBV) is a major human pathogen causing about 750 000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two α-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing. PMID:17762862

  12. Initial development of a temporal-envelope-preserving nonlinear hearing aid prescription using a genetic algorithm.

    PubMed

    Sabin, Andrew T; Souza, Pamela E

    2013-06-01

    Most hearing aid prescriptions focus on the optimization of a metric derived from the long-term average spectrum of speech, and do not consider how the prescribed values might distort the temporal envelope shape. A growing body of evidence suggests that such distortions can lead to systematic errors in speech perception, and therefore hearing aid prescriptions might benefit by including preservation of the temporal envelope shape in their rationale. To begin to explore this possibility, we designed a genetic algorithm (GA) to find the multiband compression settings that preserve the shape of the original temporal envelope while placing that envelope in the listener's audiometric dynamic range. The resulting prescription had a low compression threshold, short attack and release times, and a combination of compression ratio and gain that placed the output signal within the listener's audiometric dynamic range. Initial behavioral tests of individuals with impaired hearing revealed no difference in speech-in-noise perception between the GA and the NAL-NL2 prescription. However, gap detection performance was superior with the GA in comparison to NAL-NL2. Overall, this work is a proof of concept that consideration of temporal envelope distortions can be incorporated into hearing aid prescriptions.

  13. Cooling of neutron stars with diffusive envelopes

    NASA Astrophysics Data System (ADS)

    Beznogov, M. V.; Fortin, M.; Haensel, P.; Yakovlev, D. G.; Zdunik, J. L.

    2016-12-01

    We study the effects of heat blanketing envelopes of neutron stars on their cooling. To this aim, we perform cooling simulations using newly constructed models of the envelopes composed of binary ion mixtures (H-He, He-C, C-Fe) varying the mass of lighter ions (H, He or C) in the envelope. The results are compared with those calculated using the standard models of the envelopes which contain the layers of lighter (accreted) elements (H, He and C) on top of the Fe layer, varying the mass of accreted elements. The main effect is that the chemical composition of the envelopes influences their thermal conductivity and, hence, thermal insulation of the star. For illustration, we apply these results to estimate the internal temperature of the Vela pulsar and to study the cooling of neutron stars of ages of 105-106 yr at the photon cooling stage. The uncertainties of the cooling models associated with our poor knowledge of chemical composition of the heat insulating envelopes strongly complicate theoretical reconstruction of the internal structure of cooling neutron stars from observations of their thermal surface emission.

  14. Genetic diversity of koala retroviral envelopes.

    PubMed

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V

    2015-03-17

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

  15. Genetic Diversity of Koala Retroviral Envelopes

    PubMed Central

    Xu, Wenqin; Gorman, Kristen; Santiago, Jan Clement; Kluska, Kristen; Eiden, Maribeth V.

    2015-01-01

    Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process. PMID:25789509

  16. Methodology for determination and use of the no-escape envelope of an air-to-air-missile

    NASA Technical Reports Server (NTRS)

    Neuman, Frank

    1988-01-01

    A large gap exists between optimal control and differential-game theory and their applications. The purpose of this paper is to show how this gap may be bridged. Missile-avoidance of realistically simulated infrared heat-seeking, fire-and-forget missile is studied. In detailed simulations, sweeping out the discretized initial condition space, avoidance methods based on pilot experience are combined with those based on simplified optimal control analysis to derive an approximation to the no-escape missile envelopes. The detailed missile equations and no-escape envelopes were then incorporated into an existing piloted simulation of air-to-air combat to generate missile firing decisions as well as missile avoidance commands. The use of these envelopes was found to be effective in both functions.

  17. The joke envelope: a neglected precursor of the psychic envelope concept in Freud's writing.

    PubMed

    Spero, Moshe Halevi

    2009-01-01

    The concepts of the primeval skin ego, psychic envelope, and related pre-ego containing and wrapping functions elaborated respectively by Esther Bick, Didier Anzieu, and Francis Tustin occupy an important position in contemporary psychoanalytic theory and clinical practice. The psychic envelope begins as a virtual mental protostructure ("proto" because it is not yet based on fully symbolized representations) that holds the budding mind together pending further developments. With maturity, the enveloping functions adopt symbolized, metaphoric form (for example, the aesthetic use of cloth, the analytic framework), but can regress to more concrete and pathological forms. The aforementioned authors based their ideas on a cluster of specific allusions to the idea of a psychic covering, barrier, or envelope in Freud's work. Yet they neglected one reference, hidden in Freud's analysis of the structure ofjokes and humor: the 'joke envelope"--die witzige Einkleidung. The present essay explores Freud's use of the term Einkleidung, including his intriguing idea that a joke requires three people whereas a dream does not and the fact that Freud nowhere speaks of a "dream envelope. "I take the "joke envelope" beyond its original context and posit a relationship between laughter and the early, normative traumas of breathing, crying, and loss, and the dawn of rhythmic envelopes that enable mentalization. Jokes and joking symbolically repeat the early rupture and rapture of breathing and self-other differentiation and the internalization of maternal containing and envelopment.

  18. The MEMPACK alpha-helical transmembrane protein structure prediction server

    PubMed Central

    Nugent, Timothy; Ward, Sean; Jones, David T.

    2011-01-01

    Motivation: The experimental difficulties of alpha-helical transmembrane protein structure determination make this class of protein an important target for sequence-based structure prediction tools. The MEMPACK prediction server allows users to submit a transmembrane protein sequence and returns transmembrane topology, lipid exposure, residue contacts, helix–helix interactions and helical packing arrangement predictions in both plain text and graphical formats using a number of novel machine learning-based algorithms. Availability: The server can be accessed as a new component of the PSIPRED portal by at http://bioinf.cs.ucl.ac.uk/psipred/. Contact: d.jones@cs.ucl.ac.uk; t.nugent@cs.ucl.ac.uk PMID:21349872

  19. Structure elucidation of dimeric transmembrane domains of bitopic proteins

    PubMed Central

    Volynsky, Pavel E.; Pavlov, Konstantin V.; Efremov, Roman G.; Arseniev, Alexander S.

    2010-01-01

    The interaction between transmembrane helices is of great interest because it directly determines biological activity of a membrane protein. Either destroying or enhancing such interactions can result in many diseases related to dysfunction of different tissues in human body. One much studied form of membrane proteins known as bitopic protein is a dimer containing two membrane-spanning helices associating laterally. Establishing structure-function relationship as well as rational design of new types of drugs targeting membrane proteins requires precise structural information about this class of objects. At present time, to investigate spatial structure and internal dynamics of such transmembrane helical dimers, several strategies were developed based mainly on a combination of NMR spectroscopy, optical spectroscopy, protein engineering and molecular modeling. These approaches were successfully applied to homo- and heterodimeric transmembrane fragments of several bitopic proteins, which play important roles in normal and in pathological conditions of human organism. PMID:20421711

  20. Determination of Membrane Protein Distribution on the Nuclear Envelope by Single-Point Single-Molecule FRAP.

    PubMed

    Mudumbi, Krishna C; Yang, Weidong

    2017-09-01

    Nuclear envelope transmembrane proteins (NETs) are synthesized on the endoplasmic reticulum and then transported from the outer nuclear membrane (ONM) to the inner nuclear membrane (INM) in eukaryotic cells. The abnormal distribution of NETs has been associated with many human diseases. However, quantitative determination of the spatial distribution and translocation dynamics of NETs on the ONM and INM is still very limited in currently existing approaches. Here we demonstrate a single-point single-molecule fluorescence recovery after photobleaching (FRAP) microscopy technique that enables quick determination of distribution and translocation rates for NETs in vivo. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. Suppressive effect on polyclonal B-cell activation of a synthetic peptide homologous to a transmembrane component of oncogenic retroviruses

    SciTech Connect

    Mitani, M.; Cianciolo, G.J.; Snyderman, R.; Yasuda, M.; Good, R.A.; Day, N.K.

    1987-01-01

    Purified feline leukemia virus, UV light-inactivated feline leukemia virus, and a synthetic peptide (CKS-17) homologous to a well-conserved region of the transmembrane components of several human and animal retroviruses were each studied for their effect on IgG production by feline peripheral blood lymphocytes. Using a reverse hemolytic plaque assay, both the viable virus and the UV-inactivated feline leukemia virus, but not the CKS-17, activated B lymphocytes to secrete IgG. When staphylococcal protein A, a polyclonal B-cell activator, was used to stimulate IgG synthesis by feline lymphocytes, the viable virus, the UV-inactivated virus, and the CKS-17 peptide each strongly suppressed IgG secretion without compromising viability of the lymphocytes. These finding suggest that the immunosuppressive influences of feline leukemia virus on immunoglobulin synthesis may reside in a conserved portion of the envelope glycoprotein that includes the region homologous to CKS-17.

  2. Crystal Structures of Major Envelope Proteins VP26 and VP28 from White Spot Syndrome Virus Shed Light on Their Evolutionary Relationship

    SciTech Connect

    Tang,X.; Wu, J.; Sivaraman, J.; Hew, C.

    2007-01-01

    White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 {angstrom}, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt {beta}-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core {beta}-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.

  3. Solitary Alfven wave envelopes and the modulational instability

    SciTech Connect

    Kennel, C.F.

    1987-06-01

    The derivative nonlinear Schroedinger equation describes the modulational instability of circularly polarized dispersive Alfven wave envelopes. It also may be used to determine the properties of finite amplitude localized stationary wave envelopes. Such envelope solitons exist only in conditions of modulational stability. This leaves open the question of whether, and if so, how, the modulational instability produces envelope solitons. 12 refs.

  4. Optimizing an emperical scoring function for transmembrane protein structure determination.

    SciTech Connect

    Young, Malin M.; Sale, Kenneth L.; Gray, Genetha Anne; Kolda, Tamara Gibson

    2003-10-01

    We examine the problem of transmembrane protein structure determination. Like many other questions that arise in biological research, this problem cannot be addressed by traditional laboratory experimentation alone. An approach that integrates experiment and computation is required. We investigate a procedure which states the transmembrane protein structure determination problem as a bound constrained optimization problem using a special empirical scoring function, called Bundler, as the objective function. In this paper, we describe the optimization problem and some of its mathematical properties. We compare and contrast results obtained using two different derivative free optimization algorithms.

  5. Alternating access to the transmembrane domain of the ATP-binding cassette protein cystic fibrosis transmembrane conductance regulator (ABCC7).

    PubMed

    Wang, Wuyang; Linsdell, Paul

    2012-03-23

    The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is a member of the ATP-binding cassette (ABC) protein family, most members of which act as active transporters. Actively transporting ABC proteins are thought to alternate between "outwardly facing" and "inwardly facing" conformations of the transmembrane substrate pathway. In CFTR, it is assumed that the outwardly facing conformation corresponds to the channel open state, based on homology with other ABC proteins. We have used patch clamp recording to quantify the rate of access of cysteine-reactive probes to cysteines introduced into two different transmembrane regions of CFTR from both the intracellular and extracellular solutions. Two probes, the large [2-sulfonatoethyl]methanethiosulfonate (MTSES) molecule and permeant Au(CN)(2)(-) ions, were applied to either side of the membrane to modify cysteines substituted for Leu-102 (first transmembrane region) and Thr-338 (sixth transmembrane region). Channel opening and closing were altered by mutations in the nucleotide binding domains of the channel. We find that, for both MTSES and Au(CN)(2)(-), access to these two cysteines from the cytoplasmic side is faster in open channels, whereas access to these same sites from the extracellular side is faster in closed channels. These results are consistent with alternating access to the transmembrane regions, however with the open state facing inwardly and the closed state facing outwardly. Our findings therefore prompt revision of current CFTR structural and mechanistic models, as well as having broader implications for transport mechanisms in all ABC proteins. Our results also suggest possible locations of both functional and dysfunctional ("vestigial") gates within the CFTR permeation pathway.

  6. Enveloped and non-enveloped viral-like particles in Trypanosoma cruzi epimastigotes

    PubMed Central

    Fernández-Presas, Ana María; Padilla-Noriega, Luis; Ingeborg-Becker; Robert, Lilia; Jiménez, José Agustín; Solano, Sandra; Delgado, Jose; Tato, Patricia; Molinari, José Luis

    2017-01-01

    ABSTRACT Electron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed. These clusters appear to have been released from distended Golgi cisternae. Furthermore, a paracrystalline array of electrodense, non-enveloped VLPs (with a diameter of 32 nm) were found in distended Golgi cisternae or as smaller clusters at a distance from the RE or Golgi. We cannot rule out that the 48 nm enveloped VLPs belong to the ssRNA Flaviviridae family because they are within its size range. The localization of enveloped VLPs is consistent with the replication strategy of these viruses that transit through the Golgi to be released at the cell surface. Due to the size and shape of the 32 nm non-enveloped VLPs, we propose that they belong to the dsRNA Totiviridae family. This is the first description of cytoplasmic enveloped and non-enveloped VLPs in T. cruzi epimastigotes. PMID:28793017

  7. Enveloped and non-enveloped viral-like particles in Trypanosoma cruzi epimastigotes.

    PubMed

    Fernández-Presas, Ana María; Padilla-Noriega, Luis; Becker, Ingeborg; Robert, Lilia; Jiménez, José Agustín; Solano, Sandra; Delgado, Jose; Tato, Patricia; Molinari, José Luis

    2017-08-07

    Electron microscopy is routinely used to identify viral infections in protozoan parasites. These viruses have been described as non-enveloped and icosahedral structures with a diameter of 30-60 nm. Most of them are classified within the non-segmented dsRNA Totiviridae family. We observed virus-like particles (VLPs) through transmission electron microscopy in the cytoplasm of Trypanosoma cruzi epimastigotes grown in cultures. Clusters of electrodense enveloped VLPs having a diameter of 48 nm were also observed. These clusters appear to have been released from distended Golgi cisternae. Furthermore, a paracrystalline array of electrodense, non-enveloped VLPs (with a diameter of 32 nm) were found in distended Golgi cisternae or as smaller clusters at a distance from the RE or Golgi. We cannot rule out that the 48 nm enveloped VLPs belong to the ssRNA Flaviviridae family because they are within its size range. The localization of enveloped VLPs is consistent with the replication strategy of these viruses that transit through the Golgi to be released at the cell surface. Due to the size and shape of the 32 nm non-enveloped VLPs, we propose that they belong to the dsRNA Totiviridae family. This is the first description of cytoplasmic enveloped and non-enveloped VLPs in T. cruzi epimastigotes.

  8. Morphologically complex protostellar envelopes : structure and kinematics

    NASA Astrophysics Data System (ADS)

    Tobin, John J.

    I present an in-depth study of protostars and their surrounding envelopes of dense gas and dust, using a multitude of observational methods to reveal new details of the star formation process. I use mid-infrared imaging from the Spitzer Space Telescope, combined with photometry spanning the near-infrared to millimeter wavelengths, to construct a model of the L1527 protostellar system. I modeled both the spectral energy distribution and resolved scattered light images to determine physical properties of the protostellar system. The nature of the apparent central point source in the Spitzer images was uncertain until high-resolution L-band imaging from the Gemini observatory resolved the point source into a disk in scattered light, having a radius of 200 AU. Protostellar envelopes are also often found to cast shadows against the 8 micron Galactic background in Spitzer imaging, enabling direct probes of envelope structure. The shadow images show that the dense envelopes around twenty-two Class 0 protostars are generally morphologically complex from 0.1 pc scales down to ˜1000 AU; they are often filamentary, and frequently non-axisymmetric. The observed envelope structure indicates a likely origin in turbulent cloud structure rather than a quasi-static/equilibrium formation. The complex envelope structure also may indicate an increased likelihood of fragmentation during collapse, forming close binaries. To further characterize these envelopes, I have observed them in the dense molecular gas tracers nthp and nht, both of which closely follow the 8 micron extinction morphology. The magnitude of the velocity gradients and envelope complexity on ˜10000 AU scales indicates that the velocity structure may reflect large-scale infall in addition to the often assumed rotation. Comparisons with three-dimensional filamentary and symmetric rotating collapse models reinforce the interpretation of velocities reflecting large-scale infall, showing that the structure of the envelope

  9. Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

    PubMed

    Rogers, Jason V; Rose, Mark D

    2014-12-02

    During mating in the budding yeast Saccharomyces cerevisiae, two haploid nuclei fuse via two sequential membrane fusion steps. SNAREs (i.e., soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Prm3p mediate outer nuclear membrane fusion, but the inner membrane fusogen remains unknown. Kar5p is a highly conserved transmembrane protein that localizes adjacent to the spindle pole body (SPB), mediates nuclear envelope fusion, and recruits Prm3p adjacent to the SPB. To separate Kar5p's functions, we tested localization, Prm3p recruitment, and nuclear fusion efficiency in various kar5 mutants. All domains and the conserved cysteine residues were essential for nuclear fusion. Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment. We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p. Electron microscopy revealed a kar5 mutant that arrests with expanded nuclear envelope bridges, suggesting that Kar5p is required after outer nuclear envelope fusion. Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope. These insights suggest a mechanism by which Kar5p mediates inner nuclear membrane fusion. Copyright © 2015 Rogers and Rose.

  10. COMPLEX STRUCTURE IN CLASS 0 PROTOSTELLAR ENVELOPES

    SciTech Connect

    Tobin, John J.; Hartmann, Lee; Looney, Leslie W.; Chiang, Hsin-Fang

    2010-04-01

    We use archived Infrared Array Camera images from the Spitzer Space Telescope to show that many Class 0 protostars exhibit complex, irregular, and non-axisymmetric structure within their dusty envelopes. Our 8 {mu}m extinction maps probe some of the densest regions in these protostellar envelopes. Many of the systems are observed to have highly irregular and non-axisymmetric morphologies on scales {approx}>1000 AU, with a quarter of the sample exhibiting filamentary or flattened dense structures. Complex envelope structure is observed in regions spatially distinct from outflow cavities, and the densest structures often show no systematic alignment perpendicular to the cavities. These results indicate that mass ejection is not responsible for much of the irregular morphologies we detect; rather, we suggest that the observed envelope complexity is mostly the result of collapse from protostellar cores with initially non-equilibrium structures. The striking non-axisymmetry in many envelopes could provide favorable conditions for the formation of binary systems. We also note that protostars in the sample appear to be formed preferentially near the edges of clouds or bends in filaments, suggesting formation by gravitational focusing.

  11. Featured Image: Orbiting Stars Share an Envelope

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2016-03-01

    This beautiful series of snapshots from a simulation (click for a better look!) shows what happens when two stars in a binary system become enclosed in the same stellar envelope. In this binary system, one of the stars has exhausted its hydrogen fuel and become a red giant, complete with an expanding stellar envelope composed of hydrogen and helium. Eventually, the envelope expands so much that the companion star falls into it, where it releases gravitational potential energy into the common envelope. A team led by Sebastian Ohlmann (Heidelberg Institute for Theoretical Studies and University of Wrzburg) recently performed hydrodynamic simulations of this process. Ohlmann and collaborators discovered that the energy release eventually triggers large-scale flow instabilities, which leads to turbulence within the envelope. This process has important consequences for how these systems next evolve (for instance, determining whether or not a supernova occurs!). You can check out the authors video of their simulated stellar inspiral below, or see their paper for more images and results from their study.CitationSebastian T. Ohlmann et al 2016 ApJ 816 L9. doi:10.3847/2041-8205/816/1/L9

  12. The cell envelope proteome of Aggregatibacter actinomycetemcomitans.

    PubMed

    Smith, K P; Fields, J G; Voogt, R D; Deng, B; Lam, Y-W; Mintz, K P

    2015-04-01

    The cell envelope of gram-negative bacteria serves a critical role in maintenance of cellular homeostasis, resistance to external stress, and host-pathogen interactions. Envelope protein composition is influenced by the physiological and environmental demands placed on the bacterium. In this study, we report a comprehensive compilation of cell envelope proteins from the periodontal and systemic pathogen Aggregatibacter actinomycetemcomitans VT1169, an afimbriated serotype b strain. The urea-extracted membrane proteins were identified by mass spectrometry-based shotgun proteomics. The membrane proteome, isolated from actively growing bacteria under normal laboratory conditions, included 648 proteins representing 27% of the predicted open reading frames in the genome. Bioinformatic analyses were used to annotate and predict the cellular location and function of the proteins. Surface adhesins, porins, lipoproteins, numerous influx and efflux pumps, multiple sugar, amino acid and iron transporters, and components of the type I, II and V secretion systems were identified. Periplasmic space and cytoplasmic proteins with chaperone function were also identified. A total of 107 proteins with unknown function were associated with the cell envelope. Orthologs of a subset of these uncharacterized proteins are present in other bacterial genomes, whereas others are found exclusively in A. actinomycetemcomitans. This knowledge will contribute to elucidating the role of cell envelope proteins in bacterial growth and survival in the oral cavity.

  13. Adaptive Spectral Envelope Estimation for Doppler Ultrasound.

    PubMed

    Kathpalia, Aditi; Karabiyik, Yucel; Eik-Nes, Sturla; Tegnander, Eva; Ekroll, Ingvild; Kiss, Gabriel; Torp, Hans

    2016-07-07

    Estimation of accurate maximum velocities and spectral envelope in ultrasound Doppler blood flow spectrograms are both essential for clinical diagnostic purposes. However, obtaining accurate maximum velocity is not straightforward due to intrinsic spectral broadening and variance in the power spectrum estimate. The method proposed in this work for maximum velocity point detection has been developed by modifying an existing method - Signal Noise Slope Intersection (SNSI), incorporating in it steps from an altered version of another method called Geometric Method (GM). Adaptive noise estimation from the spectrogram ensures that a smooth spectral envelope is obtained post detection of these maximum velocity points. The method has been tested on simulated Doppler signal with scatterers possessing a parabolic flow velocity profile constant in time, steady and pulsatile string phantom recordings as well as in vivo recordings from uterine, umbilical, carotid and subclavian arteries. Results from simulation experiments indicate a bias of less than 2.5% in maximum velocities when estimated for a range of peak velocities, Doppler angles and SNR levels. Standard deviation in the envelope is low - less than 2% in case of experiments done by varying the peak velocity and Doppler angle for steady phantom and simulated flow; and also less than 2% in case of experiments done by varying SNR but keeping constant flow conditions for in vivo and simulated flow. Low variability in the envelope makes the prospect of using the envelope for automated blood flow measurements possible and is illustrated for the case of Pulsatility Index estimation in uterine and umbilical arteries.

  14. Inhibition of Enveloped Viruses Infectivity by Curcumin

    PubMed Central

    Wen, Hsiao-Wei; Ou, Jun-Lin; Chiou, Shyan-Song; Chen, Jo-Mei; Wong, Min-Liang; Hsu, Wei-Li

    2013-01-01

    Curcumin, a natural compound and ingredient in curry, has antiinflammatory, antioxidant, and anticarcinogenic properties. Previously, we reported that curcumin abrogated influenza virus infectivity by inhibiting hemagglutination (HA) activity. This study demonstrates a novel mechanism by which curcumin inhibits the infectivity of enveloped viruses. In all analyzed enveloped viruses, including the influenza virus, curcumin inhibited plaque formation. In contrast, the nonenveloped enterovirus 71 remained unaffected by curcumin treatment. We evaluated the effects of curcumin on the membrane structure using fluorescent dye (sulforhodamine B; SRB)-containing liposomes that mimic the viral envelope. Curcumin treatment induced the leakage of SRB from these liposomes and the addition of the influenza virus reduced the leakage, indicating that curcumin disrupts the integrity of the membranes of viral envelopes and of liposomes. When testing liposomes of various diameters, we detected higher levels of SRB leakage from the smaller-sized liposomes than from the larger liposomes. Interestingly, the curcumin concentration required to reduce plaque formation was lower for the influenza virus (approximately 100 nm in diameter) than for the pseudorabies virus (approximately 180 nm) and the vaccinia virus (roughly 335 × 200 × 200 nm). These data provide insights on the molecular antiviral mechanisms of curcumin and its potential use as an antiviral agent for enveloped viruses. PMID:23658730

  15. Motion of contrast envelopes: peace and noise.

    PubMed

    Cropper, S J; Johnston, A

    2001-09-01

    We examined the effect of changing the composition of the carrier on the perception of motion in a drifting contrast envelope. Human observers were required to discriminate the direction of motion of contrast modulations of an underlying carrier as a function of temporal frequency and scaled (carrier) contrast. The carriers were modulations of both color and luminance, defined within a cardinal color space. Random-noise carriers had either binary luminance profiles or flat (gray-scale-white) or 1/f (pink) spectral power functions. Independent variables investigated were the envelope spatial frequency and temporal-drift frequency and the fundamental spatial frequency, color, and temporal-update frequency of the carrier. The results show that observers were able to discriminate correctly the direction of envelope motion for binary-noise carriers at both high (16 Hz) and low (2 Hz) temporal-drift frequencies. Changing the carrier format from binary noise to a flat (gray-scale) or 1/f amplitude profile reduced discrimination performance slightly but only in the high-temporal-frequency condition. Manipulation of the fundamental frequency of the carrier elicited no change in performance at the low temporal frequencies but produced ambiguous or reversed motion at the higher temporal frequencies as soon as the fundamental frequency was higher than the envelope modulation frequency. We found that envelope motion detection was sensitive to the structure of the carrier.

  16. Transmembrane channel formation in rhodopsin-containing bilayer membranes.

    PubMed

    Montal, M; Darszon, A; Trissl, H W

    1977-05-19

    Rhodopsin has been incorporated into planar lipid bilayer membranes. The effect of light is to increase the bilayer permeability in a pattern consistent with the formation of a transmembrane channel of about 10 A diameter. A model of visual excitation based on a light-activated and voltage-sensitive channel is presented.

  17. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding

    PubMed Central

    De Marothy, Minttu T; Elofsson, Arne

    2015-01-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices—implying a dynamic relationship between membrane proteins and their environment. PMID:25970811

  18. Marginally hydrophobic transmembrane α-helices shaping membrane protein folding.

    PubMed

    De Marothy, Minttu T; Elofsson, Arne

    2015-07-01

    Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment. © 2015 The Protein Society.

  19. High Transmembrane Voltage Raised by Close Contact Initiates Fusion Pore

    PubMed Central

    Bu, Bing; Tian, Zhiqi; Li, Dechang; Ji, Baohua

    2016-01-01

    Membrane fusion lies at the heart of neuronal communication but the detailed mechanism of a critical step, fusion pore initiation, remains poorly understood. Here, through atomistic molecular dynamics simulations, a transient pore formation induced by a close contact of two apposed bilayers is firstly reported. Such a close contact gives rise to a high local transmembrane voltage that induces the transient pore formation. Through simulations on two apposed bilayers fixed at a series of given distances, the process in which two bilayers approaching to each other under the pulling force from fusion proteins for membrane fusion was mimicked. Of note, this close contact induced fusion pore formation is contrasted with previous reported electroporation under ad hoc applied external electric field or ionic charge in-balance. We show that the transmembrane voltage increases with the decrease of the distance between the bilayers. Below a critical distance, depending on the lipid composition, the local transmembrane voltage can be sufficiently high to induce the transient pores. The size of these pores is approximately 1~2 nm in diameter, which is large enough to allow passing of neurotransmitters. A resealing of the membrane pores resulting from the neutralization of the transmembrane voltage by ions through the pores was then observed. We also found that the membrane tension can either prolong the lifetime of transient pores or cause them to dilate for full collapse. This result provides a possible mechanism for fusion pore formation and regulation of pathway of fusion process. PMID:28018169

  20. High Transmembrane Voltage Raised by Close Contact Initiates Fusion Pore.

    PubMed

    Bu, Bing; Tian, Zhiqi; Li, Dechang; Ji, Baohua

    2016-01-01

    Membrane fusion lies at the heart of neuronal communication but the detailed mechanism of a critical step, fusion pore initiation, remains poorly understood. Here, through atomistic molecular dynamics simulations, a transient pore formation induced by a close contact of two apposed bilayers is firstly reported. Such a close contact gives rise to a high local transmembrane voltage that induces the transient pore formation. Through simulations on two apposed bilayers fixed at a series of given distances, the process in which two bilayers approaching to each other under the pulling force from fusion proteins for membrane fusion was mimicked. Of note, this close contact induced fusion pore formation is contrasted with previous reported electroporation under ad hoc applied external electric field or ionic charge in-balance. We show that the transmembrane voltage increases with the decrease of the distance between the bilayers. Below a critical distance, depending on the lipid composition, the local transmembrane voltage can be sufficiently high to induce the transient pores. The size of these pores is approximately 1~2 nm in diameter, which is large enough to allow passing of neurotransmitters. A resealing of the membrane pores resulting from the neutralization of the transmembrane voltage by ions through the pores was then observed. We also found that the membrane tension can either prolong the lifetime of transient pores or cause them to dilate for full collapse. This result provides a possible mechanism for fusion pore formation and regulation of pathway of fusion process.

  1. Thiourea isosteres as anion receptors and transmembrane transporters.

    PubMed

    Wenzel, Marco; Light, Mark E; Davis, Anthony P; Gale, Philip A

    2011-07-21

    Compounds containing cyanoguanidine and 3-amino-1,2,4-benzothiadiazine-1,1-dioxide have been studied as anion receptors and transporters. Significant affinity for oxo-anions was observed in organic solution and the receptors were found to function as transmembrane chloride/nitrate antiporters with transport rates enhanced in the presence of valinomycin-K(+) complex.

  2. Envelope Solitons in Acoustically Dispersive Vitreous Silica

    NASA Technical Reports Server (NTRS)

    Cantrell, John H.; Yost, William T.

    2012-01-01

    Acoustic radiation-induced static strains, displacements, and stresses are manifested as rectified or dc waveforms linked to the energy density of an acoustic wave or vibrational mode via the mode nonlinearity parameter of the material. An analytical model is developed for acoustically dispersive media that predicts the evolution of the energy density of an initial waveform into a series of energy solitons that generates a corresponding series of radiation-induced static strains (envelope solitons). The evolutionary characteristics of the envelope solitons are confirmed experimentally in Suprasil W1 vitreous silica. The value (-11.9 plus or minus 1.43) for the nonlinearity parameter, determined from displacement measurements of the envelope solitons via a capacitive transducer, is in good agreement with the value (-11.6 plus or minus 1.16) obtained independently from acoustic harmonic generation measurements. The agreement provides strong, quantitative evidence for the validity of the model.

  3. Drug design from the cryptic inhibitor envelope.

    PubMed

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J; Zhou, Pei

    2016-02-25

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

  4. Drug design from the cryptic inhibitor envelope

    PubMed Central

    Lee, Chul-Jin; Liang, Xiaofei; Wu, Qinglin; Najeeb, Javaria; Zhao, Jinshi; Gopalaswamy, Ramesh; Titecat, Marie; Sebbane, Florent; Lemaitre, Nadine; Toone, Eric J.; Zhou, Pei

    2016-01-01

    Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC—an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target—access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics. PMID:26912110

  5. Ultradonut topology of the nuclear envelope

    PubMed Central

    Torbati, Mehdi; Lele, Tanmay P.; Agrawal, Ashutosh

    2016-01-01

    The nuclear envelope is a unique topological structure formed by lipid membranes in eukaryotic cells. Unlike other membrane structures, the nuclear envelope comprises two concentric membrane shells fused at numerous sites with toroid-shaped pores that impart a “geometric” genus on the order of thousands. Despite the intriguing architecture and vital biological functions of the nuclear membranes, how they achieve and maintain such a unique arrangement remains unknown. Here, we used the theory of elasticity and differential geometry to analyze the equilibrium shape and stability of this structure. Our results show that modest in- and out-of-plane stresses present in the membranes not only can define the pore geometry, but also provide a mechanism for destabilizing membranes beyond a critical size and set the stage for the formation of new pores. Our results suggest a mechanism wherein nanoscale buckling instabilities can define the global topology of a nuclear envelope-like structure. PMID:27647910

  6. Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins

    PubMed Central

    Kobe, Bostjan; Center, Rob J.; Kemp, Bruce E.; Poumbourios, Pantelis

    1999-01-01

    Retroviral entry into cells depends on envelope glycoproteins, whereby receptor binding to the surface-exposed subunit triggers membrane fusion by the transmembrane protein (TM) subunit. We determined the crystal structure at 2.5-Å resolution of the ectodomain of gp21, the TM from human T cell leukemia virus type 1. The gp21 fragment was crystallized as a maltose-binding protein chimera, and the maltose-binding protein domain was used to solve the initial phases by the method of molecular replacement. The structure of gp21 comprises an N-terminal trimeric coiled coil, an adjacent disulfide-bonded loop that stabilizes a chain reversal, and a C-terminal sequence structurally distinct from HIV type 1/simian immunodeficiency virus gp41 that packs against the coil in an extended antiparallel fashion. Comparison of the gp21 structure with the structures of other retroviral TMs contrasts the conserved nature of the coiled coil-forming region and adjacent disulfide-bonded loop with the variable nature of the C-terminal ectodomain segment. The structure points to these features having evolved to enable the dual roles of retroviral TMs: conserved fusion function and an ability to anchor diverse surface-exposed subunit structures to the virion envelope and infected cell surface. The structure of gp21 implies that the N-terminal fusion peptide is in close proximity to the C-terminal transmembrane domain and likely represents a postfusion conformation. PMID:10200260

  7. The Atomic Structure of the HIV-1 gp41 Transmembrane Domain and Its Connection to the Immunogenic Membrane-proximal External Region.

    PubMed

    Apellániz, Beatriz; Rujas, Edurne; Serrano, Soraya; Morante, Koldo; Tsumoto, Kouhei; Caaveiro, Jose M M; Jiménez, M Ángeles; Nieva, José L

    2015-05-22

    The membrane-proximal external region (MPER) C-terminal segment and the transmembrane domain (TMD) of gp41 are involved in HIV-1 envelope glycoprotein-mediated fusion and modulation of immune responses during viral infection. However, the atomic structure of this functional region remains unsolved. Here, based on the high resolution NMR data obtained for peptides spanning the C-terminal segment of MPER and the TMD, we report two main findings: (i) the conformational variability of the TMD helix at a membrane-buried position; and (ii) the existence of an uninterrupted α-helix spanning MPER and the N-terminal region of the TMD. Thus, our structural data provide evidence for the bipartite organization of TMD predicted by previous molecular dynamics simulations and functional studies, but they do not support the breaking of the helix at Lys-683, as was suggested by some models to mark the initiation of the TMD anchor. Antibody binding energetics examined with isothermal titration calorimetry and humoral responses elicited in rabbits by peptide-based vaccines further support the relevance of a continuous MPER-TMD helix for immune recognition. We conclude that the transmembrane anchor of HIV-1 envelope is composed of two distinct subdomains: 1) an immunogenic helix at the N terminus also involved in promoting membrane fusion; and 2) an immunosuppressive helix at the C terminus, which might also contribute to the late stages of the fusion process. The unprecedented high resolution structural data reported here may guide future vaccine and inhibitor developments.

  8. BST-2/tetherin: a new component of the innate immune response to enveloped viruses

    PubMed Central

    Evans, David T.; Serra-Moreno, Ruth; Singh, Rajendra K.; Guatelli, John C.

    2010-01-01

    The interferon-inducible, transmembrane protein BST-2 (CD317, tetherin) directly holds fully formed enveloped virus particles to the cells that produce them, inhibiting their spread. BST-2 inhibits members of the retrovirus, filovirus, arenavirus, and herpesvirus families. These viruses encode a variety of proteins to degrade BST-2 and/or direct it away from its site of action at the cell surface. Viral antagonism has subjected BST-2 to positive selection, leading to species-specific differences that presented a barrier to the transmission of simian immunodeficiency viruses (SIVs) to humans. This barrier was crossed by HIV-1 when its Vpu protein acquired activity as a BST-2 antagonist. Here, we review this new host-pathogen relationship and discuss its impact on the evolution of primate lentiviruses and the origins of the HIV pandemic. PMID:20688520

  9. The photodissociation of CO in circumstellar envelopes

    NASA Technical Reports Server (NTRS)

    Mamon, G. A.; Glassgold, A. E.; Huggins, P. J.

    1988-01-01

    The CO photodissociation rate for the unshielded ISM is calculated using recent laboratory results which confirm that photodissociation occurs by way of line absorption. A value of 2.0 x 10 to the -10th/s, an order of magnitude higher than the rate used in the past, is obtained. The new rate and a treatment of the radiative transfer and shielding are used to develop a theory for the CO abundance in the circumstellar envelopes of cool, evolved stars, and results are presented on the spatial variation of CO, C, and C(+). It is shown that these distributions play important roles in determining the observational properties of circumstellar envelopes.

  10. Consumer access to utility billing envelopes

    SciTech Connect

    Anglin, M.K.

    1984-09-13

    Billing envelope inserts are a medium of advertising used by utilities for a variety of purposes, from encouraging conservation to expressing political opinions. Recently, consumer groups have begun to assert a right of access to the same medium. A constitutional right of reply has been advocated. Commissions have found regulatory authority to direct companies to provide access on the basis of several different theories. At least two states have passed legislation permitting consumer groups to use bill inserts to solicit members and contributions. When examined, these developments reveal a trend of granting organizations access to utility billing envelopes.

  11. [NESPRINS--nuclear envelope proteins ensuring integrity].

    PubMed

    Pershina, E G; Morozova, K N; Kiseleva, E V

    2014-01-01

    This review describes the nesprins (nuclear envelope spectrin-repeat proteins), which are recently discovered family of nuclear envelope proteins. These proteins play an important role in maintaining the cellular architecture and establish the link between the nucleus and other sub-cellular compartments. Many tissue-specific diseases including lipodystrophies, hearing loss, cardiac and skeletal myopathies are associated with nesprins mutations. These proteins comprise of multiple tissue specific isoforms which contain spectrin repeats providing interaction of nesprins with other nuclear membrane proteins, cytoskeleton and intranuclear matrix. We summarize recent findings and suggestions about nesprins structural organization and function inside the cell. Human diseases caused by abnormal nesprins expression are also described.

  12. Perception and coding of envelopes in weakly electric fishes.

    PubMed

    Stamper, Sarah A; Fortune, Eric S; Chacron, Maurice J

    2013-07-01

    Natural sensory stimuli have a rich spatiotemporal structure and can often be characterized as a high frequency signal that is independently modulated at lower frequencies. This lower frequency modulation is known as the envelope. Envelopes are commonly found in a variety of sensory signals, such as contrast modulations of visual stimuli and amplitude modulations of auditory stimuli. While psychophysical studies have shown that envelopes can carry information that is essential for perception, how envelope information is processed in the brain is poorly understood. Here we review the behavioral salience and neural mechanisms for the processing of envelopes in the electrosensory system of wave-type gymnotiform weakly electric fishes. These fish can generate envelope signals through movement, interactions of their electric fields in social groups or communication signals. The envelopes that result from the first two behavioral contexts differ in their frequency content, with movement envelopes typically being of lower frequency. Recent behavioral evidence has shown that weakly electric fish respond in robust and stereotypical ways to social envelopes to increase the envelope frequency. Finally, neurophysiological results show how envelopes are processed by peripheral and central electrosensory neurons. Peripheral electrosensory neurons respond to both stimulus and envelope signals. Neurons in the primary hindbrain recipient of these afferents, the electrosensory lateral line lobe (ELL), exhibit heterogeneities in their responses to stimulus and envelope signals. Complete segregation of stimulus and envelope information is achieved in neurons in the target of ELL efferents, the midbrain torus semicircularis (Ts).

  13. Aurora B–mediated localized delays in nuclear envelope formation facilitate inclusion of late-segregating chromosome fragments

    PubMed Central

    Karg, Travis; Warecki, Brandt; Sullivan, William

    2015-01-01

    To determine how chromosome segregation is coordinated with nuclear envelope formation (NEF), we examined the dynamics of NEF in the presence of lagging acentric chromosomes in Drosophila neuroblasts. Acentric chromosomes often exhibit delayed but ultimately successful segregation and incorporation into daughter nuclei. However, it is unknown whether these late-segregating acentric fragments influence NEF to ensure their inclusion in daughter nuclei. Through live analysis, we show that acentric chromosomes induce highly localized delays in the reassembly of the nuclear envelope. These delays result in a gap in the nuclear envelope that facilitates the inclusion of lagging acentrics into telophase daughter nuclei. Localized delays of nuclear envelope reassembly require Aurora B kinase activity. In cells with reduced Aurora B activity, there is a decrease in the frequency of local nuclear envelope reassembly delays, resulting in an increase in the frequency of acentric-bearing, lamin-coated micronuclei. These studies reveal a novel role of Aurora B in maintaining genomic integrity by promoting the formation of a passageway in the nuclear envelope through which late-segregating acentric chromosomes enter the telophase daughter nucleus. PMID:25877868

  14. Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene

    PubMed Central

    Sosnay, Patrick R; Siklosi, Karen R; Van Goor, Fredrick; Kaniecki, Kyle; Yu, Haihui; Sharma, Neeraj; Ramalho, Anabela S; Amaral, Margarida D; Dorfman, Ruslan; Zielenski, Julian; Masica, David L; Karchin, Rachel; Millen, Linda; Thomas, Philip J; Patrinos, George P; Corey, Mary; Lewis, Michelle H; Rommens, Johanna M; Castellani, Carlo; Penland, Christopher M; Cutting, Garry R

    2013-01-01

    Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation to clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 cystic fibrosis patients in registries and clinics in North America and Europe. Among these patients, 159 CFTR variants had an allele frequency of ≥0.01%. These variants were evaluated for both clinical severity and functional consequence with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of cystic fibrosis patients enabled assignment of 12 of the remaining 32 variants as neutral while the other 20 variants remained indeterminate. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically-relevant genomic variation. PMID:23974870

  15. Structural flexibility of the pentameric SARS coronavirus envelope protein ion channel.

    PubMed

    Parthasarathy, Krupakar; Ng, Lifang; Lin, Xin; Liu, Ding Xiang; Pervushin, Konstantin; Gong, Xiandi; Torres, Jaume

    2008-09-15

    Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane alpha-helical bundles, related by a approximately 50 degrees rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM alpha-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric alpha-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function.

  16. Structural Flexibility of the Pentameric SARS Coronavirus Envelope Protein Ion Channel

    PubMed Central

    Parthasarathy, Krupakar; Ng, Lifang; Lin, Xin; Liu, Ding Xiang; Pervushin, Konstantin; Gong, Xiandi; Torres, Jaume

    2008-01-01

    Coronaviruses contain a small envelope membrane protein with cation-selective ion channel activity mediated by its transmembrane domain (ETM). In a computational study, we proposed that ion channel activity can be explained by either of two similar ETM homopentameric transmembrane α-helical bundles, related by a ∼50° rotation of the helices. Later, we tested this prediction, using site-specific infrared dichroism of a lysine-flanked isotopically labeled ETM peptide from the virus responsible for the severe acute respiratory syndrome, SARS, reconstituted in lipid bilayers. However, the data were consistent with the presence of a kink at the center of the ETM α-helix, and it did not fit completely either computational model. Herein, we have used native ETM, without flanking lysines, and show that the helix orientation is now consistent with one of the predicted models. ETM only produced one oligomeric form, pentamers, in the lipid-mimic detergent dodecylphosphocholine and in perfluorooctanoic acid. We thus report the correct backbone model for the pentameric α-helical bundle of ETM. The disruptive effects caused by terminal lysines probably highlight the conformational flexibility required during ion channel function. PMID:18658207

  17. The Viner-Wong Envelope Theorem.

    ERIC Educational Resources Information Center

    Silberberg, Eugene

    1999-01-01

    Observes that the envelope theorem, a fundamental tool in duality analysis, is still a puzzle to many people. Argues that the essence of a solution proposed by Paul Samuelson (1947) is also unclear to many people, but can be communicated with a simple cost diagram. Presents and explains the proposed diagram. (DSK)

  18. 14 CFR 23.333 - Flight envelope.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... any combination of airspeed and load factor on and within the boundaries of a flight envelope (similar... altitudes between sea level and 20,000 feet. The gust velocity may be reduced linearly from 50 f.p.s. at 20... considered at altitudes between sea level and 20,000 feet. The gust velocity may be reduced linearly from...

  19. 14 CFR 23.333 - Flight envelope.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... any combination of airspeed and load factor on and within the boundaries of a flight envelope (similar... altitudes between sea level and 20,000 feet. The gust velocity may be reduced linearly from 50 f.p.s. at 20... considered at altitudes between sea level and 20,000 feet. The gust velocity may be reduced linearly from...

  20. The Methodology of Data Envelopment Analysis.

    ERIC Educational Resources Information Center

    Sexton, Thomas R.

    1986-01-01

    The methodology of data envelopment analysis, (DEA) a linear programming-based method, is described. Other procedures often used for measuring relative productive efficiency are discussed in relation to DEA, including ratio analysis and multiple regression analysis. The DEA technique is graphically illustrated for only two inputs and one output.…

  1. Ultraviolet Opacity and Fluorescence in Supernova Envelopes

    NASA Technical Reports Server (NTRS)

    Li, Hongwei; McCray, Richard

    1996-01-01

    By the time the expanding envelope of a Type 2 supernova becomes transparent in the optical continuum, most of the gamma-ray luminosity produced by radioactive Fe/Co/Ni clumps propagates into the hydrogen/helium envelope and is deposited there, if at all. The resulting fast electrons excite He 1 and H 1, the two- photon continua of which are the dominant internal sources of ultraviolet radiation. The UV radiation is blocked by scattering in thousands of resonance lines of metals and converted by fluorescence into optical and infrared emission lines that escape freely. We describe results of Monte Carlo calculations that simulate non-LTE scattering and fluorescence in more than five million allowed lines of Ca, Sc, Ti, V, Cr, Mn, Fe, Co, and Ni. For a model approximating conditions in the envelope of SN 1987A, the calculated emergent spectrum resembles the observed one. For the first 2 yr after explosion, the ultraviolet radiation (lambda less than or approximately equals 3000) is largely blocked and converted into a quasi continuum of many thousands of weak optical and infrared emission lines and some prominent emission features, such as the Ca 2 lambdalambda8600 triplet. Later, as the envelope cools and expands, it becomes more transparent, and an increasing fraction of the luminosity emerges in the UV band.

  2. Discriminating Dysarthria Type from Envelope Modulation Spectra

    ERIC Educational Resources Information Center

    Liss, Julie M.; LeGendre, Sue; Lotto, Andrew J.

    2010-01-01

    Purpose: Previous research demonstrated the ability of temporally based rhythm metrics to distinguish among dysarthrias with different prosodic deficit profiles (J. M. Liss et al., 2009). The authors examined whether comparable results could be obtained by an automated analysis of speech envelope modulation spectra (EMS), which quantifies the…

  3. The Methodology of Data Envelopment Analysis.

    ERIC Educational Resources Information Center

    Sexton, Thomas R.

    1986-01-01

    The methodology of data envelopment analysis, (DEA) a linear programming-based method, is described. Other procedures often used for measuring relative productive efficiency are discussed in relation to DEA, including ratio analysis and multiple regression analysis. The DEA technique is graphically illustrated for only two inputs and one output.…

  4. Discriminating Dysarthria Type from Envelope Modulation Spectra

    ERIC Educational Resources Information Center

    Liss, Julie M.; LeGendre, Sue; Lotto, Andrew J.

    2010-01-01

    Purpose: Previous research demonstrated the ability of temporally based rhythm metrics to distinguish among dysarthrias with different prosodic deficit profiles (J. M. Liss et al., 2009). The authors examined whether comparable results could be obtained by an automated analysis of speech envelope modulation spectra (EMS), which quantifies the…

  5. Ozone Reductions Using Residential Building Envelopes

    SciTech Connect

    Walker, Iain S.; Sherman, Max; Nazaroff, William W.

    2009-02-01

    Ozone is an air pollutant with that can have significant health effects and a significant source of ozone in some regions of California is outdoor air. Because people spend the vast majority of their time indoors, reduction in indoor levels of ozone could lead to improved health for many California residents. Ozone is removed from indoor air by surface reactions and can also be filtered by building envelopes. The magnitude of the envelope impact depends on the specific building materials that the air flows over and the geometry of the air flow paths through the envelope that can be changes by mechanical ventilation operation. The 2008 Residential Building Standards in California include minimum requirements for mechanical ventilation by referencing ASHRAE Standard 62.2. This study examines the changes in indoor ozone depending on the mechanical ventilation system selected to meet these requirements. This study used detailed simulations of ventilation in a house to examine the impacts of different ventilation systems on indoor ozone concentrations. The simulation results showed that staying indoors reduces exposure to ozone by 80percent to 90percent, that exhaust ventilation systems lead to lower indoor ozone concentrations, that opening of windows should be avoided at times of high outdoor ozone, and that changing the time at which mechanical ventilation occurs has the ability to halve exposure to ozone. Future work should focus on the products of ozone reactions in the building envelope and the fate of these products with respect to indoor exposures.

  6. Tegument Assembly and Secondary Envelopment of Alphaherpesviruses

    PubMed Central

    Owen, Danielle J.; Crump, Colin M.; Graham, Stephen C.

    2015-01-01

    Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called “tegument” that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei. PMID:26393641

  7. SAFEGUARDS ENVELOPE: PREVIOUS WORK AND EXAMPLES

    SciTech Connect

    Richard Metcalf; Aaron Bevill; William Charlton; Robert Bean

    2008-07-01

    The future expansion of nuclear power will require not just electricity production but fuel cycle facilities such as fuel fabrication and reprocessing plants. As large reprocessing facilities are built in various states, they must be built and operated in a manner to minimize the risk of nuclear proliferation. Process monitoring has returned to the spotlight as an added measure that can increase confidence in the safeguards of special nuclear material (SNM). Process monitoring can be demonstrated to lengthen the allowable inventory period by reducing accountancy requirements, and to reduce the false positive indications. The next logical step is the creation of a Safeguards Envelope, a set of operational parameters and models to maximize anomaly detection and inventory period by process monitoring while minimizing operator impact and false positive rates. A brief example of a rudimentary Safeguards Envelope is presented, and shown to detect synthetic diversions overlaying a measured processing plant data set. This demonstration Safeguards Envelope is shown to increase the confidence that no SNM has been diverted with minimal operator impact, even though it is based on an information sparse environment. While the foundation on which a full Safeguards Envelope can be built has been presented in historical demonstrations of process monitoring, several requirements remain yet unfulfilled. Future work will require reprocessing plant transient models, inclusion of “non-traditional” operating data, and exploration of new methods of identifying subtle events in transient processes.

  8. Right to Light: Ralph Knowles's Solar Envelope.

    ERIC Educational Resources Information Center

    Morton, David

    1979-01-01

    At the University of Southern California solar-access design research project, Barry Knowles and students have devised a solar envelope: the largest volumetric container over a land parcel that allows solar access to all adjacent neighbors within useful time constraints. (Author/MLF)

  9. Data Envelopment Analysis: Critique and Extensions.

    ERIC Educational Resources Information Center

    Sexton, Thomas R.; And Others

    1986-01-01

    Recent methodological advances are described that enable the analyst to extract additional information from the data envelopment analysis (DEA) methodology, including goal programming to develop cross-efficiencies, cluster analysis, analysis of variance, and pooled cross section time-series analysis. Some shortcomings of DEA are discussed. (LMO)

  10. Adaptive envelope protection methods for aircraft

    NASA Astrophysics Data System (ADS)

    Unnikrishnan, Suraj

    Carefree handling refers to the ability of a pilot to operate an aircraft without the need to continuously monitor aircraft operating limits. At the heart of all carefree handling or maneuvering systems, also referred to as envelope protection systems, are algorithms and methods for predicting future limit violations. Recently, envelope protection methods that have gained more acceptance, translate limit proximity information to its equivalent in the control channel. Envelope protection algorithms either use very small prediction horizon or are static methods with no capability to adapt to changes in system configurations. Adaptive approaches maximizing prediction horizon such as dynamic trim, are only applicable to steady-state-response critical limit parameters. In this thesis, a new adaptive envelope protection method is developed that is applicable to steady-state and transient response critical limit parameters. The approach is based upon devising the most aggressive optimal control profile to the limit boundary and using it to compute control limits. Pilot-in-the-loop evaluations of the proposed approach are conducted at the Georgia Tech Carefree Maneuver lab for transient longitudinal hub moment limit protection. Carefree maneuvering is the dual of carefree handling in the realm of autonomous Uninhabited Aerial Vehicles (UAVs). Designing a flight control system to fully and effectively utilize the operational flight envelope is very difficult. With the increasing role and demands for extreme maneuverability there is a need for developing envelope protection methods for autonomous UAVs. In this thesis, a full-authority automatic envelope protection method is proposed for limit protection in UAVs. The approach uses adaptive estimate of limit parameter dynamics and finite-time horizon predictions to detect impending limit boundary violations. Limit violations are prevented by treating the limit boundary as an obstacle and by correcting nominal control

  11. Effects of temporal envelope modulation on acoustic signal recognition in a vocal fish, the plainfin midshipman.

    PubMed

    McKibben, J R; Bass, A H

    2001-06-01

    Amplitude modulation is an important parameter defining vertebrate acoustic communication signals. Nesting male plainfin midshipman fish, Porichthys notatus, emit simple, long duration hums in which modulation is strikingly absent. Envelope modulation is, however, introduced when the hums of adjacent males overlap to produce acoustic beats. Hums attract gravid females and can be mimicked with continuous tones at the fundamental frequency. While individual hums have flat envelopes, other midshipman signals are amplitude modulated. This study used one-choice playback tests with gravid females to examine the role of envelope modulation in hum recognition. Various pulse train and two-tone beat stimuli resembling natural communication signals were presented individually, and the responses compared to those for continuous pure tones. The effectiveness of pulse trains was graded and depended upon both pulse duration and the ratio of pulse to gap length. Midshipman were sensitive to beat modulations from 0.5 to 10 Hz, with fewer fish approaching the beat than the pure tone. Reducing the degree of modulation increased the effectiveness of beat stimuli. Hence, the lack of modulation in the midshipman's advertisement call corresponds to the importance of envelope modulation for the categorization of communication signals even in this relatively simple system.

  12. Assessing effects of variation in global climate data sets on spatial predictions from climate envelope models

    USGS Publications Warehouse

    Romanach, Stephanie; Watling, James I.; Fletcher, Robert J.; Speroterra, Carolina; Bucklin, David N.; Brandt, Laura A.; Pearlstine, Leonard G.; Escribano, Yesenia; Mazzotti, Frank J.

    2014-01-01

    Climate change poses new challenges for natural resource managers. Predictive modeling of species–environment relationships using climate envelope models can enhance our understanding of climate change effects on biodiversity, assist in assessment of invasion risk by exotic organisms, and inform life-history understanding of individual species. While increasing interest has focused on the role of uncertainty in future conditions on model predictions, models also may be sensitive to the initial conditions on which they are trained. Although climate envelope models are usually trained using data on contemporary climate, we lack systematic comparisons of model performance and predictions across alternative climate data sets available for model training. Here, we seek to fill that gap by comparing variability in predictions between two contemporary climate data sets to variability in spatial predictions among three alternative projections of future climate. Overall, correlations between monthly temperature and precipitation variables were very high for both contemporary and future data. Model performance varied across algorithms, but not between two alternative contemporary climate data sets. Spatial predictions varied more among alternative general-circulation models describing future climate conditions than between contemporary climate data sets. However, we did find that climate envelope models with low Cohen's kappa scores made more discrepant spatial predictions between climate data sets for the contemporary period than did models with high Cohen's kappa scores. We suggest conservation planners evaluate multiple performance metrics and be aware of the importance of differences in initial conditions for spatial predictions from climate envelope models.

  13. Validating predictions from climate envelope models

    USGS Publications Warehouse

    Watling, J.; Bucklin, D.; Speroterra, C.; Brandt, L.; Cabal, C.; Romañach, Stephanie S.; Mazzotti, Frank J.

    2013-01-01

    Climate envelope models are a potentially important conservation tool, but their ability to accurately forecast species’ distributional shifts using independent survey data has not been fully evaluated. We created climate envelope models for 12 species of North American breeding birds previously shown to have experienced poleward range shifts. For each species, we evaluated three different approaches to climate envelope modeling that differed in the way they treated climate-induced range expansion and contraction, using random forests and maximum entropy modeling algorithms. All models were calibrated using occurrence data from 1967–1971 (t1) and evaluated using occurrence data from 1998–2002 (t2). Model sensitivity (the ability to correctly classify species presences) was greater using the maximum entropy algorithm than the random forest algorithm. Although sensitivity did not differ significantly among approaches, for many species, sensitivity was maximized using a hybrid approach that assumed range expansion, but not contraction, in t2. Species for which the hybrid approach resulted in the greatest improvement in sensitivity have been reported from more land cover types than species for which there was little difference in sensitivity between hybrid and dynamic approaches, suggesting that habitat generalists may be buffered somewhat against climate-induced range contractions. Specificity (the ability to correctly classify species absences) was maximized using the random forest algorithm and was lowest using the hybrid approach. Overall, our results suggest cautious optimism for the use of climate envelope models to forecast range shifts, but also underscore the importance of considering non-climate drivers of species range limits. The use of alternative climate envelope models that make different assumptions about range expansion and contraction is a new and potentially useful way to help inform our understanding of climate change effects on species.

  14. Adaptive Spectral Envelope Estimation for Doppler Ultrasound.

    PubMed

    Kathpalia, Aditi; Karabiyik, Yucel; Eik-Nes, Sturla H; Tegnander, Eva; Ekroll, Ingvild Kinn; Kiss, Gabriel; Torp, Hans

    2016-11-01

    Estimation of accurate maximum velocities and spectral envelope in ultrasound Doppler blood flow spectrograms are both essential for clinical diagnostic purposes. However, obtaining accurate maximum velocity is not straightforward due to intrinsic spectral broadening and variance in the power spectrum estimate. The method proposed in this paper for maximum velocity point detection has been developed by modifying an existing method-signal noise slope intersection, incorporating in it steps from an altered version of another method called geometric method. Adaptive noise estimation from the spectrogram ensures that a smooth spectral envelope is obtained postdetection of these maximum velocity points. The method has been tested on simulated Doppler signal with scatterers possessing a parabolic flow velocity profile constant in time, steady and pulsatile string phantom recordings, as well as in vivo recordings from uterine, umbilical, carotid, and subclavian arteries. The results from simulation experiments indicate a bias of less than 2.5% in maximum velocities when estimated for a range of peak velocities, Doppler angles, and SNR levels. Standard deviation in the envelope is low-less than 2% in the case of experiments done by varying the peak velocity and Doppler angle for steady phantom and simulated flow, and also less than 2% in the case of experiments done by varying SNR but keeping constant flow conditions for in vivo and simulated flow. Low variability in the envelope makes the prospect of using the envelope for automated blood flow measurements possible and is illustrated for the case of pulsatility index estimation in uterine and umbilical arteries.

  15. CO line emission from circumstellar envelopes

    NASA Astrophysics Data System (ADS)

    Teyssier, D.; Hernandez, R.; Bujarrabal, V.; Yoshida, H.; Phillips, T. G.

    2006-04-01

    Aims.We present the results of a multi-transition CO observational program conducted on a sample of AGB and post-AGB stars envelopes. We have collected maps and single pointing observations of these envelopes in 5 rotational transitions ranging from J = 1-0 to J = 6-5, including in particular new observations of the CO line at 691 GHz at the CSO. The use of such a set of mm and submm CO line on stellar envelopes is rare and limited to the work of some authors on IRC+10216. Methods: .Using a model for the CO emission of an AGB circumstellar envelope, in combination with a standard LVG approach, we have conducted a systematic modelling analysis using the whole set of CO data collected for a sample of 12 sources. We simultaneously fit all five transitions, taking into account the spatial information provided by the maps. Results: .We find mass-loss rates in the range 1 × 10-7 to 4 × 10-4 M_⊙/yr, and envelope temperatures ranging from 20 K to 1000 K at a radius of 1016 cm. There seem to be a general anti-correlation between mass loss rates and temperature, the high mass loss rate AGBs having low temperatures, and vice versa. We show that most AGB data can be fitted using a constant mass loss rate, at least within the calibration uncertainties associated with the data collected at different frequencies. For some cases though (e.g. CIT 6, R Hya, χ Cyg), a change in the mass loss rate history needs to be invoked to reconcile data at low- and high-J, a scenario already mentioned by several authors to explain observations of WX Psc.

  16. The Circumstellar Envelope of IRC +10216.

    NASA Astrophysics Data System (ADS)

    Keady, John Joseph

    1982-03-01

    Using recently obtained spatial and spectral line data on the circumstellar envelope of IRC +10216, we have attempted to semi-empirically probe the conditions in this envelope. The computational techniques utilized in our analysis accurately incorporate the effects of geometrical extension and velocity fields on the radiative transfer. We have also attempted to account for the non-equilibrium expected in the vibrational level populations of the gas phase species. Our modelling of the spatial distribution of the dust-produced circumstellar radiation field at 5 (mu)m and 11 (mu)m indicates that dust may be condensing in the circumstellar envelope. The dominant opacity source in our calculations, amorphous carbon, also seems to provide sufficient far-infrared flux. Modelling of the SiC emission feature confirms previous results that suggest a nonuniform particle-shape distribution for the SiC. We can produce multi-component absorption lines, very similar to the 2 (mu)m CO first overtone lines seen in IRC +10216, with continuous distributions of material. The requirement is regions of relatively low acceleration. Modelling of our high resolution, high signal-to-noise observations of the CO fundamental and first overtone indicates a mass -loss rate of 1.5(10('-4)) M(,(CIRCLE))/yr. Our calculations to date indicate that the gas reaches terminal velocity between 10 and 20 R(,*). The envelope mass within 100 R(,*) is 3(10('-2)) M(,(CIRCLE)), with the ratio (by mass) of dust to gas being 10('-3). The assumption of a constant mass-loss rate implies an envelope mass of (TURN)1 M(,(CIRCLE)) within 5000 R(,*). The computational techniques utilized are sufficiently adaptable and economical so that considerable future refinement of the modelling is possible.

  17. The envelope-based cyclic periodogram

    NASA Astrophysics Data System (ADS)

    Borghesani, P.

    2015-06-01

    Cyclostationary analysis has proven effective in identifying signal components for diagnostic purposes. A key descriptor in this framework is the cyclic power spectrum, traditionally estimated by the averaged cyclic periodogram and the smoothed cyclic periodogram. A lengthy debate about the best estimator finally found a solution in a cornerstone work by Antoni, who proposed a unified form for the two families, thus allowing a detailed statistical study of their properties. Since then, the focus of cyclostationary research has shifted towards algorithms, in terms of computational efficiency and simplicity of implementation. Traditional algorithms have proven computationally inefficient and the sophisticated "cyclostationary" definition of these estimators slowed their spread in the industry. The only attempt to increase the computational efficiency of cyclostationary estimators is represented by the cyclic modulation spectrum. This indicator exploits the relationship between cyclostationarity and envelope analysis. The link with envelope analysis allows a leap in computational efficiency and provides a "way in" for the understanding by industrial engineers. However, the new estimator lies outside the unified form described above and an unbiased version of the indicator has not been proposed. This paper will therefore extend the analysis of envelope-based estimators of the cyclic spectrum, proposing a new approach to include them in the unified form of cyclostationary estimators. This will enable the definition of a new envelope-based algorithm and the detailed analysis of the properties of the cyclic modulation spectrum. The computational efficiency of envelope-based algorithms will be also discussed quantitatively for the first time in comparison with the averaged cyclic periodogram. Finally, the algorithms will be validated with numerical and experimental examples.

  18. Shifting hydrogen bonds may produce flexible transmembrane helices.

    PubMed

    Cao, Zheng; Bowie, James U

    2012-05-22

    The intricate functions of membrane proteins would not be possible without bends or breaks that are remarkably common in transmembrane helices. The frequent helix distortions are nevertheless surprising because backbone hydrogen bonds should be strong in an apolar membrane, potentially rigidifying helices. It is therefore mysterious how distortions can be generated by the evolutionary currency of random point mutations. Here we show that we can engineer a transition between distinct distorted helix conformations in bacteriorhodopsin with a single-point mutation. Moreover, we estimate the energetic cost of the conformational transitions to be smaller than 1 kcal/mol. We propose that the low energy of distortion is explained in part by the shifting of backbone hydrogen bonding partners. Consistent with this view, extensive backbone hydrogen bond shifts occur during helix conformational changes that accompany functional cycles. Our results explain how evolution has been able to liberally exploit transmembrane helix bending for the optimization of membrane protein structure, function, and dynamics.

  19. A potential smoothing algorithm accurately predicts transmembrane helix packing.

    PubMed

    Pappu, R V; Marshall, G R; Ponder, J W

    1999-01-01

    Potential smoothing, a deterministic analog of stochastic simulated annealing, is a powerful paradigm for the solution of conformational search problems that require extensive sampling, and should be a useful tool in computational approaches to structure prediction and refinement. A novel potential smoothing and search (PSS) algorithm has been developed and applied to predict the packing of transmembrane helices. The highlight of this method is the efficient manner in which it circumvents the combinatorial explosion associated with the large number of minima on multidimensional potential energy surfaces in order to converge to the global energy minimum. Here we show how our potential smoothing and search method succeeds in finding the global minimum energy structure for the glycophorin A (GpA) transmembrane helix dimer by optimizing interhelical van der Waals interactions over rigid and semi-rigid helices. Structures obtained from our ab initio predictions are in close agreement with recent experimental data.

  20. A monodisperse transmembrane α-helical peptide barrel

    NASA Astrophysics Data System (ADS)

    Mahendran, Kozhinjampara R.; Niitsu, Ai; Kong, Lingbing; Thomson, Andrew R.; Sessions, Richard B.; Woolfson, Derek N.; Bayley, Hagan

    2017-05-01

    The fabrication of monodisperse transmembrane barrels formed from short synthetic peptides has not been demonstrated previously. This is in part because of the complexity of the interactions between peptides and lipids within the hydrophobic environment of a membrane. Here we report the formation of a transmembrane pore through the self-assembly of 35 amino acid α-helical peptides. The design of the peptides is based on the C-terminal D4 domain of the Escherichia coli polysaccharide transporter Wza. By using single-channel current recording, we define discrete assembly intermediates and show that the pore is most probably a helix barrel that contains eight D4 peptides arranged in parallel. We also show that the peptide pore is functional and capable of conducting ions and binding blockers. Such α-helix barrels engineered from peptides could find applications in nanopore technologies such as single-molecule sensing and nucleic-acid sequencing.

  1. The Lantibiotic Nisin Induces Transmembrane Movement of a Fluorescent Phospholipid

    PubMed Central

    Moll, Gert N.; Konings, Wil N.; Driessen, Arnold J. M.

    1998-01-01

    Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl}-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30°C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane. PMID:9852000

  2. Transmembrane current imaging in the heart during pacing and fibrillation.

    PubMed

    Gray, Richard A; Mashburn, David N; Sidorov, Veniamin Y; Roth, Bradley J; Pathmanathan, Pras; Wikswo, John P

    2013-10-01

    Recently, we described a method to quantify the time course of total transmembrane current (Im) and the relative role of its two components, a capacitive current (Ic) and a resistive current (Iion), corresponding to the cardiac action potential during stable propagation. That approach involved recording high-fidelity (200 kHz) transmembrane potential (Vm) signals with glass microelectrodes at one site using a spatiotemporal coordinate transformation via measured conduction velocity. Here we extend our method to compute these transmembrane currents during stable and unstable propagation from fluorescence signals of Vm at thousands of sites (3 kHz), thereby introducing transmembrane current imaging. In contrast to commonly used linear Laplacians of extracellular potential (Ve) to compute Im, we utilized nonlinear image processing to compute the required second spatial derivatives of Vm. We quantified the dynamic spatial patterns of current density of Im and Iion for both depolarization and repolarization during pacing (including nonplanar patterns) by calibrating data with the microelectrode signals. Compared to planar propagation, we found that the magnitude of Iion was significantly reduced at sites of wave collision during depolarization but not repolarization. Finally, we present uncalibrated dynamic patterns of Im during ventricular fibrillation and show that Im at singularity sites was monophasic and positive with a significant nonzero charge (Im integrated over 10 ms) in contrast with nonsingularity sites. Our approach should greatly enhance the understanding of the relative roles of functional (e.g., rate-dependent membrane dynamics and propagation patterns) and static spatial heterogeneities (e.g., spatial differences in tissue resistance) via recordings during normal and compromised propagation, including arrhythmias. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. The Psp system of Mycobacterium tuberculosis integrates envelope stress-sensing and envelope-preserving functions.

    PubMed

    Datta, Pratik; Ravi, Janani; Guerrini, Valentina; Chauhan, Rinki; Neiditch, Matthew B; Shell, Scarlet S; Fortune, Sarah M; Hancioglu, Baris; Igoshin, Oleg A; Gennaro, Maria Laura

    2015-08-01

    The bacterial envelope integrates essential stress-sensing and adaptive functions; thus, envelope-preserving functions are important for survival. In Gram-negative bacteria, envelope integrity during stress is maintained by the multi-gene Psp response. Mycobacterium tuberculosis was thought to lack the Psp system since it encodes only pspA and no other psp ortholog. Intriguingly, pspA maps downstream from clgR, which encodes a transcription factor regulated by the MprAB-σ(E) envelope-stress-signaling system. clgR inactivation lowered ATP concentration during stress and protonophore treatment-induced clgR-pspA expression, suggesting that these genes express Psp-like functions. We identified a four-gene set - clgR, pspA (rv2744c), rv2743c, rv2742c - that is regulated by clgR and in turn regulates ClgR activity. Regulatory and protein-protein interactions within the set and a requirement of the four genes for functions associated with envelope integrity and surface-stress tolerance indicate that a Psp-like system has evolved in mycobacteria. Among Actinobacteria, the four-gene module occurred only in tuberculous mycobacteria and was required for intramacrophage growth, suggesting links between its function and mycobacterial virulence. Additionally, the four-gene module was required for MprAB-σ(E) stress-signaling activity. The positive feedback between envelope-stress-sensing and envelope-preserving functions allows sustained responses to multiple, envelope-perturbing signals during chronic infection, making the system uniquely suited to tuberculosis pathogenesis.

  4. Anesthetics Target Interfacial Transmembrane Sites in Nicotinic Acetylcholine Receptors

    PubMed Central

    Forman, Stuart A.; Chiara, David C.; Miller, Keith W.

    2014-01-01

    General anesthetics are a heterogeneous group of small amphiphilic ligands that interact weakly at multiple allosteric sites on many pentameric ligand gated ion channels (pLGICs), resulting in either inhibition, potentiation of channel activity, or both. Allosteric principles imply that modulator sites must change configuration and ligand affinity during receptor state transitions. Thus, general anesthetics and related compounds are useful both as state-dependent probes of receptor structure and as potentially selective modulators of pLGIC functions. This review focuses on general anesthetic sites in nicotinic acetylcholine receptors, which were among the first anesthetic-sensitive pLGIC experimental models studied, with particular focus on sites formed by transmembrane domain elements. Structural models place many of these sites at interfaces between two or more pLGIC transmembrane helices both within subunits and between adjacent subunits, and between transmembrane helices and either lipids (the lipid-protein interface) or water (i.e. the ion channel). A single general anesthetic may bind at multiple allosteric sites in pLGICs, producing a net effect of either inhibition (e.g. blocking the ion channel) or enhanced channel gating (e.g. inter-subunit sites). Other general anesthetic sites identified by photolabeling or crystallography are tentatively linked to functional effects, including intra-subunit helix bundle sites and the lipid-protein interface. PMID:25316107

  5. Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors.

    PubMed

    Bye, Chris R; Jönsson, Marie E; Björklund, Anders; Parish, Clare L; Thompson, Lachlan H

    2015-04-14

    An important challenge for the continued development of cell therapy for Parkinson's disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a "FACS-array" approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations.

  6. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  7. Splice isoform estrogen receptors as integral transmembrane proteins.

    PubMed

    Kim, Kyung Hee; Toomre, Derek; Bender, Jeffrey R

    2011-11-01

    In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus-truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element-luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets.

  8. An analysis of oligomerization interfaces in transmembrane proteins

    PubMed Central

    2013-01-01

    Background The amount of transmembrane protein (TM) structures solved to date is now large enough to attempt large scale analyses. In particular, extensive studies of oligomeric interfaces in the transmembrane region are now possible. Results We have compiled the first fully comprehensive set of validated transmembrane protein interfaces in order to study their features and assess what differentiates them from their soluble counterparts. Conclusions The general features of TM interfaces do not differ much from those of soluble proteins: they are large, tightly packed and possess many interface core residues. In our set, membrane lipids were not found to significantly mediate protein-protein interfaces. Although no G protein-coupled receptor (GPCR) was included in the validated set, we analyzed the crystallographic dimerization interfaces proposed in the literature. We found that the putative dimer interfaces proposed for class A GPCRs do not show the usual patterns of stable biological interfaces, neither in terms of evolution nor of packing, thus they likely correspond to crystal interfaces. We cannot however rule out the possibility that they constitute transient or weak interfaces. In contrast we do observe a clear signature of biological interface for the proposed dimer of the class F human Smoothened receptor. PMID:24134166

  9. Yeast Aquaglyceroporins Use the Transmembrane Core to Restrict Glycerol Transport*

    PubMed Central

    Geijer, Cecilia; Ahmadpour, Doryaneh; Palmgren, Madelene; Filipsson, Caroline; Klein, Dagmara Medrala; Tamás, Markus J.; Hohmann, Stefan; Lindkvist-Petersson, Karin

    2012-01-01

    Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities. PMID:22593571

  10. Transmembrane chloride flux in tissue-cultured chick heart cells

    PubMed Central

    1983-01-01

    To evaluate the transmembrane movement of chloride in a preparation of cardiac muscle lacking the extracellular diffusion limitations of natural specimens, intracellular chloride concentration ( [Cl] i) and transmembrane 36Cl efflux have been determined in growth-oriented embryonic chick heart cells in tissue culture. Using the method of isotopic equilibrium, [Cl]i was 25.1 +/- 7.3 mmol x (liter cell water)- 1, comparable to the value of 24.9 +/- 5.4 mmol x (liter cell water)-1 determined by coulometric titration. Two cellular 36Cl compartments were found; one exchanged with a rate constant of 0.67 +/- 0.12 min-1 and was associated with the cardiac muscle cells; the other, attributed to the fibroblasts, exchanged with a rate constant of 0.18 +/- 0.05 min- 1. At 37 degrees C, transmembrane Cl flux of cardiac muscle under steady-state conditions was 30 pmol x cm-2 x s-1. In K-free, normal, or high-Ko solutions, the responses of the membrane potential to changes in external Cl concentration suggested that chloride conductance was low. These results indicate that Cl transport across the myocardial cell membrane is more rapid than K transport and is largely electrically silent. PMID:6864192

  11. Transmembrane allosteric coupling of the gates in a potassium channel

    PubMed Central

    Wylie, Benjamin J.; Bhate, Manasi P.; McDermott, Ann E.

    2014-01-01

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98–103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening. PMID:24344306

  12. Transmembrane Helix Assembly by Max-Min Ant System Algorithm.

    PubMed

    Sujaree, Kanon; Kitjaruwankul, Sunan; Boonamnaj, Panisak; Supunyabut, Chirayut; Sompornpisut, Pornthep

    2015-12-01

    Because of the rapid progress in biochemical and structural studies of membrane proteins, considerable attention has been given on developing efficient computational methods for solving low-to-medium resolution structures using sparse structural data. In this study, we demonstrate a novel algorithm, max-min ant system (MMAS), designed to find an assembly of α-helical transmembrane proteins using a rigid helix arrangement guided by distance constraints. The new algorithm generates a large variety with finite number of orientations of transmembrane helix bundle and finds the solution that is matched with the provided distance constraints based on the behavior of ants to search for the shortest possible path between their nest and the food source. To demonstrate the efficiency of the novel search algorithm, MMAS is applied to determine the transmembrane packing of KcsA and MscL ion channels from a limited distance information extracted from the crystal structures, and the packing of KvAP voltage sensor domain using a set of 10 experimentally determined constraints, and the results are compared with those of two popular used stochastic methods, simulated annealing Monte Carlo method and genetic algorithm. © 2015 John Wiley & Sons A/S.

  13. Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

    PubMed Central

    Jönsson, Marie E.; Björklund, Anders; Parish, Clare L.; Thompson, Lachlan H.

    2015-01-01

    An important challenge for the continued development of cell therapy for Parkinson’s disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a “FACS-array” approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations. PMID:25775569

  14. Probing transmembrane mechanical coupling and cytomechanics using magnetic twisting cytometry

    NASA Technical Reports Server (NTRS)

    Wang, N.; Ingber, D. E.

    1995-01-01

    We recently developed a magnetic twisting cytometry technique that allows us to apply controlled mechanical stresses to specific cell surface receptors using ligand-coated ferromagnetic microbeads and to simultaneously measure the mechanical response in living cells. Using this technique, we have previously shown the following: (i) beta 1 integrin receptors mediate mechanical force transfer across the cell surface and to the cytoskeleton, whereas other transmembrane receptors (e.g., scavenger receptors) do not; (ii) cytoskeletal stiffness increases in direct proportion to the level of stress applied to integrins; and (iii) the slope of this linear stiffening response differs depending on the shape of the cell. We now show that different integrins (beta 1, alpha V beta 3, alpha V, alpha 5, alpha 2) and other transmembrane receptors (scavenger receptor, platelet endothelial cell adhesion molecule) differ in their ability to mediate force transfer across the cell surface. In addition, the linear stiffening behavior previously observed in endothelial cells was found to be shared by other cell types. Finally, we demonstrate that dynamic changes in cell shape that occur during both cell spreading and retraction are accompanied by coordinate changes in cytoskeletal stiffness. Taken together, these results suggest that the magnetic twisting cytometry technique may be a powerful and versatile tool for studies analyzing the molecular basis of transmembrane mechanical coupling to the cytoskeleton as well as dynamic relations between changes in cytoskeletal structure and alterations in cell form and function.

  15. Transmembrane allosteric coupling of the gates in a potassium channel.

    PubMed

    Wylie, Benjamin J; Bhate, Manasi P; McDermott, Ann E

    2014-01-07

    It has been hypothesized that transmembrane allostery is the basis for inactivation of the potassium channel KcsA: opening the intracellular gate is spontaneously followed by ion expulsion at the extracellular selectivity filter. This suggests a corollary: following ion expulsion at neutral pH, a spontaneous global conformation change of the transmembrane helices, similar to the motion involved in opening, is expected. Consequently, both the low potassium state and the low pH state of the system could provide useful models for the inactivated state. Unique NMR studies of full-length KcsA in hydrated bilayers provide strong evidence for such a mutual coupling across the bilayer: namely, upon removing ambient potassium ions, changes are seen in the NMR shifts of carboxylates E118 and E120 in the pH gate in the hinges of the inner transmembrane helix (98-103), and in the selectivity filter, all of which resemble changes seen upon acid-induced opening and inhibition and suggest that ion release can trigger channel helix opening.

  16. Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering.

    PubMed

    Bugaj, L J; Spelke, D P; Mesuda, C K; Varedi, M; Kane, R S; Schaffer, D V

    2015-04-22

    Transmembrane receptors are the predominant conduit through which cells sense and transduce extracellular information into intracellular biochemical signals. Current methods to control and study receptor function, however, suffer from poor resolution in space and time and often employ receptor overexpression, which can introduce experimental artefacts. We report a genetically encoded approach, termed Clustering Indirectly using Cryptochrome 2 (CLICR), for spatiotemporal control over endogenous transmembrane receptor activation, enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2). CLICR offers a modular platform to enable photocontrol of the clustering of diverse transmembrane receptors including fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR) and integrins in multiple cell types including neural stem cells. Furthermore, light-inducible manipulation of endogenous receptor tyrosine kinase (RTK) activity can modulate cell polarity and establish phototaxis in fibroblasts. The resulting spatiotemporal control over cellular signalling represents a powerful new optogenetic framework for investigating and controlling cell function and fate.

  17. Nuclear membrane: nuclear envelope PORosity in fission yeast meiosis.

    PubMed

    Sazer, Shelley

    2010-11-09

    The fission yeast Schizosaccharomyces pombe undergoes closed mitosis but 'virtual nuclear envelope breakdown' at anaphase of meiosis II, in which the nuclear envelope is structurally closed but functionally open.

  18. Thermodynamics and fluid dynamics of the double shell (envelope) house

    SciTech Connect

    Reno, V.

    1980-01-01

    The concepts of the envelope house are summarized and a systems approach to the house heat energy flows is presented. Some basic principles of physics in the area of thermodynamic conduction are discussed in relation to the envelope concept. (MHR)

  19. An alternative conformation of the gp41 heptad repeat 1 region coiled coil exists in the human immunodeficiency virus (HIV-1) envelope glycoprotein precursor

    SciTech Connect

    Mische, Claudia C.; Yuan Wen; Strack, Bettina; Craig, Stewart; Farzan, Michael; Sodroski, Joseph . E-mail: joseph_sodroski@dfci.harvard.edu

    2005-07-20

    The human immunodeficiency virus (HIV-1) transmembrane envelope glycoprotein, gp41, which mediates virus-cell fusion, exists in at least three different conformations within the trimeric envelope glycoprotein complex. The structures of the prefusogenic and intermediate states are unknown; structures representing the postfusion state have been solved. In the postfusion conformation, three helical heptad repeat 2 (HR2) regions pack in an antiparallel fashion into the hydrophobic grooves on the surface of a triple-helical coiled coil formed by the heptad repeat 1 (HR1) regions. We studied the prefusogenic conformation of gp41 by mutagenic alteration of membrane-anchored and soluble forms of the HIV-1 envelope glycoproteins. Our results indicate that, in the HIV-1 envelope glycoprotein precursor, the gp41 HR1 region is in a conformation distinct from that of a trimeric coiled coil. Thus, the central gp41 coiled coil is formed during the transition of the HIV-1 envelope glycoproteins from the precursor state to the receptor-bound intermediate.

  20. Redirecting the Cyanobacterial Bicarbonate Transporters BicA and SbtA to the Chloroplast Envelope: Soluble and Membrane Cargos Need Different Chloroplast Targeting Signals in Plants

    PubMed Central

    Rolland, Vivien; Badger, Murray R.; Price, G. Dean

    2016-01-01

    Most major crops used for human consumption are C3 plants, which yields are limited by photosynthetic inefficiency. To circumvent this, it has been proposed to implement the cyanobacterial CO2-concentrating mechanism (CCM), principally consisting of bicarbonate transporters and carboxysomes, into plant chloroplasts. As it is currently not possible to recover homoplasmic transplastomic monocots, foreign genes must be introduced in these plants via nuclear transformation. Consequently, it is paramount to ensure that resulting proteins reach the appropriate sub-cellular compartment, which for cyanobacterial transporters BicA and SbtA, is the chloroplast inner-envelope membrane (IEM). At present, targeting signals to redirect large transmembrane proteins from non-chloroplastic organisms to plant chloroplast envelopes are unknown. The goal of this study was to identify such signals, using agrobacteria-mediated transient expression and confocal microscopy to determine the sub-cellular localization of ∼37 GFP-tagged chimeras. Initially, fragments of chloroplast proteins known to target soluble cargos to the stroma were tested for their ability to redirect BicA, but they proved ineffective. Next, different N-terminal regions from Arabidopsis IEM transporters were tested. We demonstrated that the N-terminus of AtHP59, AtPLGG1 or AtNTT1 (92–115 amino acids), containing a cleavable chloroplast transit peptide (cTP) and a membrane protein leader (MPL), was sufficient to redirect BicA or SbtA to the chloroplast envelope. This constitutes the first evidence that nuclear-encoded transmembrane proteins from non-chloroplastic organisms can be targeted to the envelope of plant chloroplasts; a finding which represents an important advance in chloroplast engineering by opening up the door to further manipulation of the chloroplastic envelope. PMID:26973659

  1. The action of three antiseptics/disinfectants against enveloped and non-enveloped viruses.

    PubMed

    Wood, A; Payne, D

    1998-04-01

    The antiviral action of chloroxylenol, benzalkonium chloride and cetrimide/chlorhexidine was assessed against a range of enveloped and non-enveloped human viruses using a suspension test method. Viral suspensions of 10(6)-10(7) pfu/TCID50 or sfu were prepared in each of the antiseptic/disinfectant solutions in the presence of a bovine serum/yeast extract mixture to simulate 'dirty conditions'. During incubation, aliquots were removed at predetermined timepoints up to 10 min to assess the kinetics of inactivation. Results indicate that all products were effective in inactivating the enveloped viruses herpes simplex virus type 1 and human immunodeficiency virus type 1, whilst being ineffective in inactivating human coronavirus, also enveloped, and the non-enveloped viruses. The exception to this was the benzalkonium chloride-based product (Dettol Hospital Concentrate) which was active against the non-enveloped human coxsackie virus. Four antiseptic/disinfectant solutions with chloroxylenol, benzalkonium chloride, cetrimide/chlorhexidine and povidone-iodine were also assessed for antiviral effect against human immunodeficiency virus in the presence of whole human blood. All four solutions proved to be effective within 1 min despite the cytotoxic nature of the compounds to the detection system.

  2. Effects of envelope shape on interaural envelope delay sensitivity in acoustic and electric hearing.

    PubMed

    Laback, Bernhard; Zimmermann, Inge; Majdak, Piotr; Baumgartner, Wolf-Dieter; Pok, Stefan-Marcel

    2011-09-01

    The envelope shape is important for the perception of interaural time difference (ITD) in the envelope as supported by the improved sensitivity for transposed tones compared to sinusoidally amplitude-modulated (SAM) tones. The present study investigated the effects of specific envelope parameters in nine normal-hearing (NH) and seven cochlear-implant (CI) listeners, using high-rate carriers with 27-Hz trapezoidal modulation. In NH listeners, increasing the off time (the silent interval in each modulation cycle) up to 12 ms, increasing the envelope slope from 6 to 8 dB/ms, and increasing the peak level improved ITD sensitivity. The combined effect of the off time and slope accounts for the gain in sensitivity for transposed tones relative to SAM tones. In CI listeners, increasing the off time up to 20 ms improved sensitivity, but increasing the slope showed no systematic effect. A 27-pulses/s electric pulse train, representing a special case of modulation with infinitely steep slopes and maximum possible off time, yielded considerably higher sensitivity compared to the best condition with trapezoidal modulation. Overall, the results of this study indicate that envelope-ITD sensitivity could be improved by using CI processing schemes that simultaneously increase the off time and the peak level of the signal envelope. © 2011 Acoustical Society of America

  3. Envelope-like retrotransposons in the plant kingdom: evidence of their presence in gymnosperms (Pinus pinaster).

    PubMed

    Miguel, Célia; Simões, Marta; Oliveira, Maria Margarida; Rocheta, Margarida

    2008-11-01

    Retroviruses differ from retrotransposons due to their infective capacity, which depends critically on the encoded envelope. Some plant retroelements contain domains reminiscent of the env of animal retroviruses but the number of such elements described to date is restricted to angiosperms. We show here the first evidence of the presence of putative env-like gene sequences in a gymnosperm species, Pinus pinaster (maritime pine). Using a degenerate primer approach for conserved domains of RNaseH gene, three clones from putative envelope-like retrotransposons (PpRT2, PpRT3, and PpRT4) were identified. The env-like sequences of P. pinaster clones are predicted to encode proteins with transmembrane domains. These sequences showed identity scores of up to 30% with env-like sequences belonging to different organisms. A phylogenetic analysis based on protein alignment of deduced aminoacid sequences revealed that these clones clustered with env-containing plant retrotransposons, as well as with retrotransposons from invertebrate organisms. The differences found among the sequences of maritime pine clones isolated here suggest the existence of different putative classes of env-like retroelements. The identification for the first time of env-like genes in a gymnosperm species may support the ancestrality of retroviruses among plants shedding light on their role in plant evolution.

  4. The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing.

    PubMed

    Rashmi, R N; Eckes, Beate; Glöckner, Gernot; Groth, Marco; Neumann, Sascha; Gloy, Joachim; Sellin, Lorenz; Walz, Gerd; Schneider, Maria; Karakesisoglou, Iakowos; Eichinger, Ludwig; Noegel, Angelika A

    2012-03-01

    Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing.

  5. System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics

    PubMed Central

    Zuleger, Nikolaj; Kelly, David A.; Richardson, A. Christine; Kerr, Alastair R. W.; Goldberg, Martin W.; Goryachev, Andrew B.

    2011-01-01

    The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum–inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane. PMID:21444689

  6. Structural models of the membrane anchors of envelope glycoproteins E1 and E2 from pestiviruses

    SciTech Connect

    Wang, Jimin Li, Yue; Modis, Yorgo

    2014-04-15

    The membrane anchors of viral envelope proteins play essential roles in cell entry. Recent crystal structures of the ectodomain of envelope protein E2 from a pestivirus suggest that E2 belongs to a novel structural class of membrane fusion machinery. Based on geometric constraints from the E2 structures, we generated atomic models of the E1 and E2 membrane anchors using computational approaches. The E1 anchor contains two amphipathic perimembrane helices and one transmembrane helix; the E2 anchor contains a short helical hairpin stabilized in the membrane by an arginine residue, similar to flaviviruses. A pair of histidine residues in the E2 ectodomain may participate in pH sensing. The proposed atomic models point to Cys987 in E2 as the site of disulfide bond linkage with E1 to form E1–E2 heterodimers. The membrane anchor models provide structural constraints for the disulfide bonding pattern and overall backbone conformation of the E1 ectodomain. - Highlights: • Structures of pestivirus E2 proteins impose constraints on E1, E2 membrane anchors. • Atomic models of the E1 and E2 membrane anchors were generated in silico. • A “snorkeling” arginine completes the short helical hairpin in the E2 membrane anchor. • Roles in pH sensing and E1–E2 disulfide bond formation are proposed for E1 residues. • Implications for E1 ectodomain structure and disulfide bonding pattern are discussed.

  7. The nuclear envelope protein Nesprin-2 has roles in cell proliferation and differentiation during wound healing

    PubMed Central

    Rashmi, R.N.; Eckes, Beate; Glöckner, Gernot; Groth, Marco; Neumann, Sascha; Gloy, Joachim; Sellin, Lorenz; Walz, Gerd; Schneider, Maria; Karakesisoglou, Iakowos; Eichinger, Ludwig; Noegel, Angelika A.

    2012-01-01

    Nesprin-2, a type II transmembrane protein of the nuclear envelope, is a component of the LINC complex that connects the nuclear lamina with the actin cytoskeleton. To elucidate its physiological role we studied wound healing in Nesprin-2 Giant deficient mice and found that a loss of the protein affected wound healing particularly at later stages during fibroblast differentiation and keratinocyte proliferation leading to delayed wound closure. We identified altered expression and localization of transcription factors as one of the underlying mechanisms. Furthermore, the actin cytoskeleton which surrounds the nucleus was altered and keratinocyte migration was slowed down and focal adhesion formation enhanced. We also uncovered a new activity of Nesprin-2. When we probed for an interaction of Nesprin-2 Giant with chromatin we observed in ChIP Seq experiments an association of the protein with heterochromatic and centromeric DNA. Through this activity Nesprin-2 can affect the nuclear landscape and gene regulation. Our findings suggest functions for Nesprin-2 at the nuclear envelope (NE) in gene regulation and in regulation of the actin cytoskeleton which impact on wound healing. PMID:22198684

  8. Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function

    PubMed Central

    Jara, Oscar; Acuña, Rodrigo; García, Isaac E.; Maripillán, Jaime; Figueroa, Vania; Sáez, Juan C.; Araya-Secchi, Raúl; Lagos, Carlos F.; Pérez-Acle, Tomas; Berthoud, Viviana M.; Beyer, Eric C.; Martínez, Agustín D.

    2012-01-01

    To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37–Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step. PMID:22787277

  9. Anion Gap Blood Test

    MedlinePlus

    ... Why do I need an anion gap blood test? Your health care provider may have ordered an anion gap blood ... which is a blood test. During a blood test, a health care professional uses a small needle to take a ...

  10. Solar Effective Envelope Design Advisor (SEEDA)

    NASA Astrophysics Data System (ADS)

    Mahaek, Ekkachai

    The lack of effort by mainstream architects in integrating energy-efficient strategies in architectural designing is due to the complexity in a building's energy conscious concepts and theories, the difficulties to visualize and quantify energy consumption, and the late implementing of energy consumption analysis in the conventional design process. This task would be accomplishing by a building system's engineer where results might be determined only after the basic architectural design has been completed. An effective simple tool and method should then be available to assist architects in building's energy-efficient designing at the beginning of the design. The building's energy consumption is directly and mainly influenced by the relationship of the sun, site, and its building configuration. The solar radiations will first impact on the building's envelope, which will have a direct effect on the amount of energy a building will consume. If an architect can define or map the intensity of solar energy on the site's buildable volume, and use this information to determine the levels of solar insolation, a more energy efficient building form can be proposed. This research hypothesis has shared the fundamental techniques of the Solar Envelope projection by Professor Ralph Knowles [Knowles, 1981] of the University of Southern California. However a different approach is taken by including the influence of regional restrictions and the surrounding buildings' shadows when projecting of solar volumes and solar envelope. The research methodology will discuss the development of a computer-based approach to develop a three-dimensional architectural form based on an insolation map related to the design site. The prototype computer program is referred as the Solar Effective Envelope Design Advisor (SEEDA). The solar insolation volume of the site is determined by integrating three types of computer-generated models include the Buildable Volume model based on design constraints

  11. Application of the Envelope Difference Index to Spectrally Sparse Speech

    ERIC Educational Resources Information Center

    Souza, Pamela; Hoover, Eric; Gallun, Frederick

    2012-01-01

    Purpose: Amplitude compression is a common hearing aid processing strategy that can improve speech audibility and loudness comfort but also has the potential to alter important cues carried by the speech envelope. In previous work, a measure of envelope change, the Envelope Difference Index (EDI; Fortune, Woodruff, & Preves, 1994), was moderately…

  12. Analysis of Building Envelope Construction in 2003 CBECS

    SciTech Connect

    Winiarski, David W.; Halverson, Mark A.; Jiang, Wei

    2007-06-01

    The purpose of this analysis is to determine "typical" building envelope characteristics for buildings built after 1980. We address three envelope components in this paper - roofs, walls, and window area. These typical building envelope characteristics were used in the development of DOE’s Reference Buildings .

  13. 200 Area Deactivation Project Facilities Authorization Envelope Document

    SciTech Connect

    DODD, E.N.

    2000-03-28

    Project facilities as required by HNF-PRO-2701, Authorization Envelope and Authorization Agreement. The Authorization Agreements (AA's) do not identify the specific set of environmental safety and health requirements that are applicable to the facility. Therefore, the facility Authorization Envelopes are defined here to identify the applicable requirements. This document identifies the authorization envelopes for the 200 Area Deactivation.

  14. Application of the Envelope Difference Index to Spectrally Sparse Speech

    ERIC Educational Resources Information Center

    Souza, Pamela; Hoover, Eric; Gallun, Frederick

    2012-01-01

    Purpose: Amplitude compression is a common hearing aid processing strategy that can improve speech audibility and loudness comfort but also has the potential to alter important cues carried by the speech envelope. In previous work, a measure of envelope change, the Envelope Difference Index (EDI; Fortune, Woodruff, & Preves, 1994), was moderately…

  15. Kronig-Penney-like description for band gap variation in SiC polytypes

    NASA Astrophysics Data System (ADS)

    Backes, W. H.; de Nooij, F. C.; Bobbert, P. A.; van Haeringen, W.

    1996-02-01

    A one-dimensional Kronig-Penney-like model for envelope wave functions is presented to explain the band gap variation of SiC polytypes. In this model the envelope functions obey discontinuous boundary conditions. The electronic band gaps of cubic and several hexagonal and rhombohedral SiC polytypes are calculated. The polytypic superlattices are assumed to be stackings of differently sized and orientated cubic SiC segments. The empirical Choyke-Hamilton-Patrick relation is understood and deviating trends for small hexagonalities and rhombohedral modifications are predicted.

  16. Behind the Pay Gap

    ERIC Educational Resources Information Center

    Dey, Judy Goldberg; Hill, Catherine

    2007-01-01

    Women have made remarkable gains in education during the past three decades, yet these achievements have resulted in only modest improvements in pay equity. The gender pay gap has become a fixture of the U.S. workplace and is so ubiquitous that many simply view it as normal. "Behind the Pay Gap" examines the gender pay gap for college graduates.…

  17. Dysfunction of Bovine Endogenous Retrovirus K2 Envelope Glycoprotein Is Related to Unsuccessful Intracellular Trafficking

    PubMed Central

    2014-01-01

    ABSTRACT Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. IMPORTANCE Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition

  18. Dysfunction of bovine endogenous retrovirus K2 envelope glycoprotein is related to unsuccessful intracellular trafficking.

    PubMed

    Nakaya, Yuki; Miyazawa, Takayuki

    2014-06-01

    Endogenous retroviruses (ERVs) are the remnants of retroviral infection of ancestral germ cells. Mutations introduced into ERVs halt the production of infectious agents, but their effects on the function of retroviral proteins are not fully understood. Retroviral envelope glycoproteins (Envs) are utilized in membrane fusion during viral entry, and we recently identified intact coding sequences for bovine endogenous retrovirus K1 (BERV-K1) and BERV-K2 Envs. Amino acid sequences of BERV-K1 Env (also called Fematrin-1) and BERV-K2 Env are similar, and both viruses are classified in the genus Betaretrovirus. While Fematrin-1 plays an important role in cell-to-cell fusion in bovine placenta, the BERV-K2 envelope gene is marginally expressed in vivo, and its recombinant Env protein is defective in membrane fusion due to inefficient cleavage of surface (SU) and transmembrane subunits. Here, we conducted chimeric analyses of Fematrin-1 and BERV-K2 Envs and revealed that defective maturation of BERV-K2 Env contributed to failed intracellular trafficking. Fluorescence microscopy and flow cytometric analysis suggested that in contrast to Fematrin-1 Env, BERV-K2 Env could not be transported from the endoplasmic reticulum to the trans-Golgi network, where cellular proteases required for processing retroviral Envs are localized. We also identified that one of the responsive regions of this phenomenon resided within a 65-amino-acid region of BERV-K2 SU. This is the first report to identify that retroviral Env SU is involved in the regulation of intracellular trafficking, and it may help to elucidate the maturation process of Fematrin-1 and other related Envs. Retroviruses utilize envelope glycoproteins (Envs) to enter host target cells. Mature retroviral Env is a heterodimer, which consists of surface (SU) and transmembrane (TM) subunits that are generated by the cleavage of an Env precursor protein in the trans-Golgi network. SU and TM mediate the recognition of the entry

  19. A Failure of Coalition Leadership: The Falaise-Argentan Gap

    DTIC Science & Technology

    2007-11-02

    when the Canadian advance was slowed eight miles north of Falaise on August 9th. 52 The evidence suggests thatthe Canadian attack, as the pincer ...of Argentan and sending two divisions toward the Seine River for a larger and longer pincer envelopment movement.6 This masterful decision by...Montgomery for the movement of the northern pincer (required to link the two forces and close the gap), it was doomed to disjointed failure. 67 When the

  20. Spark gap device for precise switching

    DOEpatents

    Boettcher, Gordon E.

    1984-01-01

    A spark gap device for precise switching of an energy storage capacitor into an exploding bridge wire load is disclosed. Niobium electrodes having a melting point of 2,415 degrees centrigrade are spaced apart by an insulating cylinder to define a spark gap. The electrodes are supported by conductive end caps which, together with the insulating cylinder, form a hermetically sealed chamber filled with an inert, ionizable gas, such as pure xenon. A quantity of solid radioactive carbon-14 within the chamber adjacent the spark gap serves as a radiation stabilizer. The sides of the electrodes and the inner wall of the insulating cylinder are spaced apart a sufficient distance to prevent unwanted breakdown initiation. A conductive sleeve may envelop the outside of the insulating member from the midpoint of the spark gap to the cap adjacent the cathode. The outer metallic surfaces of the device may be coated with a hydrogen-impermeable coating to lengthen the shelf life and operating life of the device. The device breaks down at about 1,700 volts for input voltage rates up to 570 volts/millisecond and allows peak discharge currents of up to 3,000 amperes from a 0.3 microfarad energy storage capacitor for more than 1,000 operations.

  1. Spark gap device for precise switching

    DOEpatents

    Boettcher, G.E.

    1984-10-02

    A spark gap device for precise switching of an energy storage capacitor into an exploding bridge wire load is disclosed. Niobium electrodes having a melting point of 2,415 degrees centigrade are spaced apart by an insulating cylinder to define a spark gap. The electrodes are supported by conductive end caps which, together with the insulating cylinder, form a hermetically sealed chamber filled with an inert, ionizable gas, such as pure xenon. A quantity of solid radioactive carbon-14 within the chamber adjacent the spark gap serves as a radiation stabilizer. The sides of the electrodes and the inner wall of the insulating cylinder are spaced apart a sufficient distance to prevent unwanted breakdown initiation. A conductive sleeve may envelop the outside of the insulating member from the midpoint of the spark gap to the cap adjacent the cathode. The outer metallic surfaces of the device may be coated with a hydrogen-impermeable coating to lengthen the shelf life and operating life of the device. The device breaks down at about 1,700 volts for input voltage rates up to 570 volts/millisecond and allows peak discharge currents of up to 3,000 amperes from a 0.3 microfarad energy storage capacitor for more than 1,000 operations. 3 figs.

  2. Development of High Specific Strength Envelope Materials

    NASA Astrophysics Data System (ADS)

    Komatsu, Keiji; Sano, Masa-Aki; Kakuta, Yoshiaki

    Progress in materials technology has produced a much more durable synthetic fabric envelope for the non-rigid airship. Flexible materials are required to form airship envelopes, ballonets, load curtains, gas bags and covering rigid structures. Polybenzoxazole fiber (Zylon) and polyalirate fiber (Vectran) show high specific tensile strength, so that we developed membrane using these high specific tensile strength fibers as a load carrier. The main material developed is a Zylon or Vectran load carrier sealed internally with a polyurethane bonded inner gas retention film (EVOH). The external surface provides weather protecting with, for instance, a titanium oxide integrated polyurethane or Tedlar film. The mechanical test results show that tensile strength 1,000 N/cm is attained with weight less than 230g/m2. In addition to the mechanical properties, temperature dependence of the joint strength and solar absorptivity and emissivity of the surface are measured. 

  3. Small carbon chains in circumstellar envelopes

    NASA Astrophysics Data System (ADS)

    Hargreaves, R. J.; Hinkle, K.; Bernath, P. F.

    2014-11-01

    Observations of carbon-rich circumstellar envelopes were made using the Phoenix spectrograph on the Gemini South telescope to determine the abundance of small carbon chain molecules. Vibration-rotation lines of the ν3 antisymmetric stretch of C3 near 2040 cm-1 (4.902 μm) have been used to determine the column density for four carbon-rich circumstellar envelopes: CRL 865, CRL 1922, CRL 2023 and IRC +10216. We additionally calculate the column density of C5 for IRC +10216, and provide an upper limit for five more objects. An upper limit estimate for the C7 column density is also provided for IRC+10216. A comparison of these column densities suggests a revision to current circumstellar chemical models may be needed.

  4. Constant envelope chirped OFDM power efficiency

    NASA Astrophysics Data System (ADS)

    Dida, Mussa A.; Hao, Huan; Anjum, M. R.; Ran, Tao

    2016-10-01

    Fractional Fourier OFDM or simply chirped OFDM performs better in time-frequency selective channel than its convectional OFDM. Although chirped OFDM outperforms OFDM it still inherits Peak to Average Power Ratio (PAPR) drawback as a convectional OFDM. To eliminate PAPR drawback Constant Envelope OFDM was developed and for better performance in time frequency selective channel Constant Envelope Fractional Fourier OFDM (CE-COFDM) is used. Its BER performance is analyzed and compared to chirped OFDM and OFDM in AWGN and Rayleigh channel. The simulations show the BER performance of CE-COFDM is the same as chirped OFDM and OFDM. The power efficiency of CE-COFDM is also studied and different simulations performed shows CE-COFDM is more power efficient than chirped OFDM and convectional OFDM for class A and class B Linear Power Amplifier (LPA).

  5. Nonlinear Analysis of Airship Envelop Aerolasticity

    NASA Astrophysics Data System (ADS)

    Liu, J. M.; Lu, C. J.; Xue, L. P.

    The large airship in flow field is a flexible body with low rigidity. The distribution of the peripheral flow field around the airship is closely related to its shape. It is essentially one of the Fluid-structure Interaction problems. Based on this, this paper aims at the numerical simulation of nonlinear airship envelop aeroelasticity by means of coupling aerodynamics and structure using an iteration method. The three-dimensional flow around the airship was studied numerically by means of SIMPLE method based on the Finite Volume Method. Two approaches, the linear method whose equilibrium equations are based on the membrance theory of thin shell and the nonlinear method which uses a nonlinear finite element method to account for the large deformation of the airship envelop, are introduced for geometrically deformation of the airship shape. A thin plate spline method is adopted as the interface of exchanging information between the fluid and structure models.

  6. Hydrogen sulfide in a circumstellar envelope

    NASA Technical Reports Server (NTRS)

    Ukita, N.; Morris, M.

    1983-01-01

    A search for hydrogen sulfide in the cool circumstellar envelopes of 25 stars was made using the 1(10)-1(01) rotational line at 1.8 mm. It was detected in the bipolar nebula/OH maser OH231.8+4.2, an object having a high rate of mass loss. An approximate analysis indicates that 1/60 of the sulfur in this outflowing envelope is in the form of H2S, a fraction which may be similar to that in the atmosphere of the central star. In addition, the shape of the observed line profile is discussed in terms of a possible variation of the outflow velocity with latitude above the system's equatorial plane.

  7. Formaldehyde in envelopes of interstellar dark clouds

    NASA Technical Reports Server (NTRS)

    Federman, S. R.; Allen, M.

    1991-01-01

    Observed formaldehyde column densities of 1 x 10 to the 12th - 3 x 10 to the 13th/sq cm in cloud envelopes along lines of sight with A(V) = 1-4 mag can not be explained with the current understanding of interstellar gas phase chemistry. However, these column densities can be reproduced by a simple time-dependent model in which H2CO is supplied to the gas phase by the erosion of icy grain mantles. The release of H2CO from the grain mantles must occur on time scales comparable to the time scales for mixing from the cloud interior to the cloud envelope. Thus, in low-density regions of clouds, it appears that formaldehyde is the second molecule whose gas phase source is primarily ejection from grains. This simple model suggests understanding gas phase steady state in clouds on macroscopic, rather than microscopic, spatial scales.

  8. Uses and misuses of bioclimatic envelope modeling.

    PubMed

    Araújo, Miguel B; Peterson, A Townsend

    2012-07-01

    Bioclimatic envelope models use associations between aspects of climate and species' occurrences to estimate the conditions that are suitable to maintain viable populations. Once bioclimatic envelopes are characterized, they can be applied to a variety of questions in ecology, evolution, and conservation. However, some have questioned the usefulness of these models, because they may be based on implausible assumptions or may be contradicted by empirical evidence. We review these areas of contention, and suggest that criticism has often been misplaced, resulting from confusion between what the models actually deliver and what users wish that they would express. Although improvements in data and methods will have some effect, the usefulness of these models is contingent on their appropriate use, and they will improve mainly via better awareness of their conceptual basis, strengths, and limitations.

  9. Inactivation of enveloped and non-enveloped viruses on seeded human tissues by gamma irradiation.

    PubMed

    Moore, Mark A

    2012-08-01

    Human tissue allografts are widely used in a variety of clinical applications with over 1.5 million implants annually in the US alone. Since the 1990s, most clinically available allografts have been disinfected to minimize risk of disease transmission. Additional safety assurance can be provided by terminal sterilization using low dose gamma irradiation. The impact of such irradiation processing at low temperatures on viruses was the subject of this study. In particular, both human tendon and cortical bone samples were seeded with a designed array of viruses and the ability of gamma irradiation to inactivate those viruses was tested. The irradiation exposures for the samples packed in dry ice were 11.6-12.9 kGy for tendon and 11.6-12.3 kGy for bone, respectively. The viruses, virus types, and log reductions on seeded tendon and bone tissue, respectively, were as follows: Human Immunodeficiency Virus (RNA, enveloped), >2.90 and >3.20; Porcine Parvovirus (DNA, non-enveloped), 1.90 and 1.58; Pseudorabies Virus (DNA, enveloped), 3.80 and 3.79; Bovine Viral Diarrhea Virus (RNA, enveloped), 2.57 and 4.56; and Hepatitis A Virus (RNA, non-enveloped), 2.54 and 2.49, respectively. While proper donor screening, aseptic technique, and current disinfection practices all help reduce the risk of viral transmission from human allograft tissues, data presented here indicate that terminal sterilization using a low temperature, low dose gamma irradiation process inactivates both enveloped and non-enveloped viruses containing either DNA or RNA, thus providing additional assurance of safety from viral transmission.

  10. Gap probability - Measurements and models of a pecan orchard

    NASA Technical Reports Server (NTRS)

    Strahler, Alan H.; Li, Xiaowen; Moody, Aaron; Liu, YI

    1992-01-01

    Measurements and models are compared for gap probability in a pecan orchard. Measurements are based on panoramic photographs of 50* by 135 view angle made under the canopy looking upwards at regular positions along transects between orchard trees. The gap probability model is driven by geometric parameters at two levels-crown and leaf. Crown level parameters include the shape of the crown envelope and spacing of crowns; leaf level parameters include leaf size and shape, leaf area index, and leaf angle, all as functions of canopy position.

  11. Gap probability - Measurements and models of a pecan orchard

    NASA Technical Reports Server (NTRS)

    Strahler, Alan H.; Li, Xiaowen; Moody, Aaron; Liu, YI

    1992-01-01

    Measurements and models are compared for gap probability in a pecan orchard. Measurements are based on panoramic photographs of 50* by 135 view angle made under the canopy looking upwards at regular positions along transects between orchard trees. The gap probability model is driven by geometric parameters at two levels-crown and leaf. Crown level parameters include the shape of the crown envelope and spacing of crowns; leaf level parameters include leaf size and shape, leaf area index, and leaf angle, all as functions of canopy position.

  12. Bound Cyclic Systems with the Envelope Theory

    NASA Astrophysics Data System (ADS)

    Semay, Claude; Buisseret, Fabien

    2017-09-01

    Approximate but reliable solutions of a quantum system with N identical particles can be easily computed with the envelope theory, also known as the auxiliary field method. This technique has been developed for Hamiltonians with arbitrary kinematics and one- or two-body potentials. It is adapted here for cyclic systems with N identical particles, that is to say systems in which a particle i has only an interaction with particles i-1 and i+1 (with N+1≡1).

  13. Envelope instability and the fourth order resonance

    NASA Astrophysics Data System (ADS)

    Li, Chao; Zhao, Ya Liang

    2014-12-01

    The well-known envelope instability or the second order even collective mode [I. Hofmann, Phys. Rev. E 57, 4 (1998)] and the fourth order resonance 4 σ =360 ° due to the nonlinear space charge effect in high intensity beams have been studied previously. A wide stop band around 15° is found in a pure periodic focusing channel. In addition, it is illustrated that the fourth order resonance dominates over the envelope instability and practically replaces it in the stop band [D. Jeon et al., Phys. Rev. ST Accel. Beams 12, 054204 (2009)]. In this paper, for a continuous beam with remarkable space charge, our 2D self-consistent particle-in-cell simulation work with the code topopic shows these two kinds of effects respectively in a periodic focusing defocusing (FD) channel. For a fixed tune depression η =0.8 , a stop band with a width of almost 15° is also demonstrated. Moreover, it is confirmed that analytical results of the rms envelope instability diagram are a valid tool to interpret the width of the stop band. Emittance growth rates in stop band are also well explained. It is found that, for a nearly rms matched beam, the emittance growth in the stop band is almost proportional to the saturation time of the nonlinear instability of the envelope, which happens in a quick manner and takes only a few FD cells. In contrast, the fourth order resonance is independent of rms matching and will be accompanied by beam evolution as "a long term effect" once the related mechanism is excited.

  14. High-performance envelopes for wood

    Treesearch

    Roger M. Rowell

    2007-01-01

    Wood can be coated with a clear finish, stained or painted to provide protection from water and ultra violet energy. In this case the coating and wood are two different phases that coexist. Another approach is to provide protection by “coating” in the surface not on the surface. Such an approach is referred to as an envelop rather than a coating. This can be done in...

  15. Fusion of Enveloped Viruses in Endosomes.

    PubMed

    White, Judith M; Whittaker, Gary R

    2016-06-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Fusion of Enveloped Viruses in Endosomes

    PubMed Central

    White, Judith M.; Whittaker, Gary R.

    2016-01-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH, and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion triggering mechanisms. A key take home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors, and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion. PMID:26935856

  17. The cell envelope glycoconjugates of Mycobacterium tuberculosis

    PubMed Central

    Angala, Shiva Kumar; Belardinelli, Juan Manuel; Huc-Claustre, Emilie; Wheat, William H.; Jackson, Mary

    2015-01-01

    Tuberculosis (TB) remains the second most common cause of death due to a single infectious agent. The cell envelope of Mycobacterium tuberculosis (Mtb), the causative agent of the disease in humans, is a source of unique glycoconjugates and the most distinctive feature of the biology of this organism. It is the basis of much of Mtb pathogenesis and one of the major causes of its intrinsic resistance to chemotherapeutic agents. At the same time, the unique structures of Mtb cell envelope glycoconjugates, their antigenicity and essentiality for mycobacterial growth provide opportunities for drug, vaccine, diagnostic and biomarker development, as clearly illustrated by recent advances in all of these translational aspects. This review focuses on our current understanding of the structure and biogenesis of Mtb glycoconjugates with particular emphasis on one of most intriguing and least understood aspect of the physiology of mycobacteria: the translocation of these complex macromolecules across the different layers of the cell envelope. It further reviews the rather impressive progress made in the last ten years in the discovery and development of novel inhibitors targeting their biogenesis. PMID:24915502

  18. High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.

    PubMed

    Takemori, Nobuaki; Takemori, Ayako; Matsuoka, Kazuhiro; Morishita, Ryo; Matsushita, Natsuki; Aoshima, Masato; Takeda, Hiroyuki; Sawasaki, Tatsuya; Endo, Yaeta; Higashiyama, Shigeki

    2015-02-01

    Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry.

  19. Structural characterization of the feline-immunodeficiency-virus envelope glycoprotein 36 ectodomain for the development of new antivirals

    PubMed Central

    2005-01-01

    In the fight against the human HIV, new targets are being explored, such as the proteins involved in the process of fusion of the virus with the host cell. Recently, the first generation of fusion inhibitors (enfuvirtide), targeting gp41 (virus envelope glycoprotein 41), has become commercially available. However, this promising class of drugs has to be improved in respect of their efficacy and bioavailability. Considering the strong homologies between HIV and FIV (feline immunodeficiency virus), as well as the highly conserved structure of the transmembrane envelope protein among species, FIV represents a relevant model of pre-screening studies for HIV. Taking into account (i) sequence homologies between the ectodomain of HIV gp41 and FIV gp36 (envelope glycoprotein 36), (ii) structural data available for gp41 and (iii) the fact that synthetic peptides derived from gp36 are effective inhibitors of FIV infection, we designed several peptides derived from gp36 sequence. We checked that these peptides had the same structural features as the corresponding peptides from gp41 HIV by CD, analytical ultracentrifugation and 1H–2H (hydrogen–deuterium) exchange combined with MS. By combining this latter technique with surface-plasmon-resonance assays, we identified the amino acid residues of the C-terminal region of the ectodomain of gp36 that are critical for interaction with the N-terminal region. This gave clues for therapy and vaccines against FIV, thus providing helpful data for treatments against HIV. PMID:15799719

  20. Equine Tetherin Blocks Retrovirus Release and Its Activity Is Antagonized by Equine Infectious Anemia Virus Envelope Protein

    PubMed Central

    Yin, Xin; Hu, Zhe; Gu, Qinyong; Wu, Xingliang; Zheng, Yong-Hui; Wei, Ping

    2014-01-01

    Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism. PMID:24227834

  1. Equine tetherin blocks retrovirus release and its activity is antagonized by equine infectious anemia virus envelope protein.

    PubMed

    Yin, Xin; Hu, Zhe; Gu, Qinyong; Wu, Xingliang; Zheng, Yong-Hui; Wei, Ping; Wang, Xiaojun

    2014-01-01

    Human tetherin is a host restriction factor that inhibits replication of enveloped viruses by blocking viral release. Tetherin has an unusual topology that includes an N-terminal cytoplasmic tail, a single transmembrane domain, an extracellular domain, and a C-terminal glycosylphosphatidylinositol anchor. Tetherin is not well conserved across species, so it inhibits viral replication in a species-specific manner. Thus, studies of tetherin activities from different species provide an important tool for understanding its antiviral mechanism. Here, we report cloning of equine tetherin and characterization of its antiviral activity. Equine tetherin shares 53%, 40%, 36%, and 34% amino acid sequence identity with feline, human, simian, and murine tetherins, respectively. Like the feline tetherin, equine tetherin has a shorter N-terminal domain than human tetherin. Equine tetherin is localized on the cell surface and strongly blocks human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and equine infectious anemia virus (EIAV) release from virus-producing cells. The antiviral activity of equine tetherin is neutralized by EIAV envelope protein, but not by the HIV-1 accessory protein Vpu, which is a human tetherin antagonist, and EIAV envelope protein does not counteract human tetherin. These results shed new light on our understanding of the species-specific tetherin antiviral mechanism.

  2. Multiscale envelope manifold for enhanced fault diagnosis of rotating machines

    NASA Astrophysics Data System (ADS)

    Wang, Jun; He, Qingbo; Kong, Fanrang

    2015-02-01

    The wavelet transform has been widely used in the field of machinery fault diagnosis for its good property of band-pass filtering. However, the filtered signal still faces the contamination of in-band noise. This paper focuses on wavelet enveloping, and proposes a new method, called multiscale envelope manifold (MEM), to extract the envelope information of fault impacts with in-band noise suppression. The MEM addresses manifold learning on the wavelet envelopes at multiple scales. Specifically, the proposed method is conducted by three following steps. First, the continuous wavelet transform (CWT) with complex Morlet wavelet base is introduced to obtain the wavelet envelopes at all scales. Second, the wavelet envelopes are restricted in one or more narrow scale bands to simply include the envelope information of fault impacts. The scale band is determined through a smoothness index-based (SI-based) selection method by considering the impulsiveness inside the power spectrum. Third, the manifold learning algorithm is conducted on the wavelet envelopes at selected scales to extract the intrinsic envelope manifold of fault-related impulses. The MEM combines the envelope information at multiple scales in a nonlinear approach, and may thus preserve the factual envelope structure of machinery fault. Simulation studies and experimental verifications confirm that the new method is effective for enhanced fault diagnosis of rotating machines.

  3. Antiviral Activity of Graphene–Silver Nanocomposites against Non-Enveloped and Enveloped Viruses

    PubMed Central

    Chen, Yi-Ning; Hsueh, Yi-Huang; Hsieh, Chien-Te; Tzou, Dong-Ying; Chang, Pai-Ling

    2016-01-01

    The discovery of novel antiviral materials is important because many infectious diseases are caused by viruses. Silver nanoparticles have demonstrated strong antiviral activity, and graphene is a potential antimicrobial material due to its large surface area, high carrier mobility, and biocompatibility. No studies on the antiviral activity of nanomaterials on non-enveloped viruses have been reported. To investigate the antiviral activity of graphene oxide (GO) sheets and GO sheets with silver particles (GO-Ag) against enveloped and non-enveloped viruses, feline coronavirus (FCoV) with an envelope and infectious bursal disease virus (IBDV) without an envelope were chosen. The morphology and sizes of GO and GO-Ag were characterized by transmission, scanning electron microscopy, and X-ray diffraction. A virus inhibition assay was used to identify the antiviral activity of GO and GO-Ag. Go-Ag inhibited 25% of infection by FCoV and 23% by IBDV, whereas GO only inhibited 16% of infection by FCoV but showed no antiviral activity against the infection by IBDV. Further application of GO and GO-Ag can be considered for personal protection equipment to decrease the transmission of viruses. PMID:27104546

  4. Antiviral Activity of Graphene-Silver Nanocomposites against Non-Enveloped and Enveloped Viruses.

    PubMed

    Chen, Yi-Ning; Hsueh, Yi-Huang; Hsieh, Chien-Te; Tzou, Dong-Ying; Chang, Pai-Ling

    2016-04-19

    The discovery of novel antiviral materials is important because many infectious diseases are caused by viruses. Silver nanoparticles have demonstrated strong antiviral activity, and graphene is a potential antimicrobial material due to its large surface area, high carrier mobility, and biocompatibility. No studies on the antiviral activity of nanomaterials on non-enveloped viruses have been reported. To investigate the antiviral activity of graphene oxide (GO) sheets and GO sheets with silver particles (GO-Ag) against enveloped and non-enveloped viruses, feline coronavirus (FCoV) with an envelope and infectious bursal disease virus (IBDV) without an envelope were chosen. The morphology and sizes of GO and GO-Ag were characterized by transmission, scanning electron microscopy, and X-ray diffraction. A virus inhibition assay was used to identify the antiviral activity of GO and GO-Ag. Go-Ag inhibited 25% of infection by FCoV and 23% by IBDV, whereas GO only inhibited 16% of infection by FCoV but showed no antiviral activity against the infection by IBDV. Further application of GO and GO-Ag can be considered for personal protection equipment to decrease the transmission of viruses.

  5. Transmembrane Peptides Influence the Affinity of Sterols for Phospholipid Bilayers

    PubMed Central

    Nyström, Joel H.; Lönnfors, Max; Nyholm, Thomas K.M.

    2010-01-01

    Abstract Cholesterol is distributed unevenly between different cellular membrane compartments, and the cholesterol content increases from the inner bilayers toward the plasma membrane. It has been suggested that this cholesterol gradient is important in the sorting of transmembrane proteins. Cholesterol has also been to shown play an important role in lateral organization of eukaryotic cell membranes. In this study the aim was to determine how transmembrane proteins influence the lateral distribution of cholesterol in phospholipid bilayers. Insight into this can be obtained by studying how cholesterol interacts with bilayer membranes of different composition in the presence of designed peptides that mimic the transmembrane helices of proteins. For this purpose we developed an assay in which the partitioning of the fluorescent cholesterol analog CTL between LUVs and mβCD can be measured. Comparison of how cholesterol and CTL partitioning between mβCD and phospholipid bilayers with different composition suggests that CTL sensed changes in bilayer composition similarly as cholesterol. Therefore, the results obtained with CTL can be used to understand cholesterol distribution in lipid bilayers. The effect of WALP23 on CTL partitioning between DMPC bilayers and mβCD was measured. From the results it was clear that WALP23 increased both the order in the bilayers (as seen from CTL and DPH anisotropy) and the affinity of the sterol for the bilayer in a concentration dependent way. Although WALP23 also increased the order in DLPC and POPC bilayers the effects on CTL partitioning was much smaller with these lipids. This indicates that proteins have the largest effect on sterol interactions with phospholipids that have longer and saturated acyl chains. KALP23 did not significantly affect the acyl chain order in the phospholipid bilayers, and inclusion of KALP23 into DMPC bilayers slightly decreased CTL partitioning into the bilayer. This shows that transmembrane proteins

  6. Molecular modelling approaches for cystic fibrosis transmembrane conductance regulator studies.

    PubMed

    Odolczyk, Norbert; Zielenkiewicz, Piotr

    2014-07-01

    Cystic fibrosis (CF) is one of the most common genetic disorders, caused by loss of function mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. CFTR is a member of ATP-binding cassette (ABC) transporters superfamily and functions as an ATP-gated anion channel. This review summarises the vast majority of the efforts which utilised molecular modelling approaches to gain insight into the various aspects of CFTR protein, related to its structure, dynamic properties, function and interactions with other protein partners, or drug-like compounds, with emphasis to its relation to CF disease.

  7. Membrane topology of transmembrane proteins: determinants and experimental tools.

    PubMed

    Lee, Hunsang; Kim, Hyun

    2014-10-17

    Membrane topology refers to the two-dimensional structural information of a membrane protein that indicates the number of transmembrane (TM) segments and the orientation of soluble domains relative to the plane of the membrane. Since membrane proteins are co-translationally translocated across and inserted into the membrane, the TM segments orient themselves properly in an early stage of membrane protein biogenesis. Each membrane protein must contain some topogenic signals, but the translocation components and the membrane environment also influence the membrane topology of proteins. We discuss the factors that affect membrane protein orientation and have listed available experimental tools that can be used in determining membrane protein topology.

  8. Transmembrane anion transport and cytotoxicity of synthetic tambjamine analogs.

    PubMed

    Hernando, Elsa; Soto-Cerrato, Vanessa; Cortés-Arroyo, Susana; Pérez-Tomás, Ricardo; Quesada, Roberto

    2014-03-21

    Ten synthetic analogs of the marine alkaloids tambjamines, bearing aromatic enamine moieties, have been synthesized. These compounds proved to be highly efficient transmembrane anion transporters in model liposomes. Changes in the electronic nature of the substituents of the aromatic enamine or the alkoxy group of the central pyrrole group did not affect this anionophore activity. The in vitro activity of these compounds has also been studied. They trigger apoptosis in several cancer cell lines with IC50 values in the low micromolar range as well as modify the intracellular pH, inducing the basification of acidic organelles.

  9. Contribution of Intrinsic Reactivity of the HIV-1 Envelope Glycoproteins to CD4-Independent Infection and Global Inhibitor Sensitivity

    PubMed Central

    Haim, Hillel; Strack, Bettina; Kassa, Aemro; Madani, Navid; Wang, Liping; Courter, Joel R.; Princiotto, Amy; McGee, Kathleen; Pacheco, Beatriz; Seaman, Michael S.; Smith, Amos B.; Sodroski, Joseph

    2011-01-01

    Human immunodeficiency virus (HIV-1) enters cells following sequential activation of the high-potential-energy viral envelope glycoprotein trimer by target cell CD4 and coreceptor. HIV-1 variants differ in their requirements for CD4; viruses that can infect coreceptor-expressing cells that lack CD4 have been generated in the laboratory. These CD4-independent HIV-1 variants are sensitive to neutralization by multiple antibodies that recognize different envelope glycoprotein epitopes. The mechanisms underlying CD4 independence, global sensitivity to neutralization and the association between them are still unclear. By studying HIV-1 variants that differ in requirements for CD4, we investigated the contribution of CD4 binding to virus entry. CD4 engagement exposes the coreceptor-binding site and increases the “intrinsic reactivity” of the envelope glycoproteins; intrinsic reactivity describes the propensity of the envelope glycoproteins to negotiate transitions to lower-energy states upon stimulation. Coreceptor-binding site exposure and increased intrinsic reactivity promote formation/exposure of the HR1 coiled coil on the gp41 transmembrane glycoprotein and allow virus entry upon coreceptor binding. Intrinsic reactivity also dictates the global sensitivity of HIV-1 to perturbations such as exposure to cold and the binding of antibodies and small molecules. Accordingly, CD4 independence of HIV-1 was accompanied by increased susceptibility to inactivation by these factors. We investigated the role of intrinsic reactivity in determining the sensitivity of primary HIV-1 isolates to inhibition. Relative to the more common neutralization-resistant (“Tier 2-like”) viruses, globally sensitive (“Tier 1”) viruses exhibited increased intrinsic reactivity, i.e., were inactivated more efficiently by cold exposure or by a given level of antibody binding to the envelope glycoprotein trimer. Virus sensitivity to neutralization was dictated both by the efficiency of

  10. Analysis of dihedral angle preferences for alanine and glycine residues in alpha and beta transmembrane regions.

    PubMed

    Saravanan, K M; Krishnaswamy, S

    2015-01-01

    For the past 50 years, the Ramachandran map has been used effectively to study the protein structure and folding. However, though extensive analysis has been done on dihedral angle preferences of residues in globular proteins, related studies and reports of membrane proteins are limited. It is of interest to explore the conformational preferences of residues in transmembrane regions of membrane proteins which are involved in several important and diverse biological processes. Hence, in the present work, a systematic comparative computational analysis has been made on dihedral angle preferences of alanine and glycine in alpha and beta transmembrane regions (the two major classes of transmembrane proteins) with the aid of the Ramachandran map. Further, the conformational preferences of residues in transmembrane regions were compared with the non-transmembrane regions. We have extracted cation-pi interacting residues present in transmembrane regions and explored the dihedral angle preferences. From our observations, we reveal the higher percentage of occurrences of glycine in alpha and beta transmembrane regions than other hydrophobic residues. Further, we noted a clear shift in ψ-angle preferences of glycine residues from negative bins in alpha transmembrane regions to positive bins in beta transmembrane regions. Also, cation-pi interacting residues in beta transmembrane regions avoid preferring ψ-angles in the range of -59° to -30°. In this article, we insist that the studies on preferences of dihedral angles in transmembrane regions, thorough understanding of structure and folding of transmembrane proteins, can lead to modeling of novel transmembrane regions towards designing membrane proteins.

  11. Transmembrane TNF-α Facilitates HIV-1 Infection of Podocytes Cultured from Children with HIV-Associated Nephropathy.

    PubMed

    Li, Jinliang; Das, Jharna R; Tang, Pingtao; Han, Zhe; Jaiswal, Jyoti K; Ray, Patricio E

    2017-03-01

    Studies have shown that podocytes and renal tubular epithelial cells from patients with HIV-associated nephropathy (HIVAN) express HIV-1 transcripts, suggesting that productive infection of renal epithelial cells precipitates development of HIVAN. However, podocytes and renal tubular epithelial cells do not express CD4 receptors, and it is unclear how these cells become productively infected in vivo We investigated the mechanisms underlying the infection by HIV-1 of podocytes cultured from the urine of children with HIVAN. We observed low-level productive infection on exposure of these cells to primary cell-free HIV-1 supernatants. However, envelope-defective recombinant HIV-1 did not infect the renal epithelial cell lines. Moreover, treatment of podocytes to inhibit endocytic transport or dynamin activity or remove cell surface heparan sulfate proteoglycans reduced infection efficiency. Transfection of CD4- 293T cells with a cDNA expression library developed from a podocyte cell line derived from a child with HIVAN led to the identification of TNF-α as a possible mediator of HIV-1 infection. Overexpression of transmembrane TNF-α in cultured CD4- renal tubular epithelial cells, 293T cells, and HeLa cells enabled the infection of these cells; exposure to soluble TNF-α did not. Immunohistochemistry showed TNF-α expression in podocytes of renal sections from children with HIVAN. Furthermore, we found that TNF-α enhanced NF-κB activation and integration of HIV-1 into the podocyte DNA. Finally, inhibition of dynamin activity blocked TNF-α-mediated infection. These data establish a role for transmembrane TNF-α in facilitating the viral entry and integration of HIV-1 into the DNA of renal epithelial cells.

  12. Structure and Mechanism of Proton Transport Through the Transmembrane Tetrameric M2 Protein Bundle of the Influenza A Virus

    SciTech Connect

    R Acharya; V Carnevale; G Fiorin; B Levine; A Polishchuk; V Balannick; I Samish; R Lamb; L Pinto; et al.

    2011-12-31

    The M2 proton channel from influenza A virus is an essential protein that mediates transport of protons across the viral envelope. This protein has a single transmembrane helix, which tetramerizes into the active channel. At the heart of the conduction mechanism is the exchange of protons between the His37 imidazole moieties of M2 and waters confined to the M2 bundle interior. Protons are conducted as the total charge of the four His37 side chains passes through 2{sup +} and 3{sup +} with a pK{sub a} near 6. A 1.65 {angstrom} resolution X-ray structure of the transmembrane protein (residues 25-46), crystallized at pH 6.5, reveals a pore that is lined by alternating layers of sidechains and well-ordered water clusters, which offer a pathway for proton conduction. The His37 residues form a box-like structure, bounded on either side by water clusters with well-ordered oxygen atoms at close distance. The conformation of the protein, which is intermediate between structures previously solved at higher and lower pH, suggests a mechanism by which conformational changes might facilitate asymmetric diffusion through the channel in the presence of a proton gradient. Moreover, protons diffusing through the channel need not be localized to a single His37 imidazole, but instead may be delocalized over the entire His-box and associated water clusters. Thus, the new crystal structure provides a possible unification of the discrete site versus continuum conduction models.

  13. pH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion.

    PubMed

    Desai, Tanay M; Marin, Mariana; Mason, Caleb; Melikyan, Gregory B

    2017-05-12

    Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Static envelope patterns in composite resonances generated by level crossing in optical toroidal microcavities.

    PubMed

    Carmon, Tal; Schwefel, Harald G L; Yang, Lan; Oxborrow, Mark; Stone, A Douglas; Vahala, Kerry J

    2008-03-14

    We study level crossing in the optical whispering-gallery (WG) modes by using toroidal microcavities. Experimentally, we image the stationary envelope patterns of the composite optical modes that arise when WG modes of different wavelengths coincide in frequency. Numerically, we calculate crossings of levels that correspond with the observed degenerate modes, where our method takes into account the not perfectly transverse nature of their field polarizations. In addition, we analyze anticrossing with a large avoidance gap between modes of the same azimuthal number.

  15. Closing the Pay Gap

    DTIC Science & Technology

    2000-10-01

    the pay gap has been narrowed, hut only to just under 10 percent. And current military compensation legislation does not close the gap until 2026. There...will continue to be a pay gap until 2026 unless the next administration and the next Congress provide more for pay above the 1999 legislated ramp- up...of .5 percent (one half of one percent) per year to attain pay equality . That means that soldiers, sailors, airmen, marines and Coast Guardsmen

  16. Highly Coarse-Grained Representations of Transmembrane Proteins

    PubMed Central

    2017-01-01

    Numerous biomolecules and biomolecular complexes, including transmembrane proteins (TMPs), are symmetric or at least have approximate symmetries. Highly coarse-grained models of such biomolecules, aiming at capturing the essential structural and dynamical properties on resolution levels coarser than the residue scale, must preserve the underlying symmetry. However, making these models obey the correct physics is in general not straightforward, especially at the highly coarse-grained resolution where multiple (∼3–30 in the current study) amino acid residues are represented by a single coarse-grained site. In this paper, we propose a simple and fast method of coarse-graining TMPs obeying this condition. The procedure involves partitioning transmembrane domains into contiguous segments of equal length along the primary sequence. For the coarsest (lowest-resolution) mappings, it turns out to be most important to satisfy the symmetry in a coarse-grained model. As the resolution is increased to capture more detail, however, it becomes gradually more important to match modular repeats in the secondary structure (such as helix-loop repeats) instead. A set of eight TMPs of various complexity, functionality, structural topology, and internal symmetry, representing different classes of TMPs (ion channels, transporters, receptors, adhesion, and invasion proteins), has been examined. The present approach can be generalized to other systems possessing exact or approximate symmetry, allowing for reliable and fast creation of multiscale, highly coarse-grained mappings of large biomolecular assemblies. PMID:28043122

  17. Transmembrane semaphorin signalling controls laminar stratification in the mammalian retina.

    PubMed

    Matsuoka, Ryota L; Nguyen-Ba-Charvet, Kim T; Parray, Aijaz; Badea, Tudor C; Chédotal, Alain; Kolodkin, Alex L

    2011-02-10

    In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes.

  18. Transmembrane semaphorin signaling controls laminar stratification in the mammalian retina

    PubMed Central

    Matsuoka, Ryota L.; Nguyen-Ba-Charvet, Kim T.; Parray, Aijaz; Badea, Tudor C.; Chédotal, Alain; Kolodkin, Alex L.

    2010-01-01

    In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells, and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL)1–3: a laminar region that is conventionally divided into five major parallel sublaminae1,2. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. Here, we show that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs), and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo with respect to the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes. PMID:21270798

  19. The Transmembrane Adaptor Protein SIT Inhibits TCR-Mediated Signaling

    PubMed Central

    Arndt, Börge; Krieger, Tina; Kalinski, Thomas; Thielitz, Anja; Reinhold, Dirk; Roessner, Albert; Schraven, Burkhart; Simeoni, Luca

    2011-01-01

    Transmembrane adaptor proteins (TRAPs) organize signaling complexes at the plasma membrane, and thus function as critical linkers and integrators of signaling cascades downstream of antigen receptors. We have previously shown that the transmembrane adaptor protein SIT regulates the threshold for thymocyte selection. Moreover, T cells from SIT-deficient mice are hyperresponsive to CD3 stimulation and undergo enhanced lymphopenia-induced homeostatic proliferation, thus indicating that SIT inhibits TCR-mediated signaling. Here, we have further addressed how SIT regulates signaling cascades in T cells. We demonstrate that the loss of SIT enhances TCR-mediated Akt activation and increased phosphorylation/inactivation of Foxo1, a transcription factor of the Forkhead family that inhibits cell cycle progression and regulates T-cell homeostasis. We have also shown that CD4+ T cells from SIT-deficient mice display increased CD69 and CD40L expression indicating an altered activation status. Additional biochemical analyses further revealed that suppression of SIT expression by RNAi in human T cells resulted in an enhanced proximal TCR signaling. In summary, the data identify SIT as an important modulator of TCR-mediated signaling that regulates T-cell activation, homeostasis and tolerance. PMID:21957439

  20. Transmembrane Domains of Attraction on the TSH Receptor

    PubMed Central

    Ali, M. Rejwan; Mezei, Mihaly; Davies, Terry F.

    2015-01-01

    The TSH receptor (TSHR) has the propensity to form dimers and oligomers. Our data using ectodomain-truncated TSHRs indicated that the predominant interfaces for oligomerization reside in the transmembrane (TM) domain. To map the potentially interacting residues, we first performed in silico studies of the TSHR transmembrane domain using a homology model and using Brownian dynamics (BD). The cluster of dimer conformations obtained from BD analysis indicated that TM1 made contact with TM4 and two residues in TM2 made contact with TM5. To confirm the proximity of these contact residues, we then generated cysteine mutants at all six contact residues predicted by the BD analysis and performed cysteine cross-linking studies. These results showed that the predicted helices in the protomer were indeed involved in proximity interactions. Furthermore, an alternative experimental approach, receptor truncation experiments and LH receptor sequence substitution experiments, identified TM1 harboring a major region involved in TSHR oligomerization, in agreement with the conclusion from the cross-linking studies. Point mutations of the predicted interacting residues did not yield a substantial decrease in oligomerization, unlike the truncation of the TM1, so we concluded that constitutive oligomerization must involve interfaces forming domains of attraction in a cooperative manner that is not dominated by interactions between specific residues. PMID:25406938

  1. Forming transmembrane channels using end-functionalized nanotubes.

    PubMed

    Dutt, Meenakshi; Kuksenok, Olga; Little, Steven R; Balazs, Anna C

    2011-01-01

    Using dissipative particle dynamics (DPD) simulations, we examine the interaction between amphiphilic nanotubes and lipid bilayer membranes. The nanotubes are represented by a hydrophobic shaft that is end-functionalized with hydrophilic groups. Nanotubes that are capped by a monolayer of hydrophilic beads or also encompass hydrophilic "hairs" on just one end of the shaft are found to spontaneously penetrate and assume a transmembrane position; the process, however, depends critically on the membrane tension. On the other hand, nanotubes that include hydrophilic hairs at both ends of the hydrophobic shaft are not observed to spontaneously self-organize into the bilayer. When the membrane is stretched to form a pore, the nanotubes with two hairy ends adsorb on the edge of the pore and become localized in the membrane, thus forming a transmembrane channel. The findings from these studies provide guidelines for creating biomimetic nanotube channels that are capable of selectively transporting molecules through the membrane in response to changes in the local environment.

  2. Transmembrane transport of peptidoglycan precursors across model and bacterial membranes.

    PubMed

    van Dam, Vincent; Sijbrandi, Robert; Kol, Matthijs; Swiezewska, Ewa; de Kruijff, Ben; Breukink, Eefjan

    2007-05-01

    Translocation of the peptidoglycan precursor Lipid II across the cytoplasmic membrane is a key step in bacterial cell wall synthesis, but hardly understood. Using NBD-labelled Lipid II, we showed by fluorescence and TLC assays that Lipid II transport does not occur spontaneously and is not induced by the presence of single spanning helical transmembrane peptides that facilitate transbilayer movement of membrane phospholipids. MurG catalysed synthesis of Lipid II from Lipid I in lipid vesicles also did not result in membrane translocation of Lipid II. These findings demonstrate that a specialized protein machinery is needed for transmembrane movement of Lipid II. In line with this, we could demonstrate Lipid II translocation in isolated Escherichia coli inner membrane vesicles and this transport could be uncoupled from the synthesis of Lipid II at low temperatures. The transport process appeared to be independent from an energy source (ATP or proton motive force). Additionally, our studies indicate that translocation of Lipid II is coupled to transglycosylation activity on the periplasmic side of the inner membrane.

  3. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  4. Retromer-Mediated Trafficking of Transmembrane Receptors and Transporters

    PubMed Central

    Klinger, Stine C.; Siupka, Piotr; Nielsen, Morten S.

    2015-01-01

    Transport between the endoplasmatic reticulum, the Golgi-network, the endo-lysosomal system and the cell surface can be categorized as anterograde or retrograde, describing traffic that goes forward or backward, respectively. Traffic going from the plasma membrane to endosomes and lysosomes or the trans-Golgi network (TGN) constitutes the major retrograde transport routes. Several transmembrane proteins undergo retrograde transport as part of a recycling mechanism that contributes to reutilization and maintenance of a steady-state protein localization. In addition, some receptors are hijacked by exotoxins and used for entry and intracellular transport. The physiological relevance of retrograde transport cannot be overstated. Retrograde trafficking of the amyloid precursor protein determines the distribution between organelles, and hence the possibility of cleavage by γ-secretase. Right balancing of the pathways is critical for protection against Alzheimer’s disease. During embryonic development, retrograde transport of Wntless to the TGN is essential for the following release of Wnt from the plasma membrane. Furthermore, overexpression of Wntless has been linked to oncogenesis. Here, we review relevant aspects of the retrograde trafficking of mammalian transmembrane receptors and transporters, with focus on the retromer-mediated transport between endosomes and the TGN. PMID:26154780

  5. Bioenergetics and mitochondrial transmembrane potential during differentiation of cultured osteoblasts

    NASA Technical Reports Server (NTRS)

    Komarova, S. V.; Ataullakhanov, F. I.; Globus, R. K.

    2000-01-01

    To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

  6. Structural Dynamics of Insulin Receptor and Transmembrane Signaling.

    PubMed

    Tatulian, Suren A

    2015-09-15

    The insulin receptor (IR) is a (αβ)2-type transmembrane tyrosine kinase that plays a central role in cell metabolism. Each αβ heterodimer consists of an extracellular ligand-binding α-subunit and a membrane-spanning β-subunit that comprises the cytoplasmic tyrosine kinase (TK) domain and the phosphorylation sites. The α- and β-subunits are linked via a single disulfide bridge, and the (αβ)2 tetramer is formed by disulfide bonds between the α-chains. Insulin binding induces conformational changes in IR that reach the intracellular β-subunit followed by a protein phosphorylation and activation cascade. Defects in this signaling process, including IR dysfunction caused by mutations, result in type 2 diabetes. Rational drug design aimed at treatment of diabetes relies on knowledge of the detailed structure of IR and the dynamic structural transformations during transmembrane signaling. Recent X-ray crystallographic studies have provided important clues about the mode of binding of insulin to IR, the resulting structural changes and their transmission to the TK domain, but a complete understanding of the structural basis underlying insulin signaling has not been achieved. This review presents a critical analysis of the current status of the structure-function relationship of IR, with a comparative assessment of the other IR family receptors, and discusses potential advancements that may provide insight into the molecular mechanism of insulin signaling.

  7. Structure of Staphylococcal α-Hemolysin, a Heptameric Transmembrane Pore

    NASA Astrophysics Data System (ADS)

    Song, Langzhou; Hobaugh, Michael R.; Shustak, Christopher; Cheley, Stephen; Bayley, Hagan; Gouaux, J. Eric

    1996-12-01

    The structure of the Staphylococcus aureus α-hemolysin pore has been determined to 1.9 overset{circ}{mathrm A} resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 overset{circ}{mathrm A} in length, that runs along the sevenfold axis and ranges from 14 overset{circ}{mathrm A} to 46 overset{circ}{mathrm A} in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel β barrel, to which each protomer contributes two β strands, each 65 overset{circ}{mathrm A} long. The interior of the β barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 overset{circ}{mathrm A} wide. The structure proves the heptameric subunit stoichiometry of the α-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of β barrel pore-forming toxins.

  8. Modeling pollutant penetration across building envelopes

    SciTech Connect

    Liu, De-Ling; Nazaroff, William W.

    2001-04-01

    As air infiltrates through unintentional openings in building envelopes, pollutants may interact with adjacent surfaces. Such interactions can alter human exposure to air pollutants of outdoor origin. We present modeling explorations of the proportion of particles and reactive gases (e.g., ozone) that penetrate building envelopes as air enters through cracks and wall cavities. Calculations were performed for idealized rectangular cracks, assuming regular geometry, smooth inner crack surface and steady airflow. Particles of 0.1-1.0 {micro}m diameter are predicted to have the highest penetration efficiency, nearly unity for crack heights of 0.25 mm or larger, assuming a pressure difference of 4 Pa or greater and a flow path length of 3 cm or less. Supermicron and ultrafine particles are significantly removed by means of gravitational settling and Brownian diffusion, respectively. In addition to crack geometry, ozone penetration depends on its reactivity with crack surfaces, as parameterized by the reaction probability. For reaction probabilities less than {approx}10{sup -5}, penetration is complete for cracks heights greater than 1 mm. However, penetration through mm scale cracks is small if the reaction probability is {approx}10{sup -4} or greater. For wall cavities, fiberglass insulation is an efficient particle filter, but particles would penetrate efficiently through uninsulated wall cavities or through insulated cavities with significant airflow bypass. The ozone reaction probability on fiberglass fibers was measured to be 10{sup -7} for fibers previously exposed to high ozone levels and 6 x 10{sup -6} for unexposed fibers. Over this range, ozone penetration through fiberglass insulation would vary from >90% to {approx}10-40%. Thus, under many conditions penetration is high; however, there are realistic circumstances in which building envelopes can provide substantial pollutant removal. Not enough is yet known about the detailed nature of pollutant penetration

  9. Surface area coefficients for airship envelopes

    NASA Technical Reports Server (NTRS)

    Diehl, W S

    1922-01-01

    In naval architecture, it is customary to determine the wetted surface of a ship by means of some formula which involves the principal dimensions of the design and suitable constants. These formulas of naval architecture may be extended and applied to the calculation of the surface area of airship envelopes by the use of new values of the constants determined for this purpose. Surface area coefficients were calculated from the actual dimensions, surfaces, and volumes of 52 streamline bodies, which form a series covering the entire range of shapes used in the present aeronautical practice.

  10. Active Monitoring Complex-Envelope Processing

    NASA Astrophysics Data System (ADS)

    Unger, R.

    2011-12-01

    Pseudo-random 3-D signal transmission followed by quadrature signal processing significantly enhances seismic Active Monitoring (AM). The algorithm produces complex-envelope seismograms consisting of the dual time series of instantaneous amplitude and phase. Minimum-standard-deviation signal arrival detection and timing from coherent instantaneous-phase seismogram ensembles yields signal time errors inversely proportional to frequency and signal-to-noise ratio. In particular, application to piezo-electric acoustic cross well monitoring as in SAFOD experiments should result in nano-second multiple-signal detection and timing precision. The method is applicable to all types (seismic, EM, acoustic) of AM.

  11. The protein translocon of the plastid envelopes.

    PubMed

    Vojta, Aleksandar; Alavi, Marcel; Becker, Thomas; Hörmann, Friederike; Küchler, Michael; Soll, Jürgen; Thomson, Rowena; Schleiff, Enrico

    2004-05-14

    The Toc and Tic translocon facilitate import of preproteins into chloroplasts. In the past, it was speculated that several translocon subunits act specifically for different types of precursor proteins or in different tissues. To generate a comprehensive picture of the expression and tissue-specific localization of the translocon subunits, their transcript levels were analyzed in roots and leaves. Certain Tocs and Tics were found to be tissue-specific. The protein composition of the transloci in the envelope membranes of chloroplasts was analyzed to describe the function and possible stoichiometry. In contrast to Tic subunits, several Toc subunits seem to have a high turnover.

  12. Low heat-leak cryogenic envelope

    DOEpatents

    DeHaan, James R.

    1976-10-19

    A plurality of cryogenic envelope sections are joined together to form a power transmission line. Each of the sections is comprised of inner and outer tubes having multilayer metalized plastic spirally wrapped within a vacuum chamber formed between the inner and outer tubes. A refrigeration tube traverses the vacuum chamber, but exits one section and enters another through thermal standoffs for reducing heat-leak from the outer tube to the refrigeration tube. The refrigeration tube passes through a spirally wrapped shield within each section's vacuum chamber in a manner so that the refrigeration tube is in close thermal contact with the shield, but is nevertheless slideable with respect thereto.

  13. Circumstellar envelopes and Asymptotic Giant Branch stars

    NASA Astrophysics Data System (ADS)

    Habing, H. J.

    Red giants are sometimes surrounded by envelopes, the result of the ejection of stellar matter at a large rate (dot M> 10^{-7}M_odot/yr) and at a low velocity (10 km/s). In this review the envelopes are discussed and the relation between stars and envelope: what stars combine with what envelopes? The envelope emits radiation by various processes and has been detected at all wavelengths between the visual and the microwave range. I review the observations of continuum radiation emitted by dust particles and of rotational transitions of molecules, where these molecules have been excited by thermal or by non thermal (“maser”) processes. I discuss mainly the oxygen rich stars, those of spectral type M, and only briefly the closely related carbon rich stars. By and large the density in the envelope is well described by spherically symmetric outflow at a constant velocity; on the time scale needed to flow from stellar surface to the outermost layers, i.e. 10^5 yr, the loss of mass is sometimes interrupted suddenly after which the envelope becomes “detached” from the star. The temperature decreases when moving outward; heat input is by friction between dust particles and gas and cooling occurs by line radiation by various molecules, especially by H_2O. The molecular composition is determined by formation in an equilibrium process deep in the atmosphere and by destruction in the outer parts of the outflow by interstellar UV radiation (H_2, CO, H_2O) or by depletion due to condensation on dust grains (SiO); dust particles of silicate material solidify where the radiation temperature is decreased to about 1000 K, and this is at a few stellar radii. The various continuum spectra produced by the dust particles in different stars are well modelled by a simple model of the density and dust temperature distribution plus the assumption that the particles consist of “dirty silicate”, i.e. silicate with Fe and Al ions added. A large range of optical depths,tau_{9.7

  14. Metal bridges illuminate transmembrane domain movements during gating of the cystic fibrosis transmembrane conductance regulator chloride channel.

    PubMed

    El Hiani, Yassine; Linsdell, Paul

    2014-10-10

    Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd(2+) bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane α-helices (TMs). Seven Cd(2+) bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd(2+) bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd(2+) bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd(2+) bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close.

  15. Metal Bridges Illuminate Transmembrane Domain Movements during Gating of the Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel*

    PubMed Central

    El Hiani, Yassine; Linsdell, Paul

    2014-01-01

    Opening and closing of the cystic fibrosis transmembrane conductance regulator are controlled by ATP binding and hydrolysis by the cytoplasmic nucleotide-binding domains. Different conformational changes in the channel pore have been described during channel opening and closing; however, the relative importance of these changes to the process of gating the pore is not known. We have used patch clamp recording to identify high affinity Cd2+ bridges formed between pairs of pore-lining cysteine residues introduced into different transmembrane α-helices (TMs). Seven Cd2+ bridges were identified forming between cysteines in TMs 6 and 12. Interestingly, each of these Cd2+ bridges apparently formed only in closed channels, and their formation stabilized the closed state. In contrast, a single Cd2+ bridge identified between cysteines in TMs 1 and 12 stabilized the channel open state. Analysis of the pattern of Cd2+ bridge formation in different channel states suggests that lateral separation and convergence of different TMs, rather than relative rotation or translation of different TMs, is the key conformational change that causes the channel pore to open and close. PMID:25143385

  16. Envelope as Climate Negotiator: Evaluating adaptive building envelope's capacity to moderate indoor climate and energy

    NASA Astrophysics Data System (ADS)

    Erickson, James

    Through manipulation of adaptable opportunities available within a given environment, individuals become active participants in managing personal comfort requirements, by exercising control over their comfort without the assistance of mechanical heating and cooling systems. Similarly, continuous manipulation of a building skin's form, insulation, porosity, and transmissivity qualities exerts control over the energy exchanged between indoor and outdoor environments. This research uses four adaptive response variables in a modified software algorithm to explore an adaptive building skin's potential in reacting to environmental stimuli with the purpose of minimizing energy use without sacrificing occupant comfort. Results illustrate that significant energy savings can be realized with adaptive envelopes over static building envelopes even under extreme summer and winter climate conditions; that the magnitude of these savings are dependent on climate and orientation; and that occupant thermal comfort can be improved consistently over comfort levels achieved by optimized static building envelopes. The resulting adaptive envelope's unique climate-specific behavior could inform designers in creating an intelligent kinetic aesthetic that helps facilitate adaptability and resiliency in architecture.

  17. On-Line Safe Flight Envelope Determination for Impaired Aircraft

    NASA Technical Reports Server (NTRS)

    Lombaerts, Thomas; Schuet, Stefan; Acosta, Diana; Kaneshige, John

    2015-01-01

    The design and simulation of an on-line algorithm which estimates the safe maneuvering envelope of aircraft is discussed in this paper. The trim envelope is estimated using probabilistic methods and efficient high-fidelity model based computations of attainable equilibrium sets. From this trim envelope, a robust reachability analysis provides the maneuverability limitations of the aircraft through an optimal control formulation. Both envelope limits are presented to the flight crew on the primary flight display. In the results section, scenarios are considered where this adaptive algorithm is capable of computing online changes to the maneuvering envelope due to impairment. Furthermore, corresponding updates to display features on the primary flight display are provided to potentially inform the flight crew of safety critical envelope alterations caused by the impairment.

  18. Tissue specificity in the nuclear envelope supports its functional complexity.

    PubMed

    de Las Heras, Jose I; Meinke, Peter; Batrakou, Dzmitry G; Srsen, Vlastimil; Zuleger, Nikolaj; Kerr, Alastair Rw; Schirmer, Eric C

    2013-01-01

    Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution.

  19. Tissue specificity in the nuclear envelope supports its functional complexity

    PubMed Central

    de las Heras, Jose I; Meinke, Peter; Batrakou, Dzmitry G; Srsen, Vlastimil; Zuleger, Nikolaj; Kerr, Alastair RW; Schirmer, Eric C

    2013-01-01

    Nuclear envelope links to inherited disease gave the conundrum of how mutations in near-ubiquitous proteins can yield many distinct pathologies, each focused in different tissues. One conundrum-resolving hypothesis is that tissue-specific partner proteins mediate these pathologies. Such partner proteins may have now been identified with recent proteome studies determining nuclear envelope composition in different tissues. These studies revealed that the majority of the total nuclear envelope proteins are tissue restricted in their expression. Moreover, functions have been found for a number these tissue-restricted nuclear envelope proteins that fit with mechanisms proposed to explain how the nuclear envelope could mediate disease, including defects in mechanical stability, cell cycle regulation, signaling, genome organization, gene expression, nucleocytoplasmic transport, and differentiation. The wide range of functions to which these proteins contribute is consistent with not only their involvement in tissue-specific nuclear envelope disease pathologies, but also tissue evolution. PMID:24213376

  20. The Parenting Gap

    ERIC Educational Resources Information Center

    Reeves, Richard V.; Howard, Kimberly

    2013-01-01

    The parenting gap is a big factor in the opportunity gap. The chances of upward social mobility are lower for children with parents struggling to do a good job--in terms of creating a supportive and stimulating home environment. Children lucky enough to have strong parents are more likely to succeed at all the critical life stages, which means…

  1. States Address Achievement Gaps.

    ERIC Educational Resources Information Center

    Christie, Kathy

    2002-01-01

    Summarizes 2 state initiatives to address the achievement gap: North Carolina's report by the Advisory Commission on Raising Achievement and Closing Gaps, containing an 11-point strategy, and Kentucky's legislation putting in place 10 specific processes. The North Carolina report is available at www.dpi.state.nc.us.closingthegap; Kentucky's…

  2. Which Achievement Gap?

    ERIC Educational Resources Information Center

    Anderson, Sharon; Medrich, Elliott; Fowler, Donna

    2007-01-01

    From the halls of Congress to the local elementary school, conversations on education reform have tossed around the term "achievement gap" as though people all know precisely what that means. As it's commonly used, "achievement gap" refers to the differences in scores on state or national achievement tests between various…

  3. Confronting the Achievement Gap

    ERIC Educational Resources Information Center

    Gardner, David

    2007-01-01

    This article talks about the large achievement gap between children of color and their white peers. The reasons for the achievement gap are varied. First, many urban minorities come from a background of poverty. One of the detrimental effects of growing up in poverty is receiving inadequate nourishment at a time when bodies and brains are rapidly…

  4. Which Achievement Gap?

    ERIC Educational Resources Information Center

    Anderson, Sharon; Medrich, Elliott; Fowler, Donna

    2007-01-01

    From the halls of Congress to the local elementary school, conversations on education reform have tossed around the term "achievement gap" as though people all know precisely what that means. As it's commonly used, "achievement gap" refers to the differences in scores on state or national achievement tests between various…

  5. The Parenting Gap

    ERIC Educational Resources Information Center

    Reeves, Richard V.; Howard, Kimberly

    2013-01-01

    The parenting gap is a big factor in the opportunity gap. The chances of upward social mobility are lower for children with parents struggling to do a good job--in terms of creating a supportive and stimulating home environment. Children lucky enough to have strong parents are more likely to succeed at all the critical life stages, which means…

  6. Confronting the Achievement Gap

    ERIC Educational Resources Information Center

    Gardner, David

    2007-01-01

    This article talks about the large achievement gap between children of color and their white peers. The reasons for the achievement gap are varied. First, many urban minorities come from a background of poverty. One of the detrimental effects of growing up in poverty is receiving inadequate nourishment at a time when bodies and brains are rapidly…

  7. Bridging a Cultural Gap

    ERIC Educational Resources Information Center

    Leviatan, Talma

    2008-01-01

    There has been a broad wave of change in tertiary calculus courses in the past decade. However, the much-needed change in tertiary pre-calculus programmes--aimed at bridging the gap between high-school mathematics and tertiary mathematics--is happening at a far slower pace. Following a discussion on the nature of the gap and the objectives of a…

  8. The National "Expertise Gap"

    ERIC Educational Resources Information Center

    Hamilton, Kendra

    2005-01-01

    This article discusses the Woodrow Wilson National Fellowship Foundation's report, "Diversity and the Ph.D.," released in May, which documents in troubling detail the exact dimensions of what the foundation's president, Dr. Robert Weisbuch, is calling the national "expertise gap." Weisbuch states that the expertise gap extends beyond the…

  9. Senseless Extravagance, Shocking Gaps

    ERIC Educational Resources Information Center

    Weissbourd, Richard; Dodge, Trevor

    2012-01-01

    Although most people in the United States believe, at least theoretically, in educational equality, fewer and fewer appear to care about the resource gaps between affluent and poor schools, says Weissbourd. He illustrates these gaps with vivid descriptions of what he calls an "opulence arms race" among affluent independent schools, but…

  10. Senseless Extravagance, Shocking Gaps

    ERIC Educational Resources Information Center

    Weissbourd, Richard; Dodge, Trevor

    2012-01-01

    Although most people in the United States believe, at least theoretically, in educational equality, fewer and fewer appear to care about the resource gaps between affluent and poor schools, says Weissbourd. He illustrates these gaps with vivid descriptions of what he calls an "opulence arms race" among affluent independent schools, but…

  11. Information Gap Activities.

    ERIC Educational Resources Information Center

    Cicekdag, Mehmet Ali

    1995-01-01

    Focuses on a real world technique used to teach language proficiency in the classroom. This method involves creating deliberate information and opinion gaps by administering pop quizzes and other communicative games and filling those gaps through cooperative action. Use of this technique generated heated discussion among students. (nine…

  12. The National "Expertise Gap"

    ERIC Educational Resources Information Center

    Hamilton, Kendra

    2005-01-01

    This article discusses the Woodrow Wilson National Fellowship Foundation's report, "Diversity and the Ph.D.," released in May, which documents in troubling detail the exact dimensions of what the foundation's president, Dr. Robert Weisbuch, is calling the national "expertise gap." Weisbuch states that the expertise gap extends beyond the…

  13. Narrowing Participation Gaps

    ERIC Educational Resources Information Center

    Hand, Victoria; Kirtley, Karmen; Matassa, Michael

    2015-01-01

    Shrinking the achievement gap in mathematics is a tall order. One way to approach this challenge is to think about how the achievement gap manifests itself in the classroom and take concrete action. For example, opportunities to participate in activities that involve mathematical reasoning and argumentation in a safe and supportive manner are…

  14. Minimum cost model energy code envelope requirements

    SciTech Connect

    Connor, C.C.; Lucas, R.G.; Turchen, S.J.

    1994-08-01

    This paper describes the analysis underlying development of the U.S. Department of Energy`s proposed revisions of the Council of American Building Officials (CABO) 1993 Model Energy Code (MEC) building thermal envelope requirements for single-family and low-rise multifamily residences. This analysis resulted in revised MEC envelope conservation levels based on an objective methodology that determined the minimum-cost combination of energy efficiency measures (EEMs) for residences in different locations around the United States. The proposed MEC revision resulted from a cost-benefit analysis from the consumer`s perspective. In this analysis, the costs of the EEMs were balanced against the benefit of energy savings. Detailed construction, financial, economic, and fuel cost data were compiled, described in a technical support document, and incorporated in the analysis. A cost minimization analysis was used to compare the present value of the total long-nm costs for several alternative EEMs and to select the EEMs that achieved the lowest cost for each location studied. This cost minimization was performed for 881 cities in the United States, and the results were put into the format used by the MEC. This paper describes the methodology for determining minimum-cost energy efficiency measures for ceilings, walls, windows, and floors and presents the results in the form of proposed revisions to the MEC. The proposed MEC revisions would, on average, increase the stringency of the MEC by about 10%.

  15. TRANSPARENT HELIUM IN STRIPPED ENVELOPE SUPERNOVAE

    SciTech Connect

    Piro, Anthony L.; Morozova, Viktoriya S.

    2014-09-01

    Using simple arguments based on photometric light curves and velocity evolution, we propose that some stripped envelope supernovae (SNe) show signs that a significant fraction of their helium is effectively transparent. The main pieces of evidence are the relatively low velocities with little velocity evolution, as are expected deep inside an exploding star, along with temperatures that are too low to ionize helium. This means that the helium should not contribute to the shaping of the main SN light curve, and thus the total helium mass may be difficult to measure from simple light curve modeling. Conversely, such modeling may be more useful for constraining the mass of the carbon/oxygen core of the SN progenitor. Other stripped envelope SNe show higher velocities and larger velocity gradients, which require an additional opacity source (perhaps the mixing of heavier elements or radioactive nickel) to prevent the helium from being transparent. We discuss ways in which similar analysis can provide insights into the differences and similarities between SNe Ib and Ic, which will lead to a better understanding of their respective formation mechanisms.

  16. Envelope Modes of Beams with Angular Momentum

    SciTech Connect

    Barnard, J J; Losic, B

    2000-08-21

    For a particle beam propagating in an alternating gradient focusing system, envelope equations are often employed to describe the evolution of the beam radii in the two directions transverse to the direction of propagation, and aligned with the principle axes of the alternating gradient system. When the beams have zero net angular momentum and when the alternating gradient focusing is approximated by a continuous focusing system, there are two normal modes to the envelope equations: the 'breathing' mode and a 'quadrupole' mode. In the former, the two radii oscillate in phase, and in the latter the radii oscillate 180 degrees out of phase. In this paper, we extend the analysis to include beams that have a finite angular momentum. We perturb the moment equations of ref. [1], wherein it was assumed that space charge is a distributed in a uniform density ellipse. Two additional modes are obtained. The breathing mode remains, but the quadrupole mode is split into two modes, and a new low frequency mode appears. We calculate the frequencies and eigenmodes of these four modes as a function of tune depression and a dimensionless net angular momentum. These modes can be excited by rotational errors of the quadrupoles in an alternating gradient focusing channel.

  17. Sensitivity to changes in amplitude envelope

    NASA Astrophysics Data System (ADS)

    Gallun, Erick; Hafter, Ervin R.; Bonnel, Anne-Marie

    2002-05-01

    Detection of a brief increment in a tonal pedestal is less well predicted by energy-detection (e.g., Macmillan, 1973; Bonnel and Hafter, 1997) than by sensitivity to changes in the stimulus envelope. As this implies a mechanism similar to an envelope extractor (Viemeister, 1979), sinusoidal amplitude modulation was used to mask a single ramped increment (10, 45, or 70 ms) added to a 1000-ms pedestal with carrier frequency (cf)=477 Hz. As in informational masking (Neff, 1994) and ``modulation-detection interference'' (Yost and Sheft, 1989), interference occurred with masker cfs of 477 and 2013 Hz. While slight masking was found with modulation frequencies (mfs) from 16 to 96 Hz, masking grew inversely with still lower mfs, being greatest for mf=4 Hz. This division is reminiscent of that said to separate sensations of ``roughness'' and ``beats,'' respectively (Terhardt, 1974), with the latter also being related to durations associated with auditory groupings in music and speech. Importantly, this result held for all of the signal durations and onset-offset ramps tested, suggesting that an increment on a pedestal is treated as a single auditory object whose detection is most difficult in the presence of other objects (in this case, ``beats'').

  18. Glycolate transporter of the pea chloroplast envelope

    SciTech Connect

    Howitz, K.T.

    1985-01-01

    The discovery of a glycolate transporter in the pea (Pisum sativum) chloroplast envelope is described. Several novel silicone oil centrifugation methods were developed to resolve the initial rate kinetics of (/sup 14/C)glycolate transport by isolated, intact pea chloroplasts. Chloroplast glycolate transport was found to be carrier mediated. Transport rates saturated with increasing glycolate concentration. N-Ethylmaleimide (NEM) pretreatment of chloroplasts inhibited transport, an inhibition prevented by glycolate. Glycolate distributed across the envelope in a way which equalized stromal and medium glycolic acid concentrations, limiting possible transport mechanisms to facilitated glycolic acid diffusion, proton symport or hydroxyl antiport. The effects of stomal and medium pH's on the K/sub m/ and V/sub max/ fit the predictions of mobile carrier kinetic models of hydroxyl antiport or proton symport (H/sup +/ binds first). The carrier mediated transport was fast enough to be consistent with in vivo rates of photorespiration. The 2-hydroxymonocarboxylates, glycerate, lactate and glyoxylate are competitive inhibitors of chloroplast glycolate uptake. Glyoxylate, D-lactate and D-glycerate cause glycolate counterflow, indicating that they are also substrates of the glycolate carrier. This finding was confirmed for D-glycerate by studies on glycolate effects on (1-/sup 14/C)D-glycerate transport.

  19. Functional organization of the HIV lipid envelope

    PubMed Central

    Huarte, Nerea; Carravilla, Pablo; Cruz, Antonio; Lorizate, Maier; Nieto-Garai, Jon A.; Kräusslich, Hans-Georg; Pérez-Gil, Jesús; Requejo-Isidro, Jose; Nieva, José L.

    2016-01-01

    The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope. PMID:27678107

  20. [Electrophysiological characterisation of envelope-following responses].

    PubMed

    Mijares Nodarse, Eleina; Pérez Abalo, María Cecilia; Torres Fortuny, Alejandro; Vega Hernández, Mayrim

    2011-01-01

    The auditory ability to discriminate rapid changes in the envelope of language sounds is essential for speech comprehension. This ability is deteriorated in some neurological diseases such as multiple sclerosis, auditory neuropathy, sensorineural hearing loss, presbycusis and primary developmental language disorder. Envelope-following responses (EFRs) in humans are useful in objective measurement of temporal processing in the auditory nervous system. To evaluate EFRs in healthy younger subjects and to investigate the effects of subject states on the EFRs recorded. Eleven young subjects were included; six of them were awake and five were asleep. EFRs were evoked by white noise carrier stimuli with a sweep of modulation frequencies from 20 to 200Hz presented at 50dB HL. The EFRs we recorded were similar in all subjects. There were two principal components. During both subject sleep and wakefulness, the first component (located between 30-50Hz) was significantly larger than the second component (located between 80-110Hz). There was also a significant effect of sleep on the EFR amplitude for the modulation frequencies between 88-110, 155-165 and 190-200Hz. However, there were no significant effects of sleep on the principal EFR components. These results corroborate the usefulness of the EFR technique for objective measurement of human auditory temporal processing. Copyright © 2011 Elsevier España, S.L. All rights reserved.

  1. Precision envelope detector and linear rectifier circuitry

    DOEpatents

    Davis, Thomas J.

    1980-01-01

    Disclosed is a method and apparatus for the precise linear rectification and envelope detection of oscillatory signals. The signal is applied to a voltage-to-current converter which supplies current to a constant current sink. The connection between the converter and the sink is also applied through a diode and an output load resistor to a ground connection. The connection is also connected to ground through a second diode of opposite polarity from the diode in series with the load resistor. Very small amplitude voltage signals applied to the converter will cause a small change in the output current of the converter, and the difference between the output current and the constant current sink will be applied either directly to ground through the single diode, or across the output load resistor, dependent upon the polarity. Disclosed also is a full-wave rectifier utilizing constant current sinks and voltage-to-current converters. Additionally, disclosed is a combination of the voltage-to-current converters with differential integrated circuit preamplifiers to boost the initial signal amplitude, and with low pass filtering applied so as to obtain a video or signal envelope output.

  2. Proteomics of the chloroplast envelope membranes from Arabidopsis thaliana.

    PubMed

    Ferro, Myriam; Salvi, Daniel; Brugière, Sabine; Miras, Stéphane; Kowalski, Solène; Louwagie, Mathilde; Garin, Jérôme; Joyard, Jacques; Rolland, Norbert

    2003-05-01

    The development of chloroplasts and the integration of their function within a plant cell rely on the presence of a complex biochemical machinery located within their limiting envelope membranes. To provide the most exhaustive view of the protein repertoire of chloroplast envelope membranes, we analyzed this membrane system using proteomics. To this purpose, we first developed a procedure to prepare highly purified envelope membranes from Arabidopsis chloroplasts. We then extracted envelope proteins using different methods, i.e. chloroform/methanol extraction and alkaline or saline treatments, in order to retrieve as many proteins as possible, from the most to least hydrophobic ones. Liquid chromatography tandem mass spectrometry analyses were then performed on each envelope membrane subfraction, leading to the identification of more than 100 proteins. About 80% of the identified proteins are known to be, or are very likely, located in the chloroplast envelope. The validation of localization in the envelope of two phosphate transporters exemplifies the need for a combination of strategies to perform the most exhaustive identification of genuine chloroplast envelope proteins. Interestingly, some of the identified proteins are found to be Nalpha-acetylated, which indicates the accurate location of the N terminus of the corresponding mature protein. With regard to function, more than 50% of the identified proteins have functions known or very likely to be associated with the chloroplast envelope. These proteins are a) involved in ion and metabolite transport, b) components of the protein import machinery, and c) involved in chloroplast lipid metabolism. Some soluble proteins, like proteases, proteins involved in carbon metabolism, or proteins involved in responses to oxidative stress, were associated with envelope membranes. Almost one-third of the proteins we identified have no known function. The present work helps understanding chloroplast envelope metabolism at

  3. Optimized measurement of gaps

    NASA Astrophysics Data System (ADS)

    Harding, Kevin; Ramamurthy, Rajesh

    2017-05-01

    Gaps are important in a wide range of measurements in manufacturing, from the fitting of critical assemblies too cosmetic features on cars. There are a variety of potential sensors that can measure a gap opening, each with aspects of gap measurements that they do well and other aspects where the technology may lack capability. This paper provides a review of a wide range of optical gages from structured light to passive systems and from line to area measurement. Each technology is considered relative to the ability to accurately measure a gap, including issues of edge effects, edge shape, surface finish, and transparency. Finally, an approach will be presented for creating an optimize measurement off gap openings for critical assembly applications.

  4. Expression, purification and crystallization of two major envelope proteins from white spot syndrome virus

    SciTech Connect

    Tang, Xuhua; Hew, Choy Leong

    2007-07-01

    The crystallization of the N-terminal transmembrane region-truncated VP26 and VP28 of white spot syndrome virus is described. White spot syndrome virus (WSSV) is a major virulent pathogen known to infect penaeid shrimp and other crustaceans. VP26 and VP28, two major envelope proteins from WSSV, have been identified and overexpressed in Escherichia coli. In order to facilitate purification and crystallization, predicted N-terminal transmembrane regions of approximately 35 amino acids have been truncated from both VP26 and VP28. Truncated VP26 and VP28 and their corresponding SeMet-labelled proteins were purified and the SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of SeMet-labelled VP26 were obtained using a reservoir consisting of 0.1 M citric acid pH 3.5, 3.0 M sodium chloride and 1%(w/v) polyethylene glycol 3350, whereas SeMet VP28 was crystallized using a reservoir solution consisting of 25% polyethylene glycol 8000, 0.2 M calcium acetate, 0.1 M Na HEPES pH 7.5 and 1.5%(w/v) 1,2,3-heptanetriol. Crystals of SeMet-labelled VP26 diffract to 2.2 Å resolution and belong to space group R32, with unit-cell parameters a = b = 73.92, c = 199.31 Å. SeMet-labelled VP28 crystallizes in space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 105.33, b = 106.71, c = 200.37 Å, and diffracts to 2.0 Å resolution.

  5. Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

    PubMed Central

    Pervushin, Konstantin; Tan, Edward; Parthasarathy, Krupakar; Lin, Xin; Jiang, Feng Li; Yu, Dejie; Vararattanavech, Ardcharaporn; Soong, Tuck Wah; Liu, Ding Xiang; Torres, Jaume

    2009-01-01

    The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target. PMID:19593379

  6. Transmembrane helix assembly and the role of salt bridges.

    PubMed

    Walther, Torsten H; Ulrich, Anne S

    2014-08-01

    Transmembrane helix-helix interactions mediate the folding and assembly of membrane proteins. Recognition motifs range from GxxxG and leucine zippers to polar side chains and salt bridges. Some canonical membrane proteins contain local charge clusters that are important for folding and function, and which have to be compatible with a stable insertion into the bilayer via the translocon. Recently, the electrostatic "charge zipper" has been described as another kind of assembly motif. The protein sequences exhibit a quasi-symmetrical pattern of complementary charges that can form extended ladders of salt bridges. Such segments can insert reversibly into membranes, or even translocate across them. Nature uses charge zippers in transport processes, and they can also be adapted in the design of cell-penetrating carriers. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  7. Transmembrane signaling by GPCRs: insight from rhodopsin and opsin structures.

    PubMed

    Choe, Hui-Woog; Park, Jung Hee; Kim, Yong Ju; Ernst, Oliver P

    2011-01-01

    G-protein-coupled receptors (GPCRs), also known as seven-transmembrane (7TM) receptors, are the largest family of membrane proteins in the human genome. As versatile signaling molecules, they mediate cellular responses to extracellular signals. Diffusible ligands like hormones and neurotransmitters bind to GPCRs to modulate GPCR activity. An extraordinary and highly specialized GPCR is the photoreceptor rhodopsin which contains the chromophore retinal as its covalently bound ligand. For receptor activation the configuration of retinal is altered by photon absorption. To date, rhodopsin is the only GPCR for which crystal structures of inactive, active and ligand-free conformations are known. Although the photochemical activation is unique to rhodopsin, many mechanistic insights from this receptor can be generalized for GPCRs. Copyright © 2010 Elsevier Ltd. All rights reserved.

  8. Atomic Structure of the Cystic Fibrosis Transmembrane Conductance Regulator.

    PubMed

    Zhang, Zhe; Chen, Jue

    2016-12-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel evolved from the ATP-binding cassette (ABC) transporter family. In this study, we determined the structure of zebrafish CFTR in the absence of ATP by electron cryo-microscopy to 3.7 Å resolution. Human and zebrafish CFTR share 55% sequence identity, and 42 of the 46 cystic-fibrosis-causing missense mutational sites are identical. In CFTR, we observe a large anion conduction pathway lined by numerous positively charged residues. A single gate near the extracellular surface closes the channel. The regulatory domain, dephosphorylated, is located in the intracellular opening between the two nucleotide-binding domains (NBDs), preventing NBD dimerization and channel opening. The structure also reveals why many cystic-fibrosis-causing mutations would lead to defects either in folding, ion conduction, or gating and suggests new avenues for therapeutic intervention. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Cystic fibrosis transmembrane conductance regulator expression in human hypothalamus.

    PubMed

    Mulberg, A E; Weyler, R T; Altschuler, S M; Hyde, T M

    1998-01-05

    We have previously characterized the expression of the cystic fibrosis transmembrane conductance regulator protein (CFTR) gene, mRNA and protein in rat brain with reverse transcriptase (RT)-PCR amplification, in situ hybridization and immunocytochemistry. We now report that the CFTR mRNA is expressed in the human anterior hypothalamus, an area involved in regulation of appetite, resting energy expenditure and sexual differentiation. Expression of CFTR in neurons localized to this region may elucidate the pathogenesis of other non-pulmonary manifestations of cystic fibrosis which commonly are observed in children with CF, including congenital absence of the vas deferens. Neuron-specific expression of CFTR in brain may be involved in the regulation of homeostatic functions including reproductive function and fertility through effects on neurosecretion, i.e. GnRH release. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain my lead to alteration in physiological function.

  10. Large-Conductance Transmembrane Porin Made from DNA Origami.

    PubMed

    Göpfrich, Kerstin; Li, Chen-Yu; Ricci, Maria; Bhamidimarri, Satya Prathyusha; Yoo, Jejoong; Gyenes, Bertalan; Ohmann, Alexander; Winterhalter, Mathias; Aksimentiev, Aleksei; Keyser, Ulrich F

    2016-09-27

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins' large ionic conductance.

  11. Hkat, a novel nutritionally regulated transmembrane protein in adipose tissues.

    PubMed

    Zhang, Ren

    2012-01-01

    White adipose tissue is an active endocrine organ regulating many aspects of whole body physiology and pathology. Adipogenesis, a process in which premature cells differentiate into adipocytes, is a complex process that includes orchestrated changes in gene expression and cell morphology in response to various nutritional and hormonal stimuli. To profile transcriptome changes in response to nutritional stimulation, we performed RNA-seq on fat in mice treated with either a high-fat diet or fasting. We identified a novel nutritionally regulated gene, Gm12824, named Hkat (heart, kidney, adipose-enriched transmembrane protein). We show that both fasting and obesity dramatically reduce Hkat in white adipose tissue, and that fasting reduces while obesity increases its expression in brown fat. Hkat is localized to the plasma membrane and induced during adipogenesis. Therefore, Hkat is a novel nutritionally regulated gene that is potentially involved in metabolism.

  12. Large-Conductance Transmembrane Porin Made from DNA Origami

    PubMed Central

    2016-01-01

    DNA nanotechnology allows for the creation of three-dimensional structures at nanometer scale. Here, we use DNA to build the largest synthetic pore in a lipid membrane to date, approaching the dimensions of the nuclear pore complex and increasing the pore-area and the conductance 10-fold compared to previous man-made channels. In our design, 19 cholesterol tags anchor a megadalton funnel-shaped DNA origami porin in a lipid bilayer membrane. Confocal imaging and ionic current recordings reveal spontaneous insertion of the DNA porin into the lipid membrane, creating a transmembrane pore of tens of nanosiemens conductance. All-atom molecular dynamics simulations characterize the conductance mechanism at the atomic level and independently confirm the DNA porins’ large ionic conductance. PMID:27504755

  13. Functional relevance of transmembrane domains in membrane fusion.

    PubMed

    Nikolaus, Jörg; Herrmann, Andreas

    2012-11-01

    Membrane fusion is ubiquitous in life. Fusion of biological membranes is mediated by specialized fusion proteins anchored to the bilayers destined to fuse. Here we describe these proteins as being instrumental in viral, intracellular and developmental fusion. Next, we review experimental and theoretical evidence that points to fusion in the different systems as following a common 'fusion through hemifusion' pathway. We also focus on the structure and dynamics of the transmembrane segment that anchors the fusion proteins to the bilayer, and its role in driving fusion. In particular, we highlight the influence of this single segment on the surrounding membrane lipids and on the overall shape of the membrane along the way to fusion.

  14. ATPase activity of the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Li, C; Ramjeesingh, M; Wang, W; Garami, E; Hewryk, M; Lee, D; Rommens, J M; Galley, K; Bear, C E

    1996-11-08

    The gene mutated in cystic fibrosis codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cyclic AMP-activated chloride channel thought to be critical for salt and water transport by epithelial cells. Plausible models exist to describe a role for ATP hydrolysis in CFTR channel activity; however, biochemical evidence that CFTR possesses intrinsic ATPase activity is lacking. In this study, we report the first measurements of the rate of ATP hydrolysis by purified, reconstituted CFTR. The mutation CFTRG551D resides within a motif conserved in many nucleotidases and is known to cause severe human disease. Following reconstitution the mutant protein exhibited both defective ATP hydrolysis and channel gating, providing direct evidence that CFTR utilizes ATP to gate its channel activity.

  15. Retrieval of transmembrane proteins to the endoplasmic reticulum

    PubMed Central

    1993-01-01

    A COOH-terminal double lysine motif maintains type I transmembrane proteins in the ER. Proteins tagged with this motif, eg., CD8/E19 and CD4/E19, rapidly receive post-translational modifications characteristic of the intermediate compartment and partially colocalized to this organelle. These proteins also received modifications characteristic of the Golgi but much more slowly. Lectin staining localized these Golgi modified proteins to ER indicating that this motif is a retrieval signal. Differences in the subcellular distribution and rate of post-translational modification of CD8 maintained in the ER by sequences derived from a variety of ER resident proteins suggested that the efficiency of retrieval was dependent on the sequence context of the double lysine motif and that retrieval may be initiated from multiple positions along the exocytotic pathway. PMID:8468349

  16. The transmembrane channel-like protein family and human papillomaviruses

    PubMed Central

    Horton, Jaime S; Stokes, Alexander J

    2014-01-01

    Epidermodysplasia verruciformis (EV) is a rare genodermatosis characterized by increased sensitivity to infection by the β-subtype of human papillomaviruses (β-HPVs), causing persistent, tinea versicolor-like dermal lesions. In a majority of affected individuals, these macular lesions progress to invasive cutaneous squamous cell carcinoma (CSCC) in sun-exposed areas. While mutations in transmembrane channel-like 6 (TMC6 / EVER1) and 8 (TMC8 / EVER2) have been causally linked to EV, their molecular functions are unclear. It is likely that their protective effects involve regulation of the β-HPV life cycle, host keratinocyte apoptosis vs. survival balance and/or T-cell interaction with infected host cells. PMID:24800179

  17. Impact of histidine residues on the transmembrane helices of viroporins.

    PubMed

    Wang, Yan; Park, Sang Ho; Tian, Ye; Opella, Stanley J

    2013-11-01

    Abstract The role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein 'u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site.

  18. Molecular Dynamics Simulation of Membranes and a Transmembrane Helix

    NASA Astrophysics Data System (ADS)

    Duong, Tap Ha; Mehler, Ernest L.; Weinstein, Harel

    1999-05-01

    Three molecular dynamics (MD) simulations of 1.5-ns length were carried out on fully hydrated patches of dimyristoyl phosphatidylcholine (DMPC) bilayers in the liquid-crystalline phase. The simulations were performed using different ensembles and electrostatic conditions: a microcanonical ensemble or constant pressure-temperature ensemble, with or without truncated electrostatic interactions. Calculated properties of the membrane patches from the three different protocols were compared to available data from experiments. These data include the resulting overall geometrical dimensions, the order characteristics of the lipid hydrocarbon chains, as well as various measures of the conformations of the polar head groups. The comparisons indicate that the simulation carried out within the microcanonical ensemble with truncated electrostatic interactions yielded results closest to the experimental data, provided that the initial equilibration phase preceding the production run was sufficiently long. The effects of embedding a non-ideal helical protein domain in the membrane patch were studied with the same MD protocols. This simulation was carried out for 2.5 ns. The protein domain corresponds to the seventh transmembrane segment (TMS7) of the human serotonin 5HT 2Areceptor. The peptide is composed of two α-helical segments linked by a hinge domain around a perturbing Asn-Pro motif that produces at the end of the simulation a kink angle of nearly 80° between the two helices. Several aspects of the TMS7 structure, such as the bending angle, backbone Φ and Ψ torsion angles, the intramolecular hydrogen bonds, and the overall conformation, were found to be very similar to those determined by NMR for the corresponding transmembrane segment of the tachykinin NK-1 receptor. In general, the simulations were found to yield structural and dynamic characteristics that are in good agreement with experiment. These findings support the application of simulation methods to the study

  19. Analysis of Structured and Intrinsically Disordered Regions of Transmembrane Proteins

    PubMed Central

    Xue, Bin; Li, Liwei; Meroueh, Samy O.; Uversky, Vladimir N.; Dunker, A. Keith

    2010-01-01

    Integral membrane proteins display two major types of transmembrane structures, helical bundles and beta barrels. The main functional roles of transmembrane proteins are the transport of small molecules and cell signaling, and sometimes these two roles are coupled. For cytosolic, water-soluble proteins, signaling and regulatory functions are often carried out by intrinsically disordered regions. Our long range goal is to determine whether integral membrane proteins likewise often use disordered regions for signaling and regulation. Here we carried out a systematic bioinformatics investigation of intrinsically disordered regions obtained from integral membrane proteins for which crystal structures have been determined, and for which the intrinsic disorder was identified as missing electron density. We found 120 disorder-containing integral membrane proteins having a total of 33,675 residues, with 3209 of the residues distributed among 240 different disordered regions. These disordered regions were compared with those obtained from water-soluble proteins with regard to their amino acid compositional biases, and with regard to accuracies of various disorder predictors. The results of these analyses show that the disordered regions from helical bundle integral membrane proteins, those from beta barrel integral membrane proteins, and those from water soluble proteins all exhibit statistically distinct amino acid compositional biases. Despite these differences in composition, current algorithms make reasonably accurate predictions of disorder for these membrane proteins. Although the small size of the current data sets are limiting, these results suggest that developing new predictors that make use of data from disordered regions in helical bundles and beta barrels, especially as these datasets increase in size, will likely lead to significantly more accurate disorder predictions for these two classes of integral membrane proteins. PMID:19585006

  20. Prediction of transmembrane helices from hydrophobic characteristics of proteins.

    PubMed

    Ponnuswamy, P K; Gromiha, M M

    1993-10-01

    Membrane proteins, requiring to be embedded into the lipid bilayers, have evolved to have amino acid sequences that will fold with a hydrophobic surface in contact with the alkane chains of the lipids and polar surface in contact with the aqueous phases on both sides of the membrane and the polar head groups of the lipids. It is generally assumed that the characteristics of the aqueous parts of the membrane proteins are similar to those of normal globular proteins, and the embedded parts are highly hydrophobic. In our earlier works, we introduced the concept of 'surrounding hydrophobicity' and developed a hydrophobicity scale for the 20 amino acid residues, and applied it successfully to the study of the family of globular proteins. In this work we use the concept of surrounding hydrophobicity to indicate quantitatively how the aqueous parts of membrane proteins compare with the normal globular proteins, and how rich the embedded parts are in their hydrophobic activity. We then develop a surrounding hydrophobicity scale applicable to membrane proteins, by mixing judicially the surrounding hydrophobicities observed in the crystals of the membrane protein, photosynthetic reaction center from the bacterium Rhodopseudomonas viridis, porin from Rhodobacter capsulatus and a set of 64 globular proteins. A predictive scheme based on this scale predicts from amino acid sequence, transmembrane segments in PRC and randomly selected 26 membrane proteins to 80% level of accuracy. This is a much higher predictive power when compared to the existing popular methods. A new procedure to measure the amphipathicity of sequence segments is proposed, and it is used to characterize the transmembrane parts of the sample membrane proteins.

  1. Photometric recording of transmembrane potential in outer hair cells

    NASA Astrophysics Data System (ADS)

    Nakagawa, Takashi; Oghalai, John S.; Saggau, Peter; Rabbitt, Richard D.; Brownell, William E.

    2006-06-01

    Cochlear outer hair cells (OHCs) are polarized epithelial cells that have mechanoelectrical transduction channels within their apical stereocilia and produce electromotile force along their lateral wall. Phase shifts, or time delays, in the transmembrane voltage occurring at different axial locations along the cell may contribute to our understanding of how these cells operate at auditory frequencies. We developed a method to optically measure the phase of the OHC transmembrane potential using the voltage-sensitive dye (VSD) di-8-ANEPPS. The exit aperture of a fibre-optic light source was driven in two dimensions so that a 24 µm spot of excitation light could be positioned along the length of the OHC. We used the whole-cell patch-clamp technique in the current-clamp mode to stimulate the OHC at the base. The photometric response and the voltage response were monitored with a photodetector and patch-clamp amplifier, respectively. The photometric response was used to measure the regional changes in the membrane potential in response to maintained (dc) and sinusoidal (ac) current stimuli applied at the base of the cell. We used a neutral density filter to lower the excitation light intensity and reduce phototoxicity. A sensitive detector and lock-in amplifier were used to measure the small ac VSD signal. This permitted measurements of the ac photometric response below the noise floor of the static fluorescence. The amplitude and phase components of the photometric response were recorded for stimuli up to 800 Hz. VSD data at 400-800 Hz show the presence of a small phase delay between the stimulus voltage at the base of the cell and the local membrane potential measured along the lateral wall. Results are consistent with the hypothesis that OHCs exhibit inhomogeneous membrane potentials that vary with position in analogy with the voltage in nerve axons.

  2. Transmembrane Pores Formed by Human Antimicrobial Peptide LL-37

    SciTech Connect

    Qian, Shuo

    2011-01-01

    Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the {alpha}-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 2333 {angstrom}. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.

  3. Transmembrane potentials during high voltage shocks in ischemic cardiac tissue.

    PubMed

    Holley, L K; Knisley, S B

    1997-01-01

    Transmembrane, voltage sensitive fluorescent dye (TMF) recording techniques have shown that high voltage shocks (HVS), typically used in defibrillation, produce either hyper- or depolarization of the transmembrane potential (TMP) when delivered in the refractory period of an action potential (AP) in normal cardiac tissue (NT). Further, HVS produce an extension of the AP, which has been hypothesized as a potential mechanism for electrical defibrillation. We examined whether HVS modify TMP of ischemic tissue (IT) in a similar manner. In seven Langendorff rabbit hearts, recordings of APs were obtained in both NT and IT with TMF using di-4-ANEPPS, and diacetylmonoxime (23 microM) to avoid motion artifacts. Local ischemia was produced by occlusion of the LAD, HVS of either biphasic (5 + 5 ms) or (3 + 2 ms) or monophasic shapes (5 ms) were delivered at varying times (20%-90%) of the paced AP. Intracardiac ECG and TMF recordings of the TMP were each amplified, recorded, and digitized at a frequency of 1 kHz. The paced AP in IT was triangular in shape with no obvious phase 3 plateau, typically seen in NT. There was normally a reduced AP amplitude (expressed as fractional fluorescence) in IT (2.6% +/- 1.79%) compared to 3.8% +/- 0.66% in NT, and shortened AP duration (137 +/- 42 vs 171 +/- 11 ms). One hundred-Volt HVS delivered during the refractory period of paced AP in IT in five rabbits, elicited a depolarization response of the TMP with an amplitude up to three times greater than the paced AP. This is in contrast to NT where the 100-V HVS produced hyperpolarization in four hearts, and only a slight depolarization response in one heart. These results suggest that HVS, typically delivered by a defibrillation shock, modify TMPs in a significantly different manner for ischemic cells, which may influence success in defibrillation.

  4. A functional protein pore with a "retro" transmembrane domain.

    PubMed Central

    Cheley, S.; Braha, O.; Lu, X.; Conlan, S.; Bayley, H.

    1999-01-01

    Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif. PMID:10386875

  5. Aquaporin-facilitated transmembrane diffusion of hydrogen peroxide.

    PubMed

    Bienert, Gerd P; Chaumont, François

    2014-05-01

    Hydrogen peroxide (H2O2) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H2O2 diffusion. This review summarizes current knowledge about aquaporin-facilitated H2O2 diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H2O2 diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes. Specific aquaporin isoforms facilitate the passive diffusion of H2O2 across biological membranes and control H2O2 membrane permeability and signaling in living organisms. Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H2O2, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H2O2 levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins. © 2013.

  6. Roles of carboxyl groups in the transmembrane insertion of peptides

    PubMed Central

    Barrera, Francisco N.; Weerakkody, Dhammika; Anderson, Michael; Andreev, Oleg A.; Reshetnyak, Yana K.; Engelman, Donald M.

    2011-01-01

    We have used the pHLIP® peptide to study the roles of carboxyl groups in transmembrane peptide insertion. The pH (low) insertion peptide (pHLIP) binds to the surface of a lipid bilayer as a disordered peptide at neutral pH, and when the pH is lowered it inserts across the membrane to form a transmembrane helix. Peptide insertion is reversed when the pH is raised above the characteristic pKa (6.0). A key event facilitating the membrane insertion is the protonation of aspartic (Asp) and/or glutamic (Glu) acid residues, since at neutral pH their negatively charged side chains hinder membrane insertion. In order to gain mechanistic understanding, we studied the membrane insertion and exit of a series of pHLIP variants where the four Asp residues were sequentially mutated to nonacidic residues, including histidine (His). Our results show that the presence of His residues does not prevent the pH-dependent peptide membrane insertion at ~pH 4 driven by the protonation of carboxyl groups at the inserting end of the peptide. A further pH drop leads to the protonation of His residues in the TM part of peptide, which induces peptide exit from the bilayer. We also find that the number of ionizable residues that undergo a change in protonation during membrane insertion correlates with the pH-dependent insertion into and exit from the lipid bilayer, and that cooperativity increases with their number. We expect that our understanding will be used to improve the targeting of acidic diseased tissue by pHLIP peptides. PMID:21888917

  7. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  8. Virtual Nuclear Envelope Breakdown and Its Regulators in Fission Yeast Meiosis.

    PubMed

    Asakawa, Haruhiko; Yang, Hui-Ju; Hiraoka, Yasushi; Haraguchi, Tokuko

    2016-01-01

    Ran, a small GTPase, is required for the spindle formation and nuclear envelope (NE) formation. After NE breakdown (NEBD) during mitosis in metazoan cells, the Ran-GTP gradient across the NE is lost and Ran-GTP becomes concentrated around chromatin, thus affecting the stability of microtubules and promoting the assembly of spindle microtubules and segregation of chromosomes. Mitosis in which chromosomes are segregated subsequent to NEBD is called "open mitosis." In contrast, many fungi undergo a process termed "closed mitosis" in which chromosome segregation and spindle formation occur without NEBD. Although the fission yeast Schizosaccharomyces pombe undergoes a closed mitosis, it exhibits a short period during meiosis (anaphase of the second meiosis; called "anaphase II") when nuclear and cytoplasmic proteins are mixed in the presence of intact NE and nuclear pore complexes (NPC). This "virtual" nuclear envelope breakdown (vNEBD) involves changes in the localization of RanGAP1, an activator of Ran-GTP hydrolysis. Recently, Nup132, a component of the structural core Nup107-160 subcomplex of the NPC, has been shown to be involved in the maintenance of the nuclear cytoplasmic barrier in yeast meiosis. In this review, we highlight the possible roles of RanGAP1 and Nup132 in vNEBD and discuss the biological significance of vNEBD in S. pombe meiosis.

  9. Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture.

    PubMed

    Gardner, Thomas J; Hernandez, Rosmel E; Noriega, Vanessa M; Tortorella, Domenico

    2016-03-30

    The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

  10. HSV-1 nucleocapsid egress mediated by UL31 in association with UL34 is impeded by cellular transmembrane protein 140

    SciTech Connect

    Guan, Ying; Guo, Lei; Yang, Erxia; Liao, Yun; Liu, Longding; Che, Yanchun; Zhang, Ying; Wang, Lichun; Wang, Jingjing; Li, Qihan

    2014-09-15

    During HSV-1 infection, the viral UL31 protein forms a complex with the UL34 protein at the cellular nuclear membrane, where both proteins play important roles in the envelopment of viral nucleocapsids and their egress into the cytoplasm. To characterize the mechanism of HSV-1 nucleocapsid egress, we screened host proteins to identify proteins that interacted with UL31 via yeast two-hybrid analysis. Transmembrane protein 140 (TMEM140), was identified and confirmed to bind to and co-localize with UL31 during viral infection. Further studies indicated that TMEM140 inhibits HSV-1 proliferation through selectively blocking viral nucleocapsid egress during the viral assembly process. The blockage function of TMEM140 is mediated by impeding the formation of the UL31–UL34 complex due to competitive binding to UL31. Collectively, these data suggest the essentiality of the UL31–UL34 interaction in the viral nucleocapsid egress process and provide a new anti-HSV-1 strategy in viral assembly process of nucleocapsid egress. - Highlights: • Cellular TMEM140 protein interacts with HSV-1 UL31 protein during viral infection. • Increasing expression of TMEM140 leads to inhibition of HSV-1 proliferation. • Increasing expression of TMEM140 blocks HSV-1 nucleocapsid egress process. • Binding to UL31 of TMEM140 impedes formation of HSV-1 UL31–UL34 complex.

  11. Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture

    PubMed Central

    Gardner, Thomas J.; Hernandez, Rosmel E.; Noriega, Vanessa M.; Tortorella, Domenico

    2016-01-01

    The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV. PMID:27026399

  12. Development of recombinant capsid antigen/transmembrane epitope fusion proteins for serological diagnosis of animal lentivirus infections.

    PubMed

    Rosati, S; Profiti, M; Lorenzetti, R; Bandecchi, P; Mannelli, A; Ortoffi, M; Tolari, F; Ciabatti, I M

    2004-10-01

    Among animal lentiviruses, Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV) and Small ruminant lentiviruses (SRLV) are important pathogens associated with a variety of clinical pictures including immunodeficiency, anaemia, arthritis, pneumonia. The detection of viral antibody response represents a practical diagnostic approach in all lentivirus infections since they remain detectable long life. Capsid antigen (CA) is the major viral core protein and specific antibodies against this antigen are usually first recognised in infected sheep, goat and horse, remaining detectable for long period. Transmembrane (TM) domain of envelope glycoprotein contains a well conserved motif known to form an immunodominant epitope in several lentiviruses. In this study a simple strategy was developed to express the entire CA and the TM epitope in a single fusion protein from equine, feline and small ruminant lentiviruses in prokaryotic system and evaluated the diagnostic utility of a purified preparation in an indirect ELISA for each of the three infections. Results demonstrate that, for FIV and SRLV infections, the combination of CA and TM fractions increases the sensitivity of diagnostic tests based only on CA. The corresponding CA/TM antigen from EIAV showed excellent agreement with Coggins test.

  13. Bright and gap solitons in membrane-type acoustic metamaterials

    NASA Astrophysics Data System (ADS)

    Zhang, Jiangyi; Romero-García, Vicente; Theocharis, Georgios; Richoux, Olivier; Achilleos, Vassos; Frantzeskakis, Dimitrios J.

    2017-08-01

    We study analytically and numerically envelope solitons (bright and gap solitons) in a one-dimensional, nonlinear acoustic metamaterial, composed of an air-filled waveguide periodically loaded by clamped elastic plates. Based on the transmission line approach, we derive a nonlinear dynamical lattice model which, in the continuum approximation, leads to a nonlinear, dispersive, and dissipative wave equation. Applying the multiple scales perturbation method, we derive an effective lossy nonlinear Schrödinger equation and obtain analytical expressions for bright and gap solitons. We also perform direct numerical simulations to study the dissipation-induced dynamics of the bright and gap solitons. Numerical and analytical results, relying on the analytical approximations and perturbation theory for solions, are found to be in good agreement.

  14. Bridging The Inferential Gap

    PubMed Central

    Stewart, Walter F.; Shah, Nirav R.; Selna, Mark J.; Paulus, Ronald A.; Walker, James M.

    2009-01-01

    Most clinical decisions involve bridging the inferential gap: Clinicians are required to “fill in” where they lack knowledge or where no knowledge yet exists. In this context we consider how the inferential gap is a product, in part, of how knowledge is created, the limits to gaining access to such knowledge, and the variable ways in which knowledge is translated into decisions. We consider how electronic health records (EHRs) will help narrow this gap by accelerating the creation of evidence relevant to everyday practice needs and facilitating real-time use of knowledge in practice. PMID:17259202

  15. SPARK GAP SWITCH

    DOEpatents

    Neal, R.B.

    1957-12-17

    An improved triggered spark gap switch is described, capable of precisely controllable firing time while switching very large amounts of power. The invention in general comprises three electrodes adjustably spaced and adapted to have a large potential impressed between the outer electrodes. The central electrode includes two separate elements electrically connected togetaer and spaced apart to define a pair of spark gaps between the end electrodes. Means are provided to cause the gas flow in the switch to pass towards the central electrode, through a passage in each separate element, and out an exit disposed between the two separate central electrode elements in order to withdraw ions from the spark gap.

  16. Defining the Core Proteome of the Chloroplast Envelope Membranes

    PubMed Central

    Simm, Stefan; Papasotiriou, Dimitrios G.; Ibrahim, Mohamed; Leisegang, Matthias S.; Müller, Bernd; Schorge, Tobias; Karas, Michael; Mirus, Oliver; Sommer, Maik S.; Schleiff, Enrico

    2013-01-01

    High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided. PMID:23390424

  17. Cricket team selection using data envelopment analysis.

    PubMed

    Amin, Gholam R; Sharma, Sujeet Kumar

    2014-01-01

    This paper suggests a new method for cricket team selection using data envelopment analysis (DEA). We propose a DEA formulation for evaluation of cricket players in different capabilities using multiple outputs. This evaluation determines efficient and inefficient cricket players and ranks them on the basis of DEA scores. The ranking can be used to choose the required number of players for a cricket team in each cricketing capability. A real dataset, Indian Premier League 4 (IPL 2011), cricket players having various capabilities is used to choose the best cricket team. The proposed method has the advantage of considering multiple factors related to the performance of players in multiple capabilities collected from IPL 4 and aggregates their scores using a linear programming DEA model. This DEA Aggregation gives the scores of players objectively instead of using subjective computations. The proposed DEA method can be used to form a national cricket team from several clubs or a team of top cricketers.

  18. Fullerenes and fulleranes in circumstellar envelopes

    NASA Astrophysics Data System (ADS)

    Zhang, Yong; Kwok, Sun; Sadjadi, SeyedAbdolreza

    2016-07-01

    Three decades of search have recently led to convincing discoveries of cosmic fullerenes. The presence of C60 and C+ 60 in both circumstellar and interstellar environments suggests that these molecules and their derivatives can be efficiently formed in circumstellar envelopes and survive in harsh conditions. Detailed analysis of the infrared bands from fullerenes and their connections with the local properties can provide valuable information on the physical conditions and chemical processes that occurred in the late stages of stellar evolution. The identification of C+ 60 as the carrier of four diffuse interstellar bands (DIBs) suggests that fullerene- related compounds are abundant in interstellar space and are essential for resolving the DIB mystery. Experiments have revealed a high hydrogenation rate when C60 is exposed to atomic hydrogen, motivating the attempt to search for cosmic fulleranes. In this paper, we present a short review of current knowledge of cosmic fullerenes and fulleranes and briefly discuss the implications on circumstellar chemistry.

  19. Envelope representations for screened Coulomb potentials

    NASA Astrophysics Data System (ADS)

    Hall, Richard L.

    1985-07-01

    We study the discrete eigenvalues Enl of the Schrödinger Hamiltonian H=-(1/2)Δ+V(r), where V(r)=g(-1/r) is an increasing concave transformation of the Coulomb potential, and n is the principal (radial) quantum number. It is demonstrated by the method of potential envelopes that upper bounds are provided by the simple formula Enl<= mins>0 \\{(1/2)s+V((n+l)/s1/2)\\}, where s is a real variable. Numerical results are compared with previous work for two specific screened Coulomb potentials. In the case of the Yukawa potential V(r)=-(v/r)exp(-λr), it is shown that the inequality (n+l)2λ/v<2/e is sufficient to guarantee the existence of the eigenvalue Enl: In the case of S states, sharp upper and lower bounds are also provided by a different method.

  20. Bilayer mechanical properties regulate transmembrane helix mobility and enzymatic state of CD39†

    PubMed Central

    Grinthal, Alison; Guidotti, Guido

    2008-01-01

    CD39 can exist in at least two distinct functional states depending on the presence and intact membrane integration of its two transmembrane helices. In native membranes, the transmembrane helices undergo dynamic rotational motions that are required for enzymatic activity and are regulated by substrate binding. In the present study we show that bilayer mechanical properties regulate conversion between the two enzymatic functional states by modulating transmembrane helix dynamics. Alteration of membrane properties by insertion of cone shaped or inverse cone shaped amphiphiles or by cholesterol removal switches CD39 to the same enzymatic state as does removing or solubilizing the transmembrane domains. The same membrane alterations increase the propensity of both transmembrane helices to rotate within the packed structure, resulting in a structure with greater mobility but not an altered primary conformation. Membrane alteration also abolishes the ability of substrate to stabilize the helices in their primary conformation, indicating a loss of coupling between substrate binding and transmembrane helix dynamics. Removal of either transmembrane helix mimics the effect of membrane alteration on the mobility and substrate sensitivity of the remaining helix, suggesting that the ends of the extracellular domain have intrinsic flexibility. We suggest that a mechanical bilayer property, potentially elasticity, regulates CD39 by altering the balance between stability and flexibility of its transmembrane helices and, in turn, of its active site. PMID:17198399

  1. Hidden markov model for the prediction of transmembrane proteins using MATLAB.

    PubMed

    Chaturvedi, Navaneet; Shanker, Sudhanshu; Singh, Vinay Kumar; Sinha, Dhiraj; Pandey, Paras Nath

    2011-01-01

    Since membranous proteins play a key role in drug targeting therefore transmembrane proteins prediction is active and challenging area of biological sciences. Location based prediction of transmembrane proteins are significant for functional annotation of protein sequences. Hidden markov model based method was widely applied for transmembrane topology prediction. Here we have presented a revised and a better understanding model than an existing one for transmembrane protein prediction. Scripting on MATLAB was built and compiled for parameter estimation of model and applied this model on amino acid sequence to know the transmembrane and its adjacent locations. Estimated model of transmembrane topology was based on TMHMM model architecture. Only 7 super states are defined in the given dataset, which were converted to 96 states on the basis of their length in sequence. Accuracy of the prediction of model was observed about 74 %, is a good enough in the area of transmembrane topology prediction. Therefore we have concluded the hidden markov model plays crucial role in transmembrane helices prediction on MATLAB platform and it could also be useful for drug discovery strategy. The database is available for free at bioinfonavneet@gmail.comvinaysingh@bhu.ac.in.

  2. Analysis of jaagsiekte sheep retrovirus (JSRV) envelope protein domains in transformation.

    PubMed

    Hull, Stacey; Lim, Joohyun; Hamil, Alexander; Nitta, Takayuki; Fan, Hung

    2012-12-01

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer in sheep. A unique feature is that JSRV envelope protein is also the oncogene for this virus. Previous studies have identified the cytoplasmic tail (CT) of the envelope transmembrane (TM) protein as critical for transformation although other regions of Env have also been implicated. In this study, the roles of other Env regions in transformation were investigated. Chimeras between JSRV Env and the Env of a related non-oncogenic endogenous retrovirus (enJSRV, 5F16) were used. A chimera containing the membrane-spanning region (MSR) of enJSRV inserted into JSRV Env showed substantially reduced transformation, indicating that the MSR plays a role in transformation. Transformation by this chimera was highly dependent on both Ras/Raf/MEK/MAPK and PI3K/Akt/mTOR signaling. A chimera containing the two amino acids in the TM ectodomain that distinguish JSRV and enJSRV showed modestly reduced transformation. Chimeras in the SU protein indicated that the amino terminal region of SU contributes to transformation, while the C-terminal part is not important. To test if Env trimerization is important for transformation, we mutated a leucine-rich sequence in the putative trimerization domain in the ectodomain of TM (Tri-M). This mutant could not transform cells and it did not oligomerize. However, Tri-M could complement a non-transforming mutant CT mutant (Y590F) so oligomerization is not necessary for at least some aspects of transformation. These experiments provide new insight into the regions and residues of JSRV Env protein necessary for oncogenic transformation.

  3. Expression of human endogenous retrovirus type K envelope glycoprotein in insect and mammalian cells.

    PubMed Central

    Tönjes, R R; Limbach, C; Löwer, R; Kurth, R

    1997-01-01

    The human endogenous retrovirus type K (HERV-K) family codes for the human teratocarcinoma-derived retrovirus (HTDV) particles. The existence of the envelope protein (ENV) of HERV-K encoded by the subgenomic env mRNA has not yet been demonstrated. To study the genetic requirements for successful expression of ENV, we have constructed a series of recombinant HERV-K env expression vectors for infection and transfection experiments in insect cells and mammalian cells, respectively. Six baculovirus constructs bearing full-length or truncated HERV-K env with or without homologous or heterologous signal peptides were used for infections of insect cells. All recombinant baculoviruses yielded ENV proteins with the expected molecular masses. The full-length 80- to 90-kDa HERV-K ENV protein including the cORF leader sequence was glycosylated in insect cells. In addition, the 14-kDa cORF protein was expressed due to splicing of the full-length env mRNA. The ENV precursor protein is not cleaved to the surface (SU) and transmembrane (TM) glycoproteins; it does not appear on the surface of infected insect cells and is not secreted into the medium. For ENV expression in COS cells, plasmid vectors harboring the cytomegalovirus immediate-early promoter/intron A element and the tissue plasminogen activator (t-PA) signal peptide or the homologous HERV-K signal peptide upstream of the env gene were employed. Glycosylated and uncleaved ENV was expressed as in GH teratocarcinoma cells but at higher levels. The heterologous t-PA signal sequence was instrumental for expression of HERV-K ENV on the cell surface. Hence, we have shown for the first time that the HERV-K env gene has the potential to be expressed as a full-length envelope protein with appropriate glycosylation. In addition, our data provide explanations for the lack of infectivity of HERV-K/HTDV particles. PMID:9060628

  4. Diversity in the fertilization envelopes of echinoderms.

    PubMed

    Oulhen, Nathalie; Reich, Adrian; Wong, Julian L; Ramos, Isabela; Wessel, Gary M

    2013-01-01

    Cell surface changes in an egg at fertilization are essential to begin development and for protecting the zygote. Most fertilized eggs construct a barrier around themselves by modifying their original extracellular matrix. This construction usually results from calcium-induced exocytosis of cortical granules, the contents of which in sea urchins function to form the fertilization envelope (FE), an extracellular matrix of cortical granule contents built upon a vitelline layer scaffold. Here, we examined the molecular mechanism of this process in sea stars, a close relative of the sea urchins, and analyze the evolutionary changes that likely occurred in the functionality of this structure between these two organisms. We find that the FE of sea stars is more permeable than in sea urchins, allowing diffusion of molecules in excess of 2 megadaltons. Through a proteomic and transcriptomic approach, we find that most, but not all, of the proteins present in the sea urchin envelope are present in sea stars, including SFE9, proteoliaisin, and rendezvin. The mRNAs encoding these FE proteins accumulated most densely in early oocytes, and then beginning with vitellogenesis, these mRNAs decreased in abundance to levels nearly undetectable in eggs. Antibodies to the SFE9 protein of sea stars showed that the cortical granules in sea star also accumulated most significantly in early oocytes, but different from sea urchins, they translocated to the cortex of the oocytes well before meiotic initiation. These results suggest that the preparation for cell surface changes in sea urchins has been shifted to later in oogenesis, and perhaps reflects the meiotic differences among the species-sea star oocytes are stored in prophase of meiosis and fertilized during the meiotic divisions, as in most animals, whereas sea urchins are one of the few taxons in which eggs have completed meiosis prior to fertilization.

  5. ASYMMETRIC ACCRETION FLOWS WITHIN A COMMON ENVELOPE

    SciTech Connect

    MacLeod, Morgan; Ramirez-Ruiz, Enrico

    2015-04-10

    This paper examines flows in the immediate vicinity of stars and compact objects dynamically inspiralling within a common envelope (CE). Flow in the vicinity of the embedded object is gravitationally focused, leading to drag and potentially to gas accretion. This process has been studied numerically and analytically in the context of Hoyle–Lyttleton accretion (HLA). Yet, within a CE, accretion structures may span a large fraction of the envelope radius, and in so doing sweep across a substantial radial gradient of density. We quantify these gradients using detailed stellar evolution models for a range of CE encounters. We provide estimates of typical scales in CE encounters that involve main sequence stars, white dwarfs, neutron stars, and black holes with giant-branch companions of a wide range of masses. We apply these typical scales to hydrodynamic simulations of three-dimensional HLA with an upstream density gradient. This density gradient breaks the symmetry that defines HLA flow, and imposes an angular momentum barrier to accretion. Material that is focused into the vicinity of the embedded object thus may not be able to accrete. As a result, accretion rates drop dramatically, by one to two orders of magnitude, while drag rates are only mildly affected. We provide fitting formulae to the numerically derived rates of drag and accretion as a function of the density gradient. The reduced ratio of accretion to drag suggests that objects that can efficiently gain mass during CE evolution, such as black holes and neutron stars, may grow less than implied by the HLA formalism.

  6. Diversity in the fertilization envelopes of echinoderms

    PubMed Central

    Oulhen, Nathalie; Reich, Adrian; Wong, Julian L.; Wessel, Gary M.

    2013-01-01

    Cell surface changes in an egg at fertilization are essential to begin development and for protecting the zygote. Most fertilized eggs construct a barrier around themselves by modifying their original extracellular matrix. This construction usually results from calcium induced exocytosis of cortical granules, the contents of which in sea urchins function to form the fertilization envelope (FE), an extracellular matrix of cortical granule contents built upon a vitelline layer scaffold. Here we examined the molecular mechanism of this process in sea stars, a close relative of the sea urchins, and analyze the evolutionary changes that likely occurred in the functionality of this structure between these two organisms. We find that the FE of sea stars is more permeable than in sea urchins, allowing diffusion of molecules in excess of 2 megadaltons. Through a proteomic and transcriptomic approach, we find that most, but not all of the proteins present in the sea urchin envelope are present in sea stars, including SFE9, proteoliaisin, rendezvin, and ovoperoxidase. The mRNAs encoding these FE proteins accumulated most densely in early oocytes, and then beginning with vitellogenesis, these mRNAs deceased in abundance to levels nearly undetectable in eggs. Antibodies to the SFE9 protein of sea stars showed that the cortical granules in sea star also accumulated most significantly in early oocytes, and different from sea urchins, they translocated to the cortex of the oocytes well before meiotic initiation. These results suggest that the preparation of the cell surface changes in sea urchins has been shifted to later in oogenesis and perhaps reflects the meiotic differences among the species–sea star oocytes are stored in prophase of meiosis and fertilized during the meiotic divisions, as in most animals, whereas sea urchins are one of the few taxa in which eggs have completed meiosis prior to fertilization. PMID:23331915

  7. Asymmetric Accretion Flows within a Common Envelope

    NASA Astrophysics Data System (ADS)

    MacLeod, Morgan; Ramirez-Ruiz, Enrico

    2015-04-01

    This paper examines flows in the immediate vicinity of stars and compact objects dynamically inspiralling within a common envelope (CE). Flow in the vicinity of the embedded object is gravitationally focused, leading to drag and potentially to gas accretion. This process has been studied numerically and analytically in the context of Hoyle-Lyttleton accretion (HLA). Yet, within a CE, accretion structures may span a large fraction of the envelope radius, and in so doing sweep across a substantial radial gradient of density. We quantify these gradients using detailed stellar evolution models for a range of CE encounters. We provide estimates of typical scales in CE encounters that involve main sequence stars, white dwarfs, neutron stars, and black holes with giant-branch companions of a wide range of masses. We apply these typical scales to hydrodynamic simulations of three-dimensional HLA with an upstream density gradient. This density gradient breaks the symmetry that defines HLA flow, and imposes an angular momentum barrier to accretion. Material that is focused into the vicinity of the embedded object thus may not be able to accrete. As a result, accretion rates drop dramatically, by one to two orders of magnitude, while drag rates are only mildly affected. We provide fitting formulae to the numerically derived rates of drag and accretion as a function of the density gradient. The reduced ratio of accretion to drag suggests that objects that can efficiently gain mass during CE evolution, such as black holes and neutron stars, may grow less than implied by the HLA formalism.

  8. Enhanced conformational sampling using enveloping distribution sampling.

    PubMed

    Lin, Zhixiong; van Gunsteren, Wilfred F

    2013-10-14

    To lessen the problem of insufficient conformational sampling in biomolecular simulations is still a major challenge in computational biochemistry. In this article, an application of the method of enveloping distribution sampling (EDS) is proposed that addresses this challenge and its sampling efficiency is demonstrated in simulations of a hexa-β-peptide whose conformational equilibrium encompasses two different helical folds, i.e., a right-handed 2.7(10∕12)-helix and a left-handed 3(14)-helix, separated by a high energy barrier. Standard MD simulations of this peptide using the GROMOS 53A6 force field did not reach convergence of the free enthalpy difference between the two helices even after 500 ns of simulation time. The use of soft-core non-bonded interactions in the centre of the peptide did enhance the number of transitions between the helices, but at the same time led to neglect of relevant helical configurations. In the simulations of a two-state EDS reference Hamiltonian that envelops both the physical peptide and the soft-core peptide, sampling of the conformational space of the physical peptide ensures that physically relevant conformations can be visited, and sampling of the conformational space of the soft-core peptide helps to enhance the transitions between the two helices. The EDS simulations sampled many more transitions between the two helices and showed much faster convergence of the relative free enthalpy of the two helices compared with the standard MD simulations with only a slightly larger computational effort to determine optimized EDS parameters. Combined with various methods to smoothen the potential energy surface, the proposed EDS application will be a powerful technique to enhance the sampling efficiency in biomolecular simulations.

  9. An improved double vaseline gap voltage clamp to study electroporated skeletal muscle fibers.

    PubMed Central

    Chen, W; Lee, R C

    1994-01-01

    An improved voltage clamp with a double vaseline gap chamber was designed to study electroporated skeletal muscle fibers. The new clamp eliminated spike overshock of membrane potential when applying step stimulation occurring in the traditional configuration. It allowed greater consistency in membrane potential distribution. After the intracellular resistances of the fiber segment at the vaseline gap area were compensated, it was possible to change membrane potential more quickly. Using this technique, strong electrical pulses used to mimic the situation of electrical shock can be delivered to the cell membrane by voltage clamp. Transmembrane currents of skeletal muscle cell were simultaneously measured during a high pulsed shock and resolved into different components. Distinct transient changes of the transmembrane current, involving the time courses of the formation of electroporation and their recovery time constants, can be recorded. Because of more even membrane potential distribution and faster response to pulsed membrane potential change, this technique is also suitable for membrane study under physiological conditions. PMID:8011901

  10. Fiber optic gap gauge

    DOEpatents

    Wood, Billy E.; Groves, Scott E.; Larsen, Greg J.; Sanchez, Roberto J.

    2006-11-14

    A lightweight, small size, high sensitivity gauge for indirectly measuring displacement or absolute gap width by measuring axial strain in an orthogonal direction to the displacement/gap width. The gap gauge includes a preferably titanium base having a central tension bar with springs connecting opposite ends of the tension bar to a pair of end connector bars, and an elongated bow spring connected to the end connector bars with a middle section bowed away from the base to define a gap. The bow spring is capable of producing an axial strain in the base proportional to a displacement of the middle section in a direction orthogonal to the base. And a strain sensor, such as a Fabry-Perot interferometer strain sensor, is connected to measure the axial strain in the base, so that the displacement of the middle section may be indirectly determined from the measurement of the axial strain in the base.

  11. Gap filler material

    NASA Image and Video Library

    2005-08-04

    S114-E-6674 (4 August 2005) --- On Discovery's middeck, one of the STS-114 crew members holds a piece of the gap filler material that had been protruding from between TPS tiles and retrieved the day before by Mission Specialist Stephen K. Robinson, during the third spacewalk of the flight. Robinson used his gloved fingers to pull out this gap filler and another one from Discovery's belly while carefully supported and maneuvered by the Canadian-built remote manipulator system, operated inside Discovery's cabin by astronauts Wendy B. Lawrence and James M. Kelly. Gap fillers like those Robinson removed are thin, coated Nextel fabric. The protruding gap fillers were identified in photos taken by Station crewmembers using telephoto lenses as Discovery did a slow back flip about 600 feet below before docking.

  12. Gaps in Oncology

    Cancer.gov

    The first plenary of the EPEC-O (Education in Palliative and End-of-Life Care for Oncology) Self-Study Original Version provides background for the curriculum and identifies gaps in current and desired comprehensive cancer care.

  13. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins

    PubMed Central

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G.; Medzihradszky, Katalin F.; Szakács, Gergely; Tusnády, Gábor E.

    2017-01-01

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins. PMID:28211907

  14. A specific interface between integrin transmembrane helices and affinity for ligand.

    PubMed

    Luo, Bing-Hao; Springer, Timothy A; Takagi, Junichi

    2004-06-01

    Conformational communication across the plasma membrane between the extracellular and intracellular domains of integrins is beginning to be defined by structural work on both domains. However, the role of the alpha and beta subunit transmembrane domains and the nature of signal transmission through these domains have been elusive. Disulfide bond scanning of the exofacial portions of the integrin alpha(IIbeta) and beta(3) transmembrane domains reveals a specific heterodimerization interface in the resting receptor. This interface is lost rather than rearranged upon activation of the receptor by cytoplasmic mutations of the alpha subunit that mimic physiologic inside-out activation, demonstrating a link between activation of the extracellular domain and lateral separation of transmembrane helices. Introduction of disulfide bridges to prevent or reverse separation abolishes the activating effect of cytoplasmic mutations, confirming transmembrane domain separation but not hinging or piston-like motions as the mechanism of transmembrane signaling by integrins.

  15. Identification of Extracellular Segments by Mass Spectrometry Improves Topology Prediction of Transmembrane Proteins.

    PubMed

    Langó, Tamás; Róna, Gergely; Hunyadi-Gulyás, Éva; Turiák, Lilla; Varga, Julia; Dobson, László; Várady, György; Drahos, László; Vértessy, Beáta G; Medzihradszky, Katalin F; Szakács, Gergely; Tusnády, Gábor E

    2017-02-13

    Transmembrane proteins play crucial role in signaling, ion transport, nutrient uptake, as well as in maintaining the dynamic equilibrium between the internal and external environment of cells. Despite their important biological functions and abundance, less than 2% of all determined structures are transmembrane proteins. Given the persisting technical difficulties associated with high resolution structure determination of transmembrane proteins, additional methods, including computational and experimental techniques remain vital in promoting our understanding of their topologies, 3D structures, functions and interactions. Here we report a method for the high-throughput determination of extracellular segments of transmembrane proteins based on the identification of surface labeled and biotin captured peptide fragments by LC/MS/MS. We show that reliable identification of extracellular protein segments increases the accuracy and reliability of existing topology prediction algorithms. Using the experimental topology data as constraints, our improved prediction tool provides accurate and reliable topology models for hundreds of human transmembrane proteins.

  16. Dissection of the role of the stable signal peptide of the arenavirus envelope glycoprotein in membrane fusion.

    PubMed

    Messina, Emily L; York, Joanne; Nunberg, Jack H

    2012-06-01

    The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition.

  17. Dissection of the Role of the Stable Signal Peptide of the Arenavirus Envelope Glycoprotein in Membrane Fusion

    PubMed Central

    Messina, Emily L.; York, Joanne

    2012-01-01

    The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition. PMID:22438561

  18. 10 CFR 434.516 - Building exterior envelope.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 false Building exterior envelope. 434.516 Section 434.516 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY CODE FOR NEW FEDERAL COMMERCIAL AND MULTI-FAMILY HIGH RISE RESIDENTIAL BUILDINGS Building Energy Cost Compliance Alternative § 434.516 Building exterior envelope. 516.1...

  19. Data Envelopment Analysis: Measurement of Educational Efficiency in Texas

    ERIC Educational Resources Information Center

    Carter, Lacy

    2012-01-01

    The purpose of this study was to examine the efficiency of Texas public school districts through Data Envelopment Analysis. The Data Envelopment Analysis estimation method calculated and assigned efficiency scores to each of the 931 school districts considered in the study. The efficiency scores were utilized in two phases. First, the school…

  20. An Inside Look at the Two Envelopes Paradox

    ERIC Educational Resources Information Center

    Falk, Ruma; Nickerson, Raymond S.

    2009-01-01

    When two sealed envelopes contain money, one twice as much as the other, a player should be indifferent between them. But when one envelope is opened, one's decision should vary as a function of the observed value and one's subjective probabilities.

  1. 14 CFR 25.333 - Flight maneuvering envelope.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Flight maneuvering envelope. 25.333 Section... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Structure Flight Maneuver and Gust Conditions § 25.333 Flight maneuvering envelope. (a) General. The strength requirements must be met at each combination of...

  2. 14 CFR 25.333 - Flight maneuvering envelope.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Flight maneuvering envelope. 25.333 Section... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY AIRPLANES Structure Flight Maneuver and Gust Conditions § 25.333 Flight maneuvering envelope. (a) General. The strength requirements must be met at each combination of...

  3. Planet formation with envelope enrichment: new insights on planetary diversity

    NASA Astrophysics Data System (ADS)

    Venturini, Julia; Alibert, Yann; Benz, Willy

    2016-12-01

    Aims: We compute for the first time self-consistent models of planet growth that include the effect of envelope enrichment. The change in envelope metallicity is assumed to be the result of planetesimal disruption or icy pebble sublimation. Methods: We solved internal structure equations taking into account global energy conservation for the envelope to compute in situ planetary growth. We considered different opacities and equations of state suited for a wide range of metallicities. Results: We find that envelope enrichment speeds up the formation of gas giants. It also explains naturally the formation of low- and intermediate-mass objects with large fractions of H-He ( 20-30% in mass). High-opacity models explain the metallicity of the giant planets of the solar system well, while low-opacity models are suited to explain the formation of low-mass objects with thick H-He envelopes and gas giants with sub-solar envelope metallicities. We find good agreement between our models and the estimated water abundance for WASP-43b. For HD 189733b, HD 209458b, and WASP-12b we predict fractions of water higher than what is estimated from observations by at least a factor 2. Conclusions: Envelope enrichment by icy planetesimals is the natural scenario to explain the formation of a wide variety of objects, ranging from mini-Neptunes to gas giants. We predict that the total and envelope metallicity decrease with planetary mass.

  4. 14 CFR 29.87 - Height-velocity envelope.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 1 2012-01-01 2012-01-01 false Height-velocity envelope. 29.87 Section 29... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Flight Performance § 29.87 Height-velocity envelope. (a) If there is any combination of height and forward velocity (including hover) under which a...

  5. 14 CFR 29.87 - Height-velocity envelope.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Height-velocity envelope. 29.87 Section 29... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Flight Performance § 29.87 Height-velocity envelope. (a) If there is any combination of height and forward velocity (including hover) under which a...

  6. 14 CFR 29.87 - Height-velocity envelope.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 1 2013-01-01 2013-01-01 false Height-velocity envelope. 29.87 Section 29... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Flight Performance § 29.87 Height-velocity envelope. (a) If there is any combination of height and forward velocity (including hover) under which a...

  7. 14 CFR 29.87 - Height-velocity envelope.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 14 Aeronautics and Space 1 2014-01-01 2014-01-01 false Height-velocity envelope. 29.87 Section 29... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Flight Performance § 29.87 Height-velocity envelope. (a) If there is any combination of height and forward velocity (including hover) under which a...

  8. 14 CFR 29.87 - Height-velocity envelope.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 1 2010-01-01 2010-01-01 false Height-velocity envelope. 29.87 Section 29... AIRWORTHINESS STANDARDS: TRANSPORT CATEGORY ROTORCRAFT Flight Performance § 29.87 Height-velocity envelope. (a) If there is any combination of height and forward velocity (including hover) under which a...

  9. Computer-Based Instruction on Skills for Addressing Envelopes.

    ERIC Educational Resources Information Center

    Humes, Ann

    The approach to computer-based instruction for third and fourth grade elementary students which is sketched teaches component placement, capitalization, and punctuation skills of addressing envelopes within the context of a simulated envelope. Part of a larger design for a complete program of composition instruction, this program comprises a set…

  10. Rolling bearing feature frequency extraction using extreme average envelope decomposition

    NASA Astrophysics Data System (ADS)

    Shi, Kunju; Liu, Shulin; Jiang, Chao; Zhang, Hongli

    2016-09-01

    The vibration signal contains a wealth of sensitive information which reflects the running status of the equipment. It is one of the most important steps for precise diagnosis to decompose the signal and extracts the effective information properly. The traditional classical adaptive signal decomposition method, such as EMD, exists the problems of mode mixing, low decomposition accuracy etc. Aiming at those problems, EAED(extreme average envelope decomposition) method is presented based on EMD. EAED method has three advantages. Firstly, it is completed through midpoint envelopment method rather than using maximum and minimum envelopment respectively as used in EMD. Therefore, the average variability of the signal can be described accurately. Secondly, in order to reduce the envelope errors during the signal decomposition, replacing two envelopes with one envelope strategy is presented. Thirdly, the similar triangle principle is utilized to calculate the time of extreme average points accurately. Thus, the influence of sampling frequency on the calculation results can be significantly reduced. Experimental results show that EAED could separate out single frequency components from a complex signal gradually. EAED could not only isolate three kinds of typical bearing fault characteristic of vibration frequency components but also has fewer decomposition layers. EAED replaces quadratic enveloping to an envelope which ensuring to isolate the fault characteristic frequency under the condition of less decomposition layers. Therefore, the precision of signal decomposition is improved.

  11. 14 CFR 29.1517 - Limiting height-speed envelope.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 1 2011-01-01 2011-01-01 false Limiting height-speed envelope. 29.1517... Operating Limitations § 29.1517 Limiting height-speed envelope. For Category A rotorcraft, if a range of heights exists at any speed, including zero, within which it is not possible to make a safe landing...

  12. Modeling Protostar Envelopes and Disks Seen With ALMA

    NASA Astrophysics Data System (ADS)

    Terebey, Susan; Flores-Rivera, Lizxandra; Willacy, Karen

    2017-01-01

    Thermal continuum emission from protostars comes from both the envelope and circumstellar disk. The dust emits on a variety of spatial scales, ranging from sub-arcseconds for disks to roughly 10 arcseconds for envelopes for nearby protostars. We present models of what ALMA should detect that incorporate a self-consistent collapse solution, radiative transfer, and realistic dust properties.

  13. Swine interferon-induced transmembrane protein, sIFITM3, inhibits foot-and-mouth disease virus infection in vitro and in vivo.

    PubMed

    Xu, Jinfang; Qian, Ping; Wu, Qunfeng; Liu, Shasha; Fan, Wenchun; Zhang, Keshan; Wang, Rong; Zhang, Huawei; Chen, Huanchun; Li, Xiangmin

    2014-09-01

    The interferon-induced transmembrane protein 3 (IFITM3) is a widely expressed potent antiviral effector of the host innate immune system. It restricts a diverse group of pathogenic, enveloped viruses, by interfering with endosomal fusion. In this report, the swine IFITM3 (sIFITM3) gene was cloned. It shares the functionally conserved CD225 domain and multiple critical amino acid residues (Y19, F74, F77, R86 and Y98) with its human ortholog, which are essential for antiviral activity. Ectopic expression of sIFITM3 significantly inhibited non-enveloped foot-and-mouth disease virus (FMDV) infection in BHK-21 cells. Furthermore, sIFITM3 blocked FMDV infection at early steps in the virus life cycle by disrupting viral attachment to the host cell surface. Importantly, inoculation of 2-day-old suckling mice with a plasmid expressing sIFITM3 conferred protection against lethal challenge with FMDV. These results suggest that sIFITM3 is a promising antiviral agent and that can safeguard the host from infection with FMDV.

  14. Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer.

    PubMed

    Ciczora, Yann; Callens, Nathalie; Montpellier, Claire; Bartosch, Birke; Cosset, François-Loïc; Op de Beeck, Anne; Dubuisson, Jean

    2005-10-01

    The envelope glycoproteins of Hepatitis C virus (HCV), E1 and E2, form a heterodimer that is retained in the endoplasmic reticulum (ER). The transmembrane (TM) domains play a major role in E1E2 heterodimerization and in ER retention. Two fully conserved charged residues in the middle of the TM domain of E2 (Asp and Arg) are crucial for these functions. Replacement of the Asp residue by a Leu impaired E1E2 heterodimerization, whereas the Arg-to-Leu mutation had a milder effect. Both Asp and Arg residues were shown to contribute to the ER retention function of E2. In addition, the entry function of HCV envelope glycoproteins was affected by these mutations. Together, these data indicate that the charged residues present in the TM domain of E2 play a major role in the biogenesis and the entry function of the E1E2 heterodimer. However, the Asp and Arg residues do not contribute equally to these functions.

  15. A Spectral Algorithm for Envelope Reduction of Sparse Matrices

    NASA Technical Reports Server (NTRS)

    Barnard, Stephen T.; Pothen, Alex; Simon, Horst D.

    1993-01-01

    The problem of reordering a sparse symmetric matrix to reduce its envelope size is considered. A new spectral algorithm for computing an envelope-reducing reordering is obtained by associating a Laplacian matrix with the given matrix and then sorting the components of a specified eigenvector of the Laplacian. This Laplacian eigenvector solves a continuous relaxation of a discrete problem related to envelope minimization called the minimum 2-sum problem. The permutation vector computed by the spectral algorithm is a closest permutation vector to the specified Laplacian eigenvector. Numerical results show that the new reordering algorithm usually computes smaller envelope sizes than those obtained from the current standard algorithms such as Gibbs-Poole-Stockmeyer (GPS) or SPARSPAK reverse Cuthill-McKee (RCM), in some cases reducing the envelope by more than a factor of two.

  16. Design reference year for development of photovoltaic envelope systems

    NASA Astrophysics Data System (ADS)

    Mihalka, Peter; Matiasovsky, Peter

    2017-07-01

    An application of photovoltaic cells on external surfaces of building envelope represents a development of new construction element. A mutual coupling between thermal behaviour of photovoltaic layer and the other layers of the structure, with special properties, requires a specific selection of the characteristic outdoor thermal boundary conditions, necessary for optimum design of the envelope from the aspect of structure, material composition and geometry. The main design criteria are the effectiveness and elimination of overheating of photovoltaic module and the optimisation of heat distribution in the envelope structure during particular year seasons. The paper contains the results of the analysis of time courses of climatic elements during a real year as the boundary conditions for simulation of photovoltaic integrated building envelope systems, used in simulations of thermal behaviour of photovoltaics integrated with building envelope systems.

  17. Designing transmembrane alpha-helices that insert spontaneously.

    PubMed

    Wimley, W C; White, S H

    2000-04-18

    Direct measurement of the free energies of transfer of hydrophobic membrane-spanning alpha-helices from water to membranes is important for the determination of an accurate experiment-based hydrophobicity scale for membrane proteins. An important objective of such a scale is to account for the presently unknown thermodynamic cost of partitioning hydrogen-bonded peptide bonds into the membrane hydrocarbon core. We describe here the physical properties of a transmembrane (TM) peptide, TMX-1, designed to test the feasibility of engineering peptides that spontaneously insert across bilayers but that have the important property of measurable monomeric water solubility. TMX-1, Ac-WNALAAVAAAL-AAVAAALAAVAAGKSKSKS-NH(2), is a 31-residue sequence with a 21-residue nonpolar core, N- and C-caps to favor helix formation, and a highly polar C-terminus to improve solubility and to control directionality of insertion into lipid vesicles. TMX-1 appeared to be soluble in water up to a concentration of at least 1 mg/mL (0.3 mM). However, fluorescence spectroscopy, fluorescence quenching, and circular dichroism (CD) spectroscopy indicated that the high solubility was due to the formation of molecular aggregates that persisted at peptide concentrations down to at least 0.1 microM peptide. Nevertheless, aqueous TMX-1 partitioned strongly into membrane vesicles with apparent mole-fraction free-energy values of -7.1 kcal mol(-1) for phosphatidylcholine (POPC) vesicles and -8.2 kcal mol(-1) for phosphatidylglycerol (POPG) vesicles. CD spectroscopy of TMX-1 in oriented multilayers formed from either lipid disclosed a very strong preference for a transmembrane alpha-helical conformation. When TMX-1 was added to preformed vesicles, it was fully helical. A novel fluorescence resonance energy transfer (FRET) method demonstrated that at least 50% of the TMX-1 insered spontaneously across the vesicle membranes. Binding and insertion were found to be fully reversible for POPC vesicles but not POPG

  18. Structural organization and interactions of transmembrane domains in tetraspanin proteins

    PubMed Central

    Kovalenko, Oleg V; Metcalf, Douglas G; DeGrado, William F; Hemler, Martin E

    2005-01-01

    Background Proteins of the tetraspanin family contain four transmembrane domains (TM1-4) linked by two extracellular loops and a short intracellular loop, and have short intracellular N- and C-termini. While structure and function analysis of the larger extracellular loop has been performed, the organization and role of transmembrane domains have not been systematically assessed. Results Among 28 human tetraspanin proteins, the TM1-3 sequences display a distinct heptad repeat motif (abcdefg)n. In TM1, position a is occupied by structurally conserved bulky residues and position d contains highly conserved Asn and Gly residues. In TM2, position a is occupied by conserved small residues (Gly/Ala/Thr), and position d has a conserved Gly and two bulky aliphatic residues. In TM3, three a positions of the heptad repeat are filled by two leucines and a glutamate/glutamine residue, and two d positions are occupied by either Phe/Tyr or Val/Ile/Leu residues. No heptad motif is apparent in TM4 sequences. Mutations of conserved glycines in human CD9 (Gly25 and Gly32 in TM1; Gly67 and Gly74 in TM2) caused aggregation of mutant proteins inside the cell. Modeling of the TM1-TM2 interface in CD9, using a novel algorithm, predicts tight packing of conserved bulky residues against conserved Gly residues along the two helices. The homodimeric interface of CD9 was mapped, by disulfide cross-linking of single-cysteine mutants, to the vicinity of residues Leu14 and Phe17 in TM1 (positions g and c) and Gly77, Gly80 and Ala81 in TM2 (positions d, g and a, respectively). Mutations of a and d residues in both TM1 and TM2 (Gly25, Gly32, Gly67 and Gly74), involved in intramolecular TM1-TM2 interaction, also strongly diminished intermolecular interaction, as assessed by cross-linking of Cys80. Conclusion Our results suggest that tetraspanin intra- and intermolecular interactions are mediated by conserved residues in adjacent, but distinct regions of TM1 and TM2. A key structural element that

  19. Envelope Enhancement Increases Cortical Sensitivity to Interaural Envelope Delays with Acoustic and Electric Hearing

    PubMed Central

    Hartley, Douglas E. H.; Isaiah, Amal

    2014-01-01

    Evidence from human psychophysical and animal electrophysiological studies suggests that sensitivity to interaural time delay (ITD) in the modulating envelope of a high-frequency carrier can be enhanced using half-wave rectified stimuli. Recent evidence has shown potential benefits of equivalent electrical stimuli to deaf individuals with bilateral cochlear implants (CIs). In the current study we assessed the effects of envelope shape on ITD sensitivity in the primary auditory cortex of normal-hearing ferrets, and profoundly-deaf animals with bilateral CIs. In normal-hearing animals, cortical sensitivity to ITDs (±1 ms in 0.1-ms steps) was assessed in response to dichotically-presented i) sinusoidal amplitude-modulated (SAM) and ii) half-wave rectified (HWR) tones (100-ms duration; 70 dB SPL) presented at the best-frequency of the unit over a range of modulation frequencies. In separate experiments, adult ferrets were deafened with neomycin administration and bilaterally-implanted with intra-cochlear electrode arrays. Electrically-evoked auditory brainstem responses (EABRs) were recorded in response to bipolar electrical stimulation of the apical pair of electrodes with singe biphasic current pulses (40 µs per phase) over a range of current levels to measure hearing thresholds. Subsequently, we recorded cortical sensitivity to ITDs (±800 µs in 80-µs steps) within the envelope of SAM and HWR biphasic-pulse trains (40 µs per phase; 6000 pulses per second, 100-ms duration) over a range of modulation frequencies. In normal-hearing animals, nearly a third of cortical neurons were sensitive to envelope-ITDs in response to SAM tones. In deaf animals with bilateral CI, the proportion of ITD-sensitive cortical neurons was approximately a fifth in response to SAM pulse trains. In normal-hearing and deaf animals with bilateral CI the proportion of ITD sensitive units and neural sensitivity to ITDs increased in response to HWR, compared with SAM stimuli. Consequently

  20. Common-envelope ejection in massive binary stars. Implications for the progenitors of GW150914 and GW151226

    NASA Astrophysics Data System (ADS)

    Kruckow, M. U.; Tauris, T. M.; Langer, N.; Szécsi, D.; Marchant, P.; Podsiadlowski, Ph.

    2016-11-01

    Context. The recently detected gravitational wave signals (GW150914 and GW151226) of the merger event of a pair of relatively massive stellar-mass black holes (BHs) calls for an investigation of the formation of such progenitor systems in general. Aims: We analyse the common-envelope (CE) stage of the traditional formation channel in binaries where the first-formed compact object undergoes an in-spiral inside the envelope of its evolved companion star and ejects the envelope in this process. Methods: We calculated envelope binding energies of donor stars with initial masses between 4 and 115M⊙ for metallicities of Z = ZMilky Way ≃ Z⊙/ 2 and Z = Z⊙/ 50, and derived minimum masses of in-spiralling objects needed to eject these envelopes. Results: In addition to producing double white dwarf and double neutron star binaries, CE evolution may also produce massive BH-BH systems with individual BH component masses of up to 50 - 60M⊙, in particular for donor stars evolved to giants beyond the Hertzsprung gap. However, the physics of envelope ejection of massive stars remains uncertain. We discuss the applicability of the energy-budget formalism, the location of the bifurcation point, the recombination energy, and the accretion energy during in-spiral as possible energy sources, and also comment on the effect of inflated helium cores. Conclusions: Massive stars in a wide range of metallicities and with initial masses of up to at least 115M⊙ may shed their envelopes and survive CE evolution, depending on their initial orbital parameters, similarly to the situation for intermediate- and low-mass stars with degenerate cores. In addition to being dependent on stellar radius, the envelope binding energies and λ-values also depend on the applied convective core-overshooting parameter, whereas these structure parameters are basically independent of metallicity for stars with initial masses below 60M⊙. Metal-rich stars ≳60M⊙ become luminous blue variables and do

  1. Functional expression of cystic fibrosis transmembrane conductance regulator in rat oviduct epithelium.

    PubMed

    Chen, Minhui; Du, Jianyang; Jiang, Weijian; Zuo, Wulin; Wang, Fang; Li, Manhui; Chan, Hsiaochang; Zhou, Wenliang

    2008-10-01

    The aim of this study was to investigate the functional expression of cystic fibrosis transmembrane conductance regulator (CFTR) with electrophysiological and molecular technique in rat oviduct epithelium. In whole-cell patch clamp, oviduct epithelial cells responded to 100 microM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) with a rise in inward current in Gap-free mode, which was inhibited successively by 5 microM CFTR(inh)-172, a CFTR specific inhibitor, and 1 mM diphenylamine-2-carboxylate (DPC), the Cl- channel blocker. The cAMP-activated current exhibited a linear current-voltage (I-V) relationship and time- and voltage-independent characteristics. The reversal potentials of the cAMP-activated currents in symmetrical Cl- solutions were close to the Cl- equilibrium, 0.5+/-0.2 mV (n=4). When Cl- concentration in the bath solution was changed from 140 mM to 70 mM and a pipette solution containing 140 mM Cl- was used, the reversal potential shifted to a value close to the new equilibrium for Cl-, 20+/-0.6 mV (n=4), as compared with the theoretic value of 18.7 mV. In addition, mRNA expression of CFTR was also detected in rat oviduct epithelium. Western blot analysis showed that CFTR protein is found in the oviduct throughout the cycle with maximal expression at estrus, and immunofluorescence and immunohistochemistry analysis revealed that CFTR is located at the apical membrane of the epithelial cells. These results showed that the cAMP-activated Cl- current in the oviduct epithelium was characteristic of CFTR, which provided direct evidence for the functional expression of CFTR in the rat oviduct epithelium. CFTR may play a role in modulating fluid transport in the oviduct.

  2. The comparison of genetic variation in the envelope protein between various immunodeficiency viruses and equine infectious anemia virus.

    PubMed

    Yuan, Qing; Liu, Chang; Liang, Zhipin; Chen, Xueqing; Diao, Danhong; Kong, Xiaohong

    2012-08-01

    The envelope protein (Env) of lentiviruses such as HIV, SIV, FIV and EIAV is larger than that of other retroviruses. The Chinese EIAV attenuated vaccine is based on Env and has helped to successfully control this virus, demonstrating that envelope is crucial for vaccine. We compared Env variation of the four kinds of lentiviruses. Phylogenetic analysis showed that the evolutionary relationship of Env between HIV and SIV was the closest and they appeared to descend from a common ancestor, and the relationship of HIV and EIAV was the furthest. EIAV had the shortest Env length and the least number of potential N-linked glycosylation sites (PNGS) as well as glycosylation density compared to various immunodeficiency viruses. However, HIV had the longest Env length and the most PNGS. Moreover, the alignment of HIV and SIV showed that PNGS were primarily distributed within extracellular membrane protein gp120 rather than transmembrane gp41. It implies that the size difference among these viruses is associated with a lentivirus specific function and also the diversity of env. There are low levels of modification of glycosylation sites of Env and selection of optimal protective epitopes might be useful for development of an effective vaccine against HIV/AIDS.

  3. Structure-based statistical analysis of transmembrane helices.

    PubMed

    Baeza-Delgado, Carlos; Marti-Renom, Marc A; Mingarro, Ismael

    2013-03-01

    Recent advances in determination of the high-resolution structure of membrane proteins now enable analysis of the main features of amino acids in transmembrane (TM) segments in comparison with amino acids in water-soluble helices. In this work, we conducted a large-scale analysis of the prevalent locations of amino acids by using a data set of 170 structures of integral membrane proteins obtained from the MPtopo database and 930 structures of water-soluble helical proteins obtained from the protein data bank. Large hydrophobic amino acids (Leu, Val, Ile, and Phe) plus Gly were clearly prevalent in TM helices whereas polar amino acids (Glu, Lys, Asp, Arg, and Gln) were less frequent in this type of helix. The distribution of amino acids along TM helices was also examined. As expected, hydrophobic and slightly polar amino acids are commonly found in the hydrophobic core of the membrane whereas aromatic (Trp and Tyr), Pro, and the hydrophilic amino acids (Asn, His, and Gln) occur more frequently in the interface regions. Charged amino acids are also statistically prevalent outside the hydrophobic core of the membrane, and whereas acidic amino acids are frequently found at both cytoplasmic and extra-cytoplasmic interfaces, basic amino acids cluster at the cytoplasmic interface. These results strongly support the experimentally demonstrated biased distribution of positively charged amino acids (that is, the so-called the positive-inside rule) with structural data.

  4. Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

    SciTech Connect

    Lin, Qingqing; Li, Huilin; Wang, Tong; London, Erwin

    2015-04-08

    Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakage assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.

  5. Hydrodynamics of bilayer membranes with diffusing transmembrane proteins.

    PubMed

    Callan-Jones, Andrew; Durand, Marc; Fournier, Jean-Baptiste

    2016-02-14

    We consider the hydrodynamics of lipid bilayers containing transmembrane proteins of arbitrary shape. This biologically-motivated problem is relevant to the cell membrane, whose fluctuating dynamics play a key role in phenomena ranging from cell migration, intercellular transport, and cell communication. Using Onsager's variational principle, we derive the equations that govern the relaxation dynamics of the membrane shape, of the mass densities of the bilayer leaflets, and of the diffusing proteins' concentration. With our generic formalism, we obtain several results on membrane dynamics. We find that proteins that span the bilayer increase the intermonolayer friction coefficient. The renormalization, which can be significant, is in inverse proportion to the protein's mobility. Second, we find that asymmetric proteins couple to the membrane curvature and to the difference in monolayer densities. For practically all accessible membrane tensions (σ > 10(-8) N m(-1)) we show that the protein density is the slowest relaxing variable. Furthermore, its relaxation rate decreases at small wavelengths due to the coupling to curvature. We apply our formalism to the large-scale diffusion of a concentrated protein patch. We find that the diffusion profile is not self-similar, owing to the wavevector dependence of the effective diffusion coefficient.

  6. Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis

    PubMed Central

    Pai, Vaibhav P.; Aw, Sherry; Shomrat, Tal; Lemire, Joan M.; Levin, Michael

    2012-01-01

    Uncovering the molecular mechanisms of eye development is crucial for understanding the embryonic morphogenesis of complex structures, as well as for the establishment of novel biomedical approaches to address birth defects and injuries of the visual system. Here, we characterize change in transmembrane voltage potential (Vmem) as a novel biophysical signal for eye induction in Xenopus laevis. During normal embryogenesis, a striking hyperpolarization demarcates a specific cluster of cells in the anterior neural field. Depolarizing the dorsal lineages in which these cells reside results in malformed eyes. Manipulating Vmem of non-eye cells induces well-formed ectopic eyes that are morphologically and histologically similar to endogenous eyes. Remarkably, such ectopic eyes can be induced far outside the anterior neural field. A Ca2+ channel-dependent pathway transduces the Vmem signal and regulates patterning of eye field transcription factors. These data reveal a new, instructive role for membrane voltage during embryogenesis and demonstrate that Vmem is a crucial upstream signal in eye development. Learning to control bioelectric initiators of organogenesis offers significant insight into birth defects that affect the eye and might have significant implications for regenerative approaches to ocular diseases. PMID:22159581

  7. Putative transmembrane transporter modulates higher-level aggression in Drosophila.

    PubMed

    Chowdhury, Budhaditya; Chan, Yick-Bun; Kravitz, Edward A

    2017-02-28

    By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems.

  8. Loss of Cystic Fibrosis Transmembrane Regulator Impairs Intestinal Oxalate Secretion.

    PubMed

    Knauf, Felix; Thomson, Robert B; Heneghan, John F; Jiang, Zhirong; Adebamiro, Adedotun; Thomson, Claire L; Barone, Christina; Asplin, John R; Egan, Marie E; Alper, Seth L; Aronson, Peter S

    2017-01-01

    Patients with cystic fibrosis have an increased incidence of hyperoxaluria and calcium oxalate nephrolithiasis. Net intestinal absorption of dietary oxalate results from passive paracellular oxalate absorption as modified by oxalate back secretion mediated by the SLC26A6 oxalate transporter. We used mice deficient in the cystic fibrosis transmembrane conductance regulator gene (Cftr) to test the hypothesis that SLC26A6-mediated oxalate secretion is defective in cystic fibrosis. We mounted isolated intestinal tissue from C57BL/6 (wild-type) and Cftr(-/-) mice in Ussing chambers and measured transcellular secretion of [(14)C]oxalate. Intestinal tissue isolated from Cftr(-/-) mice exhibited significantly less transcellular oxalate secretion than intestinal tissue of wild-type mice. However, glucose absorption, another representative intestinal transport process, did not differ in Cftr(-/-) tissue. Compared with wild-type mice, Cftr(-/-) mice showed reduced expression of SLC26A6 in duodenum by immunofluorescence and Western blot analysis. Furthermore, coexpression of CFTR stimulated SLC26A6-mediated Cl(-)-oxalate exchange in Xenopus oocytes. In association with the profound defect in intestinal oxalate secretion, Cftr(-/-) mice had serum and urine oxalate levels 2.5-fold greater than those of wild-type mice. We conclude that defective intestinal oxalate secretion mediated by SLC26A6 may contribute to the hyperoxaluria observed in this mouse model of cystic fibrosis. Future studies are needed to address whether similar mechanisms contribute to the increased risk for calcium oxalate stone formation observed in patients with cystic fibrosis.

  9. Beta-arrestin-biased ligands at seven-transmembrane receptors.

    PubMed

    Violin, Jonathan D; Lefkowitz, Robert J

    2007-08-01

    Seven-transmembrane receptors (7TMRs), the most common molecular targets of modern drug therapy, are critically regulated by beta-arrestins, which both inhibit classic G-protein signaling and initiate distinct beta-arrestin signaling. The interplay of G-protein and beta-arrestin signals largely determines the cellular consequences of 7TMR-targeted drugs. Until recently, a drug's efficacy for beta-arrestin recruitment was believed to be proportional to its efficacy for G-protein activities. This paradigm restricts 7TMR drug effects to a linear spectrum of responses, ranging from inhibition of all responses to stimulation of all responses. However, it is now clear that 'biased ligands' can selectively activate G-protein or beta-arrestin functions and thus elicit novel biological effects from even well-studied 7TMRs. Here, we discuss the current state of beta-arrestin-biased ligand research and the prospects for beta-arrestin bias as a therapeutic target. Consideration of ligand bias might have profound influences on the way scientists approach 7TMR-targeted drug discovery.

  10. A human phospholipid phosphatase activated by a transmembrane control module.

    PubMed

    Halaszovich, Christian R; Leitner, Michael G; Mavrantoni, Angeliki; Le, Audrey; Frezza, Ludivine; Feuer, Anja; Schreiber, Daniela N; Villalba-Galea, Carlos A; Oliver, Dominik

    2012-11-01

    In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.

  11. Evolution of a transcriptional regulator from a transmembrane nucleoporin

    PubMed Central

    Franks, Tobias M.; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C.N.; Young, Janet M.; Malik, Harmit S.; Gage, Fred H.; Hetzer, Martin W.

    2016-01-01

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo–cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for “soluble Pom121”) that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components. PMID:27198230

  12. Orphan Missense Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator

    PubMed Central

    Fresquet, Fleur; Clement, Romain; Norez, Caroline; Sterlin, Adélaïde; Melin, Patricia; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent; Bilan, Frédéric

    2011-01-01

    More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a three-step in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein–tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling. PMID:21708286

  13. Evolution of a transcriptional regulator from a transmembrane nucleoporin.

    PubMed

    Franks, Tobias M; Benner, Chris; Narvaiza, Iñigo; Marchetto, Maria C N; Young, Janet M; Malik, Harmit S; Gage, Fred H; Hetzer, Martin W

    2016-05-15

    Nuclear pore complexes (NPCs) emerged as nuclear transport channels in eukaryotic cells ∼1.5 billion years ago. While the primary role of NPCs is to regulate nucleo-cytoplasmic transport, recent research suggests that certain NPC proteins have additionally acquired the role of affecting gene expression at the nuclear periphery and in the nucleoplasm in metazoans. Here we identify a widely expressed variant of the transmembrane nucleoporin (Nup) Pom121 (named sPom121, for "soluble Pom121") that arose by genomic rearrangement before the divergence of hominoids. sPom121 lacks the nuclear membrane-anchoring domain and thus does not localize to the NPC. Instead, sPom121 colocalizes and interacts with nucleoplasmic Nup98, a previously identified transcriptional regulator, at gene promoters to control transcription of its target genes in human cells. Interestingly, sPom121 transcripts appear independently in several mammalian species, suggesting convergent innovation of Nup-mediated transcription regulation during mammalian evolution. Our findings implicate alternate transcription initiation as a mechanism to increase the functional diversity of NPC components.

  14. A Novel Type III Endosome Transmembrane Protein, TEMP

    PubMed Central

    Aturaliya, Rajith N.; Kerr, Markus C.; Teasdale, Rohan D.

    2012-01-01

    As part of a high-throughput subcellular localisation project, the protein encoded by the RIKEN mouse cDNA 2610528J11 was expressed and identified to be associated with both endosomes and the plasma membrane. Based on this, we have assigned the name TEMP for Type III Endosome Membrane Protein. TEMP encodes a short protein of 111 amino acids with a single, alpha-helical transmembrane domain. Experimental analysis of its membrane topology demonstrated it is a Type III membrane protein with the amino-terminus in the lumenal, or extracellular region, and the carboxy-terminus in the cytoplasm. In addition to the plasma membrane TEMP was localized to Rab5 positive early endosomes, Rab5/Rab11 positive recycling endosomes but not Rab7 positive late endosomes. Video microscopy in living cells confirmed TEMP’s plasma membrane localization and identified the intracellular endosome compartments to be tubulovesicular. Overexpression of TEMP resulted in the early/recycling endosomes clustering at the cell periphery that was dependent on the presence of intact microtubules. The cellular function of TEMP cannot be inferred based on bioinformatics comparison, but its cellular distribution between early/recycling endosomes and the plasma membrane suggests a role in membrane transport. PMID:24710541

  15. Putative transmembrane transporter modulates higher-level aggression in Drosophila

    PubMed Central

    Chowdhury, Budhaditya; Chan, Yick-Bun; Kravitz, Edward A.

    2017-01-01

    By selection of winners of dyadic fights for 35 generations, we have generated a hyperaggressive Bully line of flies that almost always win fights against the parental wild-type Canton-S stock. Maintenance of the Bully phenotype is temperature dependent during development, with the phenotype lost when flies are reared at 19 °C. No similar effect is seen with the parent line. This difference allowed us to carry out RNA-seq experiments and identify a limited number of genes that are differentially expressed by twofold or greater in the Bullies; one of these was a putative transmembrane transporter, CG13646, which showed consistent and reproducible twofold down-regulation in Bullies. We examined the causal effect of this gene on the phenotype with a mutant line for CG13646, and with an RNAi approach. In all cases, reduction in expression of CG13646 by approximately half led to a hyperaggressive phenotype partially resembling that seen in the Bully flies. This gene is a member of a very interesting family of solute carrier proteins (SLCs), some of which have been suggested as being involved in glutamine/glutamate and GABA cycles of metabolism in excitatory and inhibitory nerve terminals in mammalian systems. PMID:28193893

  16. Role of the Transmembrane Potential in the Membrane Proton Leak

    PubMed Central

    Rupprecht, Anne; Sokolenko, Elena A.; Beck, Valeri; Ninnemann, Olaf; Jaburek, Martin; Trimbuch, Thorsten; Klishin, Sergey S.; Jezek, Petr; Skulachev, Vladimir P.; Pohl, Elena E.

    2010-01-01

    Abstract The molecular mechanism responsible for the regulation of the mitochondrial membrane proton conductance (G) is not clearly understood. This study investigates the role of the transmembrane potential (ΔΨm) using planar membranes, reconstituted with purified uncoupling proteins (UCP1 and UCP2) and/or unsaturated FA. We show that high ΔΨm (similar to ΔΨm in mitochondrial State IV) significantly activates the protonophoric function of UCPs in the presence of FA. The proton conductance increases nonlinearly with ΔΨm. The application of ΔΨm up to 220 mV leads to the overriding of the protein inhibition at a constant ATP concentration. Both, the exposure of FA-containing bilayers to high ΔΨm and the increase of FA membrane concentration bring about the significant exponential Gm increase, implying the contribution of FA in proton leak. Quantitative analysis of the energy barrier for the transport of FA anions in the presence and absence of protein suggests that FA− remain exposed to membrane lipids while crossing the UCP-containing membrane. We believe this study shows that UCPs and FA decrease ΔΨm more effectively if it is sufficiently high. Thus, the tight regulation of proton conductance and/or FA concentration by ΔΨm may be key in mitochondrial respiration and metabolism. PMID:20409469

  17. Insertion of short transmembrane helices by the Sec61 translocon.

    PubMed

    Jaud, Simon; Fernández-Vidal, Mónica; Nilsson, Ingmarie; Meindl-Beinker, Nadja M; Hübner, Nadja C; Tobias, Douglas J; von Heijne, Gunnar; White, Stephen H

    2009-07-14

    The insertion efficiency of transmembrane (TM) helices by the Sec61 translocon depends on helix amino acid composition, the positions of the amino acids within the helix, and helix length. We have used an in vitro expression system to examine systematically the insertion efficiency of short polyleucine segments (L(n), n = 4 ... 12) flanked at either end by 4-residue sequences of the form XXPX-L(n)-XPXX with X = G, N, D, or K. Except for X = K, insertion efficiency (p) is <10% for n < 8, but rises steeply to 100% for n = 12. For X = K, p is already close to 100% for n = 10. A similar pattern is observed for synthetic peptides incorporated into oriented phospholipid bilayer arrays, consistent with the idea that recognition of TM segments by the translocon critically involves physical partitioning of nascent peptide chains into the lipid bilayer. Molecular dynamics simulations suggest that insertion efficiency is determined primarily by the energetic cost of distorting the bilayer in the vicinity of the TM helix. Very short lysine-flanked leucine segments can reduce the energetic cost by extensive hydrogen bonding with water and lipid phosphate groups (snorkeling) and by partial unfolding.

  18. The cystic fibrosis transmembrane conductance regulator (CFTR) and its stability.

    PubMed

    Meng, Xin; Clews, Jack; Kargas, Vasileios; Wang, Xiaomeng; Ford, Robert C

    2017-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the disease cystic fibrosis (CF). It is a membrane protein belonging to the ABC transporter family functioning as a chloride/anion channel in epithelial cells around the body. There are over 1500 mutations that have been characterised as CF-causing; the most common of these, accounting for ~70 % of CF cases, is the deletion of a phenylalanine at position 508. This leads to instability of the nascent protein and the modified structure is recognised and then degraded by the ER quality control mechanism. However, even pharmacologically 'rescued' F508del CFTR displays instability at the cell's surface, losing its channel function rapidly and it is rapidly removed from the plasma membrane for lysosomal degradation. This review will, therefore, explore the link between stability and structure/function relationships of membrane proteins and CFTR in particular and how approaches to study CFTR structure depend on its stability. We will also review the application of a fluorescence labelling method for the assessment of the thermostability and the tertiary structure of CFTR.

  19. Transmembrane channel-like (tmc) gene regulates Drosophila larval locomotion

    PubMed Central

    Guo, Yanmeng; Wang, Yuping; Zhang, Wei; Meltzer, Shan; Zanini, Damiano; Yu, Yue; Li, Jiefu; Cheng, Tong; Guo, Zhenhao; Wang, Qingxiu; Jacobs, Julie S.; Sharma, Yashoda; Eberl, Daniel F.; Göpfert, Martin C.; Jan, Lily Yeh; Jan, Yuh Nung; Wang, Zuoren

    2016-01-01

    Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC’s roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs. PMID:27298354

  20. Expression of cystic fibrosis transmembrane conductance regulator in rat ovary.

    PubMed

    Jin, Lei; Tang, Ruiling

    2008-10-01

    The protein expression of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl(-) channel, in ovarian stimulated premature female rat ovary during a cycle of follicle development and corpus luteum formation was investigated. Animals were injected with 10 U pregnant Mare's serum gonadotropin (PMSG) and subsequently 10 U hCG 48 h later. Time-dependent immunohistochemistry and Western blotting experiments were performed before and 24, 48, 72 h after hCG treatment. The immunohistochemistry revealed that administration of PMSG stimulated the CFTR expression in thecal cell layer and granulosa cell layer of mature follicles 48 h post injection, coincident with the PMSG-induced peak in follicular estradiol. However, the expression of CFTR in the granulose lutein cell layer and thecal lutein cell layer was time-dependently reduced following hCG injection, in accordance with the gradually increased progestogen level during luteum corpus formation. Western blotting analysis demonstrated that rat ovarian tissue expressed the special CFTR band at 170 kD. It is concluded that cAMP-dependent Cl(-) channels are involved in regulation of follicle development and luteum formation.

  1. Modeling of Transmembrane Potential in Realistic Multicellular Structures before Electroporation.

    PubMed

    Murovec, Tomo; Sweeney, Daniel C; Latouche, Eduardo; Davalos, Rafael V; Brosseau, Christian

    2016-11-15

    Many approaches for studying the transmembrane potential (TMP) induced during the treatment of biological cells with pulsed electric fields have been reported. From the simple analytical models to more complex numerical models requiring significant computational resources, a gamut of methods have been used to recapitulate multicellular environments in silico. Cells have been modeled as simple shapes in two dimensions as well as more complex geometries attempting to replicate realistic cell shapes. In this study, we describe a method for extracting realistic cell morphologies from fluorescence microscopy images to generate the piecewise continuous mesh used to develop a finite element model in two dimensions. The preelectroporation TMP induced in tightly packed cells is analyzed for two sets of pulse parameters inspired by clinical irreversible electroporation treatments. We show that high-frequency bipolar pulse trains are better, and more homogeneously raise the TMP of tightly packed cells to a simulated electroporation threshold than conventional irreversible electroporation pulse trains, at the expense of larger applied potentials. Our results demonstrate the viability of our method and emphasize the importance of considering multicellular effects in the numerical models used for studying the response of biological tissues exposed to electric fields. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  2. Decreasing transmembrane segment length greatly decreases perfringolysin O pore size

    DOE PAGES

    Lin, Qingqing; Li, Huilin; Wang, Tong; ...

    2015-04-08

    Perfringolysin O (PFO) is a transmembrane (TM) β-barrel protein that inserts into mammalian cell membranes. Once inserted into membranes, PFO assembles into pore-forming oligomers containing 30–50 PFO monomers. These form a pore of up to 300 Å, far exceeding the size of most other proteinaceous pores. In this study, we found that altering PFO TM segment length can alter the size of PFO pores. A PFO mutant with lengthened TM segments oligomerized to a similar extent as wild-type PFO, and exhibited pore-forming activity and a pore size very similar to wild-type PFO as measured by electron microscopy and a leakagemore » assay. In contrast, PFO with shortened TM segments exhibited a large reduction in pore-forming activity and pore size. This suggests that the interaction between TM segments can greatly affect the size of pores formed by TM β-barrel proteins. PFO may be a promising candidate for engineering pore size for various applications.« less

  3. Understanding three-dimensional damage envelopes

    NASA Astrophysics Data System (ADS)

    Browning, John; Harland, Sophie; Meredith, Philip; Healy, David; Mitchell, Tom

    2017-04-01

    Microcrack damage leading to failure in rocks evolves in response to differential loading. This loading is often visualized in a two-dimensional stress space through the use of Mohr-Coulomb diagrams. The vast majority of experimental studies investigate damage evolution and rock failure using conventional triaxial stress states (σ1 > σ2 = σ3) in which the results can be easily represented in a Mohr-Coulomb plot. However, in nature the stress state is in general truly triaxial (σ1 > σ2 > σ3) and as such comprises a 3D stress state potentially leading to more complexity. By monitoring acoustic wave velocities and acoustic emissions we have shown that damage is generated in multiple orientations depending on the loading directions and hence principal stress directions. Furthermore, crack growth is shown to be a function of differential stress regardless of the mean stress. As such, new cracks can form due to a decrease in the minimum principal stress, which reduces mean stress but increases the differential stress. Although the size of individual cracks is not affected by the intermediate principal stress it has been shown that the σ2 plays a key role in suppressing the total amount of crack growth and concentrates this damage in a single plane. Hence, the differential stress at which rocks fail (i.e. the rock strength) will be significantly increased under true triaxial stress conditions than under the much more commonly applied condition of conventional triaxial stress. Through a series of cyclic loading tests we investigated the Kaiser effect, we show that while individual stress states are important, the stress path by which this stress state is reached is equally important. Whether or not a stress state has been 'visited' before is also vitally important in determining and understanding damage envelopes. Finally, we show that damage evolution can be anisotropic and must be considered as a three-dimensional problem. It is unclear how damage envelopes

  4. Functional Characterization of the N Termini of Murine Leukemia Virus Envelope Proteins

    PubMed Central

    Lu, Chi-Wei; Roth, Monica J.

    2001-01-01

    The function of the N terminus of the murine leukemia virus (MuLV) surface (SU) protein was examined. A series of five chimeric envelope proteins (Env) were generated in which the N terminus of amphotropic 4070A was replaced by equivalent sequences from ecotropic Moloney MuLV (M-MuLV). Viral titers of these chimeras indicate that exchange with homologous sequences could be tolerated, up to V17eco/T15ampho (crossover III). Constructs encoding the first 28 amino acids (aa) of ecotropic M-MuLV resulted in Env expression and binding to the receptor; however, the virus titer was reduced 5- to 45-fold, indicating a postbinding block. Additional exchange beyond the first 28 aa of ecotropic MuLV Env resulted in defective protein expression. These N-terminal chimeras were also introduced into the AE4 chimeric Env backbone containing the amphotropic receptor binding domain joined at the hinge region to the ecotropic SU C terminus. In this backbone, introduction of the first 17 aa of the ecotropic Env protein significantly increased the titer compared to that of its parental chimera AE4, implying a functional coordination between the N terminus of SU and the C terminus of the SU and/or transmembrane proteins. These data functionally dissect the N-terminal sequence of the MuLV Env protein and identify differential effects on receptor-mediated entry. PMID:11287584

  5. Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein.

    PubMed

    Rashidan, Kianoush Khajeh; Nassoury, Nasha; Tazi, Samia; Giannopoulos, Paresa N; Guertin, Claude

    2003-09-30

    A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).

  6. A mitotic nuclear envelope tether for Gle1 also affects nuclear and nucleolar architecture

    PubMed Central

    Chemudupati, Mahesh; Osmani, Aysha H.; Osmani, Stephen A.

    2016-01-01

    During Aspergillus nidulans mitosis, peripheral nuclear pore complex (NPC) proteins (Nups) disperse from the core NPC structure. Unexpectedly, one predicted peripheral Nup, Gle1, remains at the mitotic nuclear envelope (NE) via an unknown mechanism. Gle1 affinity purification identified mitotic tether for Gle1 (MtgA), which tethers Gle1 to the NE during mitosis but not during interphase when Gle1 is at NPCs. MtgA is the orthologue of the Schizosaccharomyces pombe telomere-anchoring inner nuclear membrane protein Bqt4. Like Bqt4, MtgA has meiotic roles, but it is functionally distinct from Bqt4 because MtgA is not required for tethering telomeres to the NE. Domain analyses showed that MtgA targeting to the NE requires its C-terminal transmembrane domain and a nuclear localization signal. Of importance, MtgA functions beyond Gle1 mitotic targeting and meiosis and affects nuclear and nucleolar architecture when deleted or overexpressed. Deleting MtgA generates small, round nuclei, whereas overexpressing MtgA generates larger nuclei with altered nuclear compartmentalization resulting from NE expansion around the nucleolus. The accumulation of MtgA around the nucleolus promotes a similar accumulation of the endoplasmic reticulum (ER) protein Erg24, reducing its levels in the ER. This study extends the functions of Bqt4-like proteins to include mitotic Gle1 targeting and modulation of nuclear and nucleolar architecture. PMID:27630260

  7. Envelope protein VP24 from White spot syndrome virus: expression, purification and crystallization.

    PubMed

    Sun, Lifang; Wu, Yunkun

    2016-08-01

    White spot syndrome virus (WSSV) is a major shrimp pathogen known to infect penaeid shrimp and other crustaceans. VP24 is one of the major envelope proteins of WSSV. In order to facilitate purification, crystallization and structure determination, the predicted N-terminal transmembrane region of approximately 26 amino acids was truncated from VP24 and several mutants were prepared to increase the proportion of selenomethionine (SeMet) residues for subsequent structural determination using the SAD method. Truncated VP24, its mutants and the corresponding SeMet-labelled proteins were purified, and the native and SeMet proteins were crystallized by the hanging-drop vapour-diffusion method. Crystals of VP24 were obtained using a reservoir consisting of 0.1 M Tris-HCl pH 8.5, 2.75 M ammonium acetate with a drop volume ratio of two parts protein solution to one part reservoir solution. Notably, ATP was added as a critical additive to the drop with a final concentration of 10 mM. Crystals of SeMet-labelled VP24 mutant diffracted to 3.0 Å resolution and those of the native diffracted to 2.4 Å resolution; the crystals belonged to space group I213, with unit-cell parameters a = b = c = 140 Å.

  8. Dystonin/Bpag1 is a necessary endoplasmic reticulum/nuclear envelope protein in sensory neurons

    SciTech Connect

    Young, Kevin G.; Kothary, Rashmi

    2008-09-10

    Dystonin/Bpag1 proteins are cytoskeletal linkers whose loss of function in mice results in a hereditary sensory neuropathy with a progressive loss of limb coordination starting in the second week of life. These mice, named dystonia musculorum (dt), succumb to the disease and die of unknown causes prior to sexual maturity. Previous evidence indicated that cytoskeletal defects in the axon are a primary cause of dt neurodegeneration. However, more recent data suggests that other factors may be equally important contributors to the disease process. In the present study, we demonstrate perikaryal defects in dorsal root ganglion (DRG) neurons at stages preceding the onset of loss of limb coordination in dt mice. Abnormalities include alterations in endoplasmic reticulum (ER) chaperone protein expression, indicative of an ER stress response. Dystonin in sensory neurons localized in association with the ER and nuclear envelope (NE). A fusion protein ofthe dystonin-a2 isoform, which harbors an N-terminal transmembrane domain, associated with and reorganized the ER in cell culture. This isoform also interacts with the NE protein nesprin-3{alpha}, but not nesprin-3{beta}. Defects in dt mice, as demonstrated here, may ultimately result in pathogenesis involving ER dysfunction and contribute significantly to the dt phenotype.

  9. Hymenolepis nana: the fine structure of the embryonic envelopes.

    PubMed

    Fairweather, I; Threadgold, L T

    1981-06-01

    The fine structure of the envelopes surrounding hatched and unhatched oncospheres of Hymenolepis nana has been investigated by transmission and scanning electron microscopy (SEM), together with light microscope histochemical observations of JB-4 embedded material. The oncosphere is surrounded by 3 layers--the capsule, the outer envelope and the inner envelope, the latter giving rise to the embryophore and the 'oncospheral membrane'. An additional layer--the polar filament layer--lies between the 'oncospheral membrane' and the oncosphere. Shell material is deposited on the capsule as a thin layer. It is secreted by the outer envelope, which degenerates once shell formation is complete. The uterus may also contribute to shell formation. The embryophore forms a thin incomplete and peripheral layer within the inner envelope. In the basal region of this envelope, partial development of an 'oncospheral membrane' takes place, but it does not become detached as a separate layer. The polar filaments, which are characteristic of the oncosphere of H. nana, are derived from the epithelial covering of the oncosphere itself, which delaminates to form a separate polar filament layer. The filaments arise from knob-like projections at opposite poles of this layer. The design of the embryonic envelopes in H. nana show a number of modifications from the basic cyclophyllidean pattern, and these can be related to the demands of its 'direct' life-cycle.

  10. Cell Walls and the Convergent Evolution of the Viral Envelope

    PubMed Central

    Buchmann, Jan P.

    2015-01-01

    SUMMARY Why some viruses are enveloped while others lack an outer lipid bilayer is a major question in viral evolution but one that has received relatively little attention. The viral envelope serves several functions, including protecting the RNA or DNA molecule(s), evading recognition by the immune system, and facilitating virus entry. Despite these commonalities, viral envelopes come in a wide variety of shapes and configurations. The evolution of the viral envelope is made more puzzling by the fact that nonenveloped viruses are able to infect a diverse range of hosts across the tree of life. We reviewed the entry, transmission, and exit pathways of all (101) viral families on the 2013 International Committee on Taxonomy of Viruses (ICTV) list. By doing this, we revealed a strong association between the lack of a viral envelope and the presence of a cell wall in the hosts these viruses infect. We were able to propose a new hypothesis for the existence of enveloped and nonenveloped viruses, in which the latter represent an adaptation to cells surrounded by a cell wall, while the former are an adaptation to animal cells where cell walls are absent. In particular, cell walls inhibit viral entry and exit, as well as viral transport within an organism, all of which are critical waypoints for successful infection and spread. Finally, we discuss how this new model for the origin of the viral envelope impacts our overall understanding of virus evolution. PMID:26378223

  11. Vitelline envelope, chorion, and micropyle of Fundulus heteroclitus eggs

    SciTech Connect

    Dumont, J.N.; Brummet, A.R.

    1980-01-01

    The architecture and transformation of the vitelline envelope of the developing oocyte into the chorion of the mature egg of Fundulus heteroclitus have been examined by scanning and transmission electron microscopy. The mature vitelline envelope is structurally complex and consists of about nine strata. The envelope is penetrated by pore canals that contain microvilli arising from the oocyte and macrovilli from follicle cells. During the envelope's transformation into the chorion, the pore canals are lost and the envelope becomes more fibrous and compact and its stratified nature less apparent. The micropyle, or pore, through which the sperm gains access to the enclosed egg is located at the bottom of a small funnel-shaped depression in the envelope. Internally, the micropyle opens on the apex of a cone-like elevation of the chorion. During the development of the envelope, structured chorionic fibrils, the components of which are presumed to be synthesized by the follicle cells, become attached to its surface. These chorionic fibrils are thought to aid in the attachment of the egg to the substratum and perhaps to help prevent water loss during low tides when the egg may be exposed.

  12. Neural coding of echo-envelope disparities in echolocating bats.

    PubMed

    Borina, Frank; Firzlaff, Uwe; Wiegrebe, Lutz

    2011-05-01

    The effective use of echolocation requires not only measuring the delay between the emitted call and returning echo to estimate the distance of an ensonified object. To locate an object in azimuth and elevation, the bat's auditory system must analyze the returning echoes in terms of their binaural properties, i.e., the echoes' interaural intensity and time differences (IIDs and ITDs). The effectiveness of IIDs for echolocation is undisputed, but when bats ensonify complex objects, the temporal structure of echoes may facilitate the analysis of the echo envelope in terms of envelope ITDs. Using extracellular recordings from the auditory midbrain of the bat, Phyllostomus discolor, we found a population of neurons that are sensitive to envelope ITDs of echoes of their sonar calls. Moreover, the envelope-ITD sensitivity improved with increasing temporal fluctuations in the echo envelopes, a sonar parameter related to the spatial statistics of complex natural reflectors like vegetation. The data show that in bats envelope ITDs may be used not only to locate external, prey-generated rustling sounds but also in the context of echolocation. Specifically, the temporal fluctuations in the echo envelope, which are created when the sonar emission is reflected from a complex natural target, support ITD-mediated echolocation.

  13. CLOSE STELLAR BINARY SYSTEMS BY GRAZING ENVELOPE EVOLUTION

    SciTech Connect

    Soker, Noam

    2015-02-20

    I suggest a spiral-in process in which a stellar companion grazes the envelope of a giant star while both the orbital separation and the giant radius shrink simultaneously, forming a close binary system. The binary system might be viewed as evolving in a constant state of 'just entering a common envelope (CE) phase.' In cases where this process takes place, it can be an alternative to CE evolution where the secondary star is immersed in the giant's envelope. Grazing envelope evolution (GEE) is made possible only if the companion manages to accrete mass at a high rate and launches jets that remove the outskirts of the giant envelope, hence preventing the formation of a CE. The high accretion rate is made possible by the accretion disk launching jets which efficiently carry the excess angular momentum and energy from the accreted mass. The orbital decay itself is caused by the gravitational interaction of the secondary star with the envelope inward of its orbit, i.e., dynamical friction (gravitational tide). Mass loss through the second Lagrangian point can carry additional angular momentum and envelope mass. The GEE lasts for tens to hundreds of years. The high accretion rate, with peaks lasting from months to years, might lead to a bright object referred to as the intermediate luminosity optical transient (Red Novae; Red Transients). A bipolar nebula and/or equatorial ring are formed around the binary remnant.

  14. Precision gap particle separator

    DOEpatents

    Benett, William J.; Miles, Robin; Jones, II., Leslie M.; Stockton, Cheryl

    2004-06-08

    A system for separating particles entrained in a fluid includes a base with a first channel and a second channel. A precision gap connects the first channel and the second channel. The precision gap is of a size that allows small particles to pass from the first channel into the second channel and prevents large particles from the first channel into the second channel. A cover is positioned over the base unit, the first channel, the precision gap, and the second channel. An port directs the fluid containing the entrained particles into the first channel. An output port directs the large particles out of the first channel. A port connected to the second channel directs the small particles out of the second channel.

  15. MULTIPLE SPARK GAP SWITCH

    DOEpatents

    Schofield, A.E.

    1958-07-22

    A multiple spark gap switch of unique construction is described which will permit controlled, simultaneous discharge of several capacitors into a load. The switch construction includes a disc electrode with a plurality of protuberances of generally convex shape on one surface. A firing electrode is insulatingly supponted In each of the electrode protuberances and extends substantially to the apex thereof. Individual electrodes are disposed on an insulating plate parallel with the disc electrode to form a number of spark gaps with the protuberances. These electrodes are each connected to a separate charged capacitor and when a voltage ls applied simultaneously between the trigger electrodes and the dlsc electrode, each spark gap fires to connect its capacitor to the disc electrode and a subsequent load.

  16. Role for phosphatidylinositol in nuclear envelope formation.

    PubMed Central

    Larijani, B; Barona, T M; Poccia, D L

    2001-01-01

    PtdIns is a minor membrane phospholipid that is important in signal transduction. Recently, derivatives of PtdIns phosphorylated at the 3-position of the inositol ring have been implicated in the regulation of constitutive membrane traffic and in membrane fusion events. Assembly of the nuclear envelope (NE), a crucial step in the progress of mitosis, is also likely to involve membrane fusion reactions. We therefore investigated the role of PtdIns and phosphoinositide 3-kinase (PI-3K) activity in NE formation in vitro. GTP-induced NE formation was blocked by wortmannin and LY294002, two specific inhibitors of PI-3K, suggesting a role for PtdIns phosphorylated at the 3-position. PtdIns-specific phospholipase C mimicked GTP hydrolysis as an inducer of NE formation. This induction was dependent on a membrane vesicle subfraction (MV1) that was highly enriched in PtdIns, as determined by heteronuclear two-dimensional NMR spectroscopy. On the basis of these results, we suggest that the MV1 population serves as a source of membranes rich in PtdIns that might facilitate fusion, possibly through the production of the membrane-destabilizing lipid diacylglycerol. PMID:11368777

  17. Real-Time Flight Envelope Monitoring System

    NASA Technical Reports Server (NTRS)

    Kerho, Michael; Bragg, Michael B.; Ansell, Phillip J.

    2012-01-01

    The objective of this effort was to show that real-time aircraft control-surface hinge-moment information could be used to provide a robust and reliable prediction of vehicle performance and control authority degradation. For a given airfoil section with a control surface -- be it a wing with an aileron, rudder, or elevator -- the control-surface hinge moment is sensitive to the aerodynamic characteristics of the section. As a result, changes in the aerodynamics of the section due to angle-of-attack or environmental effects such as icing, heavy rain, surface contaminants, bird strikes, or battle damage will affect the control surface hinge moment. These changes include both the magnitude of the hinge moment and its sign in a time-averaged sense, and the variation of the hinge moment with time. The current program attempts to take the real-time hinge moment information from the aircraft control surfaces and develop a system to predict aircraft envelope boundaries across a range of conditions, alerting the flight crew to reductions in aircraft controllability and flight boundaries.

  18. Aeroelastic Model Structure Computation for Envelope Expansion

    NASA Technical Reports Server (NTRS)

    Kukreja, Sunil L.

    2007-01-01

    Structure detection is a procedure for selecting a subset of candidate terms, from a full model description, that best describes the observed output. This is a necessary procedure to compute an efficient system description which may afford greater insight into the functionality of the system or a simpler controller design. Structure computation as a tool for black-box modeling may be of critical importance in the development of robust, parsimonious models for the flight-test community. Moreover, this approach may lead to efficient strategies for rapid envelope expansion that may save significant development time and costs. In this study, a least absolute shrinkage and selection operator (LASSO) technique is investigated for computing efficient model descriptions of non-linear aeroelastic systems. The LASSO minimises the residual sum of squares with the addition of an l(Sub 1) penalty term on the parameter vector of the traditional l(sub 2) minimisation problem. Its use for structure detection is a natural extension of this constrained minimisation approach to pseudo-linear regression problems which produces some model parameters that are exactly zero and, therefore, yields a parsimonious system description. Applicability of this technique for model structure computation for the F/A-18 (McDonnell Douglas, now The Boeing Company, Chicago, Illinois) Active Aeroelastic Wing project using flight test data is shown for several flight conditions (Mach numbers) by identifying a parsimonious system description with a high percent fit for cross-validated data.

  19. Discriminating Dysarthria Type From Envelope Modulation Spectra

    PubMed Central

    Liss, Julie M.; LeGendre, Sue; Lotto, Andrew J.

    2013-01-01

    Purpose Previous research demonstrated the ability of temporally based rhythm metrics to distinguish among dysarthrias with different prosodic deficit profiles (J. M. Liss et al., 2009). The authors examined whether comparable results could be obtained by an automated analysis of speech envelope modulation spectra (EMS), which quantifies the rhythmicity of speech within specified frequency bands. Method EMS was conducted on sentences produced by 43 speakers with 1 of 4 types of dysarthria and healthy controls. The EMS consisted of the spectra of the slow-rate (up to 10 Hz) amplitude modulations of the full signal and 7 octave bands ranging in center frequency from 125 to 8000 Hz. Six variables were calculated for each band relating to peak frequency and amplitude and relative energy above, below, and in the region of 4 Hz. Discriminant function analyses (DFA) determined which sets of predictor variables best discriminated between and among groups. Results Each of 6 DFAs identified 2–6 of the 48 predictor variables. These variables achieved 84%–100% classification accuracy for group membership. Conclusions Dysarthrias can be characterized by quantifiable temporal patterns in acoustic output. Because EMS analysis is automated and requires no editing or linguistic assumptions, it shows promise as a clinical and research tool. PMID:20643800

  20. Critical point analysis of phase envelope diagram

    NASA Astrophysics Data System (ADS)

    Soetikno, Darmadi; Kusdiantara, Rudy; Puspita, Dila; Sidarto, Kuntjoro A.; Siagian, Ucok W. R.; Soewono, Edy; Gunawan, Agus Y.

    2014-03-01

    Phase diagram or phase envelope is a relation between temperature and pressure that shows the condition of equilibria between the different phases of chemical compounds, mixture of compounds, and solutions. Phase diagram is an important issue in chemical thermodynamics and hydrocarbon reservoir. It is very useful for process simulation, hydrocarbon reactor design, and petroleum engineering studies. It is constructed from the bubble line, dew line, and critical point. Bubble line and dew line are composed of bubble points and dew points, respectively. Bubble point is the first point at which the gas is formed when a liquid is heated. Meanwhile, dew point is the first point where the liquid is formed when the gas is cooled. Critical point is the point where all of the properties of gases and liquids are equal, such as temperature, pressure, amount of substance, and others. Critical point is very useful in fuel processing and dissolution of certain chemicals. Here in this paper, we will show the critical point analytically. Then, it will be compared with numerical calculations of Peng-Robinson equation by using Newton-Raphson method. As case studies, several hydrocarbon mixtures are simulated using by Matlab.