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Sample records for plasmamembrane polypeptides final

  1. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking

    PubMed Central

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael; Shales, Mike; Ulitsky, Igor; Olivera-Couto, Agustina; Braberg, Hannes; Shamir, Ron; Walter, Peter; Mann, Matthias; Ejsing, Christer S; Krogan, Nevan J; Walther, Tobias C

    2011-01-01

    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of ~400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism. PMID:20526336

  2. A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking.

    PubMed

    Aguilar, Pablo S; Fröhlich, Florian; Rehman, Michael; Shales, Mike; Ulitsky, Igor; Olivera-Couto, Agustina; Braberg, Hannes; Shamir, Ron; Walter, Peter; Mann, Matthias; Ejsing, Christer S; Krogan, Nevan J; Walther, Tobias C

    2010-07-01

    The plasma membrane delimits the cell and controls material and information exchange between itself and the environment. How different plasma-membrane processes are coordinated and how the relative abundance of plasma-membrane lipids and proteins is homeostatically maintained are not yet understood. Here, we used a quantitative genetic interaction map, or E-MAP, to functionally interrogate a set of approximately 400 genes involved in various aspects of plasma-membrane biology, including endocytosis, signaling, lipid metabolism and eisosome function. From this E-MAP, we derived a set of 57,799 individual interactions between genes functioning in these various processes. Using triplet genetic motif analysis, we identified a new component of the eisosome, Eis1, and linked the poorly characterized gene EMP70 to endocytic and eisosome function. Finally, we implicated Rom2, a GDP/GTP exchange factor for Rho1 and Rho2, in the regulation of sphingolipid metabolism.

  3. Elastomeric Polypeptides

    PubMed Central

    van Eldijk, Mark B.; McGann, Christopher L.

    2013-01-01

    Elastomeric polypeptides are very interesting biopolymers and are characterized by rubber-like elasticity, large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Their useful properties have motivated their use in a wide variety of materials and biological applications. This chapter focuses on elastin and resilin – two elastomeric biopolymers – and the recombinant polypeptides derived from them (elastin-like polypeptides and resilin-like polypeptides). This chapter also discusses the applications of these recombinant polypeptides in the fields of purification, drug delivery, and tissue engineering. PMID:21826606

  4. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J [Waukee, IA; Ellanskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Gilliam, Jacob T [Norwalk, IA; Hunter-Cevera, Jennie [Elliott City, MD; Presnail, James K [Avondale, PA; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

    2009-09-15

    The invention relates to antifungal compositions and methods for protecting a plant from a fungal pathogen. Compositions including antifungal polypeptides isolated from a fungal fermentation broth are provided.

  5. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Dahlbacka, Glen; Ellanskaya, legal representative, Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser; Ellanskaya, deceased, Irina

    2007-12-11

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  6. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J [Granger, IA; Dahlbacka, Glen [Oakland, CA; Ellanskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Herrmann, Rafael [Wilmington, DE; Hunter-Cevera, Jennie [Elliott City, MD; McCutchen, Billy F [College Station, TX; Presnail, James K [Avondale, PA; Rice, Janet A [Wilmington, DE; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

    2012-04-03

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  7. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J.; Dahlbacka, Glen; Elleskaya, Irina; Ellanskaya, legal representative; Natalia; Herrmann, Rafael; Hunter-Cevera, Jennie; McCutchen, Billy F.; Presnail, James K.; Rice, Janet A.; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-08-10

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  8. Antifungal polypeptides

    DOEpatents

    Altier, Daniel J [Waukee, IA; Dahlbacka, Glen [Oakland, CA; Elleskaya, Irina [Kyiv, UA; Ellanskaya, legal representative, Natalia; Herrmann, Rafael [Wilmington, DE; Hunter-Cevera, Jennie [Elliott City, MD; McCutchen, Billy F [College Station, IA; Presnail, James K [Avondale, PA; Rice, Janet A [Wilmington, DE; Schepers, Eric [Port Deposit, MD; Simmons, Carl R [Des Moines, IA; Torok, Tamas [Richmond, CA; Yalpani, Nasser [Johnston, IA

    2011-04-12

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include novel amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from microbial fermentation broths. Nucleic acid molecules comprising nucleotide sequences that encode the antipathogenic polypeptides of the invention are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention, or variant or fragment thereof, are also disclosed.

  9. Plasma-membrane calcium pumps and hereditary deafness.

    PubMed

    Brini, M; Di Leva, F; Domi, T; Fedrizzi, L; Lim, D; Carafoli, E

    2007-11-01

    In mammals, four different genes encode four PMCA (plasma-membrane Ca(2+)-ATPase) isoforms. PMCA1 and 4 are expressed ubiquitously, and PMCA2 and 3 are expressed predominantly in the central nervous system. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of so many isoforms is not clear, but evidently it must be related to the cell-specific demands of Ca(2+) homoeostasis. Recent studies suggest that the alternatively spliced regions in PMCA are responsible for specific targeting to plasma membrane domains, and proteins that bind specifically to the pumps could contribute to further regulation of Ca(2+) control. In addition, the combination of proteins obtained by alternative splicing occurring at two different sites could be responsible for different functional characteristics of the pumps.

  10. Heterogeneous distribution of enzymes among plasma-membrane fragments sedimenting with the microsomal fraction of rat liver

    PubMed Central

    Norris, Kenneth A.; Dobrota, Miloslav; Issa, Faiz S.; Hinton, Richard H.; Reid, Eric

    1974-01-01

    Plasma-membrane fragments recovered in the microsomal fraction of rat liver homogenates were shown to be heterogeneous in density. It was demonstrated that 5′-nucleotidase, the most commonly used plasma-membrane marker, is concentrated in the lightest subfraction. Two of the published procedures for the isolation of plasma-membrane fragments from the microsomal fraction (Touster et al., 1970; Hinton et al., 1971) are shown to give products which are not representative of all the plasma-membrane fragments of microsomal size, and it is argued that a third procedure (House & Weidemann, 1970) is likely to give a similar product. PMID:4377214

  11. Effects of heavy metals on the Ca(2+)-ATPase activity present in gill cell plasma-membrane of mussels (Mytilus galloprovincialis Lam.).

    PubMed

    Viarengo, A; Mancinelli, G; Pertica, M; Fabbri, R; Orunesu, M

    1993-11-01

    1. Heavy metals (Hg2+, Cu2+, Cd2+, Zn2+, Pb2+) at micromolar concentrations strongly inhibit the Ca(2+)-ATPase activity present in the plasma-membrane obtained from the gill cells of Mytilus galloprovincialis Lam. Heavy metals act through inhibition of the formation of the phosphorylated intermediate. 2. All the heavy metals tested inhibit the Ca(2+)-ATPase activity, the effect following the order: Hg2+ > Pb2+ > Cu2+ > Cd2+ > Zn2+; the simultaneous addition of different heavy metals causes a summatory inhibition of the enzyme activity; addition to the reaction mixture of GSH at a final concentration of 0.5 mM, reverses inhibitory effects of heavy metals. 3. The inhibitory effects of Cu2+ on Ca(2+)-ATPase are highly enhanced by addition of ascorbate to the reaction mixture. In the presence of ascorbate (100 microM), copper strongly stimulates the lipid peroxidation damage of the gill plasma-membranes, a result that may explain the high copper cytotoxicity.

  12. Characterization of the plasma-membrane calcium pump from Trypanosoma cruzi.

    PubMed Central

    Benaim, G; Moreno, S N; Hutchinson, G; Cervino, V; Hermoso, T; Romero, P J; Ruiz, F; de Souza, W; Docampo, R

    1995-01-01

    Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] suggesting that the plasma-membrane Ca(2+)-ATPases of different trypanosomatids differ from the Ca2+ pumps present in mammalian cells, Trypanosoma cruzi plasma-membrane Ca(2+)-ATPase shares several characteristics with the Ca2+ pumps present in other systems. This enzyme could be partially purified from epimastigote plasma-membrane vesicles using calmodulin-agarose affinity chromatography. The activity of the partially purified enzyme was stimulated by T. cruzi or bovine brain calmodulin. In addition, the enzyme cross-reacted with antiserum and monoclonal antibody 5F10 raised against human red-blood-cell Ca(2+)-ATPase, has a molecular mass of 140 kDa and forms Ca(2+)-dependent hydroxylamine-sensitive phosphorylated intermediates. These results, together with its high sensitivity to vanadate, indicate that this enzyme belongs to the P-type class of ionic pumps. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:7532400

  13. Targeted polypeptide degradation

    DOEpatents

    Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

    2008-05-13

    This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

  14. PTEN dephosphorylates AKT to prevent the expression of GLUT1 on plasmamembrane and to limit glucose consumption in cancer cells.

    PubMed

    Phadngam, Suratchanee; Castiglioni, Andrea; Ferraresi, Alessandra; Morani, Federica; Follo, Carlo; Isidoro, Ciro

    2016-12-20

    GLUT1 is the facilitative transporter playing the major role in the internalization of glucose. Basally, GLUT1 resides on vesicles located in a para-golgian area, and is translocated onto the plasmamembrane upon activation of the PI3KC1-AKT pathway. In proliferating cancer cells, which demand a high quantity of glucose for their metabolism, GLUT1 is permanently expressed on the plasmamembrane. This is associated with the abnormal activation of the PI3KC1-AKT pathway, consequent to the mutational activation of PI3KC1 and/or the loss of PTEN. The latter, in fact, could antagonize the phosphorylation of AKT by limiting the availability of Phosphatidylinositol (3,4,5)-trisphosphate. Here, we asked whether PTEN could control the plasmamembrane expression of GLUT1 also through its protein-phosphatase activity on AKT. Experiments of co-immunoprecipitation and in vitro de-phosphorylation assay with homogenates of cells transgenically expressing the wild type or knocked-down mutants (lipid-phosphatase, protein-phosphatase, or both) isoforms demonstrated that indeed PTEN physically interacts with AKT and drives its dephosphorylation, and so limiting the expression of GLUT1 at the plasmamembrane. We also show that growth factors limit the ability of PTEN to dephosphorylate AKT. Our data emphasize the fact that PTEN acts in two distinct steps of the PI3k/AKT pathway to control the expression of GLUT1 at the plasmamembrane and, further, add AKT to the list of the protein substrates of PTEN.

  15. Isolation and characterization of plasma-membrane glycoproteins from pig epidermis

    PubMed Central

    King, Ian A.; Tabiowo, Anne

    1982-01-01

    1. Non-desmosomal plasma membranes enriched in plasma-membrane marker enzymes and in metabolically labelled glycoproteins were isolated on a large scale from up to 500g of pig ear skin slices. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and periodic acid/Schiff staining revealed the presence of four major glycosylated components in the apparent molecular-weight range 150000–80000. 2. A large proportion of the marker enzymes, the d-[3H]glucosamine-labelled glycoproteins and the periodic acid/Schiff-stained glycoproteins were solubilized by 1% (w/v) sodium deoxycholate. However, several non-glycosylated proteins, in particular those with mol.wts. 81000, 41000 and 38000 (possibly cytoskeletal components), were relatively resistant to solubilization. 3. The deoxycholate-solubilized membranes were fractionated by lectin affinity chromatography using both concanavalin A–Sepharose 4B and lentil lectin–Sepharose 4B. From 75 to 85% of the applied glycoprotein was recovered from the columns. From 30 to 40% of the recovered glycoprotein was specifically bound by the lectins and was eluted with 2% (w/v) α-methyl d-mannoside. The enrichment of labelled glycoproteins in the material bound by the lectins (2.5-fold) was similar with both lectins, although the yield was somewhat greater when lentil lectin was used. The glycoprotein-enriched fraction was also enriched in all the plasma-membrane marker enzymes, indicating their probable glycoprotein nature. 4. The glycoprotein-enriched fraction contained the four major periodic acid/Schiff-stained bands that were detected in the original plasma membrane. They had apparent mol.wts. 147000, 130500, 108000 and 91400. The higher-molecular-weight components contained relatively more d-[3H]glucosamine, indicating differences in the sugar composition or in the metabolic turnover of the individual glycoproteins in culture. The material bound by the lectins also contained a number of lower-molecular-weight Coomassie

  16. Organic Anion Transporting Polypeptides

    PubMed Central

    Stieger, Bruno; Hagenbuch, Bruno

    2013-01-01

    Organic anion transporting polypeptides or OATPs are central transporters in the disposition of drugs and other xenobiotics. In addition, they mediate transport of a wide variety of endogenous substrates. The critical role of OATPs in drug disposition has spurred research both in academia and in the pharmaceutical industry. Translational aspects with clinical questions are the focus in academia, while the pharmaceutical industry tries to define and understand the role these transporters play in pharmacotherapy. The present overview summarizes our knowledge on the interaction of food constituents with OATPs, and on the OATP transport mechanisms. Further, it gives an update on the available information on the structure-function relationship of the OATPs, and finally, covers the transcriptional and posttranscriptional regulation of OATPs. PMID:24745984

  17. Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Cosgrove, D. J.

    1989-01-01

    Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 micromoles m-2 s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluence-response relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.

  18. Large plasma-membrane depolarization precedes rapid blue-light-induced growth inhibition in cucumber

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Cosgrove, D. J.

    1989-01-01

    Blue-light (BL)-induced suppression of elongation of etiolated Cucumis sativus L. hypocotyls began after a 30-s lag time, which was halved by increasing the fluence rate from 10 to 100 micromoles m-2 s-1. Prior to the growth suppression, the plasma-membrane of the irradiated cells depolarized by as much as 100 mV, then returned within 2-3 min to near its initial value. The potential difference measured with surface electrodes changed with an identical time course but opposite polarity. The lag time for the change in surface potential showed an inverse dependence on fluence rate, similar to the lag for the growth inhibition. Green light and red light caused neither the electrical response nor the rapid inhibition of growth. The depolarization by BL did not propagate to nonirradiated regions and exhibited a refractory period of about 10 min following a BL pulse. Fluence-response relationships for the electrical and growth responses provide correlational evidence that the plasma-membrane depolarization reflects an event in the transduction chain of this light-growth response.

  19. Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists.

    PubMed Central

    Romero, I; Maldonado, A M; Eraso, P

    1997-01-01

    Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitro effect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of approximately 1 microM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein. PMID:9148755

  20. Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species.

    PubMed

    Krauke, Yannick; Sychrova, Hana

    2008-05-20

    The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations.

  1. Functional comparison of plasma-membrane Na+/H+ antiporters from two pathogenic Candida species

    PubMed Central

    Krauke, Yannick; Sychrova, Hana

    2008-01-01

    Background The virulence of Candida species depends on many environmental conditions. Extracellular pH and concentration of alkali metal cations belong among important factors. Nevertheless, the contribution of transporters mediating the exchange of alkali metal cations for protons across the plasma membrane to the cell salt tolerance and other physiological properties of various Candida species has not been studied so far. Results The tolerance/sensitivity of four pathogenic Candida species to alkali metal cations was tested and the role of one of the cation transporters in that tolerance (presumed to be the plasma-membrane Na+/H+ antiporter) was studied. The genes encoding these antiporters in the most and least salt sensitive species, C. dubliniensis and C. parapsilosis respectively, were identified, cloned and functionally expressed in the plasma membranes of Saccharomyces cerevisiae cells lacking their own cation exporters. Both CpCnh1 and CdCnh1 antiporters had broad substrate specificity and transported Na+, K+, Li+, and Rb+. Their activity in S. cerevisiae cells differed; CpCnh1p provided cells with a much higher salt tolerance than the CdCnh1 antiporter. The observed difference in activity was confirmed by direct measurements of sodium and potassium efflux mediated by these antiporters. Conclusion We have cloned two genes encoding putative Na+/H+ antiporters in C. parapsilosis and C. dubliniensis, and characterized the transport properties of encoded proteins. Our results show that the activity of plasma-membrane Na+/H+ antiporters is one of the factors determining the tolerance of pathogenic Candida species to high external concentrations of alkali metal cations. PMID:18492255

  2. Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Cosgrove, D. J.

    1992-01-01

    A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

  3. Mechanism of blue-light-induced plasma-membrane depolarization in etiolated cucumber hypocotyls

    NASA Technical Reports Server (NTRS)

    Spalding, E. P.; Cosgrove, D. J.

    1992-01-01

    A large, transient depolarization of the plasma membrane precedes the rapid blue-light (BL)-induced growth suppression in etiolated seedlings of Cucumis sativus L. The mechanism of this voltage transient was investigated by applying inhibitors of ion channels and the plasma-membrane H(+)-ATPase, by manipulating extracellular ion concentrations, and by measuring cell input resistance and ATP levels. The depolarizing phase was not affected by Ca(2+)-channel blockers (verapamil, La3+) or by reducing extracellular free Ca2+ by treatment with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). However, these treatments did reduce the rate of repolarization, indicating an inward movement of Ca2+ is involved. No effects of the K(+)-channel blocker tetraethylammonium (TEA+) were detected. Vanadate and KCN, used to inhibit the H(+)-ATPase, reduced or completely inhibited the BL-induced depolarization. Levels of ATP increased by 11-26% after 1-2 min of BL. Input resistance of trichrome cells, measured with double-barreled microelectrodes, remained constant during the onset of the depolarization but decreased as the membrane voltage became more positive than -90 mV. The results indicate that the depolarization mechanism initially involves inactivation of the H(+)-ATPase with subsequent transient activation of one or more types of ion channels.

  4. A plasma-membrane linker for the phosphoinositide-specific phospholipase C in tobacco plants.

    PubMed

    Nakamura, Kimiyo; Sano, Hiroshi

    2009-01-01

    We previously screened genes that were transcriptionally activated during the early stage of wound response in tobacco plants (Nicotiana tabacum), and isolated a particular clone, which encoded a membrane-located protein, designated as NtC7. Upon overexpression in tobacco plants, NtC7 conferred a marked tolerance to osmotic stress, suggesting it to be involved in maintenance of osmotic adjustments. In this study, we searched for proteins which interact with NtC7 by the yeast two-hybrid screening, and isolated a clone encoding phosphoinositide-specific phospholipase C, designated as NtPI-PLC. Physical interaction between NtC7 and C2 domain of NtPI-PLC was confirmed by the pull-down assay. Expression of fused protein to green-fluorescence protein in onion epidermal cell layers indicated both proteins to predominantly localize to the plasma membrane. Their interaction in planta was shown by the bimolecular fluorescence complementation, which exhibited a clear fluorescence of reconstituted yellow fluorescence protein. Transcripts of NtC7 and NtPI-PLC were markedly increased 30 to 60 min after wounding. PI-PLC is one of key enzymes in metabolism of inositol phospholipids, which function in signal transduction and also in response to stresses including osmotic changes. It was shown to localize to plasma-membrane and, to a lesser extent, to cytosol. However, molecular mechanism of membrane localization has remained to be determined, because of the apparent lack of domains for membrane association. The present results suggest that one of such mechanisms is tethering NtPI-PLC to the plasma membrane through interaction with NtC7, which possesses a transmembrane domain at the C-terminus.

  5. Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis.

    PubMed Central

    van den Broek, P J; van Gompel, A E; Luttik, M A; Pronk, J T; van Leeuwen, C C

    1997-01-01

    Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome c oxidase as a proton-motive-force-generating system. Addition of reduced cytochrome c generated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40-50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+-glucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+-maltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilis the transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different. PMID:9020885

  6. Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis.

    PubMed

    van den Broek, P J; van Gompel, A E; Luttik, M A; Pronk, J T; van Leeuwen, C C

    1997-01-15

    Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome c oxidase as a proton-motive-force-generating system. Addition of reduced cytochrome c generated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40-50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+-glucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+-maltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilis the transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

  7. Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis.

    PubMed

    Canut, H; Baudracco, S; Cabané, M; Boudet, A M; Marigo, G

    1991-07-01

    Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg(2+)-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.

  8. A calmodulin-stimulated Ca2+ pump in plasma-membrane vesicles from Trypanosoma brucei; selective inhibition by pentamidine.

    PubMed Central

    Benaim, G; Lopez-Estraño, C; Docampo, R; Moreno, S N

    1993-01-01

    Despite previous reports [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar and Bhaduri (1990) J. Biol. Chem. 265, 11345-11351; Mazumder, Mukherjee, Ghosh, Ray and Bhaduri (1992) J. Biol. Chem. 267, 18440-18446] that the plasma membrane of different trypanosomatids only contains Ca(2+)-ATPase that does not show any demonstrable dependence on Mg2+, a high-affinity (Ca(2+)-Mg2+)-ATPase was demonstrated in the plasma membrane of Trypanosoma brucei. The enzyme became saturated with micromolar amounts of Ca2+, reaching a Vmax. of 3.45 +/- 0.66 nmol of ATP/min per mg of protein. The Km,app. for Ca2+ was 0.52 +/- 0.03 microM. This was decreased to 0.23 +/- 0.05 microM, and the Vmax. was increased to 6.36 +/- 0.22 nmol of ATP/min per mg of protein (about 85%), when calmodulin was present. T. brucei plasma-membrane vesicles accumulated Ca2+ on addition of ATP only when Mg2+ was present, and released it to addition of the Ca2+ ionophore A23187. In addition, this Ca2+ transport was stimulated by calmodulin. Addition of NaCl to Ca(2+)-loaded T. brucei plasma-membrane vesicles did not result in Ca2+ release, thus suggesting the absence of a Na+/Ca2+ exchanger in these parasites. Therefore the (Ca(2+)-Mg2+)-ATPase would be the only mechanism so far described that is responsible for the long-term fine tuning of the intracellular Ca2+ concentration of these parasites. The trypanocidal drug pentamidine inhibited the T. brucei plasma-membrane (Ca(2+)-Mg2+)-ATPase and Ca2+ transport at concentrations that had no effect on the Ca(2+)-ATPase activity of human or pig erythrocytes. In this latter case, pentamidine behaved as a weak calmodulin antagonist, since it inhibited the stimulation of the erythrocyte Ca(2+)-ATPase by calmodulin. PMID:8280074

  9. Methods for producing secreted polypeptides

    SciTech Connect

    Maiyuran, Suchindra; Fidantsef, Ana; Brody, Howard

    2008-07-01

    The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3' end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

  10. Methods for producing secreted polypeptides

    SciTech Connect

    Maiyuran, Suchindra; Fidantsef, Ana; Brody, Howard

    2013-07-30

    The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a nucleic acid construct comprising a first nucleotide sequence encoding a signal peptide operably linked to a second nucleotide sequence encoding the polypeptide, wherein the first nucleotide sequence is foreign to the second nucleotide sequence and the 3' end of the first nucleotide sequence is immediately upstream of the initiator codon of the second nucleotide sequence. The present invention also relates to the isolated signal peptide sequences and to constructs, vectors, and fungal host cells comprising the signal peptide sequences operably linked to nucleotide sequences encoding polypeptides.

  11. Surface-grafted polypeptides on flat substrates

    NASA Astrophysics Data System (ADS)

    Wang, Yuli

    In this work, we improved the vapor deposition-polymerization (VDP) technique by re-designing a new vacuum chamber, and adding two heating plates to control the temperatures of the substrate and monomer evaporation. By optimizing the reaction parameters such as monomer amount, substrate temperature and reaction time, various polypeptides with sufficiently high molecular weight have been successfully grafted onto the solid substrates. The combination of VPD with photolithography has fabricated micro-patterned polypeptides, with geometry patternable in both lateral and vertical directions. Next, the conformations of the surface-grafted polypeptides were systematically examined. New ways have been found to switch their conformations between alpha-helix and beta-sheet, or between right-handed helix and left-handed helix. Two important ionic polypeptides, poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) were the focuses of this work. The conformational transitions of surface-grafted PLGA and PLL were successfully induced by pH, surfactants and ions. In addition, a surface-grafted PLGA-block-PLL copolypeptide was studied. Their unexpected pH-responsiveness was explained by the beta-sheet formation between the PLGA and PLL blocks. The orientation of the surface-grafted alpha-helical poly(gamma-benzyl L-glutamate) (PBLG) was greatly improved by a novel "solvent quenching" method, which involves treating the film sequentially with a good solvent and a poor solvent. The average tilt angle of the PBLG rods changed from 49° to 3° by applying this "quenching". Finally, a surface-grafted PLL film was used as the template to mimic the biosilicification. Silica was spontaneously synthesized from tetraethoxysilane inside the PLL film at room temperature and at neutral pH.

  12. Hydrogenase polypeptide and methods of use

    DOEpatents

    Adams, Michael W.W.; Hopkins, Robert C.; Jenney, JR, Francis E.; Sun, Junsong

    2016-02-02

    Provided herein are polypeptides having hydrogenase activity. The polypeptide may be multimeric, and may have hydrogenase activity of at least 0.05 micromoles H.sub.2 produced min.sup.-1 mg protein.sup.-1. Also provided herein are polynucleotides encoding the polypeptides, genetically modified microbes that include polynucleotides encoding one or more subunits of the multimeric polypeptide, and methods for making and using the polypeptides.

  13. Activated FcgammaRII and signalling molecules revealed in rafts by ultra-structural observations of plasma-membrane sheets.

    PubMed

    Strzelecka-Kiliszek, Agnieszka; Korzeniowski, Marek; Kwiatkowska, Katarzyna; Mrozińska, Kazimiera; Sobota, Andrzej

    2004-01-01

    To reveal topography of FcgammaRII components of the receptor-signalling complex, large plasma-membrane sheets were obtained by cell cleavage and analysed by immuno-electron microscopy. Non-activated FcgammaRII was dispersed in the plane of the plasma membrane and only rarely was localized in the proximity of Lyn, an Src family tyrosine kinase, and CD55, a glycosylphosphatidylinositol-anchored protein. After FcgammaRII activation by cross-linking with antibodies, clusters of an electron-dense material acquiring about 86% of FcgammaRII and reaching up to 300 nm in diameter were formed within 5 min. These structures also accommodated about 85% of Lyn and 63% of CD55 labels that were located in close vicinity of gold particles attributed to the cross-linked FcgammaRII . The electron-dense structures were also abundant in tyrosine phosphorylated proteins. At their margins PIP2 was preferentially located. Based on a concentration of Lyn, CD55 and activated FcgammaRII , the electron-dense structures seem to reflect coalescent membrane rafts.

  14. A hydrophobic filter confers the cation selectivity of Zygosaccharomyces rouxii plasma-membrane Na+/H+ antiporter.

    PubMed

    Kinclova-Zimmermannova, Olga; Falson, Pierre; Cmunt, Denis; Sychrova, Hana

    2015-04-24

    Na(+)/H(+) antiporters may recognize all alkali-metal cations as substrates but may transport them selectively. Plasma-membrane Zygosaccharomyces rouxii Sod2-22 antiporter exports Na(+) and Li(+), but not K(+). The molecular basis of this selectivity is unknown. We combined protein structure modeling, site-directed mutagenesis, phenotype analysis and cation efflux measurements to localize and characterize the cation selectivity region. A three-dimensional model of the ZrSod2-22 transmembrane domain was generated based on the X-ray structure of the Escherichia coli NhaA antiporter and primary sequence alignments with homologous yeast antiporters. The model suggested a close proximity of Thr141, Ala179 and Val375 from transmembrane segments 4, 5 and 11, respectively, forming a hydrophobic hole in the putative cation pathway's core. A series of mutagenesis experiments verified the model and showed that structural modifications of the hole resulted in altered cation selectivity and transport activity. The triple ZrSod2-22 mutant T141S-A179T-V375I gained K(+) transport capacity. The point mutation A179T restricted the antiporter substrate specificity to Li(+) and reduced its transport activity, while serine at this position preserved the native cation selectivity. The negative effect of the A179T mutation can be eliminated by introducing a second mutation, T141S or T141A, in the preceding transmembrane domain. Our experimental results confirm that the three residues found through modeling play a central role in the determination of cation selectivity and transport activity in Z. rouxii Na(+)/H(+) antiporter and that the cation selectivity can be modulated by repositioning a single local methyl group. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Methods for using polypeptides having cellobiohydrolase activity

    DOEpatents

    Morant, Marc D; Harris, Paul

    2016-08-23

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Nano polypeptide particles reinforced polymer composite fibers.

    PubMed

    Li, Jiashen; Li, Yi; Zhang, Jing; Li, Gang; Liu, Xuan; Li, Zhi; Liu, Xuqing; Han, Yanxia; Zhao, Zheng

    2015-02-25

    Because of the intensified competition of land resources for growing food and natural textile fibers, there is an urgent need to reuse and recycle the consumed/wasted natural fibers as regenerated green materials. Although polypeptide was extracted from wool by alkaline hydrolysis, the size of the polypeptide fragments could be reduced to nanoscale. The wool polypeptide particles were fragile and could be crushed down to nano size again and dispersed evenly among polymer matrix under melt extrusion condition. The nano polypeptide particles could reinforce antiultraviolet capability, moisture regain, and mechanical properties of the polymer-polypeptide composite fibers.

  17. Polypeptide toxins from animal venoms.

    PubMed

    Kozlov, Sergey A

    2007-01-01

    In the course of evolution, venomous animals developed highly specialized venomous systems that provided for drastic increase in hunting and defense efficiency. Venoms of a vast number of animal species represent complex mixtures of compounds such as ions, biogenic amines, polyamines, polypeptide neurotoxins, cytolytic peptides, enzymes, etc. that exert different functions. Natural toxins are sequentially variable molecules that are very stable structurally and produce pronounced biological effects on molecular targets. High activity made them very attractive in terms of novel structure discovery and characterization. In the present review we draw attention to the structure of polypeptide molecules preferably in the 2-12 kDa molecular mass range produced by various venomous animals that were published in patent literature. The structures were reviewed on the basis of functional relation to molecular targets. We also compared the sequence information from patents with Uniprot and other protein databanks to define structures that were patented but missing from the public databases.

  18. Elastomeric polypeptide-based biomaterials

    PubMed Central

    Li, Linqing; Charati, Manoj B.; Kiick, Kristi L.

    2011-01-01

    Elastomeric proteins are characterized by their large extensibility before rupture, reversible deformation without loss of energy, and high resilience upon stretching. Motivated by their unique mechanical properties, there has been tremendous research in understanding and manipulating elastomeric polypeptides, with most work conducted on the elastins but more recent work on an expanded set of polypeptide elastomers. Facilitated by biosynthetic strategies, it has been possible to manipulate the physical properties, conformation, and mechanical properties of these materials. Detailed understanding of the roles and organization of the natural structural proteins has permitted the design of elastomeric materials with engineered properties, and has thus expanded the scope of applications from elucidation of the mechanisms of elasticity to the development of advanced drug delivery systems and tissue engineering substrates. PMID:21637725

  19. Electrospun Synthetic Polypeptide Nanofibrous Biomaterials

    NASA Astrophysics Data System (ADS)

    Khadka, Dhan; Haynie, Donald

    2011-03-01

    Water-insoluble nanofiber mats of synthetic polypeptides of defined composition have been prepared from fibers electrospun from aqueous solution in the absence of organic co-solvents. 20-50 kDa poly(L-glutamate, L-tyrosine) 4:1 (PLGY) but not 15-50 kDa or 50-100 kDa poly(L-glutamate) was spinnable at 20-55% (w/v) polymer in water. Applied voltage and needle-collector distance were crucial for spinnability. Attractive fibers were obtained at 50% polymer. Fiber diameter and mat morphology have been characterized by electron microscopy. Exposure of spun fiber mats to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), which reacts with carboxylate, decreased fiber solubility. Fluorescein-conjugated poly(L-lysine) (FITC-PLL) but not the fluorophore alone was able bind PLGY fiber mats electrostatically, judging by fluorescence microscopy. Key advances of this work are the avoidance of an animal source of peptides and of an inorganic co-solvent to achieve polypeptide spinnability. Polypeptide fiber mats are a promising type of nano-structured biomaterial for applications in biomedicine and biotechnology.

  20. Phycobilisome structure of porphyridium cruentum: polypeptide composition

    SciTech Connect

    Redlinger, T.; Gantt, E.

    1981-01-01

    Purified phycobilisomes of porphyridium cruentum were solubilized in sodium dodecyl sulfate and resolved by sodium dodecyl sulfate-acrylamide gel electrophoresis into nine colored and nine colorless polypeptides. The colored polypeptides accounted for about 84% of the total stainable protein, and the colorless polypeptides accounted for the remaining 16%. Five of the colored polypeptides ranging in molecular weight from 13,300 to 19,500 were identified as the ..cap alpha.. and ..beta.. subunits of allophycocyanin, R-phycocyanin, and phycoerythrin. Three others (29,000-30,500) were orange and are probably related to the ..gamma.. subunit of phycoerythrin. Sequential dissociation of phycobilisomes, and analysis of the polypeptides in each fraction, revealed the association of a 32,500 molecular weight colorless polypeptide with a phycoerythrin fraction. The remaining eight colorless polypeptides were in the core fraction of the phycobilisome, which also was enriched in allophycocyanin.

  1. Physical association between a novel plasma-membrane structure and centrosome orients cell division

    PubMed Central

    Negishi, Takefumi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Yasuo, Hitoyoshi; Ueno, Naoto

    2016-01-01

    In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis. DOI: http://dx.doi.org/10.7554/eLife.16550.001 PMID:27502556

  2. Polypeptide having swollenin activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elizabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica D; Damveld, Robbertus Antonius

    2015-11-04

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  3. Polypeptide having cellobiohydrolase activity and uses thereof

    DOEpatents

    Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

    2015-09-15

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 93% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  4. Bodian's Silver Method Stains Neurofilament Polypeptides

    NASA Astrophysics Data System (ADS)

    Gambetti, P.; Autilio-Gambetti, L.; Papasozomenos, S. Ch.

    1981-09-01

    Bodian's silver method was used to stain polypeptides of rat spinal cord or peripheral nerve separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bands corresponding to the three polypeptide subunits of the neurofilaments were intensely impregnated. Two other polypeptides were stained inconsistently and less intensely. The tubulin band was stained weakly or not at all; other polypeptides, including glial fibrillary acidic protein, actin, and vimentin, remained unstained. This novel application of Bodian's method provides indirect proof that neurofilaments are the neuronal subcellular structure stained by the technique.

  5. Dependence of mammalian putrescine and spermidine transport on plasma-membrane potential: identification of an amiloride binding site on the putrescine carrier.

    PubMed

    Poulin, R; Zhao, C; Verma, S; Charest-Gaudreault, R; Audette, M

    1998-03-15

    The mechanism of mammalian polyamine transport is poorly understood. We have investigated the role of plasma-membrane potential (DeltaPsipm) in putrescine and spermidine uptake in ZR-75-1 human breast cancer cells. The rate of [3H]putrescine and [3H]spermidine uptake was inversely correlated to extracellular [K+] ([K+]o) and to DeltaPsipm, as determined by the accumulation of [3H]tetraphenylphosphonium bromide (TPP). Inward transport was unaffected by a selective decrease in mitochondrial potential (DeltaPsimit) induced by valinomycin at low [K+]o, but was reduced by approximately 60% by the rheogenic protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which rapidly (<=15 min) collapsed both DeltaPsipm and DeltaPsimit. Plasma-membrane depolarization by high [K+]o or CCCP did not enhance putrescine efflux in cells pre-loaded with [3H]putrescine, suggesting that decreased uptake caused by these agents did not result from a higher excretion rate. On the other hand, the electroneutral K+/H+ exchanger nigericin (10 microM) co-operatively depressed -3H-TPP, [3H]putrescine and [3H]spermidine uptake in the presence of ouabain. Suppression of putrescine uptake by nigericin+ouabain was Na+-dependent, suggesting that plasma-membrane repolarization by the electrogenic Na+ pump was required upon acidification induced by nigericin, due to the activation of the Na+/H+ antiporter. The sole addition of 5-N, N-hexamethylene amiloride, a potent inhibitor of the Na+/H+ antiporter, strongly inhibited putrescine uptake in a competitive fashion -Ki 4.0+/-0.9 (S.D.) microM-, while being a weaker antagonist of spermidine uptake. The potency of a series of amiloride analogues to inhibit putrescine uptake was clearly different from that of the Na+/H+ antiporter, and resembled that noted for Na+ co-transport proteins. These data demonstrate that putrescine and spermidine influx is mainly unidirectional and strictly depends on DeltaPsipm, but not DeltaPsimit. This report also provides

  6. Methods for engineering polypeptide variants via somatic hypermutation and polypeptide made thereby

    DOEpatents

    Tsien, Roger Y; Wang, Lei

    2015-01-13

    Methods using somatic hypermutation (SHM) for producing polypeptide and nucleic acid variants, and nucleic acids encoding such polypeptide variants are disclosed. Such variants may have desired properties. Also disclosed are novel polypeptides, such as improved fluorescent proteins, produced by the novel methods, and nucleic acids, vectors, and host cells comprising such vectors.

  7. Ordered biological nanostructures formed from chaperonin polypeptides

    NASA Technical Reports Server (NTRS)

    Trent, Jonathan D. (Inventor); McMillan, R. Andrew (Inventor); Kagawa, Hiromi (Inventor); Paavola, Chad D. (Inventor)

    2010-01-01

    The following application relates to nanotemplates, nanostructures, nanoarrays and nanodevices formed from wild-type and mutated chaperonin polypeptides, methods of producing such compositions, methods of using such compositions and particular chaperonin polypeptides that can be utilized in producing such compositions.

  8. Polypeptides having cellobiohydrolase activitiy and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Tang, Lan; Duan, Junxin

    2015-12-15

    The present invention provides isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2016-06-28

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-10-27

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having catalase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Duan, Junxin; Zhang, Yu; Tang, Lan

    2017-05-02

    Provided are isolated polypeptides having catalase activity and polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Hybrid polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Liu, Ye; Shaghasi, Tarana

    2016-11-01

    The present invention provides hybrid polypeptides having cellobiohydrolase activity. The present invention also provides polynucleotides encoding the hybrid polypeptides; nucleic acid constructs, vectors and host cells comprising the polynucleotides; and processes of using the hybrid polypeptides.

  14. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2015-06-09

    Provided are isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. Also provided are nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-03-31

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-11-17

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Isolation of Polypeptide Sample and Measurement of Its Concentration.

    ERIC Educational Resources Information Center

    Beanan, Maureen J.

    2000-01-01

    Introduces a laboratory experiment that isolates a bacterial polypeptide sample and measures the concentration of polypeptides in the sample. Uses Escherichia coli strain MM294 and performs a bio-rad assay to determine the concentration of polypeptides. (YDS)

  19. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-02-10

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-07-14

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2015-08-18

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Duan, Junxin; Tang, Lan

    2015-09-22

    The present invention provides isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cell comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having endoglucanse activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-08

    The present invention relates to isolated polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  5. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-06-24

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  6. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-15

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-06-24

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-07-08

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-10-14

    The present invention relates to isolated polypeptides having xylanase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Zhang, Yu; Tang, Lan; Henriksen, Svend Hostgaard Bang

    2016-05-17

    The present invention provides isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also provides nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    SciTech Connect

    Spodsberg, Nikolaj

    2016-12-13

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Isolation of Polypeptide Sample and Measurement of Its Concentration.

    ERIC Educational Resources Information Center

    Beanan, Maureen J.

    2000-01-01

    Introduces a laboratory experiment that isolates a bacterial polypeptide sample and measures the concentration of polypeptides in the sample. Uses Escherichia coli strain MM294 and performs a bio-rad assay to determine the concentration of polypeptides. (YDS)

  13. Hydrophilic Silica-Polypeptide Composite Particles

    NASA Astrophysics Data System (ADS)

    Soto-Cantu, Erick; Russo, Paul

    2007-03-01

    Composite, pH-responsive particles have been synthesized by covalently attaching a simple polypeptide to a silica core. The synthesis begins with the production of organophilic poly(benzylglutamate)-coated silica particles. The particles are rendered hydrophilic by cleaving the benzyl side group by treatment with hydrogen bromide in benzene. The resulting poly(glutamic acid)-coated silica spheres exhibit a change in hydrodynamic radius in response to pH stimulus. The size transition is due to a change in the polypeptide conformation, as deduced from circular dichroism measurements. Fong,B.; Russo, P.S. Organophilic Colloidal Particles with a Synthetic Polypeptide Coating. Langmuir 1999, 15, 4421-4426.

  14. Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

    PubMed Central

    Rao, Govind S.; Rao, Marie Luise; Thilmann, Astrid; Quednau, Hans D.

    1981-01-01

    1. Influx and efflux of l-tri-[125I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23°C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3–5pm) influx into cells was saturable; an apparent Kt value of 8.6±1.6pm was obtained. 3. At 5pm-l-tri-[125I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23°C) uptake of l-tri-[125I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a Kd value of 6.1±1.3pm. 4. Efflux of l-tri-[125I]iodothyronine from cells equilibrated with the hormone (5–123pm) was constant up to 21 s; the amount that flowed out was 17.7±3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[125I]iodothyronine (556–1226pm) 66.8±5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[125I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18–22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[125I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[125I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process

  15. Restriction/modification polypeptides, polynucleotides, and methods

    DOEpatents

    Westpheling, Janet; Chung, DaeHwan; Huddleston, Jennifer; Farkas, Joel A

    2015-02-24

    The present invention relates to the discovery of a novel restriction/modification system in Caldicellulosiruptor bescii. The discovered restriction enzyme is a HaeIII-like restriction enzyme that possesses a thermophilic activity profile. The restriction/modification system also includes a methyltransferase, M.CbeI, that methylates at least one cytosine residue in the CbeI recognition sequence to m.sup.4C. Thus, the invention provides, in various aspects, isolated CbeI or M.CbeI polypeptides, or biologically active fragments thereof; isolated polynucleotides that encode the CbeI or M.CbeI polypeptides or biologically active fragments thereof, including expression vectors that include such polynucleotide sequences; methods of digesting DNA using a CbeI polypeptide; methods of treating a DNA molecule using a M.CbeI polypeptide; and methods of transforming a Caldicellulosiruptor cell.

  16. Polypeptides of the Maize Amyloplast Stroma1

    PubMed Central

    Yu, Ying; He Mu, Helen; Mu-Forster, Chen; Wasserman, Bruce P.

    1998-01-01

    In the developing endosperm of monocotyledonous plants, starch granules are synthesized and deposited within the amyloplast. A soluble stromal fraction was isolated from amyloplasts of immature maize (Zea mays L.) endosperm and analyzed for enzyme activities and polypeptide content. Specific activities of starch synthase and starch-branching enzyme (SBE), but not the cytosolic marker alcohol dehydrogenase, were strongly enhanced in soluble amyloplast stromal fractions relative to soluble extracts obtained from homogenized kernels or endosperms. Immunoblot analysis demonstrated that starch synthase I, SBEIIb, and sugary1, the putative starch-debranching enzyme, were each highly enriched in the amyloplast stroma, providing direct evidence for the localization of starch-biosynthetic enzymes within this compartment. Analysis of maize mutants shows the deficiency of the 85-kD SBEIIb polypeptide in the stroma of amylose extender cultivars and that the dull mutant lacks a >220-kD stromal polypeptide. The stromal fraction is distinguished by differential enrichment of a characteristic group of previously undocumented polypeptides. N-terminal sequence analysis revealed that an abundant 81-kD stromal polypeptide is a member of the Hsp70 family of stress-related proteins. Moreover, the 81-kD stromal polypeptide is strongly recognized by antibodies specific for an Hsp70 of the chloroplast stroma. These findings are discussed in light of implications for the correct folding and assembly of soluble, partially soluble, and granule-bound starch-biosynthetic enzymes during import into the amyloplast. PMID:9536063

  17. Polypeptides having laccase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Tang, Lan; Duan, Junxin; Zhang, Yu

    2017-08-22

    The present invention relates to isolated polypeptides having laccase activity and polynucleotides encoding the polypeptides and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptide having an amino acid replaced with N-benzylglycine

    DOEpatents

    Mitchell, Alexander R.; Young, Janis D.

    1996-01-01

    The present invention relates to one or more polypeptides having useful biological activity in a mammal, which comprise: a polypeptide related to bradykinin of four to ten amino acid residues wherein one or more specific amino acids in the polypeptide chain are replaced with achiral N-benzylglycine. These polypeptide analogues have useful potent agonist or antagonist pharmacological properties depending upon the structure. A preferred polypeptide is (N-benzylglycine.sup.7)-bradykinin.

  19. Carbohydrate degrading polypeptide and uses thereof

    DOEpatents

    Sagt, Cornelis Maria Jacobus; Schooneveld-Bergmans, Margot Elisabeth Francoise; Roubos, Johannes Andries; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide having carbohydrate material degrading activity which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional protein and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  20. Biodegradable Epoxy Networks Cured with Polypeptides

    NASA Astrophysics Data System (ADS)

    Nakamura, Shigeo; Kramer, Edward J.

    2006-03-01

    Epoxy resins are used widely for adhesives as well as coatings. However, once cured they are usually highly cross-linked and are not biodegradable. To obtain potentially biodegradable polypeptides that can cure with epoxy resins and achieve as good properties as the conventional phenol novolac hardeners, poly(succinimide-co-tyrosine) was synthesized by thermal polycondensation of L-aspartic acid and L-tyrosine with phosphoric acid under reduced pressure. The tyrosine/succinimide ratio in the polypeptide was always lower than the tyrosine/(aspartic acid) feed ratio and was influenced by the synthesis conditions. Poly(succinimide-tyrosine- phenylalanine) was also synthesized from L-aspartic acid, L- tyrosine and L-phenylalanine. The thermal and mechanical properties of epoxy resins cured with these polypeptides are comparable to those of similar resins cured with conventional hardeners. In addition, enzymatic degradability tests showed that Chymotrypsin or Subtilisin A could cleave cured films in an alkaline borate buffer.

  1. Maize mitochondria synthesize organ-specific polypeptides

    SciTech Connect

    Newton, K.J.; Walbot, V.

    1985-10-01

    The authors detected both quantitative and qualitative organ-specific differences in the total protein composition of mitochondria of maize. Labeling of isolated mitochondria from each organ demonstrated that a few protein differences are due to changes in the polypeptides synthesized by the organelle. The synthesis of developmental stage-specific mitochondrial polypeptides was found in the scutella of developing and germinating kernels. The approximately 13-kDa polypeptide synthesized by mitochondria from seedlings of the Texas (T) male-sterile cytoplasm was shown to be constitutively expressed in all organs of line B37T tested. Methomyl, an insecticide known to inhibit the growth of T sterile plants, was shown to be an effective inhibitor of protein synthesis in mitochondria from T plants.

  2. Peppytides: Interactive Models of Polypeptide Chains

    SciTech Connect

    Zuckermann, Ron; Chakraborty, Promita; Derisi, Joe

    2014-01-21

    Peppytides are scaled, 3D-printed models of polypeptide chains that can be folded into accurate protein structures. Designed and created by Berkeley Lab Researcher, Promita Chakraborty, and Berkeley Lab Senior Scientist, Dr. Ron Zuckermann, Peppytides are accurate physical models of polypeptide chains that anyone can interact with and fold intro various protein structures - proving to be a great educational tool, resulting in a deeper understanding of these fascinating structures and how they function. Build your own Peppytide model and learn about how nature's machines fold into their intricate architectures!

  3. Peppytides: Interactive Models of Polypeptide Chains

    ScienceCinema

    Zuckermann, Ron; Chakraborty, Promita; Derisi, Joe

    2016-07-12

    Peppytides are scaled, 3D-printed models of polypeptide chains that can be folded into accurate protein structures. Designed and created by Berkeley Lab Researcher, Promita Chakraborty, and Berkeley Lab Senior Scientist, Dr. Ron Zuckermann, Peppytides are accurate physical models of polypeptide chains that anyone can interact with and fold intro various protein structures - proving to be a great educational tool, resulting in a deeper understanding of these fascinating structures and how they function. Build your own Peppytide model and learn about how nature's machines fold into their intricate architectures!

  4. Polypeptides and polyaminoacids in drug delivery.

    PubMed

    González-Aramundiz, José Vicente; Lozano, María Victoria; Sousa-Herves, Ana; Fernandez-Megia, Eduardo; Csaba, Noemi

    2012-02-01

    Advances achieved over the last few years in drug delivery have provided novel and versatile possibilities for the treatment of various diseases. Among the biomaterials applied in this field, it is worth highlighting the increasing importance of polyaminoacids and polypeptides. The appealing properties of these polymers are very promising for the design of novel compositions in a variety of drug delivery applications. This review provides an overview on the general characteristics of polyaminoacids and polypeptides and briefly discusses different synthetic pathways for their production. This is followed by a detailed description of different drug delivery applications of these polymers, emphasizing those examples that already reached advanced preclinical development or have entered clinical trials. Polyaminoacids and polypeptides are gaining much attention in drug delivery due to their exceptional properties. Their application as polymers for drug delivery purposes has been sped up by the significant achievements related to their synthesis. Certainly, cancer therapy has benefited the most from these advances, although other fields such as vaccine delivery and alternative administration routes are also being successfully explored. The design of new entities based on polyaminoacids and polypeptides and the improved insight gained in drug delivery guarantee exciting findings in the near future.

  5. Polypeptide Inhibitors of Mineral Scaling and Corrosion

    DTIC Science & Technology

    1990-06-30

    and the method of synthesis are subjects of patent applications. Corrosion inhibition has been demonstrated using polyaspartate , the simplest...observed that polyaspartate caused an upward shift in the open circuit potential (Ecorr), suggesting some anodic control of corrosion. However, Tafel...the presence of polypeptides are In progress. Examination of thermal polyaspartate effects on polarization resistance corrosion of 304 stainless

  6. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Harris, Paul; Tang, Lan; Wu, Wenping

    2013-11-19

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2010-06-22

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  8. Polypeptide from a cellulolytic fungus having cellulolytic enhancing activity

    DOEpatents

    Brown, Kimberly [Elk Grove, CA; Harris, Paul [Carnation, WA; Zaretsky, Elizabeth [Reno, NV; Re, Edward [Davis, CA; Vlasenko, Elena [Davis, CA; McFarland, Keith [Davis, CA; Lopez de Leon, Alfredo [Davis, CA

    2008-04-22

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  9. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2013-10-29

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2007-07-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  11. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Liu, Ye; Tang, Lan; Harris, Paul; Wu, Wenping

    2012-10-02

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2012-10-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  13. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc D.; Harris, Paul

    2015-10-13

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  14. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Maiyuran, Suchindra; Kramer, Randall; Harris, Paul

    2014-10-21

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc

    2014-01-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase, or beta-glucosidase activity and isolated polynucleotides encoding polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Ding, Hanshu; Brown, Kimberly

    2012-06-26

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having endoglucanase activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Rey, Michael

    2015-03-10

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2010-06-22

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2015-01-27

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2012-09-18

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Schnorr, Kirk; Kramer, Randall

    2016-04-05

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2016-08-09

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Brown, Kimberly; Harris, Paul; Lopez De Leon, Alfredo; Merino, Sandra

    2007-05-22

    The present invention relates to isolated polypeptides having cellobiohydrolase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  4. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2016-08-09

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  5. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    SciTech Connect

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2014-09-30

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  6. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2017-08-08

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  7. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2016-02-23

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  8. Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

    DOEpatents

    Stringer, Mary Ann; McBrayer, Brett

    2016-11-29

    The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

  9. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj; Shagasi, Tarana

    2015-06-30

    The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

  10. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2013-04-16

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Duan, Junxin; Tang, Lan; Liu, Ye; Wu, Wenping; Quinlan, Jason; Kramer, Randall

    2013-06-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2013-06-18

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Tang, Lan; Liu, Ye; Duan, Junxin; Hanshu, Ding

    2012-10-30

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  14. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2016-06-14

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having xylanase activity and polynucleotides encoding same

    SciTech Connect

    Lopez de Leon, Alfredo; Rey, Michael

    2016-05-31

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Duan, Junxin; Tang, Lan; Wu, Wenping

    2016-11-22

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Chimeric polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark; Sweeney, Matthew; Heu, Tia

    2017-06-14

    The present invention relates to chimeric GH61 polypeptides having cellulolytic enhancing activity. The present invention also relates to polynucleotides encoding the chimeric GH61 polypeptides; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the chimeric GH61 polypeptides.

  18. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj; Shagasi, Tarana

    2017-05-30

    The present invention relates to isolated polypeptides having endoglucanase activity, catalytic domains, cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains or cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains or cellulose binding domains.

  19. Polypeptides having beta-xylosidase activity and polynucleotides encoding same

    DOEpatents

    Zhang, Yu; Liu, Ye; Duan, Junxin; Tang, Lan; McBrayer, Brett

    2017-07-04

    The present invention relates to isolated polypeptides having beta-xylosidase activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Joergensen, Christian; Kramer, Randall

    2016-11-29

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Polypeptides having xylanase activity and polynucleotides encoding the same

    DOEpatents

    Spodsberg, Nikolaj [Bagsvaed, DK

    2014-01-07

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The inventino also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  2. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Zhang, Yu; Jorgensen, Christian Isak; Kramer, Randall

    2013-12-24

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  3. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Tang, Lan; Liu, Ye; Duan, Junxin; Ding, Hanshu

    2013-04-30

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  4. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2009-05-19

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  5. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Lopez de Leon, Alfredo; Rey, Michael; Ding, Hanshu; Vlasenko, Elena

    2010-11-02

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  6. Polynucleotides encoding polypeptides having beta-glucosidase activity

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2010-03-02

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  7. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul; Golightly, Elizabeth

    2011-06-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  8. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Spodsberg, Nikolaj

    2014-10-21

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  9. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding the same

    DOEpatents

    Duan, Junxin; Schnorr, Kirk Matthew; Wu, Wenping

    2013-11-19

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  10. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    SciTech Connect

    Duan, Junxin; Liu, Ye; Tang, Lan; Wu, Wenping; Quinlan, Jason; Kramer, Randall

    2012-03-27

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  11. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc D; Patkar, Shamkant; Ding, Hanshu

    2013-11-12

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  12. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-10-14

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  13. Polypeptides having beta-glucosidase activity and polynucleotides encoding same

    SciTech Connect

    Harris, Paul; Golightly, Elizabeth

    2012-11-27

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  14. Polypeptides having xylanase activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Rey, Michael

    2010-12-14

    The present invention relates to isolated polypeptides having xylanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  15. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Dotson, William D.; Greenier, Jennifer; Ding, Hanshu

    2007-09-18

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated nucleic acids encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acids as well as methods for producing and using the polypeptides.

  16. Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

    DOEpatents

    Lopez de Leon, Alfredo; Ding, Hanshu; Brown, Kimberly

    2011-10-25

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Polypeptides having endoglucanase activity and polynucleotides encoding same

    DOEpatents

    Harris, Paul [Carnation, WA; Lopez de Leon, Alfredo [Davis, CA; Rey, Micheal [Davis, CA; Ding, Hanshu [Davis, CA; Vlasenko, Elena [Davis, CA

    2012-02-21

    The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  18. POLYPEPTIDE AND POLYSACCHARIDE PROCESSING IN HYPERTHERMOPHILIC MICROORGANISMS

    SciTech Connect

    KELLY, ROBERT M.

    2008-12-22

    This project focused on the microbial physiology and biochemistry of heterotrophic hyperthermophiles with respect to mechanisms by which these organisms process polypeptides and polysaccharides under normal and stressed conditions. Emphasis is on two model organisms, for which completed genome sequences are available: Pyrococcus furiosus (growth Topt of 98°C), an archaeon, and Thermotoga maritima (growth Topt of 80°C), a bacterium. Both organisms are obligately anaerobic heterotrophs that reduce sulfur facultatively. Whole genome cDNA spotted microarrays were used to follow transcriptional response to a variety of environmental conditions in order to identify genes encoding proteins involved in the acquisition, synthesis, processing and utilization of polypeptides and polysaccharides. This project provided new insights into the physiological aspects of hyperthermophiles as these relate to microbial biochemistry and biological function in high temperature habitats. The capacity of these microorganisms to produce biohydrogen from renewable feedstocks makes them important for future efforts to develop biofuels.

  19. Biosynthetic Polypeptides as Templates in Materials Design

    NASA Astrophysics Data System (ADS)

    Kiick, Kristi

    2007-03-01

    Biosynthetic routes to protein-based polymeric materials offer important opportunities for the production of well-defined macromolecular templates, owing to the control of sequence and molecular weight inherent in the biosynthesis of proteins. In particular, the biosynthesis of polypeptides with controlled presentation of functional groups in multiple positions, coupled with their subsequent chemical modification with biologically relevant ligands, will permit the production of well-defined, bioactive macromolecules that may provide insight into biological binding events in which multivalent binding is important. Modification of the well-defined macromolecules with ligands such as saccharides has application in the study of events such as toxin neutralization and mediation of the immune and inflammatory responses. In this work, alanine-rich polypeptides of both random coil and helical conformations, equipped with glutamic acid residues to impart chemical versatility, have been produced via biosynthetic strategies. Analysis via spectroscopic and calorimetric methods indicates that the polypeptides adopt helical, beta-sheet, or random-coil conformations that can be controlled with variations in temperature, pH, and salt concentration; the conformational behavior of the polypeptides is not compromised upon chemical modification with saccharides. The binding of these macromolecules to bacterial toxins has been characterized via immunochemical and spectroscopic methods; results indicate that specific architectural features of the glycopolymer scaffold cause changes in the binding of these molecules to multivalent receptors. Given the chemical flexibility in the design of such scaffolds, they can be modified with many different moieties in addition to saccharides, so multiple opportunities exist for their application in areas where control of active side chains is important, such as in biomaterials, electronic devices, and bioinorganic structures.

  20. Polypeptide multilayer nanofilm artificial red blood cells.

    PubMed

    Palath, Naveen; Bhad, Sujaykumar; Montazeri, Reza; Guidry, Christopher A; Haynie, Donald T

    2007-04-01

    Reliable encapsulation of hemoglobin (Hb) within polypeptide multilayer nanofilms has been achieved by a template-based approach, and protein functionality has been demonstrated postencapsulation. The method is general in scope and could be useful for many other encapsulants. Met-Hb was adsorbed onto 5 microm-diameter CaCO3 microparticles, and the Hb-coated particles were encapsulated within a multilayer nanofilm of poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) by layer-by-layer assembly. The CaCO3 templates were then dissolved within the PLGA/PLL nanofilms by addition of ethylenediaminetetraacetic acid. Encapsulation of Hb was proved by fluorescence microscopy, the pH-dependence of retention of Hb was determined by visible wavelength absorbance, and conversion of the encapsulated met-Hb to deoxy-Hb and oxy-Hb was demonstrated by spectroscopic analysis of the Soret absorption peak under various conditions. It thus has been shown that control of Hb oxygenation within polypeptide multilayer nanofilm artificial cells is possible, and that Hb thus encapsulated can bind, release, and subsequently rebind molecular oxygen. This work therefore represents an advance in the development of polypeptide multilayer film artificial red blood cells. (c) 2006 Wiley Periodicals, Inc.

  1. Heterogeneity of Glutamine Synthetase Polypeptides in Phaseolus vulgaris L. 1

    PubMed Central

    Lara, Miguel; Porta, Helena; Padilla, Jaime; Folch, Jorge; Sánchez, Federico

    1984-01-01

    Glutamine synthetases from roots, nodules, and leaves of Phaseolus vulgaris L. have been purified to homogeneity and their polypeptide composition determined. The leaf enzyme is composed of six polypeptides. The cytosolic fraction contains two 43,000 dalton polypeptides and the chloroplastic enzyme is formed by four 45,000 dalton polypeptides. Root glutamine synthetase consists only of the same two polypeptides of 43,000 dalton that are present in the leaf enzyme. The nodule enzyme is formed by two polypeptides of 43,000 dalton, one is common to the leaf and root enzyme but the other is specific for N2-fixing nodule tissue. The two glutamine synthetase forms of the nodule contain a different proportion of the 43,000 dalton polypeptides. Images Fig. 1 Fig. 2 Fig. 4 PMID:16663942

  2. Different polypeptide composition of two human rotavirus types.

    PubMed Central

    Espejo, R; Martínez, E; López, S; Muñoz, O

    1980-01-01

    Human rotaviruses, which are placed into two groups according to their ribonucleic acid patterns obtained by gel electrophoresis, were characterized both by polypeptide components from purified virions and by polypeptides translated from their denatured ribonucleic acids in rabbit reticulocyte lysates. Viruses assigned to different groups differed in the electrophoretic migration of the second largest of the polypeptides which compose the inner shell; polypeptides that had been synthetized in vitro from ribonucleic acid from each group showed this same difference, thus indicating that this is due to the genomic composition. This study suggests that there are differences in the third largest polypeptide of the inner shell and also in the three smaller polypeptides composing the outer shell. We also demonstrated that there are differences in genomic and polypeptide compositions between simian (SA11) and calf (Nebraska calf diarrhea virus) rotaviruses grown in tissue culture and human rotaviruses. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:6247284

  3. Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma-membrane calcium-ATPase in human T cells

    PubMed Central

    Bautista, Diana M; Hoth, Markus; Lewis, Richard S

    2002-01-01

    In addition to its homeostatic role of maintaining low resting levels of intracellular calcium ([Ca2+]i), the plasma-membrane calcium-ATPase (PMCA) may actively contribute to the generation of complex Ca2+ signals. We have investigated the role of the PMCA in shaping Ca2+ signals in Jurkat human leukaemic T cells using single-cell voltage-clamp and calcium-imaging techniques. Crosslinking the T-cell receptor with the monoclonal antibody OKT3 induces a biphasic elevation in [Ca2+]i consisting of a rapid overshoot to a level > 1 μM, followed by a slow decay to a plateau of ≈0.5 μM. A similar overshoot was triggered by a constant level of Ca2+ influx through calcium-release-activated Ca2+ (CRAC) channels in thapsigargin-treated cells, due to a delayed increase in the rate of Ca2+ clearance by the PMCA. Following a rise in [Ca2+]i, PMCA activity increased in two phases: a rapid increase followed by a further calcium-dependent increase of up to approximately fivefold over 10-60 s, termed modulation. After the return of [Ca2+]i to baseline levels, the PMCA recovered slowly from modulation (τ ≈4 min), effectively retaining a ‘memory’ of the previous [Ca2+]i elevation. Using a Michaelis-Menten model with appropriate corrections for cytoplasmic Ca2+ buffering, we found that modulation extended the dynamic range of PMCA activity by increasing both the maximal pump rate and Ca2+ sensitivity (reduction of KM). A simple flux model shows how pump modulation and its reversal produce the initial overshoot of the biphasic [Ca2+]i response. The modulation of PMCA activity enhanced the stability of Ca2+ signalling by adjusting the efflux rate to match influx through CRAC channels, even at high [Ca2+]i levels that saturate the transport sites and would otherwise render the cell defenceless against additional Ca2+ influx. At the same time, the delay in modulation enables small Ca2+ fluxes to transiently elevate [Ca2+]i, thus enhancing Ca2+ signalling dynamics. PMID:12068047

  4. Ordered Nanostructures Made Using Chaperonin Polypeptides

    NASA Technical Reports Server (NTRS)

    Trent, Jonathan; McMillan, Robert; Paavola, Chad; Mogul, Rakesh; Kagawa, Hiromi

    2004-01-01

    A recently invented method of fabricating periodic or otherwise ordered nanostructures involves the use of chaperonin polypeptides. The method is intended to serve as a potentially superior and less expensive alternative to conventional lithographic methods for use in the patterning steps of the fabrication of diverse objects characterized by features of the order of nanometers. Typical examples of such objects include arrays of quantum dots that would serve as the functional building blocks of future advanced electronic and photonic devices. A chaperonin is a double-ring protein structure having a molecular weight of about 60 plus or minus 5 kilodaltons. In nature, chaperonins are ubiquitous, essential, subcellular structures. Each natural chaperonin molecule comprises 14, 16, or 18 protein subunits, arranged as two stacked rings approximately 16 to 18 nm tall by approximately 15 to 17 nm wide, the exact dimensions depending on the biological species in which it originates. The natural role of chaperonins is unknown, but they are believed to aid in the correct folding of other proteins, by enclosing unfolded proteins and preventing nonspecific aggregation during assembly. What makes chaperonins useful for the purpose of the present method is that under the proper conditions, chaperonin rings assemble themselves into higher-order structures. This method exploits such higher-order structures to define nanoscale devices. The higher-order structures are tailored partly by choice of chemical and physical conditions for assembly and partly by using chaperonins that have been mutated. The mutations are made by established biochemical techniques. The assembly of chaperonin polypeptides into such structures as rings, tubes, filaments, and sheets (two-dimensional crystals) can be regulated chemically. Rings, tubes, and filaments of some chaperonin polypeptides can, for example, function as nano vessels if they are able to absorb, retain, protect, and release gases or

  5. Pulsed ELDOR in spin-labeled polypeptides

    NASA Astrophysics Data System (ADS)

    Milov, Alexander D.; Maryasov, Alexander G.; Tsvetkov, Yuri D.; Raap, Jan

    1999-04-01

    The pulsed electron-electron double-resonance (PELDOR) technique was applied to obtain information about the structure of the synthetic polypeptide-biradical in a frozen glassy solution. From the concentration dependence of the PELDOR signal, the effects of intermolecular and intramolecular interactions were separated. It was found that the intramolecular dipole-dipole interactions in the biradical peptide led to the modulation effects in the PELDOR signal decay. This may be attributed to the existence of a conformational population having a distance between the two unpaired electrons of ˜20 Å with a distribution of (˜2 Å). Its fraction is estimated as about 25%.

  6. ANTIGENICITY OF POLYPEPTIDES (POLY ALPHA AMINO ACIDS)

    PubMed Central

    Pinchuck, Paul; Maurer, Paul H.

    1965-01-01

    The response of mice to synthetic linear polypeptides of known composition but random sequence has been studied. Neither Swiss mice nor a number of inbred strains could respond to copolymers of only 2 amino acids (G60L40, G60A40, G90T10). Upon introduction of as little as 4 mole per cent of a third amino acid, good immune responses were obtained, regardless of the nature of the third amino acid. The level of the immune response to a series of glu-lys-ala polymers increased with increasing alanine content of the polymer. PMID:5849232

  7. Polypeptide having beta-glucosidase activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

    2016-09-13

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 96% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  8. Polypeptide having beta-glucosidase activity and uses thereof

    DOEpatents

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; De Jong, Rene Marcel; Damveld, Robbertus Antonius

    2015-09-01

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 70% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  9. Polypeptide having acetyl xylan esterase activity and uses thereof

    DOEpatents

    Schoonneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2015-10-20

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 82% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  10. Polypeptide having carbohydrate degrading activity and uses thereof

    DOEpatents

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Vlasie, Monica Diana; Damveld, Robbertus Antonius

    2015-08-18

    The invention relates to a polypeptide comprising the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 73% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  11. Facilitated Translocation of Polypeptides Through A Single Nanopore

    PubMed Central

    Bikwemu, Robert; Wolfe, Aaron J.; Xing, Xiangjun; Movileanu, Liviu

    2011-01-01

    The transport of polypeptides through nanopores is a key process in biology and medical biotechnology. Despite its critical importance, the underlying kinetics of polypeptide translocation through protein nanopores is not yet comprehensively understood. Here, we present a simple two-barrier, one-well kinetic model for the translocation of short positively charged polypeptides through a single transmembrane protein nanopore that is equiped with negatively charged rings, simply called traps. We demonstrate that the presence of these traps within the interior of the nanopore dramatically alters the free energy landscape for the partitioning of the polypeptide into the nanopore interior, as revealed by significant modifications in the activation free energies required for the transitions of the polypeptide from one state to other. Our kinetic model permits the calculation of the relative and absolute exit frequencies of the short cationic polypeptides through either opening of the nanopore. Moreover, this approach enabled quantitative assessment of the kinetics of translocation of the polypeptides through a protein nanopore, which is strongly dependent on several factors, including the nature of the translocating polypeptide, the position of the traps, the strength of the polypeptide-attractive trap interactions and the applied transmembrane voltage. PMID:21339604

  12. Photodynamic induction of a bacterial cell surface polypeptide.

    PubMed Central

    Hoober, J K

    1977-01-01

    The photodynamic action of several dyes on cells of a bacterium, tentatively identified as a species of Arthrobacter, resulted in remarkable stimulation of synthesis of a polypeptide 21,000 daltons in mass. This polypeptide resides on the cell surface and can be solubilized by sodium dodecyl sulfate without lysis of the cells. Chlorophyllin and rose bengal are effective in inducing synthesis of the polypeptide in proportion to their ability to sensitize the photooxidation of histidine. Etiolated cells of the alga Chlamydomonas reinhardtii y-1 excrete a substance into the medium that also sensitized the photoinduction of the polypeptide. Images PMID:885841

  13. Intracellularly Swollen Polypeptide Nanogel Assists Hepatoma Chemotherapy

    PubMed Central

    Shi, Bo; Huang, Kexin; Ding, Jianxun; Xu, Weiguo; Yang, Yu; Liu, Haiyan; Yan, Lesan; Chen, Xuesi

    2017-01-01

    Nowadays, chemotherapy is one of the principal modes of treatment for tumor patients. However, the traditional formulations of small molecule drugs show short circulation time, low tumor selectivity, and high toxicity to normal tissues. To address these problems, a facilely prepared, and pH and reduction dual-responsive polypeptide nanogel was prepared for selectively intracellular delivery of chemotherapy drug. As a model drug, doxorubicin (DOX) was loaded into the nanogel through a sequential dispersion and dialysis technique, resulting in a high drug loading efficiency (DLE) of 96.7 wt.%. The loading nanogel, defined as NG/DOX, exhibited a uniform spherical morphology with a mean hydrodynamic radius of 58.8 nm, pH and reduction dual-triggered DOX release, efficient cell uptake, and cell proliferation inhibition in vitro. Moreover, NG/DOX exhibited improved antitumor efficacy toward H22 hepatoma-bearing BALB/c mouse model compared with free DOX·HCl. Histopathological and immunohistochemical analyses were implemented to further confirm the tumor suppression activity of NG/DOX. Furthermore, the variations of body weight, histopathological morphology, bone marrow cell micronucleus rate, and white blood cell count verified that NG/DOX showed excellent safety in vivo. With these excellent properties in vitro and in vivo, the pH and reduction dual-responsive polypeptide nanogel exhibits great potential for on-demand intracellular delivery of antitumor drug, and holds good prospect for future clinical application. PMID:28255361

  14. Simplified lattice model for polypeptide fibrillar transitions

    NASA Astrophysics Data System (ADS)

    Xiao, Xuhui; Wu, Ming-Chya

    2014-10-01

    Polypeptide fibrillar transitions are studied using a simplified lattice model, modified from the three-state Potts model, where uniform residues as spins, placed on a cubic lattice, can interact with neighbors to form coil, helical, sheet, or fibrillar structure. Using the transfer matrix method and numerical calculations, we analyzed the partition function and construct phase diagrams. The model manifests phase transitions among coil, helix, sheet, and fibril through parameterizing bond coupling energy ɛh,ɛs,ɛf, structural entropies sh,ss,sf of helical, sheet, and fibrillar states, and number density ρ. The phase diagrams show the transition sequence is basically governed by ɛh, ɛs, and ɛf, while the transition temperature is determined by the competition among ɛh, ɛs, and ɛf, as well as sh, ss, sf, and ρ. Furthermore, the fibrillation is accompanied with an abrupt phase transition from coil, helix, or sheet to fibril even for short polypeptide length, resembling the feature of nucleation-growth process. The finite-size effect in specific heat at transitions for the nonfibrillation case can be described by the scaling form of lattice model. With rich phase-transition properties, our model provides a useful reference for protein aggregation experiments and modeling.

  15. Chain stiffness of elastin-like polypeptides

    PubMed Central

    Fluegel, Sabine; Fischer, Karl; McDaniel, Jonathan R.; Chilkoti, Ashutosh; Schmidt, Manfred

    2010-01-01

    The hydrodynamic radii of a series of genetically engineered monodisperse elastin like polypeptides (ELP) was determined by dynamic light scattering in aqueous solution as function of molar mass. Utilizing the known theoretical expression for the hydrodynamic radius of wormlike chains, the Kuhn statistical segment length was determined to be lk = 2.1 nm, assuming that the length of the peptide repeat unit was b = 0.365 nm, a value derived for a coiled conformation of ELP. The resulting chain stiffness is significantly larger than previously reported by force-distance curve analysis (lk < 0.4 nm). The possible occurrence of superstructures, such as hairpins or helices, would reduce the contour length of the ELP, further increasing lk. Accordingly, the value lk = 2.1 nm reported here represents a lower limit of the chain stiffness for ELP. PMID:20961120

  16. Fibrillar dimer formation of islet amyloid polypeptides

    SciTech Connect

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  17. Fibrillar dimer formation of islet amyloid polypeptides

    NASA Astrophysics Data System (ADS)

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-09-01

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 - 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 - 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  18. Fibrillar dimer formation of islet amyloid polypeptides

    SciTech Connect

    Chiu, Chi-cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  19. Protein encapsulation via polypeptide complex coacervation.

    SciTech Connect

    Black, Katie A.; Priftis, Dimitrios; Perry, Sarah L.; Yip, Jeremy; Byun, William Y.; Tirrell, Matthew

    2014-10-01

    Proteins have gained increasing success as therapeutic agents; however, challenges exist in effective and efficient delivery. In this work, we present a simple and versatile method for encapsulating proteins via complex coacervation with oppositely charged polypeptides, poly(L-lysine) (PLys) and poly(D/L-glutamic acid) (PGlu). A model protein system, bovine serum albumin (BSA), was incorporated efficiently into coacervate droplets via electrostatic interaction up to a maximum loading of one BSA per PLys/PGlu pair and could be released under conditions of decreasing pH. Additionally, encapsulation within complex coacervates did not alter the secondary structure of the protein. Lastly the complex coacervate system was shown to be biocompatible and interact well with cells in vitro. A simple, modular system for encapsulation such as the one presented here may be useful in a range of drug delivery applications.

  20. Corneal Penetrating Elastin-Like Polypeptide Carriers

    PubMed Central

    George, Eric M.; Mahdi, Fakhri; Logue, Omar C.; Robinson, Grant G.

    2016-01-01

    Abstract Purpose: Elastin-like polypeptide (ELP) is a bioengineered protein widely applied as a drug carrier due to its biocompatibility and amenability to modification with cell-penetrating peptides (CPPs) and therapeutic agents. The purpose of this study was to determine whether topically applied ELP or CPP-fused ELPs penetrate the corneal barrier. Methods: In vitro binding and cytotoxicity to human corneal epithelial (HCE) cells were determined for ELP or CPP-ELPs. Corneal binding, clearance, and penetration were assessed in a rabbit model following topical application of the fluorescently labeled proteins by quantitative fluorescence imaging and histology. Results: ELP bound to HCE cells in vitro, and binding/uptake was enhanced 2- to 3-fold by the addition of CPPs. When applied topically to rabbit eyes, ELP accumulated in the cornea at levels 7.4-fold higher than did an equivalent dose of immunoglobulin G. Both ELP and a CPP-ELP penetrated the corneal epithelium and were detectable in the stroma. Addition of CPPs to ELP, however, did not significantly enhance corneal uptake or penetration in vivo relative to ELP alone. The polypeptides cleared from the cornea over a period of 20–30 min after application, after which cornea levels reached a steady state of 15–30 μg/mL for up to 3 h. Conclusions: The ELP drug carrier can penetrate the corneal epithelium and accumulate in the stroma. Given its amenability for fusion to multiple types of therapeutic agents, ELP has the potential to serve as a drug carrier for topical ocular applications. PMID:26672799

  1. Nucleic acids encoding antifungal polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-11-02

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  2. Isolated nucleic acids encoding antipathogenic polypeptides and uses thereof

    DOEpatents

    Altier, Daniel J.; Crane, Virginia C.; Ellanskaya, Irina; Ellanskaya, Natalia; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K.; Schepers, Eric J.; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-04-20

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include amino acid sequences, and variants and fragments thereof, for antipathogenic polypeptides that were isolated from fungal fermentation broths. Nucleic acids that encode the antipathogenic polypeptides are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  3. Chirality-selected phase behaviour in ionic polypeptide complexes

    DOE PAGES

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; ...

    2015-01-14

    In this study, polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with amore » β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.« less

  4. Chirality-selected phase behaviour in ionic polypeptide complexes

    NASA Astrophysics Data System (ADS)

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

    2015-01-01

    Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

  5. Chirality-selected phase behaviour in ionic polypeptide complexes

    PubMed Central

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

    2015-01-01

    Polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation. PMID:25586861

  6. Chirality-selected phase behaviour in ionic polypeptide complexes

    SciTech Connect

    Perry, Sarah L.; Leon, Lorraine; Hoffmann, Kyle Q.; Kade, Matthew J.; Priftis, Dimitrios; Black, Katie A.; Wong, Derek; Klein, Ryan A.; Pierce, III, Charles F.; Margossian, Khatcher O.; Whitmer, Jonathan K.; Qin, Jian; de Pablo, Juan J.; Tirrell, Matthew

    2015-01-14

    In this study, polyelectrolyte complexes present new opportunities for self-assembled soft matter. Factors determining whether the phase of the complex is solid or liquid remain unclear. Ionic polypeptides enable examination of the effects of stereochemistry on complex formation. Here we demonstrate that chirality determines the state of polyelectrolyte complexes, formed from mixing dilute solutions of oppositely charged polypeptides, via a combination of electrostatic and hydrogen-bonding interactions. Fluid complexes occur when at least one of the polypeptides in the mixture is racemic, which disrupts backbone hydrogen-bonding networks. Pairs of purely chiral polypeptides, of any sense, form compact, fibrillar solids with a β-sheet structure. Analogous behaviour occurs in micelles formed from polypeptide block copolymers with polyethylene oxide, where assembly into aggregates with either solid or fluid cores, and eventually into ordered phases at high concentrations, is possible. Chirality is an exploitable tool for manipulating material properties in polyelectrolyte complexation.

  7. Clinical endocrinology and metabolism. Glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide.

    PubMed

    Meier, Juris J; Nauck, Michael A

    2004-12-01

    The 42 amino acid polypeptide glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) is released from intestinal K-cells in response to nutrient ingestion. Based on animal studies, the peptide was initially assumed to act as an endogenous inhibitor of gastric acid secretion. Later it was found that GIP is capable of augmenting glucose-stimulated insulin secretion, and subsequent studies provided evidence that, in humans, the peptide predominantly acts as an incretin hormone. A role for GIP in the regulation of lipid homeostasis and in the development of obesity has been inferred from different animal studies. While GIP strongly stimulates insulin release in healthy humans, the peptide has almost completely lost its insulinotropic effect in patients with type 2 diabetes. This is different from the actions of glucagon-like peptide 1, which stimulates insulin secretion even in the later stages of type 2 diabetes. This suggests that a diminished insulinotropic effect of GIP may contribute to the pathogenesis of type 2 diabetes. This review will summarize the actions of GIP in human physiology and discuss its role in the pathogenesis of type 2 diabetes, as well as the therapeutic options derived from these findings.

  8. Laser enhanced hydrolysis of selected polypeptides

    NASA Astrophysics Data System (ADS)

    Ouzts, Mary Paige

    This project serves as a preliminary examination of selectively enhancing bond cleavage during chemical reactions in biological molecules by using continuous wave infrared lasers. To analyze protein content, polypeptides are broken into their constituent amino acids through hydrolysis. The cleaving of the peptide bond has traditionally been accomplished under harsh conditions, 110°C in 6 N hydrochloric acid for 24 hours. In this project hydrolysis was strongly enhanced by irradiating the dipeptides, threonyl-aspartate and alanyl-alanine, for 30 minutes with coherent infrared radiation from a tunable carbon dioxide laser. The dipeptide tyrosyl-tyrosine, the chemical N- methylacetimide, and the protein BSA were successfully hydrolyzed with the laser. The effect of reaction parameters such as laser power and HCl concentration were studied, as well as the effect of the primary parameter, the beam wavelength. The samples were analyzed using standard biological methods for determining the amino acid concentration, thin layer chromatography and ion exchange chromatography. These methods gave consistent results for the irradiated samples as well as for standard amino acids and polypeptide samples. The results from these methods were used to create the hydrolysis spectra. The catalytic action of the laser was strongly wavelength dependent. The hydrolysis spectra of the molecules were compared to the absorption spectra of the samples. Laser enhanced hydrolysis occurred when the laser wavelength coincided with a line in the dipeptide spectra. This weak line in each of the dipeptide spectra is consistent both in position and strength with a line in NMA, which has been identified as a fundamental mode associated with the peptide bond. From the experimental results, the enhanced process appears to occur in the vapor phase. The initially liquid sample was progressively evaporated, and fully hydrolyzed material was carried to a collection trap by the vapor. It can, in principle

  9. Simulating Massive Conformation Changes within Polypeptide Systems

    NASA Astrophysics Data System (ADS)

    Singh, Jaspinder Paul

    In this dissertation I employ all-atom structure based models with stable energy basins to several existing and novel polypeptide systems (postulated conformation changes of the mammalian prion protein and structurally dual proteins). The common themes are finding unfolding and refolding pathways between highly dissimilar protein structures as a means of understanding exactly how and why a protein may misfold. The modeling is based on the energy funnel landscape theory of protein conformation space. The principle of minimal frustration is considered as the model includes parameters which vary the roughness of the landscape and give rise to off-pathway misfoldings. The dual basin model is applied to the C-terminal (residues 166-226) of the mammalian prion protein. One basin represents the known alpha-helical (aH) structure while the other represents the same residues in a lefthanded beta-helical (LHBH) conformation. The LHBH structure has been proposed to help describe one class of in vitro grown fibrils, as well as possibly self-templating the conversion of normal cellular prion protein to the infectious form. Yet, it is unclear how the protein may make this global rearrangement. Our results demonstrate that the conformation changes are not strongly limited by large-scale geometry modification and that there may exist an overall preference for the LHBH conformation. Furthermore, our model presents novel intermediate trapping conformations with twisted LHBH structure. Polypeptides that display structural duality have primary structures that can give rise to different potential native conformations. We apply the structure-based all-atom model to a leucine zipper protein template with a stable aH structure that has been shown in experiment to switch to a β hairpin structure when exposed to a low-pH environment. We show that the model can be used to perform large-scale temperature-dependent conformational switching by simulating this switching behavior. We augmented

  10. Aspects of structural landscape of human islet amyloid polypeptide

    SciTech Connect

    He, Jianfeng Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J.

    2015-01-28

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  11. Aspects of structural landscape of human islet amyloid polypeptide

    NASA Astrophysics Data System (ADS)

    He, Jianfeng; Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J.

    2015-01-01

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  12. Aspects of structural landscape of human islet amyloid polypeptide.

    PubMed

    He, Jianfeng; Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J

    2015-01-28

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  13. Selective posttranslational modification of phage-displayed polypeptides

    SciTech Connect

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-11-19

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2] cycloaddition reactions and Staudinger modifications.

  14. Selective posttranslational modification of phage-displayed polypeptides

    SciTech Connect

    Tsao, Meng-Lin; Tian, Feng; Schultz, Peter

    2013-02-05

    The invention relates to posttranslational modification of phage-displayed polypeptides. These displayed polypeptides comprise at least one unnatural amino acid, e.g., an aryl-azide amino acid such as p-azido-L-phenylalanine, or an alkynyl-amino acid such as para-propargyloxyphenylalanine, which are incorporated into the phage-displayed fusion polypeptide at a selected position by using an in vivo orthogonal translation system comprising a suitable orthogonal aminoacyl-tRNA synthetase and a suitable orthogonal tRNA species. These unnatural amino acids advantageously provide targets for posttranslational modifications such as azide-alkyne [3+2]cycloaddition reactions and Staudinger modifications.

  15. Fibrillar dimer formation of islet amyloid polypeptides

    DOE PAGES

    Chiu, Chi -cheng; de Pablo, Juan J.

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimentalmore » and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.« less

  16. Turnover of cytokeratin polypeptides in mouse hepatocytes

    SciTech Connect

    Denk, H.; Lackinger, E.; Zatloukal, K. ); Franke, W.W. )

    1987-11-01

    The turnover of cytokeratin polypeptides A (equivalent to No. 8 of the human cytokeratin catalog) and D (equivalent to human cytokeratin No. 18) of mouse hepatocytes was studied by pulse-labeling of mouse liver proteins after intraperitoneal injection of L-(guanido{sup 14}C)arginine and ({sup 14}C)sodium bicarbonate. With L-(guanido-{sup 14}C)arginine a rapid increase in the specific radioactivity of both cytokeratins was observed which reached a plateau between 12 and 24 h. With ({sup 14}C)sodium bicarbonate maximal specific radioactivity was obtained at 6 h followed by a rapid decrease to half maximum values within the subsequent 6 h and then a slower decrease. Half-lives were determined from the decrease of specific radioactivities after pulse-labeling by least-squares plots and found to be 84 h (for cytokeratin component A) and 104 h (component D) for arginine labeling . Values obtained after bicarbonate labeling were similar (95 h for A and 98 h for D). These results show that liver cytokeratins are relatively stable proteins and suggest that components A and D are synthesized and degraded at similar rates, probably in a coordinate way.

  17. Mechanisms of stability of electrospun polypeptide fibers

    NASA Astrophysics Data System (ADS)

    Gitnik, Alina; Khadka, Dhan; Cross, Michael; Le, Nicole; Haynie, Donald

    2013-03-01

    Electrospun nano- and microfibers made of biodegradable and absorbable polymers are of great interest in biomedical engineering for tissue engineering, wound healing and other purposes. We have investigated physical properties of fibers made of the synthetic organic polymer co-poly(L-glutamic acid4, L-tyrosine1) (PLEY). This water-soluble polypeptide has a net negative charge at neutral pH. Dehydrated fibers are crosslinked with a diimide reagent dissolved in ethanol, giving a maximum average number of crosslinks of 1 per polymer molecule. Fiber integrity has been assessed in an aqueous medium at pH 2, 7 and 12, before and after crosslinking. Non-crosslinked fibers dissolved rapidly at all pH values, on a timescale of seconds to minutes. Crosslinked fibers dissolved completely at pH 12, but not at pH 2 or pH 7, the rate depending on the concentration of crosslinking reagent and therefore the density of crosslinks. Dissolution at pH 12 is attributable to ionization of the tyrosine side chain, which has a nominal pKa of 10.4, an increase in electrostatic repulsion between side chains and the migration of counterions into the fiber. Fibers crosslinked in 50 mM EDC buckled on a timescale of minutes at pH 12 and dissolved shortly thereafter. Funding provided by the National Science Foundation

  18. Elastin-like Polypeptide Based Hydroxyapatite Bionanocomposites

    PubMed Central

    Wang, Eddie; Lee, Sang-Hyuk; Lee, Seung-Wuk

    2011-01-01

    In nature, organic matrix macromolecules play a critical role in enhancing the mechanical properties of biomineralized composites such as bone and teeth. Designing artificial matrix analogues is promising but challenging because relatively little is known about how natural matrix components function. Therefore, in lieu of using natural components, we created biomimetic matrices using genetically engineered elastin-like polypeptides (ELPs) then used them to construct mechanically robust ELP-hydroxyapatite (HAP) composites. ELPs were engineered with well-defined backbone charge distributions by periodic incorporation of negative, positive, or neutral side chains or with HAP-binding octaglutamic acid motifs at one or both protein termini. ELPs exhibited sequence-specific capacities to interact with ions, bind HAP, and disperse HAP nanoparticles. HAP-binding ELPs were incorporated into calcium phosphate cements resulting in materials with improved mechanical strength, injectability, and anti-washout properties. The results demonstrate that rational design of genetically engineered polymers is a powerful system for determining sequence-property relationships and for improving the properties of organic-inorganic composites. Our approach may be used to further develop novel, multifunctional bone cements and expanded to the design of other advanced composites. PMID:21218767

  19. Superoxide Dismutase as an Anaerobic Polypeptide 1

    PubMed Central

    Monk, Lorna S.; Fagerstedt, Kurt V.; Crawford, Robert M. M.

    1987-01-01

    The perennating organ, the rhizome, was chosen for examination of response to anoxia in the species Iris pseudacorus L., Iris germanica L. var Quechei, and Glyceria maxima (Hartm.) Holmberg. These monocots are known to differ in their tolerance of anoxia. Intact rhizomes were subjected to periods of prolonged anoxia of up to 28 days and superoxide dismutase (SOD) activity was determined in a 48 hour postanoxic recovery phase. Tests were performed to ensure the accuracy of the measured enzyme activities. In the most anoxia tolerant species, I. pseudacorus, SOD activity rose continuously during the period of imposed anoxia, and levels were maintained in the postanoxic recovery phases: 28 days brought about a 13-fold increase to 1576 U SOD per milligram protein. Small increases were found in the less anoxia tolerant I. germanica during anoxic/postanoxic phases, while a drop in activity was recorded in the least anoxia tolerant G. maxima. However, initial levels in G. maxima were more than twice as high as in the other two species. Experiments applying cycloheximide to anoxic rhizome slices of I. pseudacorus inhibited the increase in SOD activity. This indicates that SOD is, paradoxically, induced under anoxia and we suggest that in this species SOD is one of the enzymes identified as anaerobic polypeptides. The significance of the induction of an `oxygen-protecting' enzyme during complete oxygen deprivation is discussed with regard to a possible critical role during recovery from anoxic stress. PMID:16665795

  20. Gastric inhibitory polypeptide secretion after radical pancreatoduodenectomy.

    PubMed Central

    Miyata, M; Nakao, K; Tanaka, Y; Sakamoto, T; Hamaji, M; Taketani, H; Kawashima, Y

    1984-01-01

    To elucidate the role of gastric inhibitory polypeptide (GIP) in the alteration of insulin secretion following pancreatoduodenal resection, in which the main sources of GIP are removed, plasma levels of GIP were measured for 180 minutes after oral glucose administration, both before and after radical pancreatoduodenectomy in nine patients with periampullary cancer. Fasting plasma levels of GIP remained much the same before and after surgery, and were not different from those in normal controls. The levels of GIP after glucose ingestion were significantly greater in the preoperative patients than in normal controls throughout 180 minutes. After pancreatoduodenectomy, the postglucose levels significantly diminished but remained within normal limits. Changes in plasma levels of insulin early after glucose ingestion in these patients, however, were significantly less both before and after surgery than in normal controls, and were not concomitant with the initial increase in plasma GIP. On the other hand, plasma levels of insulin greatly increased immediately after glucose ingestion in accordance with a rapid elevation of plasma GIP in 11 gastrectomized patients in whom the duodenum and the pancreas were preserved intact and who served as the control group. Thus, the diminution in GIP secretion following pancreatoduodenectomy may relate to the lack of main sources of this gut hormone and not to factors involved in the reconstruction of the alimentary tract. We conclude that the impaired insulin secretion following oral glucose ingestion in patients before and after pancreatoduodenectomy does not relate to the secretion of GIP. PMID:6367676

  1. Polypeptide and RNA composition of the reticuloendotheliosis viruses.

    PubMed

    Maldonado, R L; Bose, H R

    1975-01-01

    The RNA and polypeptide composition of chick syncytial virus (CSV) and duck infectious anemia virus (DIAV) was investigated and compared to that of reticuloendotheliosis virus (REV) strain T, the prototype of the newly recognized REV group of viruses. CSV and DIAV contain genomic RNA species which cosediment with those of REV in sucrose gradients. Five or six polypeptides, two of which are glycoproteins, were consistently found in CSV and DIAV preparations. The major nonglycosylated polypeptides and glycoproteins of CSV and DIAV comigrated with the corresponding polypeptides of REV strain T. Since the genomic RNA species and the glycoproteins of avian tumor viruses fail to comigrate, this suggests that the REV complex is a more homogeneous group.

  2. Biomedical applications of polypeptide multilayer nanofilms and microcapsules

    NASA Astrophysics Data System (ADS)

    Rudra, Jai Simha S.

    The past few years have witnessed considerable growth in synthetic polymer chemistry and physics, biomaterials science, and nano-scale engineering. Research on polypeptide multilayer films, coatings, and microcapsules is located at the intersection of these areas and are promising materials for applications in medicine, biotechnology, environmental science. Most envisioned applications of polypeptide multilayers have a biomedical bent. This dissertation on polypeptide multilayer film applications covers key points of polypeptides as materials, means of polymer production, film preparation, film characterization methods, and key points of current research in basic science. Both commercial and designed peptides have been used to fabricate films for in-vitro applications such as antimicrobial coatings and cell culture coatings and also microcapsules for drug delivery applications. Other areas of product development include artificial red blood cells, anisotropic coatings, enantioselective membranes, and artificial viruses.

  3. Common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

    SciTech Connect

    Cameron, R.S.; Cameron, P.L.; Castle, J.D.

    1986-10-01

    A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues - pancreatic, lacrimal, and submandibular - from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.

  4. Beta structures of alternating polypeptides and their possible prebiotic significance

    NASA Technical Reports Server (NTRS)

    Brack, A.; Orgel, L. E.

    1975-01-01

    A survey of the commonest amino acids formed in prebiotic conditions suggests that the earliest form of genetic coding may have specified polypeptides with a strong tendency to form stable beta-sheet structures. Poly(Val-Lys), like other polypeptides in which hydrophobic and hydrophilic residues alternate, tends to form beta structures. It is shown that bilayers with a hydrophobic interior and a hydrophilic exterior may be present in aqueous solution.

  5. Controlling assembly of helical polypeptides via PEGylation strategies†

    PubMed Central

    Top, Ayben; Zhong, Sheng; Yan, Congqi

    2013-01-01

    Recent studies in our laboratories have demonstrated that a helical polypeptide (17H6), equipped with a histidine tag and a helical alanine-rich, glutamic-acid-containing domain, exhibits pH-responsive assembly behavior useful in the production of polymorphological nanostructures. In this study, the histidine tag in these polypeptides was replaced by polyethylene glycol (PEG) with different molecular masses (5 kDa, or 10 kDa), and the self-association behavior of 17H6 and the PEGylated conjugates was characterized via dynamic light scattering (DLS), small angle neutron scattering (SANS), and cryogenic transmission electron microscopy (cryo-TEM). DLS experiments illustrated that the polypeptide and its PEG-conjugates undergo reversible assembly under acidic conditions, suggesting that the aggregation state of the polypeptide and the conjugates is controlled by the charged state of the glutamic acid residues. Nanoscale aggregates were detected at polypeptide/conjugate concentrations as low as 20 μM (∼0.3–0.5 mg ml−1) at physiological and ambient temperatures. Scattering and microscopy results showed that the size, the aggregation number, and the morphology of the aggregates can be tuned by the size and the nature of the hydrophilic tag. This tunable nature of the morphology of the aggregates, along with their low critical aggregation concentration, suggests that PEG-alanine-rich polypeptide conjugates may be useful as drug delivery vehicles in which the alanine-rich block serves as a drug attachment domain. PMID:24039625

  6. Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides. Polypeptide vesicles by conformation-specific assembly. Ordered chiral macroporous hybrid silica-polypeptide composites

    NASA Astrophysics Data System (ADS)

    Bellomo, Enrico Giuseppe

    2005-07-01

    Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides . The aqueous, lyotropic liquid-crystalline phase behavior of an alpha helical polypeptide, has been studied using optical microscopy and X-ray scattering. Solutions of optically pure polypeptide were found to form cholesteric liquid crystals at volume fractions that decreased with increasing average chain length. At very high volume fractions, the formation of a hexagonal mesophase was observed. The pitch of the cholesteric phase could be varied by a mixture of enantiomeric samples, where the pitch increased as the mixture approached equimolar. The cholesteric phases could be untwisted, using either magnetic field or shear flow, into nematic phases, which relaxed into cholesterics upon removal of field or shear. We have found that the phase diagram of this polypeptide in aqueous solution parallels that of poly(gamma-benzyl glutamate) in organic solvents, thus providing a useful system for liquid-crystal applications requiring water as solvent. Polypeptide vesicles by conformation-specific assembly. We have found that block copolymers composed of polypeptide segments provide significant advantages in controlling both the function and supramolecular structure of bioinspired self-assemblies. Incorporation of the stable chain conformations found in proteins into block copolymers was found to provide an additional element of control, beyond amphiphilicity and composition that defines self-assembled architecture. The abundance of functionality present in amino acids, and the ease by which they can be incorporated into these materials, also provides a powerful mechanism to impart block copolypeptides with function. This combination of structure and function work synergistically to enable significant advantages in the preparation of therapeutic agents as well as provide insight into design of self-assemblies beginning to approach the complexity of natural structures such as virus capsids. Ordered

  7. Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.

    PubMed

    Harris, Paul W R; Brimble, Margaret A

    2015-03-01

    The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilizing tail, was assembled in a convergent manner from four unprotected peptide α-thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc solid phase peptide synthesis (SPPS) respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic C-terminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein. © 2015 Wiley Periodicals, Inc.

  8. [Antiangiogenic Effect of Oyster Polypeptide (OPP).].

    PubMed

    Wang, Zhenhua; Liu, Jincheng; Su, Ai; Sun, Mi; Wang, Chunbo

    2009-08-20

    Drugs which block tumor angiogenesis will be likely effective towards inhibiting tumor growth for angiogenesis being a prerequisite for tumor growth and metastasis. Therefore, antiangiogenesis has become a promising strategy for the treatment of cancer. Investigation on both antiangiogenic effect and mechanism(s) of oyster polypeptide (OPP) were performed via experiments of chicken embryos model in vivo and human umbilical vein endothelial cells (HUVECs) in vitro. The methods employed in experiment were chorioallantoic membrane (CAM) angiogenesis in chicken embryos in vivo, MTT cell survival assay, flat plate scarification, transwell plates assay, matrigel-induced tube formation assay and transmission electron microscope et al. and the OPP's effects on angiogenesis was observed. Study showed that treatment with OPP resulted in significant inhibition of chorioallantoic membrane (CAM) angiogenesis in chicken embryos. MTT cell survival assay showed that treatment with OPP resulted in strong inhibition of HUVECs growth, with an IC50 of 400 mug/mL. Flat plate scarification suggested that OPP (200 mug/mL, 400 mug/mL and 800 mug/mL) distinctly inhibited HUVECs' migration (18.75%, 37.93%, 74.07% respectively, treatment for 12 h). Treatment with OPP of different concentrations (200 mug/mL, 400 mug/mL and 800 mug/mL) significantly reduced the density of the migration cells by 15.5%, 37.2% and 67.24% (P<0.05) respectively. Matrigel-induced tube formation assay showed that OPP resulted in striking inhibition of tube formation of 52.43%, 84.47% and 96.12% (P<0.01) at 200 mug/mL, 400 mug/mL and 800 mug/mL (treatment for 10 h) respectively. In addition, the apoptotic analysis by transmission electron microscope showed that OPP (400 mug/mL, treatment for 48 h) distinctly induced HUVECs' apoptosis. This study strikingly showed that OPP could inhibit angiogenesis through its effects on vascular endothelial cells directly and the inhibition of their proliferation, migration

  9. Surface active complexes formed between keratin polypeptides and ionic surfactants.

    PubMed

    Pan, Fang; Lu, Zhiming; Tucker, Ian; Hosking, Sarah; Petkov, Jordan; Lu, Jian R

    2016-12-15

    Keratins are a group of important proteins in skin and hair and as biomaterials they can provide desirable properties such as strength, biocompatibility, and moisture regaining and retaining. The aim of this work is to develop water-soluble keratin polypeptides from sheep wool and then explore how their surface adsorption behaves with and without surfactants. Successful preparation of keratin samples was demonstrated by identification of the key components from gel electrophoresis and the reproducible production of gram scale samples with and without SDS (sodium dodecylsulphate) during wool fibre dissolution. SDS micelles could reduce the formation of disulphide bonds between keratins during extraction, reducing inter-molecular crosslinking and improving keratin polypeptide solubility. However, Zeta potential measurements of the two polypeptide batches demonstrated almost identical pH dependent surface charge distributions with isoelectric points around pH 3.5, showing complete removal of SDS during purification by dialysis. In spite of different solubility from the two batches of keratin samples prepared, very similar adsorption and aggregation behavior was revealed from surface tension measurements and dynamic light scattering. Mixing of keratin polypeptides with SDS and C12TAB (dodecyltrimethylammonium bromide) led to the formation of keratin-surfactant complexes that were substantially more effective at reducing surface tension than the polypeptides alone, showing great promise in the delivery of keratin polypeptides via the surface active complexes. Neutron reflection measurements revealed the coexistence of surfactant and keratin polypeptides at the interface, thus providing the structural support to the observed surface tension changes associated with the formation of the surface active complexes.

  10. Drug delivery to solid tumors by elastin-like polypeptides

    PubMed Central

    McDaniel, Jonathan R.; Callahan, Daniel J.; Chilkoti, Ashutosh

    2010-01-01

    Thermally responsive elastin-like polypeptides (ELPs) are a promising class of recombinant biopolymers for the delivery of drugs and imaging agents to solid tumors via systemic or local administration. This article reviews four applications of ELPs to drug delivery, with each delivery mechanism designed to best exploit the relationship between the characteristic transition temperature (Tt) of the ELP and body temperature (Tb). First, when Tt >> Tb, small hydrophobic drugs can be conjugated to the C-terminus of the ELP to impart the amphiphilicity needed to mediate the self-assembly of nanoparticles. These systemically delivered ELP-drug nanoparticles preferentially localize to the tumor site via the EPR effect, resulting in reduced toxicity and enhanced treatment efficacy. The remaining three approaches take direct advantage of the thermal responsiveness of ELPs. In the second strategy, where Tb < Tt < 42 °C, an ELP-drug conjugate can be injected in conjunction with external application of mild hyperthermia to the tumor to induce ELP coacervation and an increase in concentration within the tumor vasculature. The third approach utilizes hydrophilic-hydrophobic ELP block copolymers that have been designed to assemble into nanoparticles in response to hyperthermai due to the independent thermal transition of the hydrophobic block, thus resulting in multivalent ligand display of a ligand for spatially enhanced vascular targeting. In the final strategy, ELPs with Tt < Tb are conjugated with radiotherapeutics, injtect intioa tumor where they undergo coacervation to form an injectable drug depot for intratumoral delivery. These injectable coacervate ELP-radionuclide depots display a long residence in the tumor and result in inhibition of tumor growth. PMID:20546809

  11. A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

    PubMed Central

    Zeng, Xiaohua; Choudhary, Madhu; Kaplan, Samuel

    2003-01-01

    A new operon (designated the puc2BA operon) displaying a high degree of similarity to the original pucBA genes of Rhodobacter sphaeroides 2.4.1 (designated puc1) was identified and studied genetically and biochemically. The puc2B-encoded polypeptide is predicted to exhibit 94% identity with the original β-apoprotein. The puc2A-encoded polypeptide is predicted to be much larger (263 amino acids) than the 54-amino-acid puc1A-encoded polypeptide. In the first 48 amino acids of the puc2A-encoded polypeptide there is 58% amino acid sequence identity to the original puc1A-encoded polypeptide. We found that puc2BA is expressed, and DNA sequence data suggested that puc2BA is regulated by the PpsR/AppA repressor-antirepressor and FnrL. Employing genetic and biochemical approaches, we obtained evidence that the puc2B-encoded polypeptide is able to enter into LH2 complex formation, but neither the full-length puc2A-encoded polypeptide nor its N-terminal 48-amino-acid derivative is able to enter into LH2 complex formation. Thus, the sole source of α-polypeptides for the LH2 complex is puc1A. The role of the puc1C-encoded polypeptide was also determined. We found that the presence of this polypeptide is essential for normal levels of transcription and translation of the puc1 operon but not for transcription and translation of the puc2 operon. Thus, the puc1C gene product appears to have both transcriptional and posttranscriptional roles in LH2 formation. Finally, the absence of any LH2 complex when puc1B was deleted in frame was surprising since we know that in the presence of functional puc2BA, approximately 30% of the LH2 complexes normally observed contain a puc2B-encoded β-polypeptide. PMID:14526029

  12. Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L.

    PubMed

    Langhans, M; Ratajczak, R; Lützelschwab, M; Michalke, W; Wächter, R; Fischer-Schliebs, E; Ullrich, C I

    2001-05-01

    Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

  13. The salt tolerant yeast Zygosaccharomyces rouxii possesses two plasma-membrane Na+/H+-antiporters (ZrNha1p and ZrSod2-22p) playing different roles in cation homeostasis and cell physiology.

    PubMed

    Pribylova, Lenka; Papouskova, Klara; Sychrova, Hana

    2008-10-01

    Antiporters exporting Na(+) and K(+) in exchange for protons are conserved among yeast species. The only exception so far has been Zygosaccharomyces rouxii, an osmotolerant species closely related to Saccharomyces cerevisiae. Z. rouxii was described as possessing one plasma-membrane antiporter transporting only Na(+) (ZrSod2-22p in the CBS 732(T) type strain). We report the characterization of a second gene, ZrNHA1, encoding a new K(+)(Na(+))/H(+)-antiporter capable of both K(+) and Na(+) export. Synteny analyses suggested that ZrSOD2-22 originated by single duplication of the ZrNHA1 gene. Substrate specificities and transport properties of ZrNha1p and ZrSod2-22p were compared upon heterologous expression in S. cerevisiae, and then directly in Z. rouxii. Deletion mutants and phenotype analyses revealed that ZrSod2-22 antiporter is important for Na(+) detoxification, probably together with ZrEna1 ATPase; ZrNha1p is indispensable to maintain potassium homeostasis and ZrEna1p is not, in contrast to the situation in S. cerevisiae, involved in this function.

  14. Interplay between electrophoretic mobility and intrinsic viscosity of polypeptide chains.

    PubMed

    Deiber, Julio A; Peirotti, Marta B; Piaggio, María V

    2012-03-01

    The present work is motivated specifically by the need to find a simple interplay between experimental values of electrophoretic mobility and intrinsic viscosity (IV) of polypeptides. The connection between these two properties, as they are evaluated experimentally in a formulated dilute solution, may provide relevant information concerning the physicochemical characterization and separation of electrically charged chains such as polypeptides. Based on this aspect, a study on the relation between the effective electrophoretic mobility and the IV of the following globular proteins is carried out: bovine carbonic anhydrase, staphylococcal nuclease, human carbonic anhydrase, lysozyme, human serum albumin. The basic interpretation of the IV through polypeptide chain conformations involves two unknowns: one is the Flory characteristic ratio involving short-range intramolecular interactions and the other is the Mark-Houwink exponent associated with large-range intramolecular interactions. Here, it will be shown via basic and well-established electrokinetic theories and scaling concepts that the IV and global chain flexibility of polypeptides in dilute solutions may be estimated from capillary zone electrophoresis, in addition to classical transport properties. The polypeptide local chain flexibility may change due to electrostatic interactions among closer chain ionizing groups and the hindrance effect of their associated structural water.

  15. Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.

    PubMed

    Faik, A; Desveaux, D; MacLachlan, G

    2000-05-01

    Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan.

  16. Methods of increasing secretion of polypeptides having biological activity

    SciTech Connect

    Merino, Sandra

    2015-04-14

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  17. Human jagged polypeptide, encoding nucleic acids and methods of use

    DOEpatents

    Li, Linheng; Hood, Leroy

    2000-01-01

    The present invention provides an isolated polypeptide exhibiting substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the polypeptide does not have the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as JAGGED, or an active fragment thereof, provided that the nucleotide sequence does not encode the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. Also provided herein is a method of inhibiting differentiation of hematopoietic progenitor cells by contacting the progenitor cells with an isolated JAGGED polypeptide, or active fragment thereof. The invention additionally provides a method of diagnosing Alagille Syndrome in an individual. The method consists of detecting an Alagille Syndrome disease-associated mutation linked to a JAGGED locus.

  18. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2013-10-01

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  19. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2014-10-28

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  20. Methods of increasing secretion of polypeptides having biological activity

    DOEpatents

    Merino, Sandra

    2014-05-27

    The present invention relates to methods for producing a secreted polypeptide having biological activity, comprising: (a) transforming a fungal host cell with a fusion protein construct encoding a fusion protein, which comprises: (i) a first polynucleotide encoding a signal peptide; (ii) a second polynucleotide encoding at least a catalytic domain of an endoglucanase or a portion thereof; and (iii) a third polynucleotide encoding at least a catalytic domain of a polypeptide having biological activity; wherein the signal peptide and at least the catalytic domain of the endoglucanase increases secretion of the polypeptide having biological activity compared to the absence of at least the catalytic domain of the endoglucanase; (b) cultivating the transformed fungal host cell under conditions suitable for production of the fusion protein; and (c) recovering the fusion protein, a component thereof, or a combination thereof, having biological activity, from the cultivation medium.

  1. Polypeptide formation on polar mineral surfaces: possibility of complete chirality

    NASA Astrophysics Data System (ADS)

    Schrader, Malcolm E.

    2017-01-01

    In the present work, it is shown that thermodynamically feasible polymerization of cyanomethanol, which can be formed from formaldehyde and hydrogen cyanide, can lead to synthesis of polypeptides as well as to the previously reported synthesis of RNA. If the polymerization takes place on a one-dimensional feature of a mineral, such as for example a crack on its surface, the concept of quasi-chirality is introduced to describe the adsorbed polypeptide. This, in principle, would lead to formation of proteins that are completely homochiral in their alpha carbon groups. The concept of quasi-chirality can also be introduced in the condensation of glycine under similar conditions to form a polypeptide. This again leads to proteins completely chiral in their alpha carbon groups.

  2. Separation, antitumor activities, and encapsulation of polypeptide from Chlorella pyrenoidosa.

    PubMed

    Wang, Xiaoqin; Zhang, Xuewu

    2013-01-01

    Chlorella pyrenoidosa is a unicellular green algae and has been a popular foodstuff worldwide. However, no reports on the antitumor peptides from such a microalgae are available in the literature. In this study, using low-temperature high-pressure extraction, enzymatic hydrolysis, ion exchange, and gel filtration chromatography, we separated a polypeptide that exhibited inhibitory activity on human liver cancer HepG2 cells, and named the polypeptide CPAP (C. pyrenoidosa antitumor polypeptide). Furthermore, the micro- and nanoencapsulation of CPAP were investigated by using two methods: complex coacervation and ionotropic gelation. The in vitro release tests revealed that CPAP was well preserved against gastric enzymatic degradation after micro/nanoencapsulation and the slowly controlled release in the intestine could be potentially achieved. These results suggest that CPAP may be a useful ingredient in food, nutraceutical, and pharmaceutical applications. © 2013 American Institute of Chemical Engineers.

  3. Elastin-like polypeptides: biomedical applications of tunable biopolymers.

    PubMed

    MacEwan, Sarah R; Chilkoti, Ashutosh

    2010-01-01

    Artificial repetitive polypeptides have grown in popularity as a bioinspired alternative to synthetic polymers. The genetically encoded synthesis, monodispersity, potential lack of toxicity, and biocompatibility are attractive features of these biopolymers for biological applications. Elastin-like polypeptides (ELPs) are one such class of biopolymers that are of particular interest because of their "smart"-stimuli responsive-properties. Herein, we discuss the genetically encoded design and recombinant synthesis of ELPs that enable precise control of their physicochemical properties and which have led to a wide range of biomedical applications of these biopolymers in the last decade. (c) 2010 Wiley Periodicals, Inc.

  4. Reovirus-specific polypeptides: analysis using discontinuous gel electrophoresis.

    PubMed Central

    Cross, R K; Fields, B N

    1976-01-01

    The electrophoretic analysis of reovirus-specific polypeptides in infected cells using a discontinuous gel system has allowed the resolution of additional viral-specific polypeptides, including one large-sized gamma3 and two (or possibly three) medium-sized (mu3, mu4, mu5(?)) species. The proteins designated mu0, sigma1, and sigma2 based on electrophoretic mobility in gel systems containing phosphate-urea correspond to mu4, sigma2, and sigma1, respectively, when analyzed in systems containing Tris-glycine. It is likely that protein modifications (phosphorylation and glycosylation) are responsible for at least some of these differences. Images PMID:950684

  5. Atomic Layer Deposition of L-Alanine Polypeptide

    SciTech Connect

    Fu, Yaqin; Li, Binsong; Jiang, Ying-Bing; Dunphy, Darren R.; Tsai, Andy; Tam, Siu-Yue; Fan, Hongyou Y.; Zhang, Hongxia; Rogers, David; Rempe, Susan; Atanassov, Plamen; Cecchi, Joseph L.; Brinker, C. Jeffrey

    2014-10-30

    L-Alanine polypeptide thin films were synthesized via atomic layer deposition (ALD). Rather, instead of using an amino acid monomer as the precursor, an L-alanine amino acid derivatized with a protecting group was used to prevent self-polymerization, increase the vapor pressure, and allow linear cycle-by-cycle growth emblematic of ALD. Moreover, the successful deposition of a conformal polypeptide film has been confirmed by FTIR, TEM, and Mass Spectrometry, and the ALD process has been extended to polyvaline.

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

    PubMed

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

    2008-06-06

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

  7. Refined Genetic Algorithms for Polypeptide Structure Prediction.

    DTIC Science & Technology

    1996-12-01

    fo cused ev olution program. 2.5 Parallel Genetic Algorithms General information ab out parallel computing is a v ailable in App endix C. There are t w...to generate a more t \\feasible" candidate, and th us, escap e the lo cal minim a. T able 14. Final minim um energies (k cal/mol) for [Met]-enk...prop osed the tec hnique kno wn as ev olutionary programming. Ev olu- tionary programmi ng tries to generate computational biological ev olution

  8. Methods of using viral replicase polynucleotides and polypeptides

    DOEpatents

    Gordon-Kamm, William J.; Lowe, Keith S.; Bailey, Matthew A.; Gregory, Carolyn A.; Hoerster, George J.; Larkins, Brian A.; Dilkes, Brian R.; Burnett, Ronald; Woo, Young Min

    2007-12-18

    The invention provides novel methods of using viral replicase polypeptides and polynucleotides. Included are methods for increasing transformation frequencies, increasing crop yield, providing a positive growth advantage, modulating cell division, transiently modulating cell division, and for providing a means of positive selection.

  9. Porin polypeptide contributes to surface charge of gonococci.

    PubMed Central

    Swanson, J; Dorward, D; Lubke, L; Kao, D

    1997-01-01

    Each strain of Neisseria gonorrhoeae elaborates a single porin polypeptide, with the porins expressed by different strains comprising two general classes, Por1A and Por1B. In the outer membrane, each porin molecule folds into 16 membrane-spanning beta-strands joined by top- and bottom-loop domains. Por1A and Por1B have similar membrane-spanning regions, but the eight surface-exposed top loops (I to VIII) differ in length and sequence. To determine whether porins, and especially their top loop domains, contribute to bacterial cell surface charge, strain MS11 gonococci that were identical except for expressing a recombinant Por1A, Por1B, or mosaic Por1A-1B polypeptide were compared by whole-cell electrophoresis. These porin variants displayed different electrophoretic mobilities that correlated with the net numbers of charged amino acids within surface-exposed loops of their respective porin polypeptides. The susceptibilities of porin variants to polyanionic sulfated polymers correlated roughly with gonococcal surface charge; those porin variants with diminished surface negativity showed increased sensitivity to the polyanionic sulfated compounds. These observations indicate that porin polypeptides in situ contribute to the surface charge of gonococci, and they suggest that the bacterium's interactions with large sulfated compounds are thereby affected. PMID:9171398

  10. Subcellular Localization and Topology of the p7 Polypeptide of Hepatitis C Virus

    PubMed Central

    Carrère-Kremer, Séverine; Montpellier-Pala, Claire; Cocquerel, Laurence; Wychowski, Czeslaw; Penin, François; Dubuisson, Jean

    2002-01-01

    Although biological and biochemical data have been accumulated on most hepatitis C virus proteins, the structure and function of the 63-amino-acid p7 polypeptide of this virus have never been investigated. In this work, sequence analyses predicted that p7 contains two transmembrane passages connected by a short hydrophilic segment. The C-terminal transmembrane domain of p7 was predicted to function as a signal sequence, which was confirmed experimentally by analyzing the translocation of a reporter glycoprotein fused at its C terminus. The p7 polypeptide was tagged either with the ectodomain of CD4 or with a Myc epitope to study its membrane integration, its subcellular localization, and its topology. Alkaline extraction studies confirmed that p7 is an integral membrane polypeptide. The CD4-p7 chimera was detected by immunofluorescence on the surface of nonpermeabilized cells, indicating that it is exported to the plasma membrane. However, pulse-chase analyses showed that only approximately 20% of endoglycosidase H-resistant CD4-p7 was detected after long chase times, suggesting that a large proportion of p7 stays in an early compartment of the secretory pathway. Finally, by inserting a Myc epitope in several positions of p7 and analyzing the accessibility of this epitope on the plasma membrane of HepG2 cells, we showed that p7 has a double membrane-spanning topology, with both its N and C termini oriented toward the extracellular environment. Altogether, these data indicate that p7 is a polytopic membrane protein that could have a functional role in several compartments of the secretory pathway. PMID:11907211

  11. Chronic overproduction of islet amyloid polypeptide/amylin in transgenic mice: lysosomal localization of human islet amyloid polypeptide and lack of marked hyperglycaemia or hyperinsulinaemia.

    PubMed

    Höppener, J W; Verbeek, J S; de Koning, E J; Oosterwijk, C; van Hulst, K L; Visser-Vernooy, H J; Hofhuis, F M; van Gaalen, S; Berends, M J; Hackeng, W H

    1993-12-01

    Type 2 (non-insulin-dependent) diabetes mellitus is characterised by hyperglycaemia, peripheral insulin resistance, impaired insulin secretion and pancreatic islet amyloid formation. The major constituent of islet amyloid is islet amyloid polypeptide (amylin). Islet amyloid polypeptide is synthesized by islet beta cells and co-secreted with insulin. The ability of islet amyloid polypeptide to form amyloid fibrils is related to its species-specific amino acid sequence. Islet amyloid associated with diabetes is only found in man, monkeys, cats and racoons. Pharmacological doses of islet amyloid polypeptide have been shown to inhibit insulin secretion as well as insulin action on peripheral tissues (insulin resistance). To examine the role of islet amyloid polypeptide in the pathogenesis of Type 2 diabetes, we have generated transgenic mice with the gene encoding either human islet amyloid polypeptide (which can form amyloid) or rat islet amyloid polypeptide, under control of an insulin promoter. Transgenic islet amyloid polypeptide mRNA was detected in the pancreas in all transgenic mice. Plasma islet amyloid polypeptide levels were significantly elevated (up to 15-fold) in three out of five transgenic lines, but elevated glucose levels, hyperinsulinaemia and obesity were not observed. This suggests that insulin resistance is not induced by chronic hypersecretion of islet amyloid polypeptide. Islet amyloid polypeptide immunoreactivity was localized to beta-cell secretory granules in all mice. Islet amyloid polypeptide immunoreactivity in beta-cell lysosomes was seen only in mice with the human islet amyloid polypeptide gene, as in human beta cells, and might represent an initial step in intracellular formation of amyloid fibrils.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Catalytic and reactive polypeptides and methods for their preparation and use

    DOEpatents

    Schultz, Peter

    1993-01-01

    Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the bi.

  13. Interfacial energy of polypeptide complex coacervates measured via capillary adhesion.

    PubMed

    Priftis, Dimitrios; Farina, Robert; Tirrell, Matthew

    2012-06-12

    A systematic study of the interfacial energy (γ) of polypeptide complex coacervates in aqueous solution was performed using a surface forces apparatus (SFA). Poly(L-lysine hydrochloride) (PLys) and poly(L-glutamic acid sodium salt) (PGA) were investigated as a model pair of oppositely charged weak polyelectrolytes. These two synthetic polypeptides of natural amino acids have identical backbones and differ only in their charged side groups. All experiments were conducted using equal chain lengths of PLys and PGA in order to isolate and highlight effects of the interactions of the charged groups during complexation. Complex coacervates resulted from mixing very dilute aqueous salt solutions of PLys and PGA. Two phases in equilibrium evolved under the conditions used: a dense polymer-rich coacervate phase and a dilute polymer-deficient aqueous phase. Capillary adhesion, associated with a coacervate meniscus bridge between two mica surfaces, was measured upon the separation of the two surfaces. This adhesion enabled the determination of the γ at the aqueous/coacervate phase interface. Important experimental factors affecting these measurements were varied and are discussed, including the compression force (1.3-35.9 mN/m) and separation speed (2.4-33.2 nm/s). Physical parameters of the system, such as the salt concentration (100-600 mM) and polypeptide chain length (N = 30, 200, and 400) were also studied. The γ of these polypeptide coacervates was separately found to decrease with both increasing salt concentration and decreasing polypeptide chain length. In most of the above cases, γ measurements were found to be very low, <1 mJ/m(2). Biocompatible complex coacervates with low γ have a strong potential for applications in surface coatings, adhesives, and the encapsulation of a wide range of materials.

  14. Principles Governing the Self Assembly of Polypeptide Nanoparticles

    NASA Astrophysics Data System (ADS)

    Wahome, Newton

    Self assembling systems on the nanometer scale afford the advantage of being able to control submicron level events. In this study, we focus on the self-assembling polypeptide nanoparticles (SAPN). The SAPN scaffold is made up of oligomerizing domains that align along the principle rotational axes of icosahedral symmetry. By aligning them along these axes, a particle with spherical geometry can be achieved. This particle can be utilized as a vaccine, as a drug delivery vehicle, or as a biomedical imaging device. This research will try to answer why the SAPN self-assembles into distinct molecular weight ranges while mostly maintaining a spherical morphology. The first means will be theoretical and computational, where we will utilize a mathematical formalism to find out how the packing of SAPN's monomeric units can occur within symmetric space. Then molecular dynamics will be run within this symmetric space to test the per amino acid residue susceptibility of SAPN towards becoming polymorphic in nature. Means for examining the aggregation propensity of SAPN will be also be tested. Specifically, the relationship of different sequences of SAPN with pH will be elucidated. Co-assembly of SAPN to reduce the surface density of an aggregation prone epitope will be tested. Also, aggregation reduction consisting of the exchange of an anionic denaturant with a positively charged suppressor in order to mitigate a priori peptide association and misfolding, will also be attempted. SAPN has been shown to be an immunogenic platform for the presentation of pathogen derived antigens. We will attempt to show the efficacy of presenting an antigen from HIV-1 which is structurally restrained to best match the native conformation on the virus. Immunological studies will be performed to test the effect of this approach, as well testing the antigenicity of the nanoparticle in the absence of adjuvant. Finally, the antigen presenting nanoparticles will undergo formulation testing, to measure

  15. Polypeptide models to understand misfolding and amyloidogenesis and their relevance in protein design and therapeutics.

    PubMed

    Zurdo, Jesús

    2005-02-01

    The study of amyloid polypeptide models (polypeptides able to generate amyloid structures not necessarily connected with any pathology) provides an excellent tool to increase the understanding of the generic aspects of misfolding and aggregation as well as the details of the mechanism of polypeptide deposition in disease. This knowledge can be integrated and applied to different problems in therapy and biotechnology, and in particular to re-designing bio-active polypeptides (biopharmaceuticals) with improved properties.

  16. Polypeptides having beta-glucosidase activity and polynucleotides encoding the same

    DOEpatents

    Brown, Kimberly; Harris, Paul

    2013-12-17

    The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  17. Recombinant host cells and nucleic acid constructs encoding polypeptides having cellulolytic enhancing activity

    DOEpatents

    Schnorr, Kirk; Kramer, Randall

    2017-03-28

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  18. Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-05-06

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  19. Polypeptides having beta-glucosidase activity and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-04-29

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  20. Polypeptides having beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

    SciTech Connect

    Morant, Marc Dominique

    2014-05-06

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  1. Comparison of intestinal brush-border 95-Kdalton polypeptide and alpha- actinins

    PubMed Central

    1980-01-01

    To explore the suggestion that alpha-actinin cross-links actin filaments to the microvillar membrane (Mooseker and Tilney, 1975, J. Cell Biol. 67:725--743; Mooseker, 1976, J. Cell Biol. 71-417--433), we have assessed the possible relatedness of alpha-actinin and the brush- border 95-kdalton protein by four independent criteria: antigenicity, mobility on SDS gels, extractability in nonionic detergents, and peptide maps. We have found that anti-chicken gizzard alpha-actinin stains the junctional complex region of intact cells (Craig and Pardo, 1979, J. Cell Biol. 80:203--210) but does not stain isolated brush borders even though these structures contain a 95-kdalton polypeptide. Lack of staining is not caused by failure of the antibody to penetrate, as antiactin stains both the terminal web and the microvilli of isolated brush borders. By the antibody SDS gel overlay technique, we have established that anti-gizzard alpha-actinin recognizes homologous molecules in chicken skeletal and cardiac muscles, as well as in intestinal epithelial cells, but fails to recognize the brush-border 95- kdalton polypeptide. Conversely, anti-95-kdalton polypeptide does not recognize gizzard alpha-actinin. On high-resolution SDS polyacrylamide gel electrophoresis, alpha-actinin and brush-border 95-kdalton protein exhibit distinct mobilities. The two proteins also differ in their ability to be extracted in nonionic mobilities. The two proteins also differ in their ability to be extracted in nonionic detergent: epithelial cell immunoreactive alpha-actinin is soluble in NP-40, whereas 95-kdalton protein is insoluble. Finally, two-dimensional peptide mapping of iodinated tryptic peptides, as well as one- dimensional fingerprinting of partial tryptic, chymotryptic, papain, and S. aureus V8 protease digests, have revealed less than 5% homology between gizzard alpha-actinin and brush-border 95-kdalton polypeptide. The data suggest that there is no major structural homology between gizzard

  2. Three-Dimensional Polypeptide Architectures Through Tandem Catalysis and Click Chemistry

    NASA Astrophysics Data System (ADS)

    Rhodes, Allison Jane

    -defined, high-density brushes for applications in drug delivery and imaging. Here, we also report a method for the synthesis of soluble, well-defined, azido functionalized polypeptides in a straightforward, 3-step synthesis. Homo and diblock azidopolypeptides were prepared with controlled segment lengths via living polymerization using Co(PMe3)4 initiator. Through copper azide alkyne click chemistry (CuAAC) in organic solvent, azidopolypeptides were regioselectively and quantitatively modified with carboxylic acid (pH-responsive), amino acid and sugar functional groups. Finally, the advances towards well-defined hyperbranched polypeptides through alpha-amino-acid-N-thiocarboxyanhydrides (NTAs) will be discussed. Within the past 10 years, controlled NCA (alpha-amino acid-N-carboxyanhydride) ring-opening polymerization (ROP) has emerged, expanding the application of copolypeptide polymers in various drug delivery and tissue engineering motifs. Modification of NCA monomers to the corresponding alpha-amino-acid-N-thiocarboxyanhydride (NTA) will diversify ROP reactions, leading to more complex polypeptides (such as hyperbranched polymers), in addition to the possibility of performing these polymerizations under ambient conditions, which would greatly expand their potential utility. The project focuses on the preparation of hyperbranched polypeptides with well-defined architectures and controlled branching density in a one-pot reaction. This will be accomplished by taking advantage of the different selectivities of Co(PMe3)4 and depeNi(COD) polymerization initiators, and by exploiting the reactivity difference between NCA and the more stable NTA monomers.

  3. Complexes of Negatively Charged Polypeptides with Cationic Lipids

    NASA Astrophysics Data System (ADS)

    Subramanian, G.; Li, Youli; Safinya, Cyrus R.

    1997-03-01

    Complexes of cationic lipids with oppositely charged proteins are promising candidates for new biomolecular materials. In addition to being used as a direct vehicle for protein transfection, they also find applications as templates for synthesis of molecular sieves. In spite of these wide ranging applications, the structure and interactions in these complexes have largely remained unclear. Here we report on the study of complexes formed between the cationic lipid didodecyldimethylammonium bromide (DDAB) with negatively charged polypeptide poly glutamic acid (PGA) both in the presence and absence of the neutral lipid dilauroylglycerophosphocholine (DLPC). X-ray diffraction of the complexes indicates a condensed lamellar lipid structure with the polypeptide intercalated between the layers. We present a comprehensive phase diagram on this system based on X-ray diffraction data. This work is supported in part by grants NSF DMR-9624091, PRF-31352 AC7, and CU LAR STP/UC 96-118.

  4. Compositions and methods for making selenocysteine containing polypeptides

    DOEpatents

    Soll, Dieter; Aldag, Caroline; Hohn, Michael

    2016-10-11

    Non-naturally occurring tRNA.sup.Sec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNA.sup.Sec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNA.sup.Sec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNA.sup.Sec.

  5. The geometry of the ribosomal polypeptide exit tunnel.

    PubMed

    Voss, N R; Gerstein, M; Steitz, T A; Moore, P B

    2006-07-21

    The geometry of the polypeptide exit tunnel has been determined using the crystal structure of the large ribosomal subunit from Haloarcula marismortui. The tunnel is a component of a much larger, interconnected system of channels accessible to solvent that permeates the subunit and is connected to the exterior at many points. Since water and other small molecules can diffuse into and out of the tunnel along many different trajectories, the large subunit cannot be part of the seal that keeps ions from passing through the ribosome-translocon complex. The structure referred to as the tunnel is the only passage in the solvent channel system that is both large enough to accommodate nascent peptides, and that traverses the particle. For objects of that size, it is effectively an unbranched tube connecting the peptidyl transferase center of the large subunit and the site where nascent peptides emerge. At no point is the tunnel big enough to accommodate folded polypeptides larger than alpha-helices.

  6. Imparting large macroscopic changes with small changes in polypeptide composition

    NASA Astrophysics Data System (ADS)

    Sing, Michelle; McKinley, Gareth; Olsen, Bradley

    Block copolymers composed of polypeptides provide an excellent platform for exploring the underlying physics surrounding macroscopic associative network behavior. Previous work in our group has elucidated a difference in the mechanical properties of two nearly identical elastin-like polypeptide (ELP) endblocks. In poly(ELP)s, this substitution is known to result in tighter beta turns. These beta turns exhibit slower responses to changes in temperature within the material. Under shear, the modulus for the alanine-containing ELP triblock is almost three times higher than the glycine-containing ELP. Additionally, preliminary tensile tests show higher stress and strain at break for the alanine ELP triblock. We are able to explain the reasons for this behavior using a variety of spectroscopic and analytical techniques. Small angle neutron and x-ray scattering indicate differences in ordering between the alanine and glycine containing ELP materials both in shear and in stagnant flow.

  7. Interplay between Folding and Assembly of Fibril-Forming Polypeptides

    NASA Astrophysics Data System (ADS)

    Ni, Ran; Abeln, Sanne; Schor, Marieke; Cohen Stuart, Martien A.; Bolhuis, Peter G.

    2013-08-01

    Polypeptides can self-assemble into hierarchically organized fibrils consisting of a stack of individually folded polypeptides driven together by hydrophobic interaction. Using a coarse-grained model, we systematically studied this self-assembly as a function of temperature and hydrophobicity of the residues on the outside of the building block. We find the self-assembly can occur via two different pathways—a random aggregation-folding route and a templated-folding process—thus indicating a strong coupling between folding and assembly. The simulation results can explain experimental evidence that assembly through stacking of folded building blocks is rarely observed, at the experimental concentrations. The model thus provides a generic picture of hierarchical fibril formation.

  8. Side-chain and backbone ordering in a polypeptide.

    PubMed

    Wei, Yanjie; Nadler, Walter; Hansmann, Ulrich H E

    2006-10-28

    We report results from multicanonical simulations of polyglutamic acid chains of length of ten residues. For this simple polypeptide we observe a decoupling of backbone and side-chain ordering in the folding process. While the details of the two transitions vary between the peptide in gas phase and in an implicit solvent, our results indicate that, independent of the specific surroundings, upon continuously lowering the temperature side-chain ordering occurs only after the backbone topology is completely formed.

  9. An engineered coiled-coil polypeptide assembled onto quantum dots for targeted cell imaging

    NASA Astrophysics Data System (ADS)

    Yao, Ming-Hao; Yang, Jie; Song, Ji-Tao; Zhang, Lin; Fang, Bi-Yun; Zhao, Dong-Hui; Xia, Rui-Xue; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2015-12-01

    Quantum dot (QD)-polypeptide probes have been developed through the specific metal-affinity interaction between polypeptides appended with N-terminal polyhistidine sequences and CdSe/ZnS core-shell QDs. The size and charge of a QD-polypeptide can be tuned by using different coiled-coil polypeptides. Compared to glutathione-capped QDs (QD-GSH), QD-polypeptide probes showed an approximately two- to three-fold luminescence increase, and the luminescence increase was not obviously related to the charge of the polypeptide. QD-polypeptide probes with different charge have a great effect on nonspecific cellular uptake. QD-polypeptide probes with negative charge exhibited lower nonspecific cellular uptake in comparison to the QD-GSH, while positively charged QD-polypeptide probes presented higher cellular uptake than the QD-GSH. A targeted QD-ARGD probe can obviously increase targeted cellular uptake in α v β 3 overexpressing HeLa cells compared to QD-A. In addition, QD-polypeptide probes showed lower in vitro cytotoxicity compared to the original QDs. These results demonstrate that these QD-polypeptide probes with high specific cellular uptake, high fluorescence intensity and low background noise are expected to have great potential applications in targeted cell imaging.

  10. Birth, life and death of nascent polypeptide chains

    PubMed Central

    Jha, Sujata; Komar, Anton A

    2011-01-01

    The journey of nascent polypeptides from synthesis at the peptidyl transferase center of the ribosome (“birth”) to full function (“maturity”) involves multiple interactions, constraints, modifications and folding events. Each step of this journey impacts the ultimate expression level and functional capacity of the translated protein. It has become clear that the kinetics of protein translation is predominantly modulated by synonymous codon usage along the mRNA, and that this provides an active mechanism for coordinating the synthesis, maturation and folding of nascent polypeptides. Multiple quality control systems ensure that proteins achieve their native, functional form. Unproductive co-translational folding intermediates that arise during protein synthesis may undergo enhanced interaction with components of these systems, such as chaperones, and/or be subjects of co-translational degradation (“death”). This review provides an overview of our current understanding of the complex co-translational events that accompany the synthesis, maturation, folding and degradation of nascent polypeptide chains. PMID:21538896

  11. Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

    PubMed

    Walker, Candace Rae; Pushpavanam, Karthik; Nair, Divya Geetha; Potta, Thrimoorthy; Sutiyoso, Caesario; Kodibagkar, Vikram D; Sapareto, Stephen; Chang, John; Rege, Kaushal

    2013-08-13

    Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

  12. Chirality-mediated polypeptide micelles for regulated drug delivery.

    PubMed

    Ding, Jianxun; Li, Chen; Zhang, Ying; Xu, Weiguo; Wang, Jincheng; Chen, Xuesi

    2015-01-01

    Two kinds of triblock poly(ethylene glycol)-polyleucine (PEG-PLeu) copolymers were synthesized through the ring-opening polymerization of L-Leu N-carboxyanhydride (NCA), or equivalent D-Leu NCA and L-Leu NCA with amino-terminated PEG as a macroinitiator. The amphiphilic copolymers spontaneously self-assembled into spherical micellar aggregations in an aqueous environment. The micelle with a racemic polypeptide core exhibited smaller critical micelle concentration and diameter compared to those with a levorotatory polypeptide core. A model anthracycline antineoplastic agent, i.e., doxorubicin (DOX), was loaded into micelles through nanoprecipitation, and the PEG-P(D,L-Leu) micelle exhibited higher drug-loading efficacy than that with a P(L-Leu) core-this difference was attributed to the flexible and compact P(L-Leu) core. Sustained in vitro DOX release from micelles with both levorotatory and racemic polypeptide cores was observed, and the DOX-loaded PEG-P(D,L-Leu) micelle exhibited a slower release rate. More interestingly, DOX-loaded micelles exhibited chirality-mediated antitumor efficacy in vitro and in vivo, which are all better than that of free DOX. Furthermore, both enhanced tumor inhibition and excellent security in vivo were confirmed by histopathological or in situ cell apoptosis analyses. Therefore, DOX-loaded PEG-PLeu micelles appear to be an interesting nanoscale polymeric formulation for promising malignancy chemotherapy.

  13. Polypeptide Chirality Influences Multilayer Thin Film Growth and Structure

    NASA Astrophysics Data System (ADS)

    Bell, Zephra; Khadka, Dhan; Haynie, Donald

    2011-03-01

    Polypeptide multilayer thin films are being developed for a variety of applications.These include coatings for implant devices and systems for drug delivery in thebiomedical sciences, and optical coatings. Subsequent polymer adsorption steps involve polymers of opposite polarity. Here, the polymers were polypeptides. This project compared the consequences of changing polypeptide chirality on film growth and structure. The peptides were poly(L-glutamic acid), its right-handed counterpart, poly(D-glutamic acid), and poly(lysine-tyrosine). The first two are negatively charged at neutral pH, the third one is positively charged. Poly(lysine-tyrosine)/poly(L-glutamic acid) films and poly(lysine-tyrosine)/poly(D-glutamic acid) films werefabricated on 1 mm-thick quartz plates. In one experiment, films were grown to 34layers. The UV absorption spectrum was taken after each layer deposited to determinethe rate of polymer self-assembly. Separately, UV or visible wavelength spectra wereobtained for films stained with a dye cooled/heated in the range 4-65 °C. In anotherexperiment, a mixture of poly-L-glutamic acid and poly-D-glutamic acid was used as thepolyanion for film buildup. The data show that poly(lysine-tyrosine)/poly(L-glutamicacid) films built up at a higher rate than the corresponding right-handed films.

  14. Separation and nanoencapsulation of antitumor polypeptide from Spirulina platensis.

    PubMed

    Zhang, Bochao; Zhang, Xuewu

    2013-01-01

    Spirulina platensis is a multicellular edible blue-green alga with abundant proteins (∼ 60%). No report is available on the antitumor polypeptides from the whole proteins of S. platensis. In this study, for the first time, an antitumor polypeptide Y2 from trypsin digest of S. platensis proteins was obtained by using freeze-thawing plus ultrasonication extraction, hydrolysis with four enzymes (trypsin, alcalase, papain, and pepsin), and gel filtration chromatography. The results showed that the degree of hydrolysis can be ordered as: trypsin (38.5%) > alcalase (31.2%) > papain (27.8%) > pepsin (7.1%). For MCF-7 and HepG2 cells, at 250 µg/mL, the maximum inhibitory rate of Y2 was 97%, while standard drug 5-FU was 55 and 97%, respectively. Furthermore, the nanoencapsulation of Y2 with chitosan (CS) was also investigated. After nanoencapsulation, the maximum encapsulation efficiency and polypeptides contents are 49 and 15%, respectively; and the antitumor activity is basically not lost. These data demonstrated the potential of nanopolypeptides (Y2-CS) in food and pharmaceutical applications.

  15. Effects of Cationic Polypeptides on Thrombasthenic and Afibrinogenemic Blood Platelets

    PubMed Central

    White, James G.

    1972-01-01

    Cationic polypeptides are known to cause the conversion of fibrinogen to fibrin gel and the immediate aggregation of blood platelets. In the present study, the effects of the cationic polypeptides, polybrene and polylysine, on normal, afibrinogenemic and thrombasthenic platelets were examined by nephelometry and electron microscopy. Normal and afibrinogenemic platelets aggregated rapidly and irreversibly after exposure to either agent. Thus, fibrinogen was not an essential factor for the interaction, though it may facilitate the rate of clumping produced by polybrene or polylysine. Thrombasthenic platelets did not aggregate rapidly or irreversibly in the presence of either agent. Shape change and slight degrees of clumping were induced by the cationic substances, a response essentially identical to the reaction of thrombasthenic platelets to collagen. The failure of cationic polypeptides to cause immediate aggregation of thrombasthenic platelets was not due to a failure of the agents to interact with the abnormal cells. Electron microscopy revealed that normal, afibrinogenemic and thrombasthenic platelets all adsorbed polybrene and polylysine, and transferred the agents to intracellular organelles. ImagesFig 3Fig 4Fig 1Fig 2 PMID:5054252

  16. Polypeptide synthesis induced in Nicotiana clevelandii protoplasts by infection with raspberry ringspot nepovirus.

    PubMed

    Acosta, O; Mayo, M A

    1993-01-01

    Infection of Nicotiana clevelandii protoplasts by raspberry ringspot nepovirus resulted in the accumulation of about 24 polypeptides that differed in M(r) and pI from polypeptides accumulating in mock-inoculated protoplasts. Similar polypeptides accumulated in protoplasts infected with the S and E strains of RRV but different infection-specific polypeptides were detected in protoplasts infected with tobacco ringspot nepovirus. The M(r) of RRV-specific polypeptides ranged from 210,000 to 18,000 and most are presumed to be derived from others by proteolytic cleavage. No evidence was found for marked changes in polypeptide abundance with time after inoculation or for any virus-specific polypeptide becoming disproportionately abundant in the medium during culture.

  17. Honeycomb patterned surfaces functionalized with polypeptide sequences for recognition and selective bacterial adhesion.

    PubMed

    Sanz de León, Alberto; Rodríguez-Hernández, Juan; Cortajarena, Aitziber L

    2013-02-01

    We report on the preparation of functional polymer surfaces with controlled topography by using the breath figures approach. The resulting surfaces prepared from a mixture of a PS-b-PAA diblock copolymer and a homopolymer (PS) exhibit pores that are mainly composed of diblock copolymer whereas the rest of the surface is formed by homopolymer. The formation of a hexagonal assembly of pores was achieved by controlling several parameters during the casting process including relative humidity, composition of the blend and polymer concentration. A selective modification of the pore inner part by using appropriate polypeptide sequences permitted the use of these surfaces as scaffolds for pattern and display of active biomolecules, as ordered templates for specific recognition processes and finally for the micropatterning of bacterial cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Precursor Polypeptides to Structural Proteins of Visna Virus

    PubMed Central

    Vigne, Robert; Filippi, Pierre; Quérat, Gilles; Sauze, Nicole; Vitu, Christian; Russo, Pierre; Delori, Pierre

    1982-01-01

    Visna virus is a retrovirus which replicates in fibroblast-like cells of the sheep choroid plexus through a lytic cycle. Visna virions contain three major low-molecular-weight proteins (p30, p16, and p14) which, together with the genomic RNA and several molecules of reverse transcriptase, constitute the core structure of the virions. The core is surrounded by an envelope containing a major glycoprotein (gp135). By analogy with the oncoviruses, these three groups of structural proteins (i.e., the internal proteins, the envelope glycoprotein, and the reverse transcriptase) are probably encoded by the gag, env, and pol genes, respectively. To elucidate the genetic organization of the visna virus genome and its expression, we studied the synthesis of viral proteins in infected sheep choroid plexus cells. Intracellular viral proteins were detected by immunoprecipitation of pulse-labeled cell extracts with monospecific sera raised against p30, p16, and gp135 and resolution of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoprecipitation with anti-p30 and anti-p16 sera allowed the characterization of the 55,000-dalton polypeptide precursor to internal virion proteins p30, p16, and p14 (Pr55gag). Tryptic peptide mapping confirmed the precursor-product relationship between Pr55gag and the three internal proteins. In addition, a gag-related polypeptide of 150,000 daltons was also detected. This polypeptide, which was less abundant than Pr55gag, is a likely precursor to the viral reverse transcriptase (Pr150gag-pol). Pr55gag and Pr150gag-pol are not glycosylated. The precursor related to major envelope protein gp135 is a glycosylated polypeptide with an average molecular weight of 150,000 (gPr150env). Pulse-chase experiments indicated that gPr150env matures into glycoprotein gp135 intracellularly; however, gp135 was never preponderant in cell extracts. The non-glycosylated from of gPr150env, which accumulated in the presence of 2-deoxy

  19. Macromolecular Instabilities and Dynamical Coding in Brain Enzymes, Polypeptide Ligands, Polypeptide and Cholinergic Receptors, and Sodium and Cholinergic Channel Proteins

    DTIC Science & Technology

    1988-03-01

    digital computers using dynamical equations (including those of the reduced Hodgkin - Huxley variety) as well as data-analytic tools which ha-e been and are...behavior of an enzyme, a receptor, a neuron , the heart, the EEG, and even hydrostatic pressure in the kidney is a sign of loss of regulatory potential ...hydrophobic minima. Certain polypeptide families am examirud in this context and the actions of a group of equally potent corticotrophic releasing facrs

  20. Linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus. I. Isolation and characterization of phycobiliprotein-linker-polypeptide complexes.

    PubMed

    Füglistaller, P; Suter, F; Zuber, H

    1986-07-01

    Phycobilisomes from the cyanobacterium Mastigocladus laminosus cultured in white and red light were isolated and compared with respect to the phycoerythrocyanin (PEC) and linker polypeptide contents. It was verified that the production of PEC is induced by low light intensities. A PEC complex, (alpha PEC beta PEC)6LR34.5,PEC, and a phycocyanin (PC) complex, (alpha PC beta PC)6LR34.5,PC, were isolated from phycobilisomes by Cellex-D anion exchange chromatography and sucrose density gradient centrifugation. The absorption and fluorescence emission maxima of the PEC complex are at 575 and 620 nm and those of the PC complex are at 631 and 647 nm, respectively. The extinction coefficients of the two complexes were determined. From different experiments it was concluded that PEC is present as a hexameric complex, (alpha PEC beta PEC)6LR34.5,PEC, in the phycobilisome. The two linker polypeptides LR34.5,PEC and LR34.5,PC were isolated from their phycobiliprotein complexes by gel filtration on Bio-Gel P-100 in 50% formic acid. A 5-kDa terminal segment of both linker polypeptides was found to influence the hexamer formation of the phycobiliproteins. The same segments have been described to be responsible for the hexamer-hexamer linkage (Yu, M.-H. & Glazer, A.N. (1982) J. Biol. Chem. 257, 3429-3433). A 8.9-kDa linker polypeptide, LR(C)8.9, was isolated from a PEC fraction of the Cellex-D column by Bio-Gel P-100 gel filtration in 50% formic acid. Localisation of this protein within the phycobilisome was attempted. Its most probable function is to terminate the phycobilisomal rods at the end distal to the allophycocyanin core.

  1. Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes.

    PubMed Central

    Bassuk, J A; Tsichlis, P N; Sorof, S

    1987-01-01

    Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). We report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambda gt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO4 gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. Their pI values overlapped in 2-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and showed the same response to delipidation. Either polypeptide reacted with and blocked the antiserum raised against the other polypeptide. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens. Images PMID:3478711

  2. Self-association and modification of a genetically engineered polypeptide

    NASA Astrophysics Data System (ADS)

    Top, Ayben

    A genetically synthesized polypeptide and polyethylene glycol (5 kDa or 10 kDa) functionalized forms of its alanine-rich helical domain were characterized. The polypeptide composed of an N-terminal histidine tag, and an alanine-rich domain, denoted as 17H6, has a sequence of: MGH10 SSGHIHM(AAAQEAAAAQAAAQAEAAQAAQ)6AGGYGGMG. 17H6 was originally designed as a scaffold to investigate multivalent interactions after glycosylation through reactive glutamic acid residues. We speculated that the protonation of the glutamic acid residues in these sequences would afford facile opportunities to manipulate their folding and assembly behavior considering the beta-sheet propensities of similar polypeptides at acidic pH. Thus, in the first part of this study, thermal unfolding, reversible self-association, and irreversible aggregation of 17H6 were investigated. Dynamic light scattering, and thermal unfolding measurements indicate that 17H6 spontaneously and reversibly self-associates at an acidic pH and ambient temperature. The resulting multimers have an average hydrodynamic radius of ˜ 10-20 nm and reversibly dissociate to monomers upon an increase to pH 7.4. Both free monomer and 17H6 chains within the multimers are beta-helical and folded at ambient and sub-ambient temperatures. Reversible unfolding of the monomer occurs upon heating of solutions at pH 7.4. At pH 2.3, heating first causes incomplete dissociation and unfolding of the constituent chains. Further incubation at an elevated temperature (80°C) induces additional structural and morphological changes and results in fibrils with a beta-sheet structure and a width of 5-10 nm (7 nm mean) as observed via transmission electron microscopy (TEM). In the second part, the histidine tag, which imparts solubility to the alanine-rich domain at acidic pH was cleaved. Propionaldehyde-functionalized poly(ethylene glycol) (PEG) molecules (5 kDa or 10 kDa) were attached to the N-terminus of the cleaved polypeptide, c17H6, as a

  3. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    SciTech Connect

    Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1982-07-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH/sub 2/-coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X.

  4. Switching of filamin polypeptides during myogenesis in vitro

    PubMed Central

    1983-01-01

    During chicken skeletal myogenesis in vitro, the actin-binding protein filamin is present at first in association with actin filament bundles both in myoblasts and in myotubes early after fusion. Later in mature myotubes it is found in association with myofibril Z disks. These two associations of filamin are separated by a period of several days, during which the protein is absent from the cytoplasm of differentiating myotubes (Gomer, R., and E. Lazarides, 1981, Cell, 23:524-532). To characterize the two classes of filamin polypeptides we have compared, by two-dimensional peptide mapping, 125I-labeled filamin immunoprecipitated from myoblasts and fibroblasts to filamin immunoprecipitated from mature myotubes and adult skeletal myofibrils. Myoblast filamin is highly homologous to fibroblast and purified chicken gizzard filamins. Mature myotube and adult myofibril filamins are highly homologous but exhibit extensive peptide differences with respect to the other three classes of filamin. Comparison of peptide maps from immunoprecipitated 35S-methionine-labeled filamins also shows that fibroblast and myoblast filamins are highly homologous but show substantial peptide differences with respect to mature myotube filamin. Filamins from both mature myotubes and skeletal myofibrils exhibit a slightly higher electrophoretic mobility than gizzard, fibroblast, and myoblast filamins. Short pulse-labeling studies show that mature myotube filamin is synthesized as a lower molecular weight variant and is not derived from a higher molecular weight precursor. These results suggest that myoblast and mature myotube filamins are distinct gene products and that during skeletal myogenesis in vitro one class of filamin polypeptides is replaced by a new class of filamin polypeptides, and that the latter is maintained into adulthood. PMID:6833359

  5. Residue length and solvation model dependency of elastinlike polypeptides.

    PubMed

    Bilsel, Mustafa; Arkin, Handan

    2010-05-01

    We have performed exhaustive multicanonical Monte Carlo simulations of elastinlike polypeptides with a chain including amino acids (valine-proline-glycine-valine-glycine)n or in short (VPGVG)n, where n changes from 1 to 4, in order to investigate the thermodynamic and structural properties. To predict the characteristic secondary structure motifs of the molecules, Ramachandran plots were prepared and analyzed as well. In these studies, we utilized a realistic model where the interactions between all types of atoms were taken into account. Effects of solvation were also simulated by using an implicit-solvent model with two commonly used solvation parameter sets and compared with the vacuum case.

  6. Residue length and solvation model dependency of elastinlike polypeptides

    NASA Astrophysics Data System (ADS)

    Bilsel, Mustafa; Arkin, Handan

    2010-05-01

    We have performed exhaustive multicanonical Monte Carlo simulations of elastinlike polypeptides with a chain including amino acids (valine-proline-glycine-valine-glycine)n or in short (VPGVG)n , where n changes from 1 to 4, in order to investigate the thermodynamic and structural properties. To predict the characteristic secondary structure motifs of the molecules, Ramachandran plots were prepared and analyzed as well. In these studies, we utilized a realistic model where the interactions between all types of atoms were taken into account. Effects of solvation were also simulated by using an implicit-solvent model with two commonly used solvation parameter sets and compared with the vacuum case.

  7. Factors Governing Fibrillogenesis of Polypeptide Chains Revealed by Lattice Models

    NASA Astrophysics Data System (ADS)

    Li, Mai Suan; Co, Nguyen Truong; Reddy, Govardhan; Hu, Chin-Kun; Straub, J. E.; Thirumalai, D.

    2010-11-01

    Using lattice models we explore the factors that determine the tendencies of polypeptide chains to aggregate by exhaustively sampling the sequence and conformational space. The morphologies of the fibril-like structures and the time scales (τfib) for their formation depend on a balance between hydrophobic and Coulomb interactions. The extent of population of an ensemble of N* structures, which are fibril-prone structures in the spectrum of conformations of an isolated protein, is the major determinant of τfib. This observation is used to determine the aggregating sequences by exhaustively exploring the sequence space, thus providing a basis for genome wide search of fragments that are aggregation prone.

  8. Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins

    SciTech Connect

    Garrison, W.M.

    1985-01-01

    The purpose of this review is to bring together and to correlate the wide variety of experimental studies that provide information on the reaction products and reaction mechanisms involved in the radiolysis of peptides, polypeptides and proteins (including chromosomal proteins) in both aqueous and solid-state systems. The comparative radiation chemistry of these systems is developed in terms of specific reactions of the peptide main-chain and the aliphatic, aromatic-unsaturated and sulfur-containing side-chains. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis and ESR spectroscopy is included. 147 refs.

  9. Polypeptide having or assisting in carbohydrate material degrading activity and uses thereof

    DOEpatents

    Schooneveld-Bergmans, Margot Elisabeth Francoise; Heijne, Wilbert Herman Marie; Los, Alrik Pieter

    2016-02-16

    The invention relates to a polypeptide which comprises the amino acid sequence set out in SEQ ID NO: 2 or an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 1, or a variant polypeptide or variant polynucleotide thereof, wherein the variant polypeptide has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2 or the variant polynucleotide encodes a polypeptide that has at least 76% sequence identity with the sequence set out in SEQ ID NO: 2. The invention features the full length coding sequence of the novel gene as well as the amino acid sequence of the full-length functional polypeptide and functional equivalents of the gene or the amino acid sequence. The invention also relates to methods for using the polypeptide in industrial processes. Also included in the invention are cells transformed with a polynucleotide according to the invention suitable for producing these proteins.

  10. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  11. Mechanism of time-dependent inhibition of polypeptide deformylase by actinonin.

    PubMed

    Van Aller, Glenn S; Nandigama, Ravi; Petit, Chantal M; DeWolf, Walt E; Quinn, Chad J; Aubart, Kelly M; Zalacain, Magdalena; Christensen, Siegfried B; Copeland, Robert A; Lai, Zhihong

    2005-01-11

    Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*

  12. Compositions comprising a polypeptide having cellulolytic enhancing activity and a quinone compound and uses thereof

    DOEpatents

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2016-03-01

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a quinone compound. The present invention also relates to methods of using the compositions.

  13. Compositions comprising a polypeptide having cellulolytic enhancing activity and a heterocyclic compound and uses thereof

    DOEpatents

    Xu, Feng; Sweeney, Matthew; Quinlan, Jason

    2016-08-02

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a heterocyclic compound. The present invention also relates to methods of using the compositions.

  14. Compositions comprising a polypeptide having cellulolytic enhancing activity and a dioxy compound and uses thereof

    DOEpatents

    Sweeney, Matthew; Xu, Feng; Quinlan, Jason

    2016-07-19

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a dioxy compound. The present invention also relates to methods of using the compositions.

  15. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicycle compound and uses thereof

    DOEpatents

    Xu, Feng; Sweeney, Matthew; Quinlan, Jason

    2015-06-16

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a bicyclic compound. The present invention also relates to methods of using the compositions.

  16. Self-assembly of polypeptide-based copolymers into diverse aggregates.

    PubMed

    Cai, Chunhua; Wang, Liquan; Lin, Jiaping

    2011-10-28

    Recently, increasing attention has been given to the self-assembly behavior of polypeptide-based copolymers. Polypeptides can serve as either shell-forming or core-forming blocks in the formation of various aggregates. The solubility and rigidity of polypeptide blocks have been found to have a profound effect on the self-assembly behavior of polypeptide-based copolymers. Polypeptide graft copolymers combine the advantages of a grafting strategy and the characteristics of polypeptide chains and their self-assembly behavior can be easily adjusted by choosing different polymer chains and copolymer architectures. Fabricating hierarchical structures is one of the attractive topics of self-assembly research of polypeptide copolymers. These hierarchical structures are promising for use in preparing functional materials and, thus, attract increasing attention. Computer simulations have emerged as powerful tools to investigate the self-assembly behavior of polymers, such as polypeptides. These simulations not only support the experimental results, but also provide information that cannot be directly obtained from experiments. In this feature article, recent advances in both experimental and simulation studies for the self-assembly behavior of polypeptide-based copolymers are reviewed.

  17. Self-assembly of high molecular weight polypeptide copolymers studied via diffusion limited aggregation.

    PubMed

    Meier, Christoph; Wu, Yuzhou; Pramanik, Goutam; Weil, Tanja

    2014-01-13

    The assembly of high molecular weight polypeptides into complex architectures exhibiting structural complexity ranging from the nano- to the mesoscale is of fundamental importance for various protein-related diseases but also hold great promise for various nano- and biotechnological applications. Here, the aggregation of partially unfolded high molecular weight polypeptides into multiscale fractal structures is investigated by means of diffusion limited aggregation and atomic force microscopy. The zeta potential, the hydrodynamic radius, and the obtained fractal morphologies were correlated with the conformation of the polypeptide backbones as obtained from circular dichroism measurements. The polypeptides are modified with polyethylene oxide side chains to stabilize the polypeptides and to normalize intermolecular interactions. The modification with the hydrophobic thioctic acid alters the folding of the polypeptide backbone, resulting in a change in solution aggregation and fractal morphology. We found that a more compact folding results in dense and highly branched structures, whereas a less compact folded polypeptide chain yields a more directional assembly. Our results provide first evidence for the role of compactness of polypeptide folding on aggregation. Furthermore, the mesoscale-structured biofilms were used to achieve a hierarchical protein assembly, which is demonstrated by deposition of Rhodamine-labeled HSA with the preassembled fractal structures. These results contribute important insights to the fundamental understanding of the aggregation of high molecular weight polypeptides in general and provide opportunities to study nanostructure-related effects on biological systems such as adhesion, proliferation, and the development of, for example, neuronal cells.

  18. Compositions comprising a polypeptide having cellulolytic enhancing activity and a bicyclic compound and uses thereof

    SciTech Connect

    Quinlan, Jason; Xu, Feng; Sweeney, Matthew

    2016-10-04

    The present invention relates to compositions comprising: a polypeptide having cellulolytic enhancing activity and a bicyclic compound. The present invention also relates to methods of using the compositions.

  19. A Dynamic Poisson-Boltzmann Method of Simulating Polypeptides

    NASA Astrophysics Data System (ADS)

    Campbell, Victoria S.; Grayce, Christopher J.

    1998-03-01

    We present a method of performing molecular dynamics simulations of charged polymeric species in solution such as polypeptides that takes into account the instantaneous response of the ionic atmosphere to fluctuations in polymer conformation without employing explicit solvent and salt ions. Using density functional theory we write the free energy of the ionic atmosphere around the polymer as a functional of its density in the linearized Poisson-Boltzmann limit. We then add to a normal MD simulation of a charged polymer extra degrees of freedom, namely the parameters describing the instantaneous ion atomosphere density. These parameters vary dynamically under the influence of the coupled mechanical and thermodynamic forces, so that the instantaneous variations in the ionic atmosphere as the polymer conformation fluctuates are described. Using this method MD simulations were carried out on a model polypeptide system and both conformational properties as well as the electric field generated by this method were compared to results obtained by using fixed Debye-Huckel potentials.

  20. Reverse transcriptase activity of an intron encoded polypeptide.

    PubMed Central

    Fassbender, S; Brühl, K H; Ciriacy, M; Kück, U

    1994-01-01

    A number of group II introns from eukaryotic organelles and prokaryotes contain open reading frames for polypeptides with homology to retroviral reverse transcriptases (RTs). We have used the yeast transposon (Ty) system to express ORFs for RTs from eukaryotic organelles. This includes the mitochondrial coxI intron i1 from the fungus Podospora anserina, the plastid petD intron from the alga Scenedesmus obliquus and the mitochondrial RTL gene from the alga Chlamydomonas reinhardtii. The ORFs were fused with the TYA ORF from the yeast retrotransposon Ty to produce virus-like particles in the recipient strains with detectable amounts of the RT-like polypeptides. Analysis of the heterologous gene products revealed biochemical evidence that the P. anserina intron encodes an RNA-directed DNA polymerase with properties typically found for RTs of viral or retrotransposable origin. In vitro assays showed that the intron encoded RT is sensitive to RT inhibitors such as N-ethylmaleimide and dideoxythymidine triphosphate but is insensitive against the DNA polymerase inhibitor aphidicolin. The direct biochemical evidence provided here supports the idea that intron encoded RTs are involved in intron transposition events. Images PMID:7514530

  1. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 12 figs.

  2. Effects of snake venom polypeptides on central nervous system.

    PubMed

    Osipov, Alexey; Utkin, Yuri

    2012-12-01

    The nervous system is a primary target for animal venoms as the impairment of its function results in the fast and efficient immobilization or death of a prey. There are numerous evidences about effects of crude snake venoms or isolated toxins on peripheral nervous system. However, the data on their interactions with the central nervous system (CNS) are not abundant, as the blood-brain barrier (BBB) impedes penetration of these compounds into brain. This updated review presents the data about interaction of snake venom polypeptides with CNS. Such data will be described according to three main modes of interactions: - Direct in vivo interaction of CNS with venom polypeptides either capable to penetrate BBB or injected into the brain. - In vitro interactions of cell or sub-cellular fractions of CNS with crude venoms or purified toxins. - Indirect effects of snake venoms or their components on functioning of CNS under different conditions. Although the venom components penetrating BBB are not numerous, they seem to be the most suitable candidates for the leads in drug design. The compounds with other modes of action are more abundant and better studied, but the lack of the data about their ability to penetrate BBB may substantially aggravate the potentials for their medical perspectives. Nevertheless, many such compounds are used for research of CNS in vitro. These investigations may give invaluable information for understanding the molecular basis of CNS diseases and thus lay the basis for targeted drug design. This aspect also will be outlined in the review.

  3. Structural analysis of photosystem I polypeptides using chemical crosslinking

    NASA Technical Reports Server (NTRS)

    Armbrust, T. S.; Odom, W. R.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Thylakoid membranes, obtained from leaves of 14 d soybean (Glycine max L. cv. Williams) plants, were treated with the chemical crosslinkers glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to investigate the structural organization of photosystem I. Polypeptides were resolved using lithium dodecyl sulfate polyacrylamide gel electrophoresis, and were identified by western blot analysis using a library of polyclonal antibodies specific for photosystem I subunits. An electrophoretic examination of crosslinked thylakoids revealed numerous crosslinked products, using either glutaraldehyde or EDC. However, only a few of these could be identified by western blot analysis using subunit-specific polyclonal antibodies. Several glutaraldehyde dependent crosslinked species were identified. A single band was identified minimally composed of PsaC and PsaD, documenting the close interaction between these two subunits. The most interesting aspect of these studies was a crosslinked species composed of the PsaB subunit observed following EDC treatment of thylakoids. This is either an internally crosslinked species, which will provide structural information concerning the topology of the complex PsaB protein, a linkage with a polypeptide for which we do not yet have an immunological probe, or a masking of epitopes by the EDC linkage at critical locations in the peptide which is linked to PsaB.

  4. Prediction of the absolute aggregation rates of amyloidogenic polypeptide chains.

    PubMed

    DuBay, Kateri F; Pawar, Amol P; Chiti, Fabrizio; Zurdo, Jesús; Dobson, Christopher M; Vendruscolo, Michele

    2004-08-27

    Protein aggregation is associated with a variety of pathological conditions, including Alzheimer's and Creutzfeldt-Jakob diseases and type II diabetes. Such degenerative disorders result from the conversion of the normal soluble state of specific proteins into aggregated states that can ultimately form the characteristic amyloid fibrils found in diseased tissue. Under appropriate conditions it appears that many, perhaps all, proteins can be converted in vitro into amyloid fibrils. The aggregation propensities of different polypeptide chains have, however, been observed to vary substantially. Here, we describe an approach that uses the knowledge of the amino acid sequence and of the experimental conditions to reproduce, with a correlation coefficient of 0.92 and over five orders of magnitude, the in vitro aggregation rates of a wide range of unstructured peptides and proteins. These results indicate that the formation of protein aggregates can be rationalised to a considerable extent in terms of simple physico-chemical parameters that describe the properties of polypeptide chains and their environment.

  5. Characterization of dopuin, a polypeptide with special residue distributions.

    PubMed

    Chen, Z W; Bergman, T; Ostenson, C G; Efendic, S; Mutt, V; Jörnvall, H

    1997-10-15

    A 62-residue polypeptide, dopuin, has been isolated from pig small intestine. It is distinguished by an N-terminal part with a high content of proline (7 in a 26-residue segment), a C-terminal part with a high proportion of histidine (3 in a 9-residue segment), and six half-cystine residues in three intrachain disulphide bridges (connecting positions 22-25, 23-54 and 35-44). The Cys and Pro distributions suggest a tight and special conformation. In contrast to PEC-60 and somatostatin, it has no established inhibitory effect on insulin secretion. At 10 nM concentration, a weak inhibitory tendency is less than half of that of the other two peptides. Like gastrointestinal trefoil peptides, dopuin has three disulphide bridges, Ala-Pro segments, and many charged residues, but they are differently distributed and dopuin belongs to a separate, apparently novel family. However, dopuin is similar to a peptide corresponding to an expressed-sequence-tag cDNA of human fetal liver and spleen, establishing the nature of the mature form of the product of this cDNA, and showing a general tissue, age, and species distribution of this peptide. A truncated form of vimentin, composed of its C-terminal 37 residues, vimentin-C37, was also purified and structurally characterized. These two peptides increase the complexity of known intestinal polypeptides and at least dopuin has properties compatible with specific biofunctions.

  6. CDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  7. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1999-05-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  8. Vibrational neutron spectroscopy of collagen and model polypeptides.

    PubMed Central

    Middendorf, H D; Hayward, R L; Parker, S F; Bradshaw, J; Miller, A

    1995-01-01

    A pulsed source neutron spectrometer has been used to measure vibrational spectra (20-4000 cm-1) of dry and hydrated type I collagen fibers, and of two model polypeptides, polyproline II and (prolyl-prolyl-glycine)10, at temperatures of 30 and 120 K. the collagen spectra provide the first high resolution neutron views of the proton-dominated modes of a protein over a wide energy range from the low frequency phonon region to the rich spectrum of localized high frequency modes. Several bands show a level of fine structure approaching that of optical data. The principal features of the spectra are assigned. A difference spectrum is obtained for protein associated water, which displays an acoustic peak similar to pure ice and a librational band shifted to lower frequency by the influence of the protein. Hydrogen-weighted densities of states are extracted for collagen and the model polypeptides, and compared with published calculations. Proton mean-square displacements are calculated from Debye-Waller factors measured in parallel quasi-elastic neutron-scattering experiments. Combined with the collagen density of states function, these yield an effective mass of 14.5 a.m.u. for the low frequency harmonic oscillators, indicating that the extended atom approximation, which simplifies analyses of low frequency protein dynamics, is appropriate. PMID:8527680

  9. Structural analysis of photosystem I polypeptides using chemical crosslinking.

    PubMed

    Armbrust, T S; Odom, W R; Guikema, J A

    1994-07-01

    Thylakoid membranes, obtained from leaves of 14 d soybean (Glycine max L. cv. Williams) plants, were treated with the chemical crosslinkers glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to investigate the structural organization of photosystem I. Polypeptides were resolved using lithium dodecyl sulfate polyacrylamide gel electrophoresis, and were identified by western blot analysis using a library of polyclonal antibodies specific for photosystem I subunits. An electrophoretic examination of crosslinked thylakoids revealed numerous crosslinked products, using either glutaraldehyde or EDC. However, only a few of these could be identified by western blot analysis using subunit-specific polyclonal antibodies. Several glutaraldehyde dependent crosslinked species were identified. A single band was identified minimally composed of PsaC and PsaD, documenting the close interaction between these two subunits. The most interesting aspect of these studies was a crosslinked species composed of the PsaB subunit observed following EDC treatment of thylakoids. This is either an internally crosslinked species, which will provide structural information concerning the topology of the complex PsaB protein, a linkage with a polypeptide for which we do not yet have an immunological probe, or a masking of epitopes by the EDC linkage at critical locations in the peptide which is linked to PsaB.

  10. Analysis of the delocalized Raman modes of conformationally disordered polypeptides.

    PubMed Central

    Chen, L X; Strauss, H L; Snyder, R G

    1993-01-01

    Bands associated with delocalized vibrational modes were identified in the isotropic Raman spectra of a series of polyglycine oligomers in aqueous solution as zwitterions and as cations. The dependence of these bands on conformational disorder and chain length was determined. The observed dependence is closely mimicked in spectra calculated for a series of corresponding model polypeptides. The simulated spectra were calculated in a skeletal approximation for ensembles of conformationally disordered chains. As the chain length of the conformationally disordered polypeptides increases, the observed isotropic spectra rapidly approach the spectrum of the infinitely long disordered chain. Convergence is nearly complete at the tripeptide for both the zwitterion and the cation. The stimulated spectra behave in essentially the same way. Convergence to the spectrum of the infinitely long chain is much more rapid for the conformationally disordered polyglycines than for the ordered polyglycines because of the mode localization that results from disorder. In the low-frequency region the bands in the calculated spectra have frequencies that are systematically dependent on chain length. These bands are related to the longitudinal acoustic modes of the ordered chain. PMID:8324189

  11. Membrane Permeation Induced by Aggregates of Human Islet Amyloid Polypeptides

    PubMed Central

    Poojari, Chetan; Xiao, Dequan; Batista, Victor S.; Strodel, Birgit

    2013-01-01

    Several neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases as well as nonneuropathic diseases such as type II diabetes and atrial amyloidosis are associated with aggregation of amyloid polypeptides into fibrillar structures, or plaques. In this study, we use molecular dynamics simulations to test the stability and orientation of membrane-embedded aggregates of the human islet amyloid polypeptide (hIAPP) implicated in type II diabetes. We find that in both monolayers and bilayers of dipalmitoylphosphatidylglycerol (DPPG) hIAPP trimers and tetramers remain inside the membranes and preserve their β-sheet secondary structure. Lipid bilayer-inserted hIAPP trimers and tetramers orient inside DPPG at 60° relative to the membrane/water interface and lead to water permeation and Na+ intrusion, consistent with ion-toxicity in islet β-cells. In particular, hIAPP trimers form a water-filled β-sandwich that induce water permeability comparable with channel-forming proteins, such as aquaporins and gramicidin-A. The predicted disruptive orientation is consistent with the amphiphilic properties of the hIAPP aggregates and could be probed by chiral sum frequency generation (SFG) spectroscopy, as predicted by the simulated SFG spectra. PMID:24268144

  12. cDNA encoding a polypeptide including a hevein sequence

    DOEpatents

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1,018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli. 11 figures.

  13. Elastin-like Polypeptides as Models of Intrinsically Disordered Proteins

    PubMed Central

    Roberts, Stefan; Dzuricky, Michael; Chilkoti, Ashutosh

    2015-01-01

    Elastin-like polypeptides (ELPs) are a class of stimuli-responsive biopolymers inspired by the intrinsically disordered domains of tropoelastin that are composed of repeats of the VPGXG pentapeptide motif, where X is a “guest residue”. They undergo a reversible, thermally triggered lower critical solution temperature (LCST) phase transition, which has been utilized for a variety of applications including protein purification, affinity capture, immunoassays, and drug delivery. ELPs have been extensively studied as protein polymers and as biomaterials, but their relationship to other disordered proteins has heretofore not been established. The biophysical properties of ELPs that lend them their unique material behavior are similar to the properties of many intrinsically disordered proteins (IDP). Their low sequence complexity, phase behavior, and elastic properties make them an interesting “minimal” artificial IDP, and the study of ELPs can hence provide insights into the behavior of other more complex IDPs. Motivated by this emerging realization of the similarities between ELPs and IDPs, this review discusses the biophysical properties of ELPs, their biomedical utility, and their relationship to other disordered polypeptide sequences. PMID:26325592

  14. Reversible Deficiency of Antimicrobial Polypeptides in Bacterial Vaginosis

    PubMed Central

    Valore, Erika V.; Wiley, Dorothy J.; Ganz, Tomas

    2006-01-01

    Bacterial vaginosis is a common condition associated with increased risk of sexually transmitted diseases, including human immunodeficiency virus infections. In contrast, vulvovaginal candidiasis has a much weaker association with sexually transmitted diseases. We found that vaginal lavage fluid from women with bacterial vaginosis is deficient in antimicrobial polypeptides and antimicrobial activity compared to fluid from healthy women or women with vulvovaginal candidiasis. Effective treatment normalized the concentrations of antimicrobial polypeptides in both bacterial vaginosis and in vulvovaginal candidiasis, suggesting that the abnormalities were a result of the diseases. Unlike in vulvovaginal candidiasis, the neutrophil attractant chemokine interleukin-8 (IL-8) was not increased in bacterial vaginosis, accounting for low concentrations of neutrophil-derived defensins in vaginal fluid. In organotypic cultures of human vaginal epithelium containing dendritic cells, treatment with Lactobacillus jensenii, a typical vaginal resident, induced the synthesis of IL-8 mRNA and the epithelial human β-defensin-2 mRNA, but a typical bacterial vaginosis pathogen, Gardnerella vaginalis, had no effect. When the two bacteria were combined, Gardnerella vaginalis did not interfere with the immunostimulatory effect of Lactobacillus jensenii. The loss of normal immunostimulatory flora in bacterial vaginosis is thus associated with a local deficiency of multiple innate immune factors, and this deficiency could predispose individuals to sexually transmitted diseases. PMID:16988245

  15. Polypeptide profiles of South Indian isolate of Trypanosoma evansi.

    PubMed

    Sivajothi, S; Rayulu, V C; Bhaskar Reddy, B V; Malakondaiah, P; Sreenivasulu, D; Sudhakara Reddy, B

    2016-09-01

    The field isolates of Trypanosoma evansi was collected from the infected cattle and it was propagated in rats. Trypanosoma evansi parasites were separated from the blood of infected rats by using diethylaminoethyl cellulose column chromatography. Whole cell lysate antigen (WCL) was prepared from purified trypanosomes by ultrasonication and centrifugation. The prepared WCL antigen was further purified by 50 % ammonium sulphate precipitation. Protein concentration of WCL antigen of T. evansi was 60 mg/ml. Protein concentration was adjusted to 1.0 mg/ml in PBS, pH 8.0 and stored at -20(0) C.   Polypeptide profiles of WCL antigen of T. evansi was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A total of eight polypeptide bands of the size ranging from 25 to 85 kDa in WCL antigen of T. evansi were obtained. Five prominent bands with molecular weight of 74, 60, 53, 42 and 37 kDa and three light bands with molecular weight of 85, 34 and 25 kDa were observed.

  16. Synthesis and incorporation of myelin polypeptides into CNS myelin

    PubMed Central

    1982-01-01

    The distribution of newly synthesized proteolipid protein (PLP, 23 kdaltons) and myelin basic proteins (MBPs, 14-21.5 kdaltons) was determined in microsomal and myelin fractions prepared from the brainstems o1 10-30 d-old rats sacrificed at different times after an intracranial injection of 35S-methionine. Labeled MBPs were found in the myelin fraction 2 min after the injection, whereas PLP appeared first in the rough microsomal fraction and only after a lag of 30 min in the myelin fraction. Cell-free translation experiments using purified mRNAs demonstrated that PLP and MBPs are synthesized in bound and free polysomes, respectively. A mechanism involving the cotranslational insertion into the ER membrane and subsequent passage of the polypeptides through the Golgi apparatus is consistent with the lag observed in the appearance of the in vivo-labeled PLP in the myelin membrane. Newly synthesized PLP and MBPs are not proteolytically processed, because the primary translation products synthesized in vitro had the same electrophoretic mobility and N-terminal amino acid sequence as the mature PLP and MBP polypeptides. It was found that crude myelin fractions are highly enriched in mRNAs coding for the MBPs but not in mRNA coding for PLP. This suggests that whereas the bound polysomes synthesizing PLP are largely confined to the cell body, free polysomes synthesizing MBPs are concentrated in oligodendrocyte processes involved in myelination, which explains the immediate incorporation of MBPs into the developing myelin sheath. PMID:6183276

  17. Mechanically Controlled Electron Transfer in a Single-Polypeptide Transistor

    PubMed Central

    Sheu, Sheh-Yi; Yang, Dah-Yen

    2017-01-01

    Proteins are of interest in nano-bio electronic devices due to their versatile structures, exquisite functionality and specificity. However, quantum transport measurements produce conflicting results due to technical limitations whereby it is difficult to precisely determine molecular orientation, the nature of the moieties, the presence of the surroundings and the temperature; in such circumstances a better understanding of the protein electron transfer (ET) pathway and the mechanism remains a considerable challenge. Here, we report an approach to mechanically drive polypeptide flip-flop motion to achieve a logic gate with ON and OFF states during protein ET. We have calculated the transmission spectra of the peptide-based molecular junctions and observed the hallmarks of electrical current and conductance. The results indicate that peptide ET follows an NC asymmetric process and depends on the amino acid chirality and α-helical handedness. Electron transmission decreases as the number of water molecules increases, and the ET efficiency and its pathway depend on the type of water-bridged H-bonds. Our results provide a rational mechanism for peptide ET and new perspectives on polypeptides as potential candidates in logic nano devices. PMID:28051140

  18. Mechanically Controlled Electron Transfer in a Single-Polypeptide Transistor

    NASA Astrophysics Data System (ADS)

    Sheu, Sheh-Yi; Yang, Dah-Yen

    2017-01-01

    Proteins are of interest in nano-bio electronic devices due to their versatile structures, exquisite functionality and specificity. However, quantum transport measurements produce conflicting results due to technical limitations whereby it is difficult to precisely determine molecular orientation, the nature of the moieties, the presence of the surroundings and the temperature; in such circumstances a better understanding of the protein electron transfer (ET) pathway and the mechanism remains a considerable challenge. Here, we report an approach to mechanically drive polypeptide flip-flop motion to achieve a logic gate with ON and OFF states during protein ET. We have calculated the transmission spectra of the peptide-based molecular junctions and observed the hallmarks of electrical current and conductance. The results indicate that peptide ET follows an NC asymmetric process and depends on the amino acid chirality and α-helical handedness. Electron transmission decreases as the number of water molecules increases, and the ET efficiency and its pathway depend on the type of water-bridged H-bonds. Our results provide a rational mechanism for peptide ET and new perspectives on polypeptides as potential candidates in logic nano devices.

  19. Polypeptide Expression in Prostate Hyperplasia and Prostate Adenocarcinoma

    PubMed Central

    Alaiya, Ayodele; Roblick, Uwe; Egevad, Lars; Carlsson, Adelaide; Franzén, Bo; Volz, Daniela; Huwendiek, Sören; Linder, Stig; Auer, Gert

    2000-01-01

    Cells were collected from prostate hyperplasias (n=6) and prostate carcinomas (n=6) and subjected to two‐dimensional gel electrophoresis (2‐DE). The resulting polypeptide patterns were analysed with the PDQUEST computer software. Malignant tumors showed significant increases in the level of expression of proliferating cell nuclear antigen (PCNA), calreticulin, HSP 90 and pHSP 60, oncoprotein 18(v), elongation factor 2, glutathione‐S‐transferase π (GST‐π), superoxide dismutase and triose phosphate isomerase. In addition, decreases in the levels of tropomyosin‐1 and 2 and cytokeratin 18 were observed in prostate carcinomas compared to prostate hyperplasias. This pattern of alterations is similar to that observed in other carcinomas in our previous studies. All malignant tumors showed simultaneous alterations in 5 or more of 9 markers studied, whereas only one case of benign hyperplasia showed alterations in 5 markers. The EST‐data base for prostate tumors available from NCI (CGAP) was searched for the expression of the mRNAs corresponding to proteins identified in our gels. Large differences in the relative expression of mRNAs and proteins were observed. Our data show alterations in the pattern of polypeptide expression in prostate carcinomas which are similar to those observed in other carcinomas. PMID:11254220

  20. cDNA encoding a polypeptide including a hevein sequence

    SciTech Connect

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  1. Investigation of genetically-engineered beta-sheet polypeptides for nanoelectronics

    NASA Astrophysics Data System (ADS)

    Rana, Narender

    2007-12-01

    Ongoing miniaturization in integrated circuit (IC) device fabrication via conventional lithography faces increasing technical challenges and imposes significant performance limitations on devices and interconnects stemming from the fundamental physics of electron transport. This drives the need to explore other nanofabrication approaches, such as self-assembly, and alternate device or interconnect structures with novel electron transport mechanisms, such as ballistic electron transport. Molecular self-assembly, ubiquitous in biology and bio-inspired materials, might have tremendous potential for nanoelectronic applications. Specifically, genetically-engineered beta-sheet polypeptides offer certain key attributes for nanoelectronic applications. These attributes include: controllable self-assembly, potential to form one dimensional quantum channels for ballistic electron transport, and substrate-specific interactions for interfacial engineering. This dissertation explores and evaluates the nanowire self-assembly characteristics of several de novo genetically-engineered beta-sheet polypeptides (synthesized by our group) on various substrates for applications in nanoelectronic interconnect schemes. In addition, substrate-attachment of the beta-sheet polypeptide nanowire structures is investigated and preliminary electrical testing of a polypeptide nanowire fibril is presented. Chapters 1 and 2 provide an overall introduction and discuss the characterization techniques utilized in the experimental work. Chapter 3 describes a detailed self-assembly study of various polypeptides and documents the formulation and deposition of controlled, linear self-assemblies of polypeptides. It was determined that control of the concentration and deposition-time enables the deposition of linear ordered polypeptide assemblies on substrates. A predominance of bilayer stacking of polypeptide sheets in the solution-formed linear assemblies has been observed. Template-directed self

  2. Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

    DOEpatents

    Waldo, Geoffrey S.

    2007-09-18

    The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

  3. Thymus Polypeptide Preparation Tactivin Restores Learning and Memory in Thymectomied Rats.

    PubMed

    Novoseletskaya, A V; Kiseleva, N M; Zimina, I V; Bystrova, O V; Belova, O V; Inozemtsev, A N; Arion, V Ya; Sergienko, V I

    2015-09-01

    We studied the effects of tactivin and splenic polypeptides on learning and memory of thymectomized animals. In 3-week rats, thymectomy blocked active avoidance conditioning. Injections of tactivin (0.5 mg/kg) during 1 month after surgery restored learning capacity; splenic polypeptides were ineffective.

  4. Design of a single-chain polypeptide tetrahedron assembled from coiled-coil segments.

    PubMed

    Gradišar, Helena; Božič, Sabina; Doles, Tibor; Vengust, Damjan; Hafner-Bratkovič, Iva; Mertelj, Alenka; Webb, Ben; Šali, Andrej; Klavžar, Sandi; Jerala, Roman

    2013-06-01

    Protein structures evolved through a complex interplay of cooperative interactions, and it is still very challenging to design new protein folds de novo. Here we present a strategy to design self-assembling polypeptide nanostructured polyhedra based on modularization using orthogonal dimerizing segments. We designed and experimentally demonstrated the formation of the tetrahedron that self-assembles from a single polypeptide chain comprising 12 concatenated coiled coil-forming segments separated by flexible peptide hinges. The path of the polypeptide chain is guided by a defined order of segments that traverse each of the six edges of the tetrahedron exactly twice, forming coiled-coil dimers with their corresponding partners. The coincidence of the polypeptide termini in the same vertex is demonstrated by reconstituting a split fluorescent protein in the polypeptide with the correct tetrahedral topology. Polypeptides with a deleted or scrambled segment order fail to self-assemble correctly. This design platform provides a foundation for constructing new topological polypeptide folds based on the set of orthogonal interacting polypeptide segments.

  5. Thermodynamic stability of polypeptides folding within modeled ribosomal exit tunnel: a density functional study.

    PubMed

    Xu, Xiaofei; Cao, Dapeng

    2010-07-01

    The mechanism of polypeptide folding, especially for the formation of tertiary structures, within the ribosomal exit tunnel, remains one of the most important unsolved problems in biophysical chemistry and molecular biology. In this work, we use a density functional theory (DFT) to explore the polypeptide folding within a modified nanopore, which mimics the confined environment of ribosomal exit tunnel. Results indicate that too long polypeptides (N>100 cannot fold into a helix state within the nanopore, and the helix polypeptides favor folding into a negative coiled coil rather than a positive one, because the negative coiled coil has a lower grand potential than the positive one, and the polypeptide folding into the negative coiled coil therefore needs less driving force than the positive one. To fold into the positive coiled coil, the helix polypeptides must have a small minor radius or a short chain length, which provides helpful insights into the design of nanodevices for manipulating the positive coiled coil. In the presence of attractive interaction, helices need more driving force to fold into coiled coil. Importantly, we have also proposed a scaling relation to understand the folding behavior. The scaling relation gives a good estimate for the computational results, and provides a reasonable explanation for the folding behavior. In summary, it is expected that the proposed DFT approach and the scaling relation provide alternative means for the investigation of polypeptide folding in confined environment, and these impressive results could give useful insights into nascent polypeptide folding.

  6. Nodule-Specific Polypeptides from Effective Alfalfa Root Nodules and from Ineffective Nodules Lacking Nitrogenase 1

    PubMed Central

    Lang-Unnasch, Naomi; Ausubel, Frederick M.

    1985-01-01

    In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664146

  7. Polypeptide synthesis in alphavirus-infected Aedes albopictus cells during the establishment of persistent infection.

    PubMed

    Richardson, M A; Boulton, R W; Raghow, R S; Dalgarno, L

    1980-01-01

    Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cell, RRV reached peak titres at 34--48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and less than 5 per cent of cells assayed as infected. There was no shut-down of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s). The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persitently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK celos host protein synthesis was severly inhibited and by 9--11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity.

  8. Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum.

    PubMed

    Janusz, M; Starościk, K; Zimecki, M; Wieczorek, Z; Lisowski, J

    1981-10-01

    A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

  9. Expansin polynucleotides, related polypeptides and methods of use

    DOEpatents

    Cosgrove, Daniel J.; Wu, Yajun

    2006-02-21

    The present invention relates to beta expansin polypeptides, nucleotide sequences encoding the same and regulatory elements and their use in altering cell wall structure in plants. Nucleic acid constructs comprising a beta expansin sequence operably linked to a promoter, or other regulatory sequence are disclosed as well as vectors, plant cells, plants, and transformed seeds containing such constructs are provided. Methods for the use of such constructs in repressing or inducing expression of a beta expansin sequences in a plant are also provided as well as methods for harvesting transgenic expansin proteins. In addition, methods are provided for inhibiting or improving cell wall structure in plants by repression or induction of expansin sequences in plants.

  10. Identification of antigenically related polypeptides at centrioles and basal bodies.

    PubMed

    Lin, W; Fung, B; Shyamala, M; Kasamatsu, H

    1981-04-01

    An antigen localized at the centriolar region has been identified by indirect immunofluorescence studies in African green monkey kidney, human, hamster, rat, and mouse cells. The antigen consists of two polypeptides of 14,000 and 17,000 daltons. A related antigen is also present at the basal body region in ciliated cells from chicken, cat, mouse, pig, steer, and rabbit trachea and from rabbit fimbria. Immunoelectron microscopy shows that the immunoreactive antigen is indeed located in the region around the basal bodies of ciliated cat tracheal cells. Thus, we have found an antigen that is common to a variety of cell types from many different animal sources and is specifically associated with both centrioles and basal bodies. The possible role of the antigen in differentiation is discussed.

  11. Hippocampal asymmetry in exploratory behavior to vasoactive intestinal polypeptide.

    PubMed

    Ivanova, Margarita; Ternianov, Alexandar; Belcheva, Stiliana; Tashev, Roman; Negrev, Negrin; Belcheva, Iren

    2008-06-01

    The effects of vasoactive intestinal polypeptide (VIP) microinjected uni- or bilaterally into the CA1 hippocampal area of male Wistar rats at a dose of 10, 50 and 100 ng on exploratory behavior were examined. VIP microinjected bilaterally at a high dose (100 ng) significantly decreased the horizontal movements, while at low doses (10 and 50 ng) had no effect on the exploratory activity. Microinjections of VIP into the left hippocampal CA1 area at doses 50 and 100 ng suppressed the exploratory activity, while right-side VIP administration at a dose 100 ng significantly increased horizontal movements compared to the respective controls. Vertical activity was stimulated only by VIP administered into the right hippocampal CA1 area at the three doses used. Neither bilateral nor left injections of VIP induced changes in the vertical movements. The main finding was the presence of hippocampal asymmetry in exploratory behavior to unilateral microinjections of VIP depending on the dose and the microinjected hemisphere.

  12. Discovery and Characterization of smORF-Encoded Bioactive Polypeptides

    PubMed Central

    Saghatelian, Alan; Couso, Juan Pablo

    2016-01-01

    Analysis of genomes, transcriptomes, and proteomes reveals the existence of hundreds to thousands of translated, yet non-annotated short open reading frames (small ORFs or smORFs). The discovery of smORFs, and their protein products, smORF-encoded polypeptides (SEPs), reveals a fundamental gap in our knowledge of protein-coding genes. Different studies have identified central roles for smORFs in metabolism, apoptosis, and development. The discovery of these bioactive SEPs emphasizes the functional potential of this unexplored class of biomolecules. Here, we provide an overview of this emerging field and highlight the opportunities for chemical biology to answer fundamental questions about these novel genes. Such studies will provide new insights into the protein-coding potential of genomes and identify functional genes with roles in biology and disease. PMID:26575237

  13. Identification of antigenically related polypeptides at centrioles and basal bodies.

    PubMed Central

    Lin, W; Fung, B; Shyamala, M; Kasamatsu, H

    1981-01-01

    An antigen localized at the centriolar region has been identified by indirect immunofluorescence studies in African green monkey kidney, human, hamster, rat, and mouse cells. The antigen consists of two polypeptides of 14,000 and 17,000 daltons. A related antigen is also present at the basal body region in ciliated cells from chicken, cat, mouse, pig, steer, and rabbit trachea and from rabbit fimbria. Immunoelectron microscopy shows that the immunoreactive antigen is indeed located in the region around the basal bodies of ciliated cat tracheal cells. Thus, we have found an antigen that is common to a variety of cell types from many different animal sources and is specifically associated with both centrioles and basal bodies. The possible role of the antigen in differentiation is discussed. Images PMID:6166008

  14. Applications of elastin-like polypeptides in drug delivery

    PubMed Central

    MacEwan, Sarah R; Chilkoti, Ashutosh

    2014-01-01

    Elastin-like polypeptides (ELPs) are biopolymers inspired by human elastin. Their lower critical solution temperature phase transition behavior and biocompatibility make them useful materials for stimulus-responsive applications in biological environments. Due to their genetically encoded design and recombinant synthesis, the sequence and size of ELPs can be exactly defined. These design parameters control the structure and function of the ELP with a precision that is unmatched by synthetic polymers. Due to these attributes, ELPs have been used extensively for drug delivery in a variety of different embodiments—as soluble macromolecular carriers, self-assembled nanoparticles, cross-linked microparticles, or thermally coacervated depots. These ELP systems have been used to deliver biologic therapeutics, radionuclides, and small molecule drugs to a variety of anatomical sites for the treatment of diseases including cancer, type 2 diabetes, osteoarthritis, and neuroinflammation. PMID:24979207

  15. Polypeptides in alpha-helix conformation perform as diodes.

    PubMed

    Cristancho, Dahiyana; Seminario, Jorge M

    2010-02-14

    Molecules that resemble a semiconductor diode depletion zone are those with an intrinsic electric dipole, which were suggested as potential electronic devices. However, so far, no single molecule has met such a goal because any electron donor-acceptor linker strongly diminishes any possibility of diode behavior. We find an intrinsic diode behavior in polypeptides such as poly(L-alanine) and polyglycine in alpha-helix conformation, explained in terms of molecular orbital theory using ab initio methods. The application of an antiparallel electric field with respect to the molecular dipole yields a gradual increase in current through the junction because the valence and conduction orbitals approach each other reducing their gap as the bias increases. However, a parallel field makes the gap energy increase, avoiding the pass of the electrons.

  16. [Periodontal regeneration: the use of polypeptide growth factors].

    PubMed

    Di Genio, M; Barone, A; Ramaglia, L; Sbordone, L

    1994-10-01

    Polypeptide growth factors are a class of potent natural biologic mediators which regulate many of the activities of wound healing including cell proliferation, migration and metabolism. Periodontal regeneration is thought to require the migration and proliferation of periodontal ligament cells on the root surface. In fact, repopulation of the detached root surface by cells from periodontal ligament (PDL) is a prerequisite for new attachment formation. Many studies suggested that Polypeptide Growth Factors (PGF) such as Insulin-like Growth Factor I (IGF-I), Platelet Derived Growth Factor (PDGF), Transforming Growth Factor B (TGF-B), Epidermal Growth Factor (EGF), are important mediators of cellular events in wound healing. Studies in vitro analysed the mitogenic effects determined on periodontal ligament cells by growth factors using (3H) Thymidine incorporation during DNA synthesis. The results suggested that recombinant human PDGF and IGF-I stimulate the proliferation of PDL fibroblastic cells and the combination of these growth factors showed a synergistic effect revealing the highest mitogenic effect among all individual growth factors as well as any combination of the growth factors tested. Furthermore these studies demonstrated that rh-PDGF and IGF-I stimulate chemotaxis of PDL fibroblastic cells, and supported a role for TGF-B as a regulator of the mitogenic response to PDGF in these cells. Other studies in vivo showed periodontal tissues regeneration introducing mixtures of recombinant human platelet derived growth factor and insulin-like growth factor into lesions of experimentally induced periodontitis in beagle dogs and monkeys.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Structural features that distinguish kinetically distinct biomineralization polypeptides.

    PubMed

    Collino, Sebastiano; Evans, John Spencer

    2007-05-01

    AP7 and AP24 are mollusk shell proteins which are responsible for aragonite polymorph formation and stabilization within the nacre layer of the Pacific red abalone, Haliotis rufescens. It is known that the 30-AA N-terminal mineral modification domains of both proteins (AP7N, AP24N) possess identical multifunctional mineralization capabilities within in vitro assays but differ in terms of rate kinetics, with AP24N > AP7N. In this report, we identify previously unreported molecular features of AP24N and contrast the lowest energy polypeptide backbone structures of AP24N (planar configuration) with that of AP7N ("bent paper clip" configuration) using NMR data and simulated annealing molecular dynamics structure refinement. Like AP7N, we find that AP24N possesses an unfolded conformation, can sequester Ca(II) and other multivalent metal ions, can adsorb onto or within calcite crystals, and possesses anionic and cationic electrostatic "pocket" regions on its molecular surfaces. However, AP24N has some unique features: greater conformational responsiveness to Ca(II), the tendency to form a more planar backbone configuration, and longer anionic and hydrogen-bonding donor/acceptor sequence blocks. We conclude that the presence of unfolded polypeptide conformation, electrostatic surface pockets, and interactive sequence clustering endow both AP7N and AP24N with similar features that lead to comparable effects on crystal morphology and nucleation. However, AP24N possesses longer anionic and hydrogen-bonding sequence clusters and exhibits a tendency to adopt a more planar backbone configuration than AP7N does. We believe that these features facilitate peptide-mineral, peptide-ion, or water cluster interactions, thereby enhancing the mineralization kinetics of AP24N over AP7N.

  18. Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

    PubMed Central

    Norris, S J

    1993-01-01

    Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide

  19. Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

    PubMed

    Norris, S J

    1993-09-01

    Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide

  20. [Mechanisms of formation and action of polypeptide bio-regulators-- cytomedins ].

    PubMed

    Kaĭdashev, I P

    1994-01-01

    Possible mechanism of synthesis and effects of polypeptide bioregulative molecules (cytomedines) have been shown. This polypeptide has been obtained from different organs and tissues of animals. Its effects are supposed to be based on the processes of intercellular exchange and the cross-membrane transference of information signals. Synthesis of these molecules is considered as the limited proteolysis of protein structures. The information molecules of this kind will be found both inside and outside the cells. The evolution of regulative polypeptides was shown from the organisms of prebiotic era till contemporary organisms. The possibility of interaction between entigenic--endogenic peptides and regulative peptides is described. A hypothetic scheme of the effect of polypeptide molecules on the cell populations is suggested. Space interactions between proteins and polypeptides on the basis of the recognition codes of aminoacids can be most important factors.

  1. Mechanism(s) of heat killing: accumulation of nascent polypeptides in the nucleus?

    PubMed

    Lee, Y J; Borrelli, M J; Corry, P M

    1991-05-15

    To investigate the possibility that nascent polypeptides released from polysomes by heat shock accumulate in the nucleus, cells were pulse labeled with [35S]methionine for two minutes and heated immediately thereafter at 45.5 degrees C for 10 minutes. When isolated nuclei were subjected to gel electrophoresis and subsequently autoradiographed, heated nuclei exhibited an approximately 10-fold increase in radioactive polypeptides in comparison to nonheated controls. These nascent polypeptides were nonspecific molecules covering a wide range of molecular weights. It is plausible that the accumulation of polypeptides in the nucleus results in hyperthermic cytotoxicity. Therefore, we propose that a potential target for heat killing is within the nucleus, at sites where nascent polypeptides accumulate after heat shock.

  2. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating

    PubMed Central

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1–5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure. PMID:27152720

  3. Ice Growth Inhibition in Antifreeze Polypeptide Solution by Short-Time Solution Preheating.

    PubMed

    Nishi, Naoto; Miyamoto, Takuya; Waku, Tomonori; Tanaka, Naoki; Hagiwara, Yoshimichi

    2016-01-01

    The objective of this study is to enhance the inhibition of ice growth in the aqueous solution of a polypeptide, which is inspired by winter flounder antifreeze protein. We carried out measurements on unidirectional freezing of the polypeptide solution. The thickness of the solution was 0.02 mm, and the concentration of polypeptide was varied from 0 to 2 mg/mL. We captured successive microscopic images of ice/solution interfaces, and measured the interface velocity from the locations of tips of the pectinate interface in the images. We also simultaneously measured the temperature by using a small thermocouple. The ice/solution interface temperature was defined by the temperature at the tips. It was found that the interface temperature was decreased with an increasing concentration of polypeptide. To try varying the activity of the polypeptide, we preheated the polypeptide solution and cooled it before carrying out the measurements. Preheating for 1-5 hours was found to cause a further decrease in the interface temperature. Furthermore, wider regions of solution and ice with inclined interfaces in the pectinate interface structure were observed, compared with the case where the solution was not preheated. Thus, the ice growth inhibition was enhanced by this preheating. To investigate the reason for this enhancement, we measured the conformation and aggregates of polypeptide in the solution. We also measured the local concentration of polypeptide. It was found that the polypeptide aggregates became larger as a result of preheating, although the polypeptide conformation was unchanged. These large aggregates caused both adsorption to the interface and the wide regions of supercooled solution in the pectinate interface structure.

  4. Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

    PubMed

    Shi, Lei; Chen, Huai; Zhang, Si-Yu; Chu, Ting-Ting; Zhao, Yu-Fen; Chen, Yong-Xiang; Li, Yan-Mei

    2017-06-01

    Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

  5. Domed Silica Microcylinders Coated with Oleophilic Polypeptides and Their Behavior in Lyotropic Cholesteric Liquid Crystals of the Same Polypeptide.

    PubMed

    Rosu, Cornelia; Jacobeen, Shane; Park, Katherine; Reichmanis, Elsa; Yunker, Peter; Russo, Paul S

    2016-12-13

    Liquid crystals can organize dispersed particles into useful and exotic structures. In the case of lyotropic cholesteric polypeptide liquid crystals, polypeptide-coated particles are appealing because the surface chemistry matches that of the polymeric mesogen, which permits a tighter focus on factors such as extended particle shape. The colloidal particles developed here consist of a magnetic and fluorescent cylindrically symmetric silica core with one rounded, almost hemispherical end. Functionalized with helical poly(γ-stearyl-l-glutamate) (PSLG), the particles were dispersed at different concentrations in cholesteric liquid crystals (ChLC) of the same polymer in tetrahydrofuran (THF). Defects introduced by the particles to the director field of the bulk PSLG/THF host led to a variety of phases. In fresh mixtures, the cholesteric mesophase of the PSLG matrix was distorted, as reflected in the absence of the characteristic fingerprint pattern. Over time, the fingerprint pattern returned, more quickly when the concentration of the PSLG-coated particles was low. At low particle concentration the particles were "guided" by the PSLG liquid crystal to organize into patterns similar to that of the re-formed bulk chiral nematic phase. When their concentration increased, the well-dispersed PSLG-coated particles seemed to map onto the distortions in the bulk host's local director field. The particles located near the glass vial-ChLC interfaces were stacked lengthwise into architectures with apparent two-dimensional hexagonal symmetry. The size of these "crystalline" structures increased with particle concentration. They displayed remarkable stability toward an external magnetic field; hydrophobic interactions between the PSLG polymers in the shell and those in the bulk LC matrix may be responsible. The results show that bio-inspired LCs can assemble suitable colloidal particles into soft crystalline structures.

  6. Peptides and polypeptides as scaffolds for optoelectronics and biomaterials applications

    NASA Astrophysics Data System (ADS)

    Charati, Manoj B.

    Peptides and polypeptides are emerging as a new class of biomaterials due to their unique structural, physiochemical, mechanical, and biological properties. The development of peptide and protein-based biomaterials is driven by the convergence of convenient techniques for peptide/protein engineering and its importance in applications as smart biomaterials. The thesis is divided in two parts; the first part highlights the importance of incorporation of non-natural amino acids into peptides and proteins. In particular, incorporation on p-bromophenylalanine in short alpha-helical peptide templates to control the association of chromophores is discussed. In the second part, design of a multi-component, biocompatible polypeptide with superior elasticity is discussed. Part 1. Novel peptide templates to control association of chromophores. Tailor made peptide and protein materials have many versatile applications, as both conformation and functional group position can be controlled. Such control may have intriguing applications in the development of hybrid materials for electroactive applications. A critical need in fabricating devices from organic semiconducting materials is to achieve control over the conformation and distance between two conjugated chains. Controlling chromophore spacing and orientation with required precision over nanometer length scale poses a greater challenge. Here we propose a peptide based template to control the alignment of the methylstilbene and Oxa-PPV chromophores with desired orientations and spacing. The hybrid peptides were characterized via CD, exciton coupled CD, 1H NMR and photoluminescence experiments. It is observed that slight change in the orientation of molecules has pronounced effect on the photo-physical behavior of the molecules. Characterization of the hybrid peptides via circular dichroism (CD) confirmed the helical character of the designed peptides and indicated that inclusion of non-natural amino acids has significant

  7. Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Yao, Ming-Hao; Wen, Lang; Song, Ji-Tao; Zhang, Ming-Zhen; Zhao, Yuan-Di; Liu, Bo

    2014-09-01

    A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy.A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron

  8. Polypeptide folding-mediated tuning of the optical and structural properties of gold nanoparticle assemblies.

    PubMed

    Aili, Daniel; Gryko, Piotr; Sepulveda, Borja; Dick, John A G; Kirby, Nigel; Heenan, Richard; Baltzer, Lars; Liedberg, Bo; Ryan, Mary P; Stevens, Molly M

    2011-12-14

    Responsive hybrid nanomaterials with well-defined properties are of significant interest for the development of biosensors with additional applications in tissue engineering and drug delivery. Here, we present a detailed characterization using UV-vis spectroscopy and small angle X-ray scattering of a hybrid material comprised of polypeptide-decorated gold nanoparticles with highly controllable assembly properties. The assembly is triggered by a folding-dependent bridging of the particles mediated by the heteroassociation of immobilized helix-loop-helix polypeptides and a complementary nonlinear polypeptide present in solution. The polypeptides are de novo designed to associate and fold into a heterotrimeric complex comprised of two disulfide-linked four-helix bundles. The particles form structured assemblies with a highly defined interparticle gap (4.8±0.4 nm) that correlates to the size of the folded polypeptides. Transitions in particle aggregation dynamics, mass-fractal dimensions and ordering, as a function of particle size and the concentration of the bridging polypeptide, are observed; these have significant effects on the optical properties of the assemblies. The assembly and ordering of the particles are highly complex processes that are affected by a large number of variables including the number of polypeptides bridging the particles and the particle mobility within the aggregates. A fundamental understanding of these processes is of paramount interest for the development of novel hybrid nanomaterials with tunable structural and optical properties and for the optimization of nanoparticle-based colorimetric biodetection strategies.

  9. Photochromic polypeptides as synthetic models of biological photoreceptors: a spectroscopic study.

    PubMed Central

    Angelini, N; Corrias, B; Fissi, A; Pieroni, O; Lenci, F

    1998-01-01

    L-Glutamic acid polypeptides containing photochromic nitrospiropyran bound to the side chains at various percentages ("local" concentration) have been synthesized and investigated as possible artificial models of biological photoreceptors. Absorption and fluorescence spectroscopy have been utilized to investigate the photophysical and photochemical properties of nitrospiropyrans, both inserted in the polypeptide chain and in solution as "free" dye. Conformational variations produced by dark storage and light exposure of the photochromic polypeptides have been studied by means of circular dichroism. Dark-kept "free" dyes in hexafluoro-2-propanol solution in the merocyanine form ("open" form) give rise to molecular aggregates, which have been characterized as merocyanine dimers. The equilibrium constant between the monomer and the dimer, K, and their molar extinction coefficients, epsilon, at several wavelengths have been determined. Fluorescence measurements on "free" and polypeptide-bound nitrospiropyrans suggest that the dimerization process between merocyanines is favored when the photochromic units are inserted in the polypeptide chain and that under these conditions an efficient energy transfer from the monomer (donor) to the dimer (acceptor) occurs. By varying "local" as well as total nitrospiropyran concentration, it has been shown that the dimeric species result from intermolecular interactions between photochromic groups inserted in the same polypeptide chain. The alpha-helix --> random coil transition of the polypeptide structure after dark storage has eventually been shown to be the result of the dimerization process and not of the dark isomerization per se from the "closed" spiropyran form to the "open" merocyanine form of the dye. PMID:9591684

  10. Polypeptides and functions of antigens from human coronaviruses 229E and OC43.

    PubMed Central

    Schmidt, O W; Kenny, G E

    1982-01-01

    Coronaviruses possess three major size classes of polypeptides as judged by molecular weight: approximately 180,000, approximately 50,000, and approximately 23,000. Human coronaviruses 229E and OC43 possess not only three similar size classes of polypeptides but also three distinct antigens, none of which cross-react with the heterologous strain. Polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were reacted in rocket immunoelectrophoresis with antiserum monospecific to each of the three strain-specific antigens (excised precipitin lines from crossed immunoelectrophoresis profiles were used for immunogens). Monospecific antiserum with neutralizing ability reacted with a polypeptide of 186,000 daltons for 229E and a polypeptide of 190,000 daltons for OC43. The antigen which elicited neutralizing antibody response was located at the surface, associated with the corona of the virion, glycosylated, and bound by concanavalin A. Another less prominent surface antigen was represented by size classes of 23,000 daltons for 229E and 24,000 for OC43. The core antigens of the viruses had molecular weights of 49,000 and 229E and 52,000 and OC43 virus. Thus, the molecular weights and functions of the antigens of human coronaviruses are similar to those of animal coronaviruses. The polypeptides of coronaviruses 229E and OC43 are nearly identical as judged by molecular weight, but the similar polypeptides of the two viruses represent different immunological specificities. Images PMID:6173324

  11. Composition and biosynthesis of thylakoid membrane polypeptides in the red alga Cyanidium caldarium: Comparison with the thylakoid polypeptide composition of higher plants and cyanobacteria.

    PubMed

    Yurina, N P; Karakashev, G V; Karapetyan, N V; Odintsova, M S

    1991-10-01

    The polypeptide composition of thylakoid membranes of the red alga Cyanidium caldarium was studied by PAGE in the presence of lithium dodecyl sulfate. The thylakoid membranes were shown to contain 65 polypeptides with mol wt from 110 to 10 kDa. PS I isolated from C. caldarium cells is composed of at least 5 components, one of which is the chlorophyll-protein complex with mol wt of 110 kDa typical of higher plants. Cyt f, c 552, b 6 and b 559 were identified. Inhibition of carotenoid biosynthesis with norflurazon caused no changes in the polypeptide composition of thylakoid membranes of the algae grown in dark. The suppression of the biosynthesis rate of some thylakoid polypeptides in the algae grown with norflurazon in light is a result of membrane photodestruction. Thylakoid membranes from C. caldarium cells are more similar in the number of protein components to thylakoid membranes from cells of the cyanobacterium Anacystis nidulans than to those of higher plants (Pisum sativum), which was proved by immune-blotting assays: Thylakoid membranes of the red alga and cyanobacteria contain 28 homologous polypeptides, while thylakoid membranes of the alga and pea, only 15.

  12. Glucose-dependent insulinotropic polypeptide induces cytokine expression, lipolysis, and insulin resistance in human adipocytes.

    PubMed

    Timper, Katharina; Grisouard, Jean; Sauter, Nadine S; Herzog-Radimerski, Tanja; Dembinski, Kaethi; Peterli, Ralph; Frey, Daniel M; Zulewski, Henryk; Keller, Ulrich; Müller, Beat; Christ-Crain, Mirjam

    2013-01-01

    Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1β, and the IL-1 receptor antagonist IL-1Ra, whereas TNFα, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-κB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1β, and TNFα. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-κB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-κB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes.

  13. Purification of Escherichia coli RNA polymerase using a self-cleaving elastin-like polypeptide tag

    PubMed Central

    Fong, Baley A; Gillies, Alison R; Ghazi, Iraj; LeRoy, Gary; Lee, Kathleen C; Westblade, Lars F; Wood, David W

    2010-01-01

    A self-cleaving elastin-like polypeptide (ELP) tag was used to purify the multisubunit Escherichia coli RNA polymerase (RNAP) via a simple, nonchromatographic method. To accomplish this, the RNAP α subunit was tagged with a self-cleaving ELP-intein tag and coexpressed with the β, β′, and ω subunits. The assembled RNAP was purified with its associated subunits, and was active and acquired at reasonable yield and purity. To remove residual polynucleotides bound to the purified RNAP, two polymer precipitation methods were investigated: polyethyleneimine (PEI) and polyethylene (PEG) precipitation. The PEG procedure was shown to enhance purity and was compatible with downstream ELP-intein purification. Thus, this simple ELP-based method should be applicable for the nonchromatographic purification of other recombinant, in vivo-assembled multisubunit complexes in a single step. Further, the simplicity and low cost of this method will likely facilitate scale up for large-scale production of additional multimeric protein targets. Finally, this technique may have utility in isolating protein interaction partners that associate with a given target. PMID:20512976

  14. Glucose-Dependent Insulinotropic Polypeptide Receptor Deficiency Leads to Impaired Bone Marrow Hematopoiesis.

    PubMed

    Mantelmacher, Fernanda Dana; Fishman, Sigal; Cohen, Keren; Pasmanik Chor, Metsada; Yamada, Yuichiro; Zvibel, Isabel; Varol, Chen

    2017-04-15

    The bone marrow (BM) contains controlled specialized microenvironments, or niches, that regulate the quiescence, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPC). The glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone that mediates postprandial insulin secretion and has anabolic effects on adipose tissue. Previous studies demonstrated altered bone microarchitecture in mice deficient for GIP receptor (Gipr(-/-) ), as well as the expression of high-affinity GIP receptor by distinct cells constructing the BM HSPC niche. Nevertheless, the involvement of GIP in the process of BM hematopoiesis remains elusive. In this article, we show significantly reduced representation and proliferation of HSPC and myeloid progenitors in the BM of Gipr(-/-) mice. This was further manifested by reduced levels of BM and circulating differentiated immune cells in young and old adult mice. Moreover, GIP signaling was required for the establishment of supportive BM HSPC niches during HSPC repopulation in radioablated BM chimera mice. Finally, molecular profiling of various factors involved in retention, survival, and expansion of HSPC revealed significantly lower expression of the Notch-receptor ligands Jagged 1 and Jagged 2 in osteoblast-enriched bone extracts from Gipr(-/-) mice, which are important for HSPC expansion. In addition, there was increased expression of CXCL12, a factor important for HSPC retention and quiescence, in whole-BM extracts from Gipr(-/-) mice. Collectively, our data suggest that the metabolic hormone GIP plays an important role in BM hematopoiesis.

  15. Pituitary adenylate cyclase-activating polypeptide is reduced in Alzheimer disease.

    PubMed

    Han, Pengcheng; Liang, Winnie; Baxter, Leslie C; Yin, Junxiang; Tang, Zhiwei; Beach, Thomas G; Caselli, Richard J; Reiman, Eric M; Shi, Jiong

    2014-05-13

    There is growing evidence that pituitary adenylate cyclase-activating polypeptide (PACAP) is associated with Alzheimer disease (AD) pathology in animal models, but human studies are needed. We studied the brains of patients with pathologically confirmed late-onset AD and age-matched cognitively normal (CN) subjects to investigate the expression of PACAP messenger RNA (34 AD and 14 CN) and protein (12 AD and 11 CN) in a case-control study. We report that PACAP levels are reduced in multiple brain regions, including the entorhinal cortex, the middle temporal gyrus, the superior frontal gyrus, and the primary visual cortex. This reduction is correlated with higher amyloid burden (CERAD plaque density) in the entorhinal cortex and superior frontal gyrus but not in the primary visual cortex, a region spared in most cases of AD. PACAP expression is lower in advanced Braak stages (V and VI) than in moderate stages (III and IV). Increased PACAP levels are associated with decreased scores on the Dementia Rating Scale, a global cognitive measure. Finally, CSF levels paralleled brain levels in AD but not in Parkinson dementia or frontotemporal dementia brains. The close relationship between PACAP reduction and the severity of AD pathology suggests that downregulation of PACAP may contribute to AD pathogenesis.

  16. Fabrication and characterization of non-volatile transistor memory based on polypeptide as gate dielectric

    NASA Astrophysics Data System (ADS)

    Liang, Lijuan; Li, LianFang; Wei, Xianfu; Huang, Beiqing; Wei, Yen

    2017-01-01

    The organic thin film transistor (OTFT) fabricated with the polypeptide as a dielectric layer shows memory function. In order to investigate the effect of polypeptide structure on the performance of non-volatile transistor memory, the Fourier-transform IR (FT- IR) and Circular Dichiroism (CD) spectral of PMLG film has been applied, respectively. In conclusion, the memory transistor device fabricated with polypeptide as the ferroelectric exhibit promising behavior such as a large memory window, and the dipole moment of the amide group was considered as the main source of the memory function.

  17. The Research on the Impact of Maca Polypeptide on Sport Fatigue.

    PubMed

    Miao, Hua

    2015-01-01

    In order to study the effect of maca polypeptide on sport fatigue, this paper selected 40 male mice, and they were randomly divided into group A, B, C and D. group A, B and C were fed food with different concentrations of maca polypeptide, and group D was control group. After two weeks of feeding, measured physiological indexes of mice, including blood glucose, urea nitrogen and creatinine. At last gived the experimental results, as well as the analysis. Experimental results show that maca polypeptide can improve the ability of anti-fatigue mice, and in a certain concentration range, the higher the concentration, the better the resistance to fatigue.

  18. Characterization of an amidated form of pancreatic polypeptide from the daddy sculpin (Cottus scorpius).

    PubMed

    Conlon, J M; Schmidt, W E; Gallwitz, B; Falkmer, S; Thim, L

    1986-12-30

    The primary structure of pancreatic polypeptide from the teleostean fish, Cottus scorpius (daddy sculpin) was established as: YPPQPESPGGNASPEDWAKYHAAVRHYVNLITRQRYNH2 The presence of a COOH-terminally alpha-amidated amino acid was established using an HPLC method of general applicability. Although the peptide shows strong homology towards anglerfish pancreatic polypeptide (86%), homology towards porcine peptide YY (PYY) (61%) and porcine neuropeptide Y (NPY) (61%) was greater than towards porcine pancreatic polypeptide (PP) (47%). This result supports suggestions that the gene duplication events which led to PP, NPY and PYY formation took place after the time of divergence of fish and mammals.

  19. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  20. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  1. Vasoactive intestinal polypeptide entrains circadian rhythms in astrocytes

    PubMed Central

    Marpegan, Luciano; Krall, Thomas J.; Herzog, Erik D.

    2009-01-01

    Many mammalian cell types show daily rhythms in gene expression driven by a circadian pacemaker. For example, cultured astrocytes display circadian rhythms in Period1 and Period2 expression. It is not known, however, how or which intercellular factors synchronize and sustain rhythmicity in astrocytes. Because astrocytes are highly sensitive to vasoactive intestinal polypeptide (VIP), a neuropeptide released by neurons and important for the coordination of daily cycling, we hypothesized that VIP entrains circadian rhythms in astrocytes. We used astrocyte cultures derived from knock-in mice containing a bioluminescent reporter of PERIOD2 (PER2) protein, to assess the effects of VIP on the rhythmic properties of astrocytes. VIP induced a dose-dependent increase in the peak-to-trough amplitude of the ensemble rhythms of PER2 expression with maximal effects near 100nM VIP and threshold values between 0.1 and 1 nM. VIP also induced dose- and phase-dependent shifts in PER2 rhythms and daily VIP administration entrained bioluminescence rhythms of astrocytes to a predicted phase angle. This is the first demonstration that a neuropeptide can entrain glial cells to a phase predicted by a phase response curve. We conclude that VIP potently entrains astrocytes in vitro and is a candidate for coordinating daily rhythms among glia in the brain. PMID:19346450

  2. HUMAN PANCREATIC POLYPEPTIDE IN A PHOSPHOLIPID BASED MICELLAR FORMULATION

    PubMed Central

    Banerjee, Amrita; Onyuksel, Hayat

    2012-01-01

    Purpose Pancreatic polypeptide (PP) has important glucoregulatory functions and thereby holds significance in the treatment of diabetes and obesity. However, short plasma half-life and aggregation propensity of PP in aqueous solution, limits its therapeutic application. To address these issues, we prepared and characterized a formulation of PP in sterically stabilized micelles (SSM) that protects and stabilizes PP in its active conformation. Methods PP-SSM was prepared by incubating PP with SSM dispersion in buffer. Peptide-micelle association and freeze-drying efficacy of the formulation was characterized in phosphate buffers with or without sodium chloride using dynamic light scattering, fluorescence spectroscopy and circular dichroism. The degradation kinetics of PP-SSM in presence of proteolytic enzyme was determined using HPLC and bioactivity of the formulation was evaluated by in vitro cAMP inhibition. Results PP self-associated with SSM and this interaction was influenced by presence/absence of sodium chloride in the buffer. The formulation was effectively lyophilized, demonstrating feasibility for its long-term storage. The stability of peptide against proteolytic degradation was significantly improved and PP in SSM retained its bioactivity in vitro. Conclusions Self-association of PP with phospholipid micelles addressed the delivery issues of the peptide. This PP nanomedicine should be further developed for the treatment of diabetes. PMID:22399387

  3. Free radical scavenging abilities of polypeptide from Chlamys farreri

    NASA Astrophysics Data System (ADS)

    Han, Zhiwu; Chu, Xiao; Liu, Chengjuan; Wang, Yuejun; Mi, Sun; Wang, Chunbo

    2006-09-01

    We investigated the radical scavenging effect and antioxidation property of polypeptide extracted from Chlamys farreri (PCF) in vitro using chemiluminescence and electron spin resonance (ESR) methods. We examined the scavenging effects of PCF on superoxide anions (O{2/-}), hydroxyl radicals (OH·), peroxynitrite (ONOO-) and the inhibiting capacity of PCF on peroxidation of linoleic acid. Our experiment suggested that PCF could scavenge oxygen free radicals including superoxide anions (O{2/-}) (IC50=0.3 mg/ml), hydroxyl radicals (OH·) (IC50=0.2 μg/ml) generated from the reaction systems and effectively inhibit the oxidative activity of ONOO- (IC50=0.2 mg/ml). At 1.25 mg/ml of PCF, the inhibition ratio on lipid peroxidation of linoleic acid was 43%. The scavenging effect of PCF on O{2/-}, OH· and ONOO- free radicals were stronger than those of vitamin C but less on lipid peroxidation of linoleic acid. Thus PCF could scavenge free radicals and inhibit the peroxidation of linoleic acid in vitro. It is an antioxidant from marine products and potential for industrial production in future.

  4. Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.

    PubMed

    Westermark, Per; Andersson, Arne; Westermark, Gunilla T

    2011-07-01

    Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of β-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

  5. Aqueous cholesteric liquid crystals using uncharged rodlike polypeptides.

    PubMed

    Bellomo, Enrico G; Davidson, Patrick; Impéror-Clerc, Marianne; Deming, Timothy J

    2004-07-28

    The aqueous, lyotropic liquid-crystalline phase behavior of the alpha-helical polypeptide, poly(N(epsilon)-2-[2-(2-methoxyethoxy)ethoxy]acetyl-lysine) (1), has been studied using optical microscopy and X-ray scattering. Solutions of optically pure 1 were found to form cholesteric liquid crystals at volume fractions that decreased with increasing average chain length. At very high volume fractions, the formation of a hexagonal mesophase was observed. The pitch of the cholesteric phase could be varied by a mixture of enantiomeric samples L-1 and D-1, where the pitch increased as the mixture approached equimolar. The cholesteric phases could be untwisted, using either magnetic field or shear flow, into nematic phases, which relaxed into cholesterics upon removal of field or shear. We have found that the phase diagram of 1 in aqueous solution parallels that of poly(gamma-benzyl glutamate) in organic solvents, thus providing a useful system for liquid-crystal applications requiring water as solvent.

  6. Atrial natriuretic polypeptide-like material in rat lung

    SciTech Connect

    Chang, J.K.; Chang, D.; Xie, C.W.; Song, D.L.; Li, X.R.; Zhang, S.X.; Wang, T.L.; Tang, J.

    1986-03-05

    Atrial natriuretic polypeptide-like immunoreactive material (ANP-IR) was found in rat lung by radioimmunoassay, with the concentration ranging from 0.6-1.2 pmol/g of tissue in each lobe. PAP-immunohistochemical study demonstrated that specific staining of granules for ..cap alpha..-human ANP are mainly located in the muscular layer of the pulmonary vein. Fractionation of lung extract by gel filtration and reserve phase HPLC revealed the presence of multiple forms of ANP-IR, which possibly possessed molecular structure partially different from rat ANP, atriopeptin I and III. Intravenous injection of lung extract induced potent diuresis and natriuresis in rats. These responses could be abolished when the lung extract was preincubated with antiserum for ..cap alpha..-human ANP. Specific binding sites for /sup 125/I-labeled rat ANP were also found in lung membrane preparation by radioreceptor assay. Incubation of synthetic atriopeptin III (10/sup -9/ to 10/sup -6/M) with lung tissue induced 1-28 fold increase in lung cGMP content. The results suggest that ANP-IR and its receptors existing in rat lung may be involved in the regulation of pulmonary function and have a synergic effect with ANP of cardiac origin in the control of water-electrolytes balance.

  7. Crystal structures of a polypeptide processing and secretion transporter.

    PubMed

    Lin, David Yin-wei; Huang, Shuo; Chen, Jue

    2015-07-23

    Bacteria secrete peptides and proteins to communicate, to poison competitors, and to manipulate host cells. Among the various protein-translocation machineries, the peptidase-containing ATP-binding cassette transporters (PCATs) are appealingly simple. Each PCAT contains two peptidase domains that cleave the secretion signal from the substrate, two transmembrane domains that form a translocation pathway, and two nucleotide-binding domains that hydrolyse ATP. In Gram-positive bacteria, PCATs function both as maturation proteases and exporters for quorum-sensing or antimicrobial polypeptides. In Gram-negative bacteria, PCATs interact with two other membrane proteins to form the type 1 secretion system. Here we present crystal structures of PCAT1 from Clostridium thermocellum in two different conformations. These structures, accompanied by biochemical data, show that the translocation pathway is a large α-helical barrel sufficient to accommodate small folded proteins. ATP binding alternates access to the transmembrane pathway and also regulates the protease activity, thereby coupling substrate processing to translocation.

  8. Idealized models of protofilaments of human islet amyloid polypeptide

    PubMed Central

    Li, Yiyu; Hatmal, Ma'mon M.; Langen, Ralf; Haworth, Ian S.

    2012-01-01

    Fibrils formed by assembly of human islet amyloid polypeptide (hIAPP) are found in most patients with type II diabetes. Structurally, these fibrils are composed of multiple protofilaments and are characterized by extended beta sheets, variable helical twists, and different morphologies. We have previously derived models for the hIAPP protofilament using simulations constrained by data from EPR spectroscopy. In the current work, these models were used as a basis for generating idealized hIAPP protofilaments with symmetrical geometrical properties using a new algorithm, MFIBRIL. We show good agreement of the idealized protofilaments with experimental data for amino acid side chain orientations and geometrical features including the inter-beta sheet distance and the protofilament radius. These idealized protofilaments can be used in MFIBRIL to generate fibril models that may be experimentally testable at the molecular level. MFIBRIL can also be used for building structures of any repetitive molecular assembly starting with a single building block obtained from any source. PMID:23116372

  9. Structural organization and polypeptide composition of the avian adenovirus core.

    PubMed Central

    Li, P; Bellett, A J; Parish, C R

    1984-01-01

    CELO virus (fowl adenovirus 1) contained three core polypeptides of molecular weights 20,000, 12,000, and 9,500. The core was similar to that of human adenoviruses, with some evidence of compact subcore domains. Micrococcal nuclease digestion of CELO virus cores produced a smear of DNA fragments of gradually decreasing size, with no nucleosome subunit or repeat pattern. Moreover, when digested cores were analyzed without protease treatment, there was again no evidence of a nucleosome substructure; neither DNA fragments nor core proteins entered a 4% polyacrylamide gel. The organization of the core is thus quite unlike that of chromatin. Restriction endonuclease analysis of the DNA from digested cores showed that the right end was on the outside of the core. We suggest that adenovirus DNA is condensed into the core by cross-linking and neutralization by the core proteins, beginning with the packaging sequence at the center of the core and ending with the right end of the DNA on the outside. Images PMID:6092686

  10. Characterization of a baculovirus gene encoding a small conotoxinlike polypeptide.

    PubMed Central

    Eldridge, R; Li, Y; Miller, L K

    1992-01-01

    We identified a gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) that encodes a small cysteine-rich polypeptide which has size and sequence similarity to omega-conotoxins, a class of calcium ion (Ca2+) channel inhibitors, found in the venom of cone snails. Transcriptional analysis indicated that the 159-bp open reading frame, which we named ctl, and a downstream 984-bp open reading frame are transcribed as a single 1.3-kb bicistronic late RNA. The mature ctl gene product was identified as a small secreted protein by high-pressure liquid chromatography fractionation of extracellular fluid. Viruses with a site-specific deletion in ctl appeared normal with regard to the kinetics and virulence of infection, both in vitro and in vivo. Although we studied the behavior of wild-type and mutant virus-infected insects in some detail, a biological role for ctl in AcMNPV infection remains to be established. Images PMID:1404603

  11. Parametric sensitivity analysis of avian pancreatic polypeptide (APP).

    PubMed

    Zhang, H; Wong, C F; Thacher, T; Rabitz, H

    1995-10-01

    Computer simulations utilizing a classical force field have been widely used to study biomolecular properties. It is important to identify the key force field parameters or structural groups controlling the molecular properties. In the present paper the sensitivity analysis method is applied to study how various partial charges and solvation parameters affect the equilibrium structure and free energy of avian pancreatic polypeptide (APP). The general shape of APP is characterized by its three principal moments of inertia. A molecular dynamics simulation of APP was carried out with the OPLS/Amber force field and a continuum model of solvation energy. The analysis pinpoints the parameters which have the largest (or smallest) impact on the protein equilibrium structure (i.e., the moments of inertia) or free energy. A display of the protein with its atoms colored according to their sensitivities illustrates the patterns of the interactions responsible for the protein stability. The results suggest that the electrostatic interactions play a more dominant role in protein stability than the part of the solvation effect modeled by the atomic solvation parameters.

  12. Bovine pancreatic polypeptide as an antagonist of muscarinic cholinergic receptors

    SciTech Connect

    Pan, G.Z.; Lu, L.; Qian, J.; Xue, B.G.

    1987-03-01

    In dispersed acini from rat pancreas, it was found that bovine pancreatic polypeptide (BPP) and its C-fragment hexapeptide amide (PP-6), at concentrations of 0.1 and 30 ..mu..M, respectively, could significantly inhibit amylase secretion stimulated by carbachol, and this inhibition by BPP was dose dependent. /sup 45/Ca outflux induced by carbachol was also inhibited by BPP or PP-6, but they had no effect on cholecystokinin octapeptide- (CCK-8) or A23187-stimulated /sup 45/Ca outflux. BPP was also capable of displacing the specific binding of (/sup 3/H)-quinuclidinyl benzilate to its receptors, and it possessed a higher affinity (K/sub i/35nM) than carbachol (K/sub i/ 1.8 ..mu..M) in binding with M-receptors. It is concluded from this study that BPP acts as an antagonist of muscarinic cholinergic receptors in rat pancreatic acini. In addition, BPP inhibited the potentiation of amylase secretion caused by the combination of carbachol plus secretin or vasoactive intestinal peptide. This may be a possible explanation of the inhibitory effect of BPP on secretin-induced pancreatic enzyme secretion shown in vivo, since pancreatic enzyme secretion stimulated by secretin under experimental conditions may be the result of potentiation of enzyme release produced by the peptide in combination with a cholinergic stimulant.

  13. Coarse-grained, foldable, physical model of the polypeptide chain

    PubMed Central

    Chakraborty, Promita; Zuckermann, Ronald N.

    2013-01-01

    Although nonflexible, scaled molecular models like Pauling–Corey’s and its descendants have made significant contributions in structural biology research and pedagogy, recent technical advances in 3D printing and electronics make it possible to go one step further in designing physical models of biomacromolecules: to make them conformationally dynamic. We report here the design, construction, and validation of a flexible, scaled, physical model of the polypeptide chain, which accurately reproduces the bond rotational degrees of freedom in the peptide backbone. The coarse-grained backbone model consists of repeating amide and α-carbon units, connected by mechanical bonds (corresponding to φ and ψ) that include realistic barriers to rotation that closely approximate those found at the molecular scale. Longer-range hydrogen-bonding interactions are also incorporated, allowing the chain to readily fold into stable secondary structures. The model is easily constructed with readily obtainable parts and promises to be a tremendous educational aid to the intuitive understanding of chain folding as the basis for macromolecular structure. Furthermore, this physical model can serve as the basis for linking tangible biomacromolecular models directly to the vast array of existing computational tools to provide an enhanced and interactive human–computer interface. PMID:23898168

  14. Vasoactive intestinal polypeptide provokes acetylcholine release from the myenteric plexus

    SciTech Connect

    Kusunoki, M.; Tsai, L.H.; Taniyama, K.; Tanaka, C.

    1986-07-01

    Effects of vasoactive intestinal polypeptide (VIP) on the release of acetylcholine (ACh) from longitudinal muscle strips with myenteric plexus (LM) preparations were examined in the guinea pig small intestine. VIP (10 to 10 W M) induced a concentration-dependent contraction of LM preparation. The VIP-induced contractions seem to be related to three components, the scopolamine-sensitive, the scopolamine-insensitive, the tetrodotoxin-sensitive, and the tetrodotoxin-insensitive contractions. VIP (10 to 10 W M) induced a concentration-dependent increase in the release of (TH)ACh from LM preparations preloaded with (TH)choline. The VIP-evoked (TH)ACh release was inhibited by removal of CaS from the perfusion medium and by treatment with tetrodotoxin but not by scopolamine and hexamethonium. The spontaneous and VIP-evoked (TH)ACh release was not affected by phentolamine, propranolol, methysergide, diphenhydramine, cimetidine, bicuculline, or (D-ProS, D-Trp/sup 7,9/)substance P. The result demonstrates that VIP induces contractions of longitudinal smooth muscle directly and indirectly by the stimulation of both cholinergic neurons and noncholinergic excitatory neurons.

  15. Polypeptide multilayer self-assembly studied by ellipsometry.

    PubMed

    Craig, Marina; Holmberg, Krister; Le Ru, Eric; Etchegoin, Pablo

    2014-01-01

    A polypeptide nanofilm made by layer-by-layer (LbL) self-assembly was built on a surface that mimics nonwoven, a material commonly used in wound dressings. Poly-L-lysine (PLL) and poly-L-glutamic acid (PLGA) are the building blocks of the nanofilm, which is intended as an enzymatically degradable lid for release of bactericides to chronic wounds. Chronic wounds often carry infection originating from bacteria such as Staphylococcus aureus and a release system triggered by the degree of infection is of interest. The dry nanofilm was studied with ellipsometry. The thickness of the nanofilm was 60% less in its dry state than in its wet state. The measurements showed that a primer was not necessary to build a stable nanofilm, which is practically important in our case because a nondegradable primer is highly unwanted in a wound care dressing. Added V8 (glutamyl endopeptidase) enzymes only showed adsorption on the nanofilm at room temperature, indicating that the PLL/PLGA "lid" may remain intact until the dressing has been filled with wound exudate at the elevated temperature typical of that of the wound.

  16. Volumetric properties of human islet amyloid polypeptide in liquid water.

    PubMed

    Brovchenko, I; Andrews, M N; Oleinikova, A

    2010-04-28

    The volumetric properties of human islet amyloid polypeptide (hIAPP) in water were studied in a wide temperature range by computer simulations. The intrinsic density rho(p) and the intrinsic thermal expansion coefficient alpha(p) of hIAPP were evaluated by taking into account the difference between the volumetric properties of hydration and bulk water. The density of hydration water rho(h) was found to decrease almost linearly with temperature upon heating and its thermal expansion coefficient was found to be notably higher than that of bulk water. The peptide surface exposed to water is more hydrophobic and its rho(h) is smaller in conformation with a larger number of intrapeptide hydrogen bonds. The two hIAPP peptides studied (with and without disulfide bridge) show negative alpha(p), which is close to zero at 250 K and decreases to approximately -1.5 x 10(-3) K(-1) upon heating to 450 K. The analysis of various structural properties of peptides shows a correlation between the intrinsic peptide volumes and the number of intrapeptide hydrogen bonds. The obtained negative values of alpha(p) can be attributed to the shrinkage of the inner voids of the peptides upon heating.

  17. Low-dose pancreatic polypeptide inhibits food intake in man.

    PubMed

    Jesudason, David R; Monteiro, Mariana P; McGowan, Barbara M C; Neary, Nicola M; Park, Adrian J; Philippou, Elena; Small, Caroline J; Frost, Gary S; Ghatei, Mohammad A; Bloom, Stephen R

    2007-03-01

    Pancreatic polypeptide (PP) is a gut hormone released from the pancreas in response to food ingestion and remains elevated for up to 6 h postprandially. Plasma levels are elevated in patients with pancreatic tumours. An intravenous infusion of PP has been reported to reduce food intake in man, suggesting that PP is a satiety hormone. We investigated whether a lower infusion rate of PP would induce significant alterations in energy intake. The study was randomised and double-blinded. Fourteen lean fasted volunteers (five men and nine women) received 90 min infusions of PP (5 pmol/kg per min) and saline on two separate days. The dose chosen was half that used in a previous human study which reported a decrease in appetite but at supra-physiological levels of PP. One hour after the end of the infusion, a buffet lunch was served and energy intake measured. PP infusion was associated with a significant 11 % reduction in energy intake compared with saline (2440 (se 200) v. 2730 (se 180) kJ; P<0 x 05). Preprandial hunger as assessed by a visual analogue score was decreased in the PP-treated group compared to saline. These effects were achieved with plasma levels of PP within the pathophysiological range of pancreatic tumours.

  18. Human pancreatic polypeptide in a phospholipid-based micellar formulation.

    PubMed

    Banerjee, Amrita; Onyuksel, Hayat

    2012-06-01

    Pancreatic polypeptide (PP) has important glucoregulatory functions and thereby holds significance in the treatment of diabetes and obesity. However, short plasma half-life and aggregation propensity of PP in aqueous solution, limits its therapeutic application. To address these issues, we prepared and characterized a formulation of PP in sterically stabilized micelles (SSM) that protects and stabilizes PP in its active conformation. PP-SSM was prepared by incubating PP with SSM dispersion in buffer. Peptide-micelle association and freeze-drying efficacy of the formulation was characterized in phosphate buffers with or without sodium chloride using dynamic light scattering, fluorescence spectroscopy and circular dichroism. The degradation kinetics of PP-SSM in presence of proteolytic enzyme was determined using HPLC and bioactivity of the formulation was evaluated by in vitro cAMP inhibition study. PP self-associated with SSM and this interaction was influenced by presence/absence of sodium chloride in the buffer. The formulation was effectively lyophilized, demonstrating feasibility for its long-term storage. The stability of peptide against proteolytic degradation was significantly improved and PP in SSM retained its bioactivity in vitro. Self-association of PP with phospholipid micelles addressed the delivery issues of the peptide. This nanomedicine should be further developed for the treatment of diabetes.

  19. Tuning calcium carbonate growth through physical confinement and templating with amyloid-like polypeptide aggregates

    NASA Astrophysics Data System (ADS)

    Colaco, Martin Francis

    that this methodology does not extend to three-dimensional confined systems, as the water has no method of escape. Through the addition of an insoluble hydroscopic polymer to our microreactors, amorphous calcium carbonate of controllable sizes can be grown. However, crystalline calcium carbonate cannot be grown without some type of templating. Studies of calcium carbonate templating have predominantly been performed on SAMs or in poorly characterized gels or protein films. The use of ordered protein or polypeptide aggregates for templating permits both geometry and charge surface density to be varied. We have studied the kinetics and final morphology of ordered aggregates of poly-L-glutamic acid and a copolymer of glutamic acid and alanine through experiments and simulations. Electrostatics, not structure, of the monomer appeared to be the dominating factor in the aggregation, as pH and salt concentration changes led to dramatic changes in the kinetics. Examining our experimental with existing models provided inconsistent results, so we developed a new model that yielded physically realistic rate constants, while generating better fits with longer lag phases and faster growths. However, despite the similarity of aggregation conditions, the two polypeptides yielded vastly different morphologies, with the PEA forming typical amyloid-like fibrils and PE forming larger, twisted lamellar aggregates. Templating with these aggregates also yielded dramatically different patterns. Polycrystalline rhombohedral calcite with smooth faces and edges grew on PEA fibrils, with minimal templating in evidence. However, on PE, numerous calcite crystals with triangular projections tracked the surface of the aggregate. The PE lamellae are characterized by extensive beta-sheet structure. In this conformation, the glutamic acid spacings on the surface of the aggregates can mimic the spacings of the carboxylates in the calcite lattice. In addition, the high negative charge density on the

  20. Hepatitis B e antigen polypeptides isolated from sera of individuals infected with hepatitis B virus: comparison with HBeAg polypeptide derived from Dane particles.

    PubMed

    Takahashi, K; Imai, M; Gotanda, T; Sano, T; Oinuma, A; Mishiro, S; Miyakawa, Y; Mayumi, M

    1980-09-01

    Hepatitis B e antigen (HBeAg) occurs in the serum of individuals infected with hepatitis B virus both free and in association with IgG. Utilizing a succession of steps involving salt precipitation, affinity chromatography, ion-exchange chromatography and isoelectrofocusing, we isolated free and IgG-bound forms of HBeAg from the sera of infected individuals with an overall gain in specific activity of 3000-fold and 540-fold, respectively. Polypeptide profiles of purified HBeAg preparations were studied by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Both free and IgG-bound preparations revealed polypeptides with mol. wt. of 15500 (P15.5) and 16 500 (P16.5), and HBeAg activity was detected corresponding to their positions. The HBeAg polypeptides (P15.5/16.5) derived from sera were physicochemically different from the two polypeptides with HBeAg activity (P19 and P45) liberated from Dane particle cores by the conventional method involving incubation with Nonidet P40 and 2-mercaptoethanol. However, when core particles were prepared in the presence of a proteolytic enzyme, in addition to Nonidet P40 and 2-mercaptoethanol, they gave rise to HBeAg polypeptides with mol. wt. of 31000 (P31) and 15 500. Furthermore, P31 split into P15.5 when heated at 100 degrees C for 2 min. On the basis of these results, P15.5 may be assumed to be the essential polypeptide bearing HBeAg activity in the serum and also in Dane particles.

  1. Plastid development in Pisum sativum leaves during greening. I. A comparison of plastid polypeptide composition and in organello translation characteristics

    SciTech Connect

    Dietz, K.J.; Bogorad, L.

    1987-11-01

    Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twenty-three of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A/sup +/) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.

  2. Biosynthesis of metal-binding polypeptides and their precursors in response to cadmium in Datura innoxia

    SciTech Connect

    Jackson, P.J.; Delhaize, E.; Kuske, C.R.

    1991-01-01

    Metal-tolerant Datura innoxia cells synthesize large amounts of a class of metal-binding polypeptides, poly({gamma}-glutamylcysteinyl) glycines (({gamma}-EC){sub n}G, n=2-5), when exposed to Cd. These polypeptides have a high affinity for Cd (2) and certain other metal ions and are thought to play a role in metal tolerance in higher plants. ({gamma}-EC){sub n}G is biosynthetically derived from glutathione. Therefore, the response of Datura cells to Cd must include an increase in production of glutathione and its precursors, since cells rapidly accumulate very high concentrations of these metal-binding polypeptides. The biosynthesis of ({gamma}-EC){sub n}Gs, glutathione, and cysteine in response to Cd exposure is described. The physiological significance of the synthesis of these polypeptides and their precursors and its relevance to Cd tolerance and metal homeostasis are discussed. 34 refs., 6 figs., 1 tab.

  3. Characterization of the Large Picornaviral Polypeptides Produced in the Presence of Zinc Ion

    PubMed Central

    Butterworth, Byron E.; Korant, Bruce D.

    1974-01-01

    Zinc ion inhibits the posttranslational cleavages of human rhinovirus-1A, encephalomyocarditis virus, and poliovirus polypeptides. Each virus displayed a different susceptibility to zinc. However, in each case the cleavages of the capsid precursor and the cleavages analogous to the C → D → E conversion in encephalomyocarditis virus were most sensitive to zinc. Higher concentrations of zinc resulted in the buildup of even larger precursor polypeptides of a size between 106,000 and 214,000 daltons. The sizes of these polypeptides and the relative position of their gene loci on the viral RNA were determined. These data were used to place these polypeptides in the over-all scheme of viral protein processing. PMID:4367904

  4. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  5. Isolation of proteins related to the Rh polypeptides from nonhuman erythrocytes.

    PubMed Central

    Saboori, A M; Denker, B M; Agre, P

    1989-01-01

    It is thought that the Rh antigens may be important in maintaining normal erythrocyte membrane integrity. Despite their name, Rh antigens are serologically present only on human erythrocytes. Rh structural polymorphisms are known to reside within a family of nonglycosylated Mr 32,000 integral membrane proteins that can be purified by hydroxylapatite chromatography. Mr 32,000 integral membrane proteins were purified similarly from erythrocyte membrane vesicles prepared from rhesus monkeys, cows, cats, and rats, but could not be purified from human Rhmod erythrocytes, a rare syndrome lacking Rh antigens. The purified Mr 32,000 polypeptides were labeled with 125I, digested with chymotrypsin, and found to be 30-60% identical to human Rh polypeptides when compared by two-dimensional iodopeptide mapping. The physiologic function of the Rh polypeptides remains to be identified; however, the existence of related proteins in nonhuman erythrocytes supports the concept that the Rh polypeptides are erythrocyte membrane components of fundamental significance. Images PMID:2492035

  6. Theoretical investigations on model ternary polypeptides using genetic algorithm—Some new results

    NASA Astrophysics Data System (ADS)

    Arora, Vinita; Bakhshi, A. K.

    2011-04-01

    Using genetic algorithm (GA) model ternary polypeptides containing glycine, alanine and serine in β-pleated conformation have been theoretically investigated. In designing, the criterion to attain the optimum solution at the end of GA run is minimum band gap and maximum delocalization in the polypeptide chain. Ab initio results obtained using Clementi's minimal basis set are used as input. Effects of (i) change of basis set from minimal to double zeta, (ii) change in secondary structure from β-pleated to α-helical, (iii) presence of solvation shell and (iv) binding of H + and Li + ions to peptide group on the resulting solution as well as on electronic structure and conduction properties of polypeptides are investigated. A comparison is drawn between results obtained for the two cationic adducts. The protonated adduct is expected to withdraw more negative charge from the polypeptide chain due to smaller size of H + and is found to have high electron affinity compared to Li + adduct.

  7. Pericarp polypeptides and SRAP markers associated with fruit quality traits in an interspecific tomato backcross.

    PubMed

    Pereira da Costa, J H; Rodríguez, G R; Pratta, G R; Picardi, L A; Zorzoli, R

    2014-01-24

    The aim of this study was to detect polypeptides and genomic regions associated with fruit quality traits in a backcross generation using as parent the Argentinean cultivated tomato Caimanta of Solanum lycopersicum and the wild accession LA722 of S. pimpinellifolium. We tested two types of molecular marker: polypeptide profile (at two ripening stages, mature green and red ripe) and SRAP (sequence-related amplified polymorphism). A polypeptide of 45 kDa present in the wild parents at the mature green stage was associated with larger fruit and long shelf life. Some amplification fragments from SRAP markers were associated with more than one quality trait such as fruit color, firmness, titratable acidity, and fruit soluble solids content. This study demonstrated for the first time the usefulness of the polypeptide profiles of pericarp and SRAP markers in finding associations with quality fruit traits in a tomato backcross generation.

  8. Polypeptide synthesis in columnar and squamous explants of human uterine cervix.

    PubMed

    Cowan, M E; Ward, K; Woodman, C B; Skinner, G R

    1982-10-01

    There were quantitative and qualitative differences in the in-vitro synthesis of 3 polypeptides between squamous and columnar epithelial explants of human cervix. One cross-linked keratin-like polypeptide of mol. wt 50,000 was synthesized and phosphorylated by squamous but not by columnar explants; a second cross-linked keratin-like polypeptide of mol. wt 52,000, which was present in larger amounts in squamous than columnar explants, was both glycosylated and phosphorylated during in-vitro explantation of squamous tissue; a third polypeptide of mol. wt 25,200 which was keratin-like but not cross-linked, was synthesized in squamous-tissue explants but in only 4% of columnar-tissue explants.

  9. Polypeptide synthesis in columnar and squamous explants of human uterine cervix.

    PubMed Central

    Cowan, M. E.; Ward, K.; Woodman, C. B.; Skinner, G. R.

    1982-01-01

    There were quantitative and qualitative differences in the in-vitro synthesis of 3 polypeptides between squamous and columnar epithelial explants of human cervix. One cross-linked keratin-like polypeptide of mol. wt 50,000 was synthesized and phosphorylated by squamous but not by columnar explants; a second cross-linked keratin-like polypeptide of mol. wt 52,000, which was present in larger amounts in squamous than columnar explants, was both glycosylated and phosphorylated during in-vitro explantation of squamous tissue; a third polypeptide of mol. wt 25,200 which was keratin-like but not cross-linked, was synthesized in squamous-tissue explants but in only 4% of columnar-tissue explants. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:6184064

  10. Identification and characterization of salt-inducible polypeptide in Paenibacillus sp., a moderately halophilic bacterium.

    PubMed

    Sokhansanj, Ashrafaddin; Karkhane, Ali Asghar; Jazii, Ferdous Rastgar

    2005-11-01

    In response to salt, Paenibacillus sp. strain XII expresses a 21.4 kDa polypeptide. N-terminal sequencing and sequence homology analysis indicate homology between the N-terminal sequence of the polypeptide and a segment of the N-terminus of the spore coat associated protein CotN of Oceanobacillus iheyensis, an extremely halotolerant bacteria of the deep-sea.

  11. Vasoactive Intestinal Polypeptide and Muscarinic Receptors: Supersensitivity Induced by Long-Term Atropine Treatment

    NASA Astrophysics Data System (ADS)

    Hedlund, Britta; Abens, Janis; Bartfai, Tamas

    1983-04-01

    Long-term treatment of rats with atropine induced large increases in the numbers of muscarinic receptors and receptors for vasoactive intestinal polypeptide in the salivary glands. Since receptors for vasoactive intestinal polypeptide coexist with muscarinic receptors on the same neurons in this preparation, the results suggest that a drug that alters the sensitivity of one receptor may also affect the sensitivity of the receptor for a costored transmitter and in this way contribute to the therapeutic or side effects of the drug.

  12. EXPRESSED PROTEIN LIGATION. A NEW TOOL FOR THE BIOSYNTHESIS OF CYCLIC POLYPEPTIDES

    SciTech Connect

    Kimura, R; Camarero, J A

    2004-11-11

    The present paper reviews the use of expressed protein ligation for the biosynthesis of backbone cyclic polypeptides. This general method allows the in vivo and in vitro biosynthesis of cyclic polypeptides using recombinant DNA expression techniques. Biosynthetic access to backbone cyclic peptides opens the possibility to generate cell-based combinatorial libraries that can be screened inside living cells for their ability to attenuate or inhibit cellular processes.

  13. Islet amyloid polypeptide (IAPP): a second amyloid in Alzheimer's disease.

    PubMed

    Fawver, Janelle N; Ghiwot, Yonatan; Koola, Catherine; Carrera, Wesley; Rodriguez-Rivera, Jennifer; Hernandez, Caterina; Dineley, Kelly T; Kong, Yu; Li, Jianrong; Jhamandas, Jack; Perry, George; Murray, Ian V J

    2014-01-01

    Amyloid formation is the pathological hallmark of type 2 diabetes (T2D) and Alzheimer's disease (AD). These diseases are marked by extracellular amyloid deposits of islet amyloid polypeptide (IAPP) in the pancreas and amyloid β (Aβ) in the brain. Since IAPP may enter the brain and disparate amyloids can cross-seed each other to augment amyloid formation, we hypothesized that pancreatic derived IAPP may enter the brain to augment misfolding of Aβ in AD. The corollaries for validity of this hypothesis are that IAPP [1] enters the brain, [2] augments Aβ misfolding, [3] associates with Aβ plaques, and most importantly [4] plasma levels correlate with AD diagnosis. We demonstrate the first 3 corollaries that: (1) IAPP is present in the brain in human cerebrospinal fluid (CSF), (2) synthetic IAPP promoted oligomerization of Aβ in vitro, and (3) endogenous IAPP localized to Aβ oligomers and plaques. For the 4th corollary, we did not observe correlation of peripheral IAPP levels with AD pathology in either an African American cohort or AD transgenic mice. In the African American cohort, with increased risk for both T2D and AD, peripheral IAPP levels were not significantly different in samples with no disease, T2D, AD, or both T2D and AD. In the Tg2576 AD mouse model, IAPP plasma levels were not significantly elevated at an age where the mice exhibit the glucose intolerance of pre-diabetes. Based on this negative data, it appears unlikely that peripheral IAPP cross-seeds or "infects" Aβ pathology in AD brain. However, we provide novel and additional data which demonstrate that IAPP protein is present in astrocytes in murine brain and secreted from primary cultured astrocytes. This preliminary report suggests a potential and novel association between brain derived IAPP and AD, however whether astrocytic derived IAPP cross-seeds Aβ in the brain requires further research.

  14. Characterization of Mixed Polypeptide Colloidal Particles by Light Scattering

    NASA Astrophysics Data System (ADS)

    Shuman, Hannah E.; Gaeckle, Grace K.; Gavin, John; Holland, Nolan B.; Streletzky, Kiril A.

    2014-03-01

    Temperature-dependent polymer surfactants have been developed by connecting three elastin-like polypeptide (ELP) chains to a charged protein domain (foldon), forming a three-armed star polymer. At low temperatures the polymer is soluble, while at higher temperatures it forms micelles. The behavior of mixtures of the three-armed star ELP (E20-Foldon) and H40-Linear ELP chains was analyzed under different salt and protein concentrations and various foldon to linear ELP ratio using Depolarized Dynamic Light Scattering. It was expected that under certain conditions the pure E20-Foldon would form spherical micelles, which upon adding the linear ELP would change in size and possibly shape. The pure E20-Foldon indeed formed largely spherical micelles with Rh of 10-20nm in solutions with 15-100mM salt and protein concentration between 10 μM and 100 μM. For the mixtures of 50 μM E20-Foldon and varying concentrations of H40-Linear in 25mM of salt, it was discovered that low and high H40-Linear concentration (4 μM and 50 μM) had only one transition. For the mixtures with of 10 and 25 μM of H40-Linear the two distinct transition temperatures were observed by spectrophotometry. The first transition corresponded to significantly elongated diffusive particles of apparent Rh of 30-50nm, while the second transition corresponded to slightly anisotropic diffusive particles with apparent Rh of about 20nm. At all H40-Linear concentrations studied, diffusive particles were seen above the second transition. Their radius and ability to depolarize light increased with the increase of H40-Linear concentration.

  15. New Kunitz-Type HCRG Polypeptides from the Sea Anemone Heteractis crispa.

    PubMed

    Gladkikh, Irina; Monastyrnaya, Margarita; Zelepuga, Elena; Sintsova, Oksana; Tabakmakher, Valentin; Gnedenko, Oksana; Ivanov, Alexis; Hua, Kuo-Feng; Kozlovskaya, Emma

    2015-09-24

    Sea anemones are a rich source of Kunitz-type polypeptides that possess not only protease inhibitor activity, but also Kv channels toxicity, analgesic, antihistamine, and anti-inflammatory activities. Two Kunitz-type inhibitors belonging to a new Heteractis crispa RG (HCRG) polypeptide subfamily have been isolated from the sea anemone Heteractis crispa. The amino acid sequences of HCRG1 and HCRG2 identified using the Edman degradation method share up to 95% of their identity with the representatives of the HCGS polypeptide multigene subfamily derived from H. crispa cDNA. Polypeptides are characterized by positively charged Arg at the N-terminus as well as P1 Lys residue at their canonical binding loop, identical to those of bovine pancreatic trypsin inhibitor (BPTI). These polypeptides are shown by our current evidence to be more potent inhibitors of trypsin than the known representatives of the HCGS subfamily with P1Thr. The kinetic and thermodynamic characteristics of the intermolecular interactions between inhibitors and serine proteases were determined by the surface plasmon resonance (SPR) method. Residues functionally important for polypeptide binding to trypsin were revealed using molecular modeling methods. Furthermore, HCRG1 and HCRG2 possess anti-inflammatory activity, reducing tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) secretions, as well as proIL-1β expression in lipopolysaccharide (LPS)-activated macrophages. However, there was no effect on nitric oxide (NO) generation.

  16. Polypeptide Point Modifications with Fatty Acid and Amphiphilic Block Copolymers for Enhanced Brain Delivery

    PubMed Central

    Batrakova, Elena V.; Vinogradov, Serguei V.; Robinson, Sandra M.; Niehoff, Michael L.; Banks, William A.; Kabanov, Alexander V.

    2009-01-01

    There is a tremendous need to enhance delivery of therapeutic polypeptides to the brain to treat disorders of the central nervous system (CNS). The brain delivery of many polypeptides is severely restricted by the blood—brain barrier (BBB). The present study demonstrates that point modifications of a BBB-impermeable polypeptide, horseradish peroxidase (HRP), with lipophilic (stearoyl) or amphiphilic (Pluronic block copolymer) moieties considerably enhance the transport of this polypeptide across the BBB and accumulation of the polypeptide in the brain in vitro and in vivo. The enzymatic activity of the HRP was preserved after the transport. The modifications of the HRP with amphiphilic block copolymer moieties through degradable disulfide links resulted in the most effective transport of the HRP across in vitro brain microvessel endothelial cell monolayers and efficient delivery of HRP to the brain. Stearoyl modification of HRP improved its penetration by about 60% but also increased the clearance from blood. Pluronic modification using increased penetration of the BBB and had no significant effect on clearance so that uptake by brain was almost doubled. These results show that point modification can improve delivery of even highly impermeable polypeptides to the brain. PMID:16029020

  17. [Determination of Ca2+ content in human enamel by electroprobe after polypeptide tooth paste usage].

    PubMed

    Zhang, Bin; Liu, Zheng; Zhang, Wei-guo

    2002-03-01

    The purpose of this study was to investigate the capability of polypeptide tooth paste for remineralization of human enamel. 8 enamel slabs,which were taken from extracted tooth for orthodontics reason, were immersed in artificial cariogenic solution for 0.5h. Then,the slabs were treated by tooth paste with polypeptide and tooth paste without polypeptide for 5 min. After tooth paste were washed away by warm water, the slabs were immersed in artificial saliva. The experiment were carried out twice per day for 15 days. Two slabs were taken from test group(treated by tooth paste with polypeptide) and control group (treated by tooth paste without polypeptide) on the twelfth day and fifteenth day, respectively. The Ca(2+) content in enamel slabs were determined by electroprobe and analyzed by image process. The content of Ca(2+) in test group was higher than that in control group significantly. The polypeptide tooth paste has certain capability of inhibiting demineralization for human enamel.

  18. A polypeptide from shark troponin I can inhibit angiogenesis and tumor growth.

    PubMed

    Xie, Qiuling; Yao, Sheng; Chen, Xiaojia; Xu, Lihui; Peng, Wendan; Zhang, Ling; Zhang, Qihao; Liang, Xu-Fang; Hong, An

    2012-02-01

    The shark troponin I gene (TnI) was found for the first time in this study to inhibit endothelial cell proliferation and angiogenesis. This shark TnI had 68.9% amino acid homology with human TnI, whereas the polypeptide from Lys91 to Leu123, which is thought to be the active site of TnI, had 78.8% homology with the corresponding fragment of human TnI. However, the polypeptide of shark had higher activity to inhibit the proliferation of HUVEC and tumor cell lines than that of human TnI. To investigate the anti-angiogenesis and anti-tumor effect of the shark TnI polypeptide, the DNA sequence of polypeptide (Lys91-Leu123) of white-spot catshark TnI(psTnI) was cloned and fused with the His-SUMO cDNA, followed by expression in Escherichia coli. After its purification by Ni(2+) affinity chromatography, the fusion His-SUMO-psTnI protein was digested with the SUMO enzyme to release psTnI. The inhibitory ability of this recombinant shark TnI polypeptide for angiogenesis was confirmed by chicken embryo allantoic membrane (CAM) test and IHC analysis. It was also found by breast carcinoma xenograft study in Balb/c mice that this polypeptide could inhibit tumor growth in vivo.

  19. Collagen and keratin polypeptide models for assessing the natural and artificial protein decay of organic materials.

    PubMed

    Fotou, Evmorfia; Sakarellos-Daitsiotis, Maria; Ioakeimoglou, Eleni; Tziamourani, Eleni; Malea, Ekaterini; Panayiaris, George; Panou-Pomonis, Eugenia

    2016-11-01

    Among the materials constituting the natural and cultural heritage, organic materials of proteinaceous origin as bone (collagen), parchment and woolen textiles (keratin) are the most susceptible to damage and decay because of their exposure to air pollution, inappropriate values of ambient temperature, humidity and light. Aiming at contributing to the development of a reliable and reproducible immunoassay for the evaluation of collagen and keratin decay, three polypeptide models of these proteins were designed, synthesized and studied. Polypeptide [Pro-Ser(OBzl)-Gly]n incorporates the typical motif Pro-X-Gly of collagen; polypeptide [Pro-Cys(Acm)-Gly]n is a model of the C-terminal domain of type I keratin, corresponding to the repeating unit Pro-Cys-X of keratin, while polypeptide Ac-YRSGGGFGYRSGGGFGYRS-βAla-NH2 encloses the characteristic repeating sequence GGGFGYRS of the N-terminal part of Type II keratin. These polypeptides may be considered as simplified models that mimic fragments of collagen and keratin resulting from artificial and natural ageing or decay. It is concluded that high recognition of anti-polypeptide antibodies, produced after immunizations, by the bone, parchment and textile samples is indicative of high deterioration, while high anti-collagen or anti-keratin recognition is indicative of low deterioration. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  20. Synthesis of Tacaribe virus polypeptides in an in vitro coupled transcription and translation system.

    PubMed

    Boersma, D P; Compans, R W

    1985-04-01

    We have analyzed polypeptides synthesized in a coupled in vitro transcription and translation system in response to detergent-disrupted Tacaribe virus. Analysis of the major Tacaribe virus-specified product by two-dimensional polyacrylamide gel electrophoresis indicated that it had an isoelectric point similar to that of the Tacaribe nucleocapsid polypeptide N; however, the in vitro product had an approximate mol. wt. of 73 000, compared to a mol. wt. of 68 000 for the N protein. The 73 000 dalton product was found to yield proteolytic cleavage products with similar electrophoretic mobilities to those obtained from the virion P and N proteins. These results, as well as pulse-chase experiments in Tacaribe virus-infected cells, suggest that a 73 000 dalton polypeptide may be processed to yield the N polypeptide. The polypeptides synthesized in the coupled system depended on the amount and type of virus added; addition of purified Shark River (SR) virus, a member of the Patois group of bunyaviruses, resulted in synthesis of a polypeptide of mol. wt. 22 000 which corresponds to the SR nucleocapsid protein.

  1. Construction and characterization of an experimental ISCOMS-based hepatitis B polypeptide vaccine

    PubMed Central

    Guan, Xiao-Ju; Guan, Xiao-Jun; Wu, Yu-Zhang; Jia, Zheng-Cai; Shi, Tong-Dong; Tang, Yan

    2002-01-01

    AIM: To characterize the biochemical and immunological properties of an experimental ISCOMS vaccine prepared from a novel therapeutic polypeptide based on T cell epitopes of HBsAg, and a heptatis B-ISCOMS was prepared and investigated. METHODS: An immunostimulating complexes (ISCOMS)-based vaccine containing a novel therapeutic hepatits B polypeptide was prepared by dialysis method, and its formation was visualized by electron microscopy and biochemically verified by SDS-polyacrylamide gel electrophoresis. Amount of the peptide within ISCOMS was determined by Bradford assay, and specific CTL response was detected by ELISPOT assay. RESULTS: Typical cage-like structures of submicroparticle with a diameter of about 40 nm were observed by electron microscopy. Results from Bradford assay showed that the level of peptide incorporation was about 0.33 g•L⁻¹. At the paralleled position close to the sixth band of the molecular weight marker (3480 kDa) a clear band was shown in SDS-PAGE analysis, indicating successful incorporation of polypeptide into ISCOMS. It is suggested that ISCOMS delivery system could efficiently improve the immunogenicity of polypeptide and elicit specific immune responses in vivo by the results of ELISPOT assay, which showed that IFN-γ producing cells (specific CTL responses) were increased (spots of ISCOMS-treated group: 47 ± 5, n = 3; control group: 5 ± 2, n = 3). CONCLUSION: ISCOMS-based hepatitis B polypeptide vaccine is successfully constructed and it induces a higher CTL response compared with short polypeptides vaccine in vivo. PMID:11925610

  2. Polypeptide composition of urea- and heat-resistant mutants of poliovirus types 1 and 2.

    PubMed

    Fennell, R; Phillips, B A

    1974-10-01

    Five urea-resistant and two heat-resistant mutants of poliovirus types 1 and 2 were isolated and their structural and nonstructural polypeptides compared to those of their wild-type, parental strains in an attempt to correlate mutant phenotypes with alterations in specific capsid polypeptides. Four of the seven mutants were found to contain polypeptides which differed in molecular weight from their respective parental viruses. However, resistance of virions to heat- or urea-inactivation could not be attributed to changes in particular capsid polypeptides because alterations were detected in all but one of the capsid components. For two of the urea-resistant mutants and one heat-resistant mutant, no differences were found in the molecular weights of the capsid and noncapsid polypeptides. These results, and the fact that at least 12 selective treatments were required to obtain stable mutants, indicate that: (i) such phenotypes probably can be expressed by mutations affecting one or more of the larger capsid polypeptides, and (ii) such phenotypes reflect multiple mutational steps.

  3. Proline-rich polypeptides in Alzheimer's disease and neurodegenerative disorders -- therapeutic potential or a mirage?

    PubMed

    Gladkevich, A; Bosker, F; Korf, J; Yenkoyan, K; Vahradyan, H; Aghajanov, M

    2007-10-01

    The development of effective and safe drugs for a growing Alzheimer disease population is an increasing need at present. Both experimental and clinical evidence support a beneficial effect of proline-rich polypeptides in a number of neurodegenerative diseases, including Alzheimer disease. Experimental data have shown that proline-rich polypeptides isolated from bovine neurohypophisis possess neuroprotective and neuromodulatory properties in mice with aluminum neurotoxicosis or neuronal damage caused by venoms and toxins. Proline-rich polypeptides from ovine colostrums, so called Colostrinin, have been shown to produce cognitive improvement in an experimental model and in patients with Alzheimer disease. However, the precise mechanism underlying the neuroprotective action of proline-rich polypeptides is not very well established. Moreover, studies pointing at a neuroprotective effect of proline-rich polypeptides from bovine neurohypophisis in humans have not been reported thus far. The authors conclude that more detailed information on the mode of action of proline-rich polypeptides is needed as well as confirmation of their efficacy in broad clinical trials before this approach can really show its potential in the treatment of neurodegenerative disorders.

  4. Interaction of Actinomyces Organisms with Cationic Polypeptides I. Histochemical Studies of Infected Human and Animal Tissues

    PubMed Central

    Crawford, James J.

    1971-01-01

    Histochemical techniques were used to study the nature of acidophilic hyaline clubs arranged radially at the peripheries of Actinomyces colonies in infected lung tissues of two persons. Concentrations of arginine-rich polypeptides were demonstrated in the acidophilic areas and in the cytoplasm of granulocytic leukocytes surrounding the colonies. Exposure of Actinomyces organisms to strongly cationic polypeptides (protamine, histone) in vitro killed the organisms and caused them to develop acidophilic staining. Weakly cationic proteins, ribonuclease, and hemoglobin produced no such effects. No acidophilic component could be detected in fresh broth-grown organisms themselves. Viable and nonviable colonies of the test strain lacking hyaline clubs were injected beneath the skin of guinea pigs. Agrinine-rich cationic polypeptides were evident in the cytoplasm of surrounding leukocytes and permeating the microbial colonies. In light of current evidence pertaining to leukocyte lysosomes and capsule production by Actinomyces and related organisms, the acidophilic hyaline clubs observed in human tissues appear to be a combination of a capsular component of the actinomycete and a cationic polypeptide component of host leukocytes. Organisms deeper in the human tissue colonies retained their normal basophilic reaction, suggesting a protective role for the peripheral hyaline club matrix. The acidophilic club complexes serve to indicate the reaction of cationic polypeptides in response of the human host to infecting Actinomyces organisms. These observations also support a broader concept that antimicrobial polypeptides of leukocyte lysosomes are an important factor in response of both the human and animal host to infecting bacteria. Images PMID:4117293

  5. The design, synthesis, and characterization of novel alanine-rich polypeptides with varied functional group density

    NASA Astrophysics Data System (ADS)

    Farmer, Robin S.

    Protein engineering methods have proven valuable for the synthesis of protein-based polymers with controlled conformational properties and functional group placement for use in a variety of biological and materials applications. These strategies were employed to produce alanine-rich polypeptides with the general sequence [(AAAQ)y(AAAE)(AAAQ)y]x, which utilizes the high helical propensity of alanine and chemical functionality of glutamic acid. Modifications to the general sequence allow for variations in both the spacing between and the number of glutamic acid residues along the protein backbone. Three families of alanine-rich polypeptides with similar amino acid compositions were designed with glutamic acid residues displayed at nominal distances of 17A, 35A, and 65A. From these three families, four of these polypeptides were focused on for this work, 17-H-3, 17-H-6, 35-H-6 and 65-H-2. Understanding the conformational and thermal behavior of the polypeptides can give insight into how these molecules will behave after functionalization. The conformational behavior of the four polypeptides from the three alanine-rich families have been investigated via circular dichroic spectroscopy under multiple solution conditions; pH 2.3, 10 mM phosphate, pH 2.3, 10 mM phosphate, 150 mM NaCl, and pH 7.4 PBS. All the polypeptides adopt an alpha-helical conformation under all solution conditions and exhibit an alpha-helical to non-alpha-helical transition with increasing temperature. In pH 2.3, 10 mM phosphate buffer, the conformation differs between sequences at high temperature and high polypeptide concentration. Although the compositions of the three families are similar, changes in the amino acid sequences result in variations in hydrophobicity. The most hydrophobic sequence, 65-H-2 , undergoes the helix-to-coil transition but at high polypeptide concentrations and temperatures above 45°C, the polypeptide irreversibly adopts a beta-sheet structure. The less hydrophobic

  6. Identification of origin of two polypeptides of 4 and 5 kD isolated from human lenses.

    PubMed

    Srivastava, O P; Srivastava, K; Silney, C

    1994-01-01

    To purify crystallin fragments (degraded polypeptides molecular weight < 18 kD) and identify their parent crystallins. The purification of polypeptides with apparent molecular weights of 4 and 5 kD was carried out using three sequential steps: Sephadex G-50 chromatography under denaturing conditions, preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and high-performance liquid chromatography using a C-18 column. The parent crystallins of the two polypeptides were identified by the Western blotting method using polyclonal antibodies raised against individual 4 and 5 kD polypeptides and by comparing N-terminal amino acid sequences of the polypeptides with crystallins. Two polypeptides of 4 and 5 kD were purified by the three sequential steps as described from water-soluble proteins of lenses from 60-80-year-old donors. Both purified polypeptides showed a single major peak during high-performance liquid chromatography on a C-18 column and also a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Western blot analyses showed maximum immunoreactivity of the anti-4 kD polypeptide antibody to a 22 kD species of beta-crystallin, whereas the anti-5 kD polypeptide antibody showed maximum reactivity to only the alpha B crystallin. These results were further confirmed during comparison of the N-terminal amino acid sequences of the two polypeptides with crystallins. Such comparison showed that the 4 kD polypeptide originated from beta A 3/A1 crystallin after cleavage at His187-His188 bond. Further, the 5 kD polypeptide was a fragment of alpha B crystallin that originated after cleavage at Val145-Asn146 bond. These results showed that specific bonds of beta A3/A1 and alpha B crystallins are posttranslationally cleaved in vivo to produce 4 kD and 5 kD polypeptides, respectively.

  7. Interaction of Silymarin Flavonolignans with Organic Anion-Transporting Polypeptides

    PubMed Central

    Köck, Kathleen; Xie, Ying; Oberlies, Nicholas H.; Brouwer, Kim L. R.

    2013-01-01

    Organic anion-transporting polypeptides (OATPs) are multispecific transporters mediating the uptake of endogenous compounds and xenobiotics in tissues that are important for drug absorption and elimination, including the intestine and liver. Silymarin is a popular herbal supplement often used by patients with chronic liver disease; higher oral doses than those customarily used (140 mg three times/day) are being evaluated clinically. The present study examined the effect of silymarin flavonolignans on OATP1B1-, OATP1B3-, and OATP2B1-mediated transport in cell lines stably expressing these transporters and in human hepatocytes. In overexpressing cell lines, OATP1B1- and OATP1B3-mediated estradiol-17β-glucuronide uptake and OATP2B1-mediated estrone-3-sulfate uptake were inhibited by most of the silymarin flavonolignans investigated. OATP1B1-, OATP1B3-, and OATP2B1-mediated substrate transport was inhibited efficiently by silymarin (IC50 values of 1.3, 2.2 and 0.3 µM, respectively), silybin A (IC50 values of 9.7, 2.7 and 4.5 µM, respectively), silybin B (IC50 values of 8.5, 5.0 and 0.8 µM, respectively), and silychristin (IC50 values of 9.0, 36.4, and 3.6 µM, respectively). Furthermore, silymarin, silybin A, and silybin B (100 µM) significantly inhibited OATP-mediated estradiol-17β-glucuronide and rosuvastatin uptake into human hepatocytes. Calculation of the maximal unbound portal vein concentrations/IC50 values indicated a low risk for silymarin-drug interactions in hepatic uptake with a customary silymarin dose. The extent of silymarin-drug interactions depends on OATP isoform specificity and concentrations of flavonolignans at the site of drug transport. Higher than customary doses of silymarin, or formulations with improved bioavailability, may increase the risk of flavonolignan interactions with OATP substrates in patients. PMID:23401473

  8. Isolation of two polypeptides comprising the neutrophil-immobilizing factor of human leucocytes.

    PubMed Central

    Watt, K W; Brightman, I L; Goetzl, E J

    1983-01-01

    Human leucocyte lysosomal polypeptides of mol. wt 4000-5000, which constitute the neutrophil-immobolizing factor (NIF), were isolated from the 22,000 g supernate of sonicates of human neutrophils by filtration on Sephadex G-75. The larger (NIF-1) and smaller (NIF-2) of the polypeptides were resolved by filtration on Bio-Gel P6 and purified to homogeneity by sequential reverse-phase high performance liquid chromatography and paper electrophoresis. The results of analyses of amino acid composition indicated that NIF-1 and NIF-2 are distinct polypeptides composed of an apparent total of 41 and 38 amino acids, respectively. Both NIF polypeptides contain one cysteine and one methionine, lack isoleucine, tyrosine and phenylalanine, and are rich in histidine and proline. The sequence of 20 of the amino-terminal amino acids of both NIF polypeptides is identical, but NIF-2 possesses an additional alanine at the amino-terminus. Highly purified NIF-1 and NIF-2 inhibited human neutrophil random migration and chemotaxis to diverse stimuli in a concentration-dependent manner, with 50% inhibition of chemotaxis by 0.31-1 x 10(-8) M NIF-1 and 1-3 x 10(-7) M NIF-2. Neither NIF polypeptide was cytotoxic for neutrophils, altered neutrophil phagocytosis or release of lysosomal enzymes, or inhibited mononuclear leucocyte chemotaxis. The leucocyte and functional specificity of the NIF polypeptides and the quantitites released upon stimulation of the human leucocytes suggest that the transition to a mononuclear leucocyte population in chronic inflammation may be attributable in part to the NIF derived from the leucocyte infiltrates of acute responses. PMID:6848456

  9. The mining of toxin-like polypeptides from EST database by single residue distribution analysis.

    PubMed

    Kozlov, Sergey; Grishin, Eugene

    2011-01-31

    Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening. Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined. The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed.

  10. Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide.

    PubMed

    Yang, Jie; Koruza, Katarina; Fisher, Zoë; Knecht, Wolfgang; Baltzer, Lars

    2017-10-15

    The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3nM, whereas that of the 4-C10L17-AZM conjugate was 90pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to μM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. The mining of toxin-like polypeptides from EST database by single residue distribution analysis

    PubMed Central

    2011-01-01

    Background Novel high throughput sequencing technologies require permanent development of bioinformatics data processing methods. Among them, rapid and reliable identification of encoded proteins plays a pivotal role. To search for particular protein families, the amino acid sequence motifs suitable for selective screening of nucleotide sequence databases may be used. In this work, we suggest a novel method for simplified representation of protein amino acid sequences named Single Residue Distribution Analysis, which is applicable both for homology search and database screening. Results Using the procedure developed, a search for amino acid sequence motifs in sea anemone polypeptides was performed, and 14 different motifs with broad and low specificity were discriminated. The adequacy of motifs for mining toxin-like sequences was confirmed by their ability to identify 100% toxin-like anemone polypeptides in the reference polypeptide database. The employment of novel motifs for the search of polypeptide toxins in Anemonia viridis EST dataset allowed us to identify 89 putative toxin precursors. The translated and modified ESTs were scanned using a special algorithm. In addition to direct comparison with the motifs developed, the putative signal peptides were predicted and homology with known structures was examined. Conclusions The suggested method may be used to retrieve structures of interest from the EST databases using simple amino acid sequence motifs as templates. The efficiency of the procedure for directed search of polypeptides is higher than that of most currently used methods. Analysis of 39939 ESTs of sea anemone Anemonia viridis resulted in identification of five protein precursors of earlier described toxins, discovery of 43 novel polypeptide toxins, and prediction of 39 putative polypeptide toxin sequences. In addition, two precursors of novel peptides presumably displaying neuronal function were disclosed. PMID:21281459

  12. Biochemical basis of prolidase deficiency. Polypeptide and RNA phenotypes and the relation to clinical phenotypes.

    PubMed Central

    Endo, F; Tanoue, A; Kitano, A; Arata, J; Danks, D M; Lapière, C M; Sei, Y; Wadman, S K; Matsuda, I

    1990-01-01

    Cultured skin fibroblasts or lymphoblastoid cells from eight patients with clinical symptoms of prolidase deficiency were analyzed in terms of enzyme activity, presence of material crossreacting with specific antibodies, biosynthesis of the polypeptide, and mRNA corresponding to the enzyme. There are at least two enzymes that hydrolyze imidodipeptides in these cells and these two enzymes could be separated by an immunochemical procedure. The specific assay for prolidase showed that the enzyme activity was virtually absent in six cell strains and was markedly reduced in two (less than 3% of controls). The activities of the labile enzyme that did not immunoprecipitate with the anti-prolidase antibody were decreased in the cells (30-60% of controls). Cell strains with residual activities of prolidase had immunological polypeptides crossreacting with a Mr 56,000, similar to findings in the normal enzyme. The polypeptide biosynthesis in these cells and the controls was similar. Northern blot analyses revealed the presence of mRNA in the polypeptide-positive cells, yet it was absent in the polypeptide-negative cells. The substrate specificities analyzed in the partially purified enzymes from the polypeptide-positive cell strains differed, presumably due to different mutations. Thus, there seems to be a molecular heterogeneity in prolidase deficiency. There was no apparent relation between the clinical symptoms and the biochemical phenotypes, except that mental retardation was present in the polypeptide-negative patients. The activities of the labile enzyme may not be a major factor in modifying the clinical symptoms. Images PMID:1688567

  13. Evaluation of membrane models and their composition for islet amyloid polypeptide-membrane aggregation.

    PubMed

    Caillon, Lucie; Lequin, Olivier; Khemtémourian, Lucie

    2013-09-01

    Human islet amyloid polypeptide (IAPP) forms amyloid fibrils in the pancreatic islets of patients suffering from type 2 diabetes mellitus (T2DM). The formation of IAPP fibrils has been shown to cause membrane damage which most likely is responsible for the death of pancreatic islet β-cells during the pathogenesis of T2DM. Several studies have demonstrated a clear interaction between IAPP and lipid membranes. However the effect of different lipid compositions and of various membrane mimetics (including micelles, bicelles, SUV and LUV) on fibril formation kinetics and fibril morphology has not yet systematically been analysed. Here we report that the interaction of IAPP with various membrane models promoted different processes of fibril formation. Our data reveal that in SDS and DPC micelles, IAPP adopts a stable α-helical structure for several days, suggesting that the micelle models may stabilize monomeric or small oligomeric species of IAPP. In contrast, zwitterionic DMPC/DHPC bicelles and DOPC SUV accelerate the fibril formation compared to zwitterionic DOPC LUV, indicating that the size of the membrane model and its curvature influence the fibrillation process. Negatively charged membranes decrease the lag-time of the fibril formation kinetics while phosphatidylethanolamine and cholesterol have an opposite effect, probably due to the modulation of the physical properties of the membrane and/or due to direct interactions with IAPP within the membrane core. Finally, our results show that the modulation of lipid composition influences not only the growth of fibrils at the membrane surface but also the interactions of β-sheet oligomers with membranes. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Inhibitory Mechanism of EGCG on Fibrillation and Aggregation of Amidated Human Islet Amyloid Polypeptide.

    PubMed

    Xu, Zhixue; Ma, Gongli; Zhang, Qiang; Chen, Congheng; He, Yanming; Xu, Lihui; Zhou, Guangrong; Li, Zhenhua; Yang, Hongjie; Zhou, Ping

    2017-03-15

    The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) is associated with development of T2DM. EGCG can bind amyloid proteins to inhibit the fibrillation of these proteins. Here, we sought to investigate the effect of EGCG on amidated hIAPP (hIAPP-NH2) fibrillation and aggregation by using spectroscopic and microscopic techniques, and also sought to view insight into the interaction of EGCG and hIAPP22-27 by using spectroscopic experiments and quantum chemical calculations. ThT fluorescence, real-time NMR and TEM studies demonstrated that EGCG could inhibit the formation of hIAPP-NH2 fibrils, while promote the formation of hIAPP-NH2 amorphous aggregates. Phenylalanine intrinsic fluorescence and NMR studies of EGCG/hIAPP22-27 complex revealed three important binding sites including A-ring of EGCG, residue Phe23 and residue Ile26. DFT calculation identified the dominant binding structures of EGCG/Phe23 and EGCG/Ile26 complexes, named Structure I and Structure II, respectively. Our study demonstrates the inhibitory mechanism of EGCG on fibrillation and aggregation of hIAPP-NH2 in which EGCG interacts with hIAPP-NH2 through hydrogen bonding and π-π interaction between A-ring and residue Phe23 as well as hydrophobic interactions between A-ring and residue Ile26, thus can inhibit the inter-peptide interaction between hIAPP-NH2 monomers and finally inhibit fibrillation of hIAPP-NH2. This study offers an intuitive explanation at molecular level.

  15. Long-acting glucose-dependent insulinotropic polypeptide ameliorates obesity-induced adipose tissue inflammation.

    PubMed

    Varol, Chen; Zvibel, Isabel; Spektor, Lior; Mantelmacher, Fernanda Dana; Vugman, Milena; Thurm, Tamar; Khatib, Marian; Elmaliah, Elinor; Halpern, Zamir; Fishman, Sigal

    2014-10-15

    Obesity induces low-grade chronic inflammation, manifested by proinflammatory polarization of adipose tissue innate and adaptive resident and recruited immune cells that contribute to insulin resistance (IR). The glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that mediates postprandial insulin secretion and has anabolic effects on the adipose tissue. Importantly, recent evidence suggested that GIP is a potential suppressor of inflammation in several metabolic models. In this study, we aimed to investigate the immunoregulatory role of GIP in a murine model of diet-induced obesity (DIO) using the long-acting GIP analog [d-Ala(2)]GIP. Administration of [d-Ala(2)]GIP resulted in adipocytes of increased size, increased levels of adipose tissue lipid droplet proteins, indicating better lipid storage capacity, and reduced adipose tissue inflammation. Flow cytometry analysis revealed reduced numbers of inflammatory Ly6C(hi) monocytes and F4/80(hi)CD11c(+) macrophages, associated with IR. In addition, [d-Ala(2)]GIP reduced adipose tissue infiltration of IFN-γ-producing CD8(+) and CD4(+) T cells. Furthermore, [d-Ala(2)]GIP treatment induced a favorable adipose tissue adipokine profile, manifested by a prominent reduction in key inflammatory cytokines (TNF-α, IL-1β, IFN-γ) and chemokines (CCL2, CCL8, and CCL5) and an increase in adiponectin. Notably, [d-Ala(2)]GIP also reduced the numbers of circulating neutrophils and proinflammatory Ly6C(hi) monocytes in mice fed regular chow or a high-fat diet. Finally, the beneficial immune-associated effects were accompanied by amelioration of IR and improved insulin signaling in liver and adipose tissue. Collectively, our results describe key beneficial immunoregulatory properties for GIP in DIO and reveal that its augmentation ameliorates adipose tissue inflammation and improves IR. Copyright © 2014 by The American Association of Immunologists, Inc.

  16. Investigation of phase separation behavior and formation of plasmonic nanocomposites from polypeptide-gold nanorod nanoassemblies.

    PubMed

    Huang, Huang-Chiao; Nanda, Alisha; Rege, Kaushal

    2012-04-24

    Genetically engineered elastin-like polypeptides (ELP) can be interfaced with cetyltrimethyl ammonium bromide (CTAB)-stabilized gold nanorods (GNRs) resulting in the formation of stable dispersions (nanoassemblies). Increasing the dispersion temperature beyond the ELP transition temperature results in phase separation and formation of solid-phase ELP-GNR matrices (nanocomposites). Here, we investigated different physicochemical conditions that influence nanocomposite formation from temperature-induced phase separation of ELP-GNR nanoassemblies. The presence of cetyltrimethyl ammonium bromide (CTAB), used to template the formation of gold nanorods, plays a significant role in the phase separation behavior, with high concentrations of the surfactant leading to dramatic enhancements in ELP transition temperature. Nanocomposites could be generated at 37 °C in the presence of low CTAB concentrations (<1.5 mM); higher concentrations of CTAB necessitated higher temperatures (60 °C) due to elevated transition temperatures. The concentration of gold nanorods, however, had minimal influence on the phase separation behavior and nanocomposite formation. Further analysis of the kinetics of nanocomposite formation using a mathematical model indicated that CTAB largely influenced the early event of coacervation of ELP-GNR nanoassemblies leading to nanocomposites, but had minimal effect on nanocomposite maturation, which is a later-stage longer event. Finally, nanocomposites prepared in the presence of low CTAB concentrations demonstrated a superior photothermal response following laser irradiation compared to those generated using higher CTAB concentrations. Our results on understanding the formation of plasmonic/photothermal ELP-GNR nanocomposites have significant implications for tissue engineering, regenerative medicine, and drug delivery.

  17. Differentiation between Phycobiliprotein and Colorless Linker Polypeptides by Fluorescence in the Presence of ZnSO41

    PubMed Central

    Raps, Shirley

    1990-01-01

    Microcystis aeruginosa, a unicellular cyanobacterium, contains small phycobilisomes consisting of C-phycocyanin, allophycocyanin, and linker polypeptides. SDS-polyacrylamide gels of the phycobilisomes were examined for fluorescent bands before and after spraying with a solution of ZnSO4, followed by Coomassie brilliant blue staining for protein. This procedure provides a rapid and sensitive method for detecting small amounts of phycobilin-containing polypeptides and distinguishing them from other tetrapyrrole-containing polypeptides and from `colorless' ones. Three polypeptide bands, in addition to the α and β phycobiliprotein subunits, have been detected under these conditions. An 85 kilodalton polypeptide was identified as a phycobiliprotein due to its enhanced fluorescence in the presence of ZnSO4. The other polypeptides do not contain chromophores and are colorless. They are approximately 34.5 and 30 kilodaltons in size. Images Figure 1 Figure 2 Figure 3 PMID:16667282

  18. Export is the default pathway for soluble unfolded polypeptides that accumulate during expression in Escherichia coli

    SciTech Connect

    Scotto-Lavino, E.; Freimuth, P.; Bai, M.; Zhang, Y.-B.

    2011-09-01

    Several E. coli endogenous, cytoplasmic proteins that are known clients of the chaperonin GroEL were overexpressed to examine the fate of accumulated unfolded polypeptides. Substantial fractions of about half of the proteins formed insoluble aggregates, consistent with the hypothesis that these proteins were produced at rates or in amounts that exceeded the protein-folding capacity of GroEL. In addition, large fractions of three overexpressed GroEL client proteins were localized in an extra-cytoplasmic, osmotically-sensitive compartment, suggesting they had initially accumulated in the cytoplasm as soluble unfolded polypeptides and thus were able to access a protein export pathway. Consistent with this model, an intrinsically unfoldable, hydrophilic, non-secretory polypeptide was quantitatively exported from the E. coli cytoplasm into an osmotically-sensitive compartment. Our results support the conclusion that a soluble, unfolded conformation alone may be sufficient to direct non-secretory polypeptides into a protein export pathway for signal peptide-independent translocation across the inner membrane, and that export rather than degradation by cytoplasmic proteases is the preferred fate for newly-synthesized, soluble, unfolded polypeptides that accumulate in the cytoplasm. The stable folded conformation of exported GroEL client proteins further suggests that the requirement for GroEL may be conditional on protein folding in the molecularly-crowded environment of the cytoplasm.

  19. Exposure of salivary gland cells to low-frequency electromagnetic fields alters polypeptide synthesis.

    PubMed Central

    Goodman, R; Henderson, A S

    1988-01-01

    This study demonstrates that exposure of cells to extremely low-frequency electromagnetic fields can cause measurable changes in protein synthesis. Sciara coprophila salivary gland cells were exposed to five low-frequency (1.5-72 Hz) electromagnetic signals: three signals (1.5, 15, and 72 Hz) produced pulsed asymmetric electromagnetic fields and two signals (60 and 72 Hz) were sinusoidal. Subsequent analyses of two-dimensional gels showed that cell exposure to either type of low-frequency electromagnetic field resulted in both qualitative and quantitative changes in patterns of protein synthesis. Thus, signals producing diverse waveform characteristics induced previously undetectable polypeptides, some of which were signal specific and augmented or suppressed other polypeptides as compared with nonexposed cells. The pattern of polypeptide synthesis differed from that seen with heat shock: only five polypeptides in cells exposed to electromagnetic signals overlap those polypeptides exposed to heat shock, and the suppression of protein synthesis characteristic of heat shock does not occur. Images PMID:3375247

  20. Is There a Role for the 42 Kilodalton Polypeptide in Inorganic Carbon Uptake by Cyanobacteria? 1

    PubMed Central

    Schwarz, Rakefet; Friedberg, Devorah; Kaplan, Aaron

    1988-01-01

    Cyanobacterial cells accumulate substantial amounts of a membrane-associated 42 kilodalton polypeptide during adaptation to low CO2 conditions. The role of this polypeptide in the process of adaptation and in particular in the large increase in the ability to accumulate inorganic carbon (Ci), which accompanies this process, is not yet understood. We have isolated a mutant Synechococcus PCC7942 that does not accumulate the 42 kilodalton polypeptide. The mutant requires a high-CO2 concentration for growth and exhibits a very low apparent photosynthetic affinity for extracellular Ci. The latter might be attributable to the observed defective ability of the mutant to utilize the intracellular Ci pool for photosynthesis. The 42 kilodalton polypeptide does not appear to participate directly in the active transport of Ci, since the difference between the observed capabilities for CO2 and HCO3− uptake of the mutant and the wild type is not sufficient to account for their different growth and photosynthetic performance. Furthermore, high CO2-grown wild-type cells, where we could not detect the 42 kilodalton polypeptide, transported CO2 faster than the mutant. An analysis of the curves relating the rate of accumulation of Ci to the concentration of CO2 or HCO3− supplied, in the presence or absence of carbonic anhydrase, indicated that under the experimental conditions used here, CO2 was the preferred Ci species taken up by Synechococcus. Images Fig. 1 PMID:16666296

  1. Fabrication of genetically engineered polypeptide@quantum dots hybrid nanogels for targeted imaging

    NASA Astrophysics Data System (ADS)

    Yang, Jie; Yao, Ming-Hao; Zhao, Dong-Hui; Zhang, Xiao-Shuai; Jin, Rui-Mei; Zhao, Yuan-Di; Liu, Bo

    2017-08-01

    Nanogels have been widely used as multifunctional drug delivery carriers because of high water content, biocompatibility, and high loading capability. We designed and biosynthesized two triblock artificial polypeptides PC10A and PC10ARGD as vehicles for encapsulating hydrophobic materials. These polypeptides can form nanogels by self-assembly when the concentration is below 2% ( w/ v). The physical properties of nanogels, including size, surface potential, and targeting domain, are able to be tuned. Hydrophobic materials from molecular size to nano-size can be loaded into the polypeptide nanogels to form hybrid nanogels. Hydrophobic quantum dots CdSe@ZnS below 10 nM were loaded into the polypeptide nanogels by ultrasonic treatment. Encapsulation endows hydrophobic QDs with good tunability of size, water solubility, stability, targeting, and biocompatibility. PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels showed excellent biocompatibility, which the cellular viabilities of HeLa and MCF-7 cells treated with 1% PC10ARGD nanogels and PC10ARGD@QDs hybrid nanogels contained 20 nM QDs were above 90 and 80%, respectively. PC10ARGD@QDs hybrid nanogels with an arginine-glycine-aspartic acid motif present efficient receptor-mediated endocytosis in α v β 3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy. These results demonstrate that such polypeptide nanogels as nanocarriers are expected to have great potential applications in biomedicine.

  2. Application of Statistical Thermodynamics To Predict the Adsorption Properties of Polypeptides in Reversed-Phase HPLC.

    PubMed

    Tarasova, Irina A; Goloborodko, Anton A; Perlova, Tatyana Y; Pridatchenko, Marina L; Gorshkov, Alexander V; Evreinov, Victor V; Ivanov, Alexander R; Gorshkov, Mikhail V

    2015-07-07

    The theory of critical chromatography for biomacromolecules (BioLCCC) describes polypeptide retention in reversed-phase HPLC using the basic principles of statistical thermodynamics. However, whether this theory correctly depicts a variety of empirical observations and laws introduced for peptide chromatography over the last decades remains to be determined. In this study, by comparing theoretical results with experimental data, we demonstrate that the BioLCCC: (1) fits the empirical dependence of the polypeptide retention on the amino acid sequence length with R(2) > 0.99 and allows in silico determination of the linear regression coefficients of the log-length correction in the additive model for arbitrary sequences and lengths and (2) predicts the distribution coefficients of polypeptides with an accuracy from 0.98 to 0.99 R(2). The latter enables direct calculation of the retention factors for given solvent compositions and modeling of the migration dynamics of polypeptides separated under isocratic or gradient conditions. The obtained results demonstrate that the suggested theory correctly relates the main aspects of polypeptide separation in reversed-phase HPLC.

  3. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    PubMed Central

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-01-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex. Images PMID:3100577

  4. Congenital deficiency of two polypeptide subunits of the iron-protein fragment of mitochondrial complex I.

    PubMed

    Moreadith, R W; Cleeter, M W; Ragan, C I; Batshaw, M L; Lehninger, A L

    1987-02-01

    Recently, we described a patient with severe lactic acidosis due to congenital complex I (NADH-ubiquinone oxidoreductase) deficiency. We now report further enzymatic and immunological characterizations. Both NADH and ferricyanide titrations of complex I activity (measured as NADH-ferricyanide reductase) were distinctly altered in the mitochondria from the patient's tissues. In addition, antisera against complex I immunoprecipitated NADH-ferricyanide reductase from the control but not the patient's mitochondria. However, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of complex I polypeptides demonstrated that the majority of the 25 polypeptides comprising complex I were present in the affected mitochondria. A more detailed analysis using subunit selective antisera against the main polypeptides of the iron-protein fragments of complex I revealed a selective absence of the 75- and 13-kD polypeptides. These findings suggest that the underlying basis for this patient's disease was a congenital deficiency of at least two polypeptides comprising the iron-protein fragment of complex I, which resulted in the inability to correctly assemble a functional enzyme complex.

  5. Polypeptide multilayer film co-delivers oppositely-charged drug molecules in sustained manners.

    PubMed

    Jiang, Bingbing; Defusco, Elizabeth; Li, Bingyun

    2010-12-13

    The current state-of-the-art for drug-carrying biomedical devices is mostly limited to those that release a single drug. Yet there are many situations in which more than one therapeutic agent is needed. Also, most polyelectrolyte multilayer films intended for drug delivery are loaded with active molecules only during multilayer film preparation. In this paper, we present the integration of capsules as vehicles within polypeptide multilayer films for sustained release of multiple oppositely charged drug molecules using layer-by-layer nanoassembly technology. Calcium carbonate (CaCO(3)) particles were impregnated with polyelectrolytes, shelled with polyelectrolyte multilayers, and then assembled onto polypeptide multilayer films using glutaraldehyde. Capsule-integrated polypeptide multilayer films were obtained after decomposition of CaCO(3) templates. Two oppositely charged drugs were loaded into capsules within polypeptide multilayer films postpreparation based on electrostatic interactions between the drugs and the polyelectrolytes impregnated within capsules. We determined that the developed innovative capsule-integrated polypeptide multilayer films could be used to load multiple drugs of very different properties (e.g., opposite charges) any time postpreparation (e.g., minutes before surgical implantation inside an operating room), and such capsule-integrated films allowed simultaneous delivery of two oppositely charged drug molecules and a sustained (up to two weeks or longer) and sequential release was achieved.

  6. Novel folded protein domains generated by combinatorial shuffling of polypeptide segments

    PubMed Central

    Riechmann, Lutz; Winter, Greg

    2000-01-01

    It has been proposed that the architecture of protein domains has evolved by the combinatorial assembly and/or exchange of smaller polypeptide segments. To investigate this proposal, we fused DNA encoding the N-terminal half of a β-barrel domain (from cold shock protein CspA) with fragmented genomic Escherichia coli DNA and cloned the repertoire of chimeric polypeptides for display on filamentous bacteriophage. Phage displaying folded polypeptides were selected by proteolysis; in most cases the protease-resistant chimeric polypeptides comprised genomic segments in their natural reading frames. Although the genomic segments appeared to have no sequence homologies with CspA, one of the originating proteins had the same fold as CspA, but another had a different fold. Four of the chimeric proteins were expressed as soluble polypeptides; they formed monomers and exhibited cooperative unfolding. Indeed, one of the chimeric proteins contained a set of very slowly exchanging amides and proved more stable than CspA itself. These results indicate that native-like proteins can be generated directly by combinatorial segment assembly from nonhomologous proteins, with implications for theories of the evolution of new protein folds, as well as providing a means of creating novel domains and architectures in vitro. PMID:10954734

  7. DVL, a novel class of small polypeptides: overexpression alters Arabidopsis development.

    PubMed

    Wen, Jiangqi; Lease, Kevin A; Walker, John C

    2004-03-01

    Small polypeptides can act as important regulatory molecules that coordinate cellular responses required for differentiation, growth, and development. In a gain-of-function genetic screen for genes that influence fruit development in Arabidopsis, we identified a novel gene -DEVIL1 (DVL1) - encoding a small protein. Overexpression of DVL1 results in pleiotropic phenotypes featured by shortened stature, rounder rosette leaves, clustered inflorescences, shortened pedicles, and siliques with pronged tips. cDNA analysis indicates that DVL1 has a 153-nucleotide (nt) open-reading frame (ORF) encoding a 51-amino acid polypeptide that shares no significant similarity to previously identified proteins. Sequence alignment shows that DVL1 belongs to a family of related genes that are limited to angiosperm plants. Ectopic overexpression of each of the five closely related Arabidopsis DVL genes causes similar phenotypic changes, suggesting overlapping function in the DVL gene family. Point mutations of conserved amino acids in the C-terminal region of the DVL1 polypeptide reveal that these conserved residues are required for DVL1-overexpression phenotypes. Our results show that the DVL family is a novel class of small polypeptides and the overexpression phenotypes suggest that these polypeptides may have a role in plant development.

  8. Fluorescence probe of polypeptide conformational dynamics in gas phase and in solution

    NASA Astrophysics Data System (ADS)

    Iavarone, Anthony T.; Meinen, Jan; Schulze, Susanne; Parks, Joel H.

    2006-07-01

    Fluorescence measurements of polypeptides derivatized with the fluorescent dye BODIPY TMR have been used to probe the polypeptide conformational dynamics as a function of temperature and charge state. Measurements of (BODIPY TMR)-[Pro]n-Arg-Trp and (BODIPY TMR)-[Gly-Ser]m-Arg-Trp have been performed for charge states 1+ and 2+ of n = 4 and 10 and m = 2 and 5. The 2+ charge states of both of these polypeptides exhibit similar temperature dependences for equal chain lengths (n = 4, m = 2 and n = 10, m = 5) and suggest conformations dominated by Coulomb repulsion. In the absence of such Coulomb repulsion, the 1+ charge state conformations appear to be characterized by the flexibility of the polypeptide chain for which [Gly-Ser]m > [Pro]n. Comparisons of these gas phase polypeptide measurements with corresponding measurements in solution provide a direct measure of the effects of solvent on the conformational dynamics. The change in fluorescence as a function of temperature in the gas phase is two orders of magnitude greater than that in solution, a dramatic result we attribute to the restrictions on intramolecular dynamics imposed by diffusion-limited kinetics and the lack of shielding by solvent. Measurements were also made of unsolvated Pron peptides without the tryptophan (Trp) residue to isolate the interaction of the fluorescent dye with charges.

  9. Oligosaccharyltransferase Subunits Bind Polypeptide Substrate to Locally Enhance N-glycosylation*

    PubMed Central

    Jamaluddin, M. Fairuz B.; Bailey, Ulla-Maja; Schulz, Benjamin L.

    2014-01-01

    Oligosaccharyltransferase is a multiprotein complex that catalyzes asparagine-linked glycosylation of diverse proteins. Using yeast genetics and glycoproteomics, we found that transient interactions between nascent polypeptide and Ost3p/Ost6p, homologous subunits of oligosaccharyltransferase, were able to modulate glycosylation efficiency in a site-specific manner in vivo. These interactions were driven by hydrophobic and electrostatic complementarity between amino acids in the peptide-binding groove of Ost3p/Ost6p and the sequestered stretch of substrate polypeptide. Based on this dependence, we used in vivo scanning mutagenesis and in vitro biochemistry to map the precise interactions that affect site-specific glycosylation efficiency. We conclude that transient binding of substrate polypeptide by Ost3p/Ost6p increases glycosylation efficiency at asparagines proximal and C-terminal to sequestered sequences. We detail a novel mode of interaction between translocating nascent polypeptide and oligosaccharyltransferase in which binding to Ost3p/Ost6p segregates a short flexible loop of glycosylation-competent polypeptide substrate that is delivered to the oligosaccharyltransferase active site for efficient modification. PMID:25118247

  10. Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes.

    PubMed Central

    Fujiki, Y; Rachubinski, R A; Lazarow, P B

    1984-01-01

    The manner of synthesis and assembly of the peroxisomal membrane proteins is unknown. Understanding these processes is essential to an understanding of the formation of the organelle. We have investigated the biogenesis of the previously identified major 21.7-kDa integral peroxisomal membrane polypeptide [Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, P. B. (1982) J. Cell Biol. 93, 103-110]. This protein was purified to apparent homogeneity and used to elicit a rabbit antiserum. In immunoblotting analysis, antibody bound only to the 22-kDa membrane polypeptide present exclusively in peroxisomal membranes. Total rat liver RNA was translated in a nuclease-treated rabbit reticulocyte cell-free protein-synthesizing system. The in vitro translation product, isolated by means of the antibody and Staphylococcus aureus cells, comigrated with the mature 22-kDa polypeptide in NaDodSO4/PAGE. Analysis of the translation products of RNAs from free and membrane-bound polysomes indicated that the mRNA for the 22-kDa membrane polypeptide is found predominantly in free polysomes. The results imply post-translational insertion of the membrane polypeptide into the peroxisomal membrane without proteolytic processing and suggest that peroxisomes, like mitochondria and chloroplasts, form by fission from preexisting organelles. Images PMID:6594687

  11. Competition between surface adsorption and folding of fibril-forming polypeptides

    NASA Astrophysics Data System (ADS)

    Ni, Ran; Kleijn, J. Mieke; Abeln, Sanne; Cohen Stuart, Martien A.; Bolhuis, Peter G.

    2015-02-01

    Self-assembly of polypeptides into fibrillar structures can be initiated by planar surfaces that interact favorably with certain residues. Using a coarse-grained model, we systematically studied the folding and adsorption behavior of a β -roll forming polypeptide. We find that there are two different folding pathways depending on the temperature: (i) at low temperature, the polypeptide folds in solution into a β -roll before adsorbing onto the attractive surface; (ii) at higher temperature, the polypeptide first adsorbs in a disordered state and folds while on the surface. The folding temperature increases with increasing attraction as the folded β -roll is stabilized by the surface. Surprisingly, further increasing the attraction lowers the folding temperature again, as strong attraction also stabilizes the adsorbed disordered state, which competes with folding of the polypeptide. Our results suggest that to enhance the folding, one should use a weakly attractive surface. They also explain the recent experimental observation of the nonmonotonic effect of charge on the fibril formation on an oppositely charged surface [C. Charbonneau et al., ACS Nano 8, 2328 (2014), 10.1021/nn405799t].

  12. Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

    NASA Astrophysics Data System (ADS)

    Munegumi, Toratane; Tanikawa, Naoya

    2017-09-01

    Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

  13. Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere

    NASA Astrophysics Data System (ADS)

    Munegumi, Toratane; Tanikawa, Naoya

    2017-07-01

    Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

  14. Polycondensation of Asparagine-comprising Dipeptides in Aqueous Media-A Simulation of Polypeptide Formation in Primordial Earth Hydrosphere.

    PubMed

    Munegumi, Toratane; Tanikawa, Naoya

    2017-07-26

    Asparagine and aspartic acid might have mutually transformed in the primordial hydrosphere of the earth, if ammonia and aspartic acid had existed in equilibrium. These amino acids seem to contribute to polypeptides, while the simple amino acids glycine and alanine easily form cyclic dipeptides and do not achieve long peptide chains. Asparagine-comprising dipeptides contribute some kinds of activation forms of dipeptides because these can polymerize faster than asparagine only. The new finding of polypeptide formation suggests a pathway of sequential polypeptides to evolve a diversity of polypeptides.

  15. mim3 and nam3 omnipotent suppressor genes similarly affect the polypeptide composition of yeast mitoribosomes.

    PubMed

    Mieszczak, M; Zagórski, W

    1987-05-01

    Yeast informational suppressors of mit- mutations coded for by nuclear (nam3-1, nam3-2) or by mitochondrial DNA (mim3-1) affect the mitoribosome. Nuclear mutations result in the appearance of an additional polypeptide called SI in the small mitoribosomal subunit. An identical polypeptide, not detected in the wild type 37S subunit, is present in crude preparations of mitoribosomes isolated from a mim3-1 suppressor carrying strain. Traces of the SI polypeptide may be found in highly purified small subunits from the mim3-1 strain. Therefore, mutations affecting either mitochondrial rRNA (mim3-1) or mitochondrial r-proteins (nam3-1, nam3-2) could be followed by similar changes in overall mitoribosome structure. This may explain the functional similarity of nuclear and mitochondrially coded suppressors.

  16. Identification of the polypeptide encoded by the URF-1 gene of Neurospora crassa mtDNA.

    PubMed

    Zauner, R; Christner, J; Jung, G; Borchart, U; Machleidt, W; Videira, A; Werner, S

    1985-08-01

    Two peptides, potentially representing antigenic determinants of a proposed gene product, were synthesized. The peptide sequences were deduced from the nucleotide sequence of the unidentified reading frame (URF)1 of the Neurospora crassa mitochondrial genome. Specific antisera to the synthetic peptides were produced. The antibodies recognized a single polypeptide species with an apparent relative molecular mass of about 30 000. The mitochondrial origin of this polypeptide was verified by in vivo labelling experiments in the presence of cycloheximide, as well as by in vitro translation using isolated mitochondria. The chemical identification of the protein was performed by partial radiosequencing of the N-terminal portion of the immunoprecipitated URF-1 product. The amount of URF-1 polypeptide present in N. crassa mitochondria is in the range of 1-2%. The protein is a constituent of the inner envelope of the organelle and probably part of a more complex membrane unit.

  17. Characterization of human red cell Rh (rhesus-)specific polypeptides by limited proteolysis.

    PubMed

    Krahmer, M; Prohaska, R

    1987-12-21

    Human red cells of various Rh phenotypes were surface-labelled with 125I and the Rh-specific labelled polypeptides were isolated by preparative SDS-PAGE. The polypeptides were subjected to limited proteolysis and the resulting fragments were analysed by SDS-PAGE and autoradiography. Chymotryptic peptide maps of proteins obtained from Rh(D)-positive and -negative types appeared completely identical, whereas tryptic peptide maps revealed a difference: a fragment of Mr 17,500 was associated with the Rh(D) antigen, and one of Mr 19,000 with the Rh(C/c,E/e) antigens. Treatment of Rh polypeptides with carboxypeptidase Y prior to tryptic digestion resulted in a shift of nearly all tryptic fragments, including a fragment of Mr 8,000, indicating that the surface label was incorporated into the C-terminal part of the molecule.

  18. Structure-function relationship in the antifreeze activity of synthetic alanine-lysine antifreeze polypeptides.

    PubMed

    Wierzbicki, A; Knight, C A; Rutland, T J; Muccio, D D; Pybus, B S; Sikes, C S

    2000-01-01

    Recently antifreeze proteins (AFP) have been the subject of many structure-function relationship studies regarding their antifreeze activity. Attempts have been made to elucidate the structure-function relationship by various amino acid substitutions, but to our knowledge there has been no successful from first principles design of a polypeptide that would bind to designated ice planes along a specific direction. In this paper we show the results of our first attempt on an entirely de novo design of an alanine-lysine-rich antifreeze polypeptide. This 43 residue alanine-lysine peptide exhibits characteristic nonequilibrium freezing point depression and binds to the designated (210) planes of ice along the [122] vector. The structural and thermodynamic properties of this polypeptide were determined using circular dichroism spectroscopy and its nonequilibrium antifreeze properties were investigated using an ice-etching method and nanoliter osmometry.

  19. Calcium binding peptide motifs from calmodulin confer divalent ion selectivity to elastin-like polypeptides

    PubMed Central

    Hassouneh, Wafa; Nunalee, Michelle L.; Shelton, M. Coleman; Chilkoti, Ashutosh

    2013-01-01

    Calcium sensitive elastin-like polypeptides (CELPs) were synthesized by periodically interspersing a calcium-binding peptide sequence from calmodulin within an elastin-like polypeptide (ELP), with the goal of creating thermal and calcium responsive peptide polymers. The CELPs exhibit high sensitivity to calcium compared to monovalent cations but do not exhibit the exquisite selectivity for calcium over other divalent cations such as magnesium that is displayed by calmodulin. The CELPs were further used as a building block for the synthesis of calcium sensitive nanoparticles by fusing a hydrophilic, non-calcium sensitive ELP block with a CELP block that becomes more hydrophobic upon calcium binding. We show that addition of calcium at concentrations between 50–500 mM imparts sufficient amphiphilicity to the diblock polypeptide between 33 and 46 °C to trigger its self-assembly into monodisperse spherical micelles with a hydrodynamic radius of ~50 nm. PMID:23705904

  20. Magnetite loaded Polypeptide-PLGA multifunctional microbubbles for dual-mode US/MR imaging.

    PubMed

    Sun, Ying; Zhu, Yunkai; Huang, Can; Li, Rongxin; Chen, Yaqing; Duan, Yourong

    2016-01-01

    Magnetite loaded Polypeptide-PLGA multifunctional microbubbles (Fe3O4 /Polypeptide-PLGA MMBs) that show superparamagnetic properties were prepared by a modified double emulsion method and employed as imaging agent for dual-mode Ultrasound/Magnetic resonance (US/MR) imaging of prostatic cancer. The successful synthesis of MMBs was determined by Fourier Transform Infrared Spectrometer (FTIR), X-ray diffraction (XRD), Transmission Electron Microscope (TEM), Scanning Electron Microscope (SEM), Atomic Absorption Spectroscopy (AAS) and vibrating sample magnetometer (VSM). The as-prepared MMBs had a diameter of 700 nm and were quite safe as confirmed by MTT assays. Prussian Blue Staining showed that targeted Fe3O4 /Polypeptide-PLGA MMBs enhanced the cellular uptake efficiency. In cell attachment study, adherence of MMBs was significantly higher to LNCaP cells compared with negative control PC3 cells. The in vitro results demonstrated that these MMBs could enhance both US and MR imaging of prostatic cancer.

  1. Comparison between the polypeptide profile of halophilic bacteria and salt tolerant plants.

    PubMed

    Muñoz, G; González, C; Flores, P; Prado, B; Campos, V

    1997-12-01

    Changes in the polypeptide profile induced by salt stress in halotolerant and halophilic bacteria, isolated from the Atacama desert (northern Chile), were compared with those in the cotyledons of Prosopis chilensis (Leguminoseae) seedlings, a salt tolerant plant. SDS-PAGE analyses show the presence of four predominant polypeptides, with molecular weights around 78, 70, 60 and 44 kDa respectively, both in bacteria and in cotyledons from P. chilensis seedlings raised under salt stress conditions. Moreover, the 60 and 44 kDa polypeptides seem to be salt responsive, since their concentration increases with increasing NaCl in the growth medium. Our results suggest a common mechanism for salt tolerance in prokaryotes and in eukaryotes.

  2. Polypeptides with Quaternary Phosphonium Side Chains: Synthesis, Characterization, and Cell-Penetrating Properties

    PubMed Central

    2015-01-01

    Polypeptides bearing quaternary phosphonium side chains were synthesized via controlled ring-opening polymerization of chlorine-functionalized amino acid N-carboxyanhydride monomers followed by one-step nucleophilic substitution reaction with triethylphosphine. The conformation of the resulting polypeptides can be controlled by modulating the side-chain length and α-carbon stereochemistry. The phosphonium-based poly(l-glutamate) derivatives with 11 σ-bond backbone-to-charge distance adopt stable α-helical conformation against pH and ionic strength changes. These helical, quaternary phosphonium-bearing polypeptides exhibit higher cell-penetrating capability than their racemic and random-coiled analogues. They enter cells mainly via an energy-independent, nonendocytic cell membrane transduction mechanism and exhibit low cytotoxicity, substantiating their potential use as a safe and effective cell-penetrating agent. PMID:24635536

  3. Selective covalent bond formation in polypeptide ions via gas-phase ion/ion reaction chemistry.

    PubMed

    Han, Hongling; McLuckey, Scott A

    2009-09-16

    Primary amines present in protonated polypeptides can be covalently modified via gas-phase ion/ion reactions using bifunctional reagent ions. The use of reagent anions with a charge-bearing site that leads to strong interactions with the polypeptide, such as sulfonic acid, gives rise to the formation of a long-lived adduct. A distinct reactive functional group, an aldehyde in the present case, can then undergo reaction with the peptide. Collisional activation of the adduct ion formed from a reagent with an aldehyde group and a peptide ion with a primary amine gives rise to water loss in conjunction with imine (Schiff base) formation. The covalently bound modification is retained upon subsequent collisional activation. This work demonstrates the ability to selectively modify polypeptide ions in the gas phase within the context of a multistage mass spectrometry experiment.

  4. Involvement of Rh blood group polypeptides in the maintenance of aminophospholipid asymmetry

    SciTech Connect

    Schroit, A.J.; Connor, J. ); Bloy, C.; Carton, J-P. )

    1990-11-01

    The human erythrocyte (RBC) Rh blood group system consists of a complex of distinct integral membrane polypeptides with physical properties common to the aminophospholipid transporter responsible for the transbilayer movement of phosphatidylserine (PS) in RBC. To assess the involvement of Rh polypeptides in PS translocation, the aminophospholipid translocase was labeled with a photoactivatable PS analogue, {sup 125}I-azido-PS, and with an inhibitor of PS transport, {sup 125}I-labeled 2-(2-pyridyldithio)ethylamine. The ability of monoclonal Rh antibodies to immunoprecipitate the labeled transporter was determined. Immunoprecipitated Rh polypeptides were found to be labeled with the aminophospholipid translocase markers, suggesting that Rh proteins are involved in the transbilayer movement of PS.

  5. Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus.

    PubMed Central

    Lightfoote, M M; Coligan, J E; Folks, T M; Fauci, A S; Martin, M A; Venkatesan, S

    1986-01-01

    Automated N-terminal microsequencing of immune affinity-purified acquired immunodeficiency syndrome retrovirus polypeptides from infected cells was used to locate the N termini of 64-, 51-, and 34-kilodalton (kDa) polypeptides within the pol open reading frame (ORF) of the proviral DNA. The 64- and 51-kDa proteins had identical N termini (Pro-Ile-Ser-Pro-IIe-Glu-Thr-Val-) positioned 156 residues from the beginning of the pol ORF. The N terminus of the 34-kDa pol gene product, Phe-Leu-Asp-Gly-Ile-Asp-Lys-, mapped 716 residues into the pol ORF. These polypeptides were absent in an RT-negative, CD4-negative, persistently infected cell line (8E5) carrying a single defective copy of a constitutively expressed, integrated proviral DNA. Images PMID:2430111

  6. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

    NASA Astrophysics Data System (ADS)

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

    2017-09-01

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

  7. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

    NASA Astrophysics Data System (ADS)

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

    2017-05-01

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states.

  8. Medial septal and median raphe innervation of vasoactive intestinal polypeptide-containing interneurons in the hippocampus.

    PubMed

    Papp, E C; Hajos, N; Acsády, L; Freund, T F

    1999-05-01

    Vasoactive intestinal polypeptide-immunoreactive interneurons are known to form three anatomically and neurochemically well-characterized neuron populations in the hippocampus. Two of these establish synaptic contacts selectively with other GABAergic cells (interneuron-selective cells), whereas the third type innervates pyramidal cell bodies and proximal dendrites like a conventional basket cell. Our aim was to examine which of the vasoactive intestinal polypeptide-containing interneuron populations are among the targets of GABAergic septohippocampal and serotonergic raphe-hippocampal pathways. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with double immunocytochemistry for vasoactive intestinal polypeptide was used at the light and electron microscopic levels. Our results show that both interneuron-selective cells and vasoactive intestinal polypeptide-containing basket cells receive synaptic input from the medial septum and median raphe nucleus. The GABAergic component of the septohippocampal pathway establishes multiple contacts on both cell types. In the case of the raphe-hippocampal projection, single or double contacts were more frequent on vasoactive intestinal polypeptide-positive interneuron selective cells (76%), whereas multiple contacts predominated on basket cells (83%). The extrinsic GABAergic innervation of interneuron-selective cells in the hippocampus indicates a complex interaction among GABAergic systems, which might ensure the timing and rhythmic synchronization of inhibitory processes in the hippocampus. On the other hand, our results suggest that the serotonergic effect on perisomatic inhibition is exerted via vasoactive intestinal polypeptide-containing basket cells that are functionally distinct from their parvalbumin-positive relatives, which appear to escape control of serotonergic as well as local interneuron-selective cells.

  9. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages.

    PubMed

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A

    2017-05-11

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. Graphical Abstract ᅟ.

  10. Chlorophyll-Protein Complexes from Euglena gracilis and Mutants Deficient in Chlorophyll b: II. Polypeptide Composition.

    PubMed

    Cunningham, F X; Schiff, J A

    1986-01-01

    Chlorophyll-protein complexes (CPs) obtained from thylakoids of Euglena gracilis Klebs var bacillaris Cori contain the following polypeptides (listed in parentheses in order of prominence after Coomassie R-250 staining of polyacrylamide gels): CP Ia (66, 18, 22, 22.5, 27.5, 21, 28, 24, 25.5, and 26 kilodaltons [kD]); CP I (66 kD); CPx (41 kD); LHCP(2) (an oligomer of LHCP) (26.5, 28, and 26 kD); CPy (27 and 19 kD); CPa (54 kD); and LHCP (26.5, 28, and 26 kD). Mutants of bacillaris low in chlorophyll b (Gr(1)BSL, G(1)BU, and O(4)BSL; Chl a/b [mol/mol] = 50-100) which lack CP Ia, LHCP(2), and LHCP also lack or are deficient in polypeptides associated with these complexes in wild-type cells. Mutants G(1) and O(4), which also lack CPy, lack the CPy-associated polypeptides found in wild-type and Gr(1). Using an antiserum which was elicited by and reacts strongly and selectively with the SDS-treated major polypeptide (26.5 kD) of the LHCP complexes of wild-type, this polypeptide is undetectable in the mutants (<0.25% of the level in wild-type on a cell basis); the antiserum does not react with the SDS-treated 28 kD polypeptide of the Euglena LHCP complexes and cross-reacts only very weakly with components in SDS-treated cells of Chlamydomonas reinhardtii Dangeard and chloroplasts of Spinacia oleracea L. cv Winter Bloomsdale. Rates of photosynthesis of the wild-type and mutant cells of Euglena are approximately equal on a cell basis when measured at light saturation, consistent with the selective loss of major antenna components but not CP I or CPa from the mutants.

  11. Catalytic and reactive polypeptides and methods for their preparation and use

    DOEpatents

    Schultz, Peter

    1994-01-01

    Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like.

  12. Pancreatic vasoactive intestinal polypeptide-oma as a cause of secretory diarrhoea.

    PubMed

    Masel, S L; Brennan, B A; Turner, J H; Cullingford, G L; Cullen, D J

    2000-04-01

    A 42-year-old woman presented with a 4-year history of worsening diarrhoea that was watery, profuse and confirmed to be secretory in nature. She had tested positive for phenolphthalein on urinary laxative screening but continued to deny laxative usage. Her vasoactive intestinal polypeptide (VIP) level was subsequently found to be markedly elevated. Despite a normal abdominal ultrasound, a computed tomography scan revealed a 5-cm pancreatic tail mass. Octreotide scanning was used to exclude metastatic disease and she went on to have surgical removal of a localized pancreatic vasoactive intestinal polypeptide-oma which resulted in the complete resolution of her diarrhoea.

  13. Final Report

    SciTech Connect

    Gurney, Kevin R.

    2015-01-12

    This document constitutes the final report under DOE grant DE-FG-08ER64649. The organization of this document is as follows: first, I will review the original scope of the proposed research. Second, I will present the current draft of a paper nearing submission to Nature Climate Change on the initial results of this funded effort. Finally, I will present the last phase of the research under this grant which has supported a Ph.D. student. To that end, I will present the graduate student’s proposed research, a portion of which is completed and reflected in the paper nearing submission. This final work phase will be completed in the next 12 months. This final workphase will likely result in 1-2 additional publications and we consider the results (as exemplified by the current paper) high quality. The continuing results will acknowledge the funding provided by DOE grant DE-FG-08ER64649.

  14. Final Report

    SciTech Connect

    DeTar, Carleton

    2012-12-10

    This document constitutes the Final Report for award DE-FC02-06ER41446 as required by the Office of Science. It summarizes accomplishments and provides copies of scientific publications with significant contribution from this award.

  15. TOPOFOLD, the designed modular biomolecular folds: polypeptide-based molecular origami nanostructures following the footsteps of DNA.

    PubMed

    Kočar, Vid; Božič Abram, Sabina; Doles, Tibor; Bašić, Nino; Gradišar, Helena; Pisanski, Tomaž; Jerala, Roman

    2015-01-01

    Biopolymers, the essential components of life, are able to form many complex nanostructures, and proteins in particular are the material of choice for most cellular processes. Owing to numerous cooperative interactions, rational design of new protein folds remains extremely challenging. An alternative strategy is to design topofolds-nanostructures built from polypeptide arrays of interacting modules that define their topology. Over the course of the last several decades DNA has successfully been repurposed from its native role of information storage to a smart nanomaterial used for nanostructure self-assembly of almost any shape, which is largely because of its programmable nature. Unfortunately, polypeptides do not possess the straightforward complementarity as do nucleic acids. However, a modular approach can nevertheless be used to assemble polypeptide nanostructures, as was recently demonstrated on a single-chain polypeptide tetrahedron. This review focuses on the current state-of-the-art in the field of topological polypeptide folds. It starts with a brief overview of the field of structural DNA and RNA nanotechnology, from which it draws parallels and possible directions of development for the emerging field of polypeptide-based nanotechnology. The principles of topofold strategy and unique properties of such polypeptide nanostructures in comparison to native protein folds are discussed. Reasons for the apparent absence of such folds in nature are also examined. Physicochemical versatility of amino acid residues and cost-effective production makes polypeptides an attractive platform for designed functional bionanomaterials. © 2014 Wiley Periodicals, Inc.

  16. lncRNA-Encoded Polypeptide SPAR(s) with mTORC1 to Regulate Skeletal Muscle Regeneration.

    PubMed

    Tajbakhsh, Shahragim

    2017-04-06

    Although prematurely baptized as non-coding, some lncRNAs encode polypeptides with regulatory functions that are implicated in various biological processes. Matsumoto et al. (2017) recently report in Nature that LINC00961 generates SPAR polypeptide that acts via the lysosome to suppress amino-acid-mediated mTORC1 activity, thereby modulating skeletal muscle regenerative response following injury.

  17. Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

    PubMed Central

    Chilkoti, Ashutosh

    2014-01-01

    Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products. PMID:24961229

  18. Production of Recombinant Polypeptides Containing One GA-Module and Analysis of Their Ability to Bind to Human Albumin.

    PubMed

    Bormotova, E A; Gupalova, T V

    2016-11-01

    Surface proteins of many bacterial species interact with human serum albumin (HSA) via a special region of amino acid sequence termed GA module. For instance, surface peptostreptococcal albumin-binding protein of anaerobic bacteria Peptostreptococcus magnus contains one HSA-binding GA-module. Protein G from group G and C Streptococcus strains isolated from humans has HSA-binding region consisting of three GA-modules. HSA-binding protein containing two GA-modules was found in strains of group G Streptococcus of animal origin. We obtained two recombinant polypeptides GA1 and GA2 congaing one GA-module each. Recombinant polypeptide with two GA-modules binds HSA with a much higher affinity than polypeptides GA1 and GA2 containing one GA-module. Polypeptide with the second GAmodule more effectively binds HSA than polypeptides with the GA-module.

  19. Coordinating electrical activity of the heart: ankyrin polypeptides in human cardiac disease.

    PubMed

    Curran, Jerry; Mohler, Peter J

    2011-07-01

    Over the past ten years, ankyrin polypeptides have emerged as players in cardiac excitation-contraction coupling. Once thought to solely play a structural role, loss-of-function variants of genes encoding ankyrin polypeptides have highlighted how this protein mediates subcellular localization of various electrical components of the excitation-contraction coupling machinery. Evidence has revealed how disruption of this localization is the primary cause of various cardiomyopathies, ranging from long-QT syndrome 4, to sinus node disease, to more common forms of arrhythmias. The roles of ankyrin polypeptides in excitation-contraction coupling in the heart and the development of ankyrin-specific cardiomyopathies. How ankyrin polypeptides may be involved in structural and electrical remodeling of the heart, post-myocardial infarct. How ankyrin interactions with membrane-bound ion channels may regulate these channels' response to stimuli. New data, which offers the potential for unique therapies, for not only combating heart disease, but also for wider applications to various disease states. The ankyrin family of adapter proteins is emerging as an intimate player in cardiac excitation-contraction coupling. Until recently, these proteins have gone largely unappreciated for their importance in proper cardiac function. New insights into how these proteins function within the heart are offering potentially new avenues for therapies against cardiomyopathy.

  20. Coordinating Electrical Activity of the Heart: Ankyrin Polypeptides in Human Cardiac Disease

    PubMed Central

    Curran, Jerry; Mohler, Peter J

    2011-01-01

    Introduction Over the past ten years, ankyrin polypeptides have emerged as critical players in cardiac excitation-contraction coupling. Once thought to solely play only a structural role, loss-of-function variants in genes encoding ankyrin polypeptides have highlighted how this protein mediates the proper subcellular localization of the various electrical components of the excitation-contraction coupling machinery. A large body of evidence has revealed how the disruption of this localization is the primary cause of various cardiomyopathies, ranging from long QT syndrome 4, to sinus node disease, to more common forms of arrhythmias. Areas Covered This review details the varied roles that ankyrin polypeptides play in excitation-contraction coupling in the heart and the development of ankyrin-specific cardiomyopathies. It will further discuss how ankyrin polypeptides may be involved in structural and electrical remodeling of the heart, post-myocardial infarct. Attention is given to how ankyrin interactions with membrane bound ion channels may regulate these channels’ response to stimuli. Special attention is given to exciting new data, which may offer the potential for unique therapies, for not only combating heart disease, but which also holds promise for wider applications to various disease states. Expert Opinion The ankyrin family of adapter proteins is emerging as an intimate player in cardiac excitation-contraction coupling. Until recently, these proteins have gone largely unappreciated for their importance in proper cardiac function. New insights into how these proteins function within the heart are offering potentially new avenues for therapies against cardiomyopathy. PMID:21457127

  1. Simple bioseparations using self-cleaving elastin-like polypeptide tags.

    PubMed

    Banki, Mahmoud Reza; Feng, Liang; Wood, David W

    2005-09-01

    We introduce a new method for the purification of recombinant proteins expressed in Escherichia coli using self-cleaving elastin-like polypeptide (ELP) fusion tags without the need for affinity chromatography or proteolytic tag removal. Using this method we obtained high purity, activity and reasonable yields for ten diverse target proteins.

  2. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    NASA Astrophysics Data System (ADS)

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-02-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring.

  3. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    NASA Technical Reports Server (NTRS)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  4. Novel antibacterial polypeptide produced by Lactobacillus paracasei strain NRRL B-50314

    USDA-ARS?s Scientific Manuscript database

    This study reports the production and characterization of a novel antibacterial polypeptide, designated as laparaxin, which is secreted by Lactobacillus paracasei NRRL B-50314. The crude laparaxin has antibacterial activity against a range of Gram-positive bacteria including the following: lactic a...

  5. Dynamics of the formation of a hydrogel by a pathogenic amyloid peptide: islet amyloid polypeptide

    PubMed Central

    Jean, Létitia; Lee, Chiu Fan; Hodder, Peter; Hawkins, Nick; Vaux, David J.

    2016-01-01

    Many chronic degenerative diseases result from aggregation of misfolded polypeptides to form amyloids. Many amyloidogenic polypeptides are surfactants and their assembly can be catalysed by hydrophobic-hydrophilic interfaces (an air-water interface in-vitro or membranes in-vivo). We recently demonstrated the specificity of surface-induced amyloidogenesis but the mechanisms of amyloidogenesis and more specifically of adsorption at hydrophobic-hydrophilic interfaces remain poorly understood. Thus, it is critical to determine how amyloidogenic polypeptides behave at interfaces. Here we used surface tensiometry, rheology and electron microscopy to demonstrate the complex dynamics of gelation by full-length human islet amyloid polypeptide (involved in type II diabetes) both in the bulk solution and at hydrophobic-hydrophilic interfaces (air-water interface and phospholipids). We show that the hydrogel consists of a 3D supramolecular network of fibrils. We also assessed the role of solvation and dissected the evolution over time of the assembly processes. Amyloid gelation could have important pathological consequences for membrane integrity and cellular functions. PMID:27535008

  6. Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.)

    NASA Technical Reports Server (NTRS)

    Cai, X.; Henry, R. L.; Takemoto, L. J.; Guikema, J. A.; Wong, P. P.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.

  7. Structural variation and inhibitor binding in polypeptide deformylase from four different bacterial species.

    PubMed

    Smith, Kathrine J; Petit, Chantal M; Aubart, Kelly; Smyth, Martin; McManus, Edward; Jones, Jo; Fosberry, Andrew; Lewis, Ceri; Lonetto, Michael; Christensen, Siegfried B

    2003-02-01

    Polypeptide deformylase (PDF) catalyzes the deformylation of polypeptide chains in bacteria. It is essential for bacterial cell viability and is a potential antibacterial drug target. Here, we report the crystal structures of polypeptide deformylase from four different species of bacteria: Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli. Comparison of these four structures reveals significant overall differences between the two Gram-negative species (E. coli and H. influenzae) and the two Gram-positive species (S. pneumoniae and S. aureus). Despite these differences and low overall sequence identity, the S1' pocket of PDF is well conserved among the four enzymes studied. We also describe the binding of nonpeptidic inhibitor molecules SB-485345, SB-543668, and SB-505684 to both S. pneumoniae and E. coli PDF. Comparison of these structures shows similar binding interactions with both Gram-negative and Gram-positive species. Understanding the similarities and subtle differences in active site structure between species will help to design broad-spectrum polypeptide deformylase inhibitor molecules.

  8. Identification of immunogenic polypeptides from a Mycoplasma hyopneumoniae genome library by phage display.

    PubMed

    Kügler, Jonas; Nieswandt, Simone; Gerlach, Gerald F; Meens, Jochen; Schirrmann, Thomas; Hust, Michael

    2008-09-01

    The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.

  9. Self-assemble nanoparticles based on polypeptides containing C-terminal luminescent Pt-cysteine complex

    PubMed Central

    Vlakh, E. G.; Grachova, E. V.; Zhukovsky, D. D.; Hubina, A. V.; Mikhailova, A. S.; Shakirova, J. R.; Sharoyko, V. V.; Tunik, S. P.; Tennikova, T. B.

    2017-01-01

    The growing attention to the luminescent nanocarriers is strongly stimulated by their potential application as drug delivery systems and by the necessity to monitor their distribution in cells and tissues. In this communication we report on the synthesis of amphiphilic polypeptides bearing C-terminal phosphorescent label together with preparation of nanoparticles using the polypeptides obtained. The approach suggested is based on a unique and highly technological process where the new phosphorescent Pt-cysteine complex serves as initiator of the ring-opening polymerization of α-amino acid N-carboxyanhydrides to obtain the polypeptides bearing intact the platinum chromophore covalently bound to the polymer chain. It was established that the luminescent label retains unchanged its emission characteristics not only in the polypeptides but also in more complicated nanoaggregates such as the polymer derived amphiphilic block-copolymers and self-assembled nanoparticles. The phosphorescent nanoparticles display no cytotoxicity and hemolytic activity in the tested range of concentrations and easily internalize into living cells that makes possible in vivo cell visualization, including prospective application in time resolved imaging and drug delivery monitoring. PMID:28155880

  10. Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

    PubMed

    Butterworth, A G; Lord, J M

    1983-12-01

    Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

  11. The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

    ERIC Educational Resources Information Center

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

    During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

  12. The Beads of Translation: Using Beads to Translate mRNA into a Polypeptide Bracelet

    ERIC Educational Resources Information Center

    Dunlap, Dacey; Patrick, Patricia

    2012-01-01

    During this activity, by making beaded bracelets that represent the steps of translation, students simulate the creation of an amino acid chain. They are given an mRNA sequence that they translate into a corresponding polypeptide chain (beads). This activity focuses on the events and sites of translation. The activity provides students with a…

  13. Novel antibacterial polypeptide laparaxin produced by Lactobacillus paracasei strain NRRL B-50314 via fermentation

    USDA-ARS?s Scientific Manuscript database

    This study reports the production and characterization of a novel antibacterial polypeptide, designated laparaxin, which is secreted by Lactobacillus paracasei NRRL B-50314. Crude laparaxin has antibacterial activity against a wide variety of Gram-positive bacteria, including: lactic acid bacteria ...

  14. Noncanonical Self-Assembly of Highly Asymmetric Genetically Encoded Polypeptide Amphiphiles into Cylindrical Micelles

    PubMed Central

    2015-01-01

    Elastin-like polypeptides (ELPs) are a class of biopolymers consisting of the pentameric repeat (VPGαG)n based on the sequence of mammalian tropoelastin that display a thermally induced soluble-to-insoluble phase transition in aqueous solution. We have discovered a remarkably simple approach to driving the spontaneous self-assembly of high molecular weight ELPs into nanostructures by genetically fusing a short 1.5 kDa (XGy)z assembly domain to one end of the ELP. Classical theories of self-assembly based on the geometric mass balance of hydrophilic and hydrophobic block copolymers suggest that these highly asymmetric polypeptides should form spherical micelles. Surprisingly, when sufficiently hydrophobic amino acids (X) are presented in a periodic sequence such as (FGG)8 or (YG)8, these highly asymmetric polypeptides self-assemble into cylindrical micelles whose length can be tuned by the sequence of the morphogenic tag. These nanostructures were characterized by light scattering, tunable resistive pulse sensing, fluorescence spectrophotometry, and thermal turbidimetry, as well as by cryogenic transmission electron microscopy (cryo-TEM) and small-angle neutron scattering (SANS). These short assembly domains provide a facile strategy to control the size, shape, and stability of stimuli responsive polypeptide nanostructures. PMID:25268037

  15. Anthracycline photoaffinity labeling of a mitochondrial polypeptide in P388 murine leukemic cell lines

    SciTech Connect

    Averbuch, S.D.; Glover, C.J.; Felsted, R.L.

    1986-12-01

    N-(p-Azido(3,5-/sup 3/H)benzoyl)daunorubicin ((/sup 3/H)NABD), a radioactive photoactive anthracycline analogue, was used to photoaffinity label anthracycline binding polypeptides in P388 murine leukemic cell lines. Whole cell homogenates were mixed with 6 X 10(-8) M (/sup 3/H)NABD, exposed to ultraviolet light, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for radiolabel incorporation. Autoradiofluorography showed incorporation of radioactivity into a Mr 18,000 component independent of polypeptides prominently stained with Coomassie blue. Photolabeling of subcellular fractions showed predominant mitochondrial localization of the Mr 18,000 radiolabel. The protein composition of the photolabeled constituents was confirmed by treatment with proteinase K, DNase and RNase, or by lipid extraction with organic solvent. (/sup 3/H)NABD photolabeling of homogenates from anthracycline sensitive and resistant cells resulted in Mr 18,000 radiolabel incorporation of 3966 +/- 355 and 6487 +/- 533 dpm per 50 micrograms cellular protein for anthracycline sensitive and resistant cells, respectively (P less than 0.005). These studies characterize the photoaffinity labeling of a low molecular weight mitochondrial polypeptide using a photoactive anthracycline analogue. The role for this polypeptide as a mediator of anthracycline activity remains to be determined.

  16. Acetylation in vitro of constituent polypeptides by smooth endoplasmic reticulum (SER) and Golgi membrane fractions

    SciTech Connect

    Sambasivam, H.; Murray, R.K.

    1986-05-01

    Many polypeptides of the membranes of the ER are phosphorylated. To determine if any such polypeptides are acetylated, microsomal and other classical subcellular fractions were incubated with (/sup 3/H) acetyl-CoA; the specific activity of the microsomal fraction (MF) was the greatest. SDS-PAGE revealed that some 20 polypeptides of the MF were acetylated; 2-D electrophoretograms extended this number to approximately 60. Separation of the MF into smooth (S) and rough (R) fractions showed that the great majority of the labelled polypeptides belonged to the former. Isolation of a Golgi fraction revealed that its acetylation activity was approximately 3-fold greater than the SER fraction. Extensive proteolytic digestion of the MF followed by radiochromatography disclosed some 9 components whose precise nature (acetylated amino acids and/or sialic acids, etc.) is under study. Assuming that the majority of the radioactivity is in the former components and that a similar process occurs in vivo, the authors suggest that the Golgi apparatus may be a major site of acetylation of membrane and possibly other proteins.

  17. A role for helical intermediates in amyloid formation by natively unfolded polypeptides?

    NASA Astrophysics Data System (ADS)

    Abedini, Andisheh; Raleigh, Daniel P.

    2009-03-01

    Amyloid formation and aberrant protein aggregation have been implicated in more than 15 different human diseases and an even wider range of proteins form amyloid in vitro. From a structural perspective the proteins which form amyloid can be divided into two classes: those which adopt a compact globular fold and must presumably at least partially unfold to form amyloid and those which are unstructured in their monomeric state. Important examples of the latter include the Aβ peptide of Alzheimer's disease, atrial natriuretic factor, calcitonin, pro-calcitonin, islet amyloid polypeptide (IAPP, amylin), α-synuclein and the medin polypeptide. The kinetics of amyloid assembly are complex and typically involve a lag phase during which little or no fibril material is formed, followed by a rapid growth stage leading to the β-sheet-rich amyloid structure. Increasing evidence suggests that some natively unfolded polypeptides populate a helical intermediate during the lag phase. We propose a model in which early oligomerization is linked to helix formation and is promoted by helix-helix association. Recent work has highlighted the potential importance of polypeptide membrane interactions in amyloid formation and helical intermediates appear to play an important role here as well. Characterization of helical intermediates is experimentally challenging but new spectroscopic techniques are emerging which hold considerable promise and even have the potential to provide residue specific information.

  18. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon.

    PubMed

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S

    2015-06-15

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome-Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S-RQC and 80S-Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome-translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel.

  19. The ribosome quality control pathway can access nascent polypeptides stalled at the Sec61 translocon

    PubMed Central

    von der Malsburg, Karina; Shao, Sichen; Hegde, Ramanujan S.

    2015-01-01

    Cytosolic ribosomes that stall during translation are split into subunits, and nascent polypeptides trapped in the 60S subunit are ubiquitinated by the ribosome quality control (RQC) pathway. Whether the RQC pathway can also target stalls during cotranslational translocation into the ER is not known. Here we report that listerin and NEMF, core RQC components, are bound to translocon-engaged 60S subunits on native ER membranes. RQC recruitment to the ER in cultured cells is stimulated by translation stalling. Biochemical analyses demonstrated that translocon-targeted nascent polypeptides that subsequently stall are polyubiquitinated in 60S complexes. Ubiquitination at the translocon requires cytosolic exposure of the polypeptide at the ribosome–Sec61 junction. This exposure can result from either failed insertion into the Sec61 channel or partial backsliding of translocating nascent chains. Only Sec61-engaged nascent chains early in their biogenesis were relatively refractory to ubiquitination. Modeling based on recent 60S–RQC and 80S–Sec61 structures suggests that the E3 ligase listerin accesses nascent polypeptides via a gap in the ribosome–translocon junction near the Sec61 lateral gate. Thus the RQC pathway can target stalled translocation intermediates for degradation from the Sec61 channel. PMID:25877867

  20. Biomimetic assembly of polypeptide-stabilized CaCO(3) nanoparticles.

    PubMed

    Zhang, Zhongping; Gao, Daming; Zhao, Hui; Xie, Chenggen; Guan, Guijian; Wang, Dapeng; Yu, Shu-Hong

    2006-05-04

    In this paper, we report a simple polypeptide-directed strategy for fabricating large spherical assembly of CaCO(3) nanoparticles. Stepwise growth and assembly of a large number of nanoparticles have been observed, from the formation of an amorphous liquidlike CaCO(3)-polypeptide precursor, to the crystallization and stabilization of polypeptide-capped nanoparticles, and eventually, the spherical assembly of nanoparticles. The "soft" poly(aspartate)-capping layer binding on a nanoparticle surface resulted in the unusual soft nature of nanoparticle assembly, providing a reservoir of primary nanoparticles with a moderate mobility, which is the basis of a new strategy for reconstructing nanoparticle assembly into complex nanoparticle architectures. Moreover, the findings of the secondary assembly of nanoparticle microspheres and the morphology transformation of nanoparticle assembly demonstrate a flexible and controllable pathway for manipulating the shapes and structures of nanoparticle assembly. In addition, the combination of the polypeptide with a double hydrophilic block copolymer (DHBC) allows it to possibly further control the shape and complexity of the nanoparticle assembly. A clear perspective is shown here that more complex nanoparticle materials could be created by using "soft" biological proteins or peptides as a mediating template at the organic-inorganic interface.

  1. Cloning, expression and properties of porcine trachea UDP-galnac: polypeptide N-acetylgalactosaminyl transferase.

    PubMed

    Sangadala, Sreedhara; Swain, Ja Baris; McNear, Adrian; Mendicino, Joseph

    2004-11-01

    A UDP-GalNAc:polypeptide N-acetyl-galactosaminyl transferase which catalyses the transfer of GalNAc from UDP-GalNAc to serine and threonine residues in mucin polypeptide chains was purified to homogeneity from swine trachea epithelium (Mendicino J, Sangadala S: Mol Cell Biochem 185: 135-145, 1998). Peptides obtained by proteolysis of the purified enzyme were isolated, sequenced and used to prepare degenerate oligonucleotide primers. Amplified segments of a gene encoding GalNAc transferase were synthesised using the primers and a swine trachea epithelial cDNA library. Selected cDNA fragments were then used to screen the cDNA library, and a clone containing an open reading frame encoding 559 amino acids was isolated. The predicted amino acid sequence contains type II transmembrane region, three potential N-glycosylation sites as well as all of the isolated peptide sequences. The nucleotide sequence and predicted primary protein structure of the transferase were very similar to those of type T-1 GalNAc transferases. The isolated clone was transiently expressed in COS 7 cells and the recombinant enzyme, which contained an N-terminal hexa-histidine tag, was purified to homogeneity and its enzymatic properties were examined. The Vmax of the recombinant enzyme, 2.08 micromol/(min mg), was nearly the same as the native enzyme, 2.12 micromol/(min mg), when assayed with partially deglycosylated mucins as glycosyl acceptors. Both enzymes showed much higher activities when assayed with peptides prepared by limited acid hydrolysis of incompletely deglycosylated Cowper's gland, swine, and human respiratory mucins and tryptic peptides isolated from deglycosylated mucin polypeptide chains. However, as noted earlier (Mendicino J, Sangadala S: Mol Cell Biochem 185: 135-145, 1998), these enzymes showed very little activity with completely deglycosylated mucin polypeptide chains. When completely deglycosylated polypeptide chains were partially glycosylated by incubation with microsome

  2. A two-step enzymatic glycosylation of polypeptides with complex N-glycans

    PubMed Central

    Lomino, Joseph V.; Naegeli, Andreas; Orwenyo, Jared; Amin, Mohammed N.; Aebi, Markus; Wang, Lai-Xi

    2013-01-01

    A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc–Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc–Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc–Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc–Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery. PMID:23477942

  3. [Clinical analysis of Cervus and Cucumis Polypeptide injection based on real world hospital information system].

    PubMed

    Sun, Shuai-Ling; Xie, Yan-Ming; Li, Yuan-Yuan; Zhang, Yin; Yi, Dan-Hui; Zhuang, Yan

    2016-11-01

    To analyze the clinical application of Cervus and Cucumis Polypeptide injection in the real world, in order to define the characteristics of clinical drug use and correlation, and provide reference for risk management and further study for Cervus and Cucumis Polypeptide injection. Descriptive analysis and association rules analysis were performed on 37 721 cases using Cervus and Cucumis Polypeptide injection in 26 hospitals nationwide. Cervus and Cucumis Polypeptide injection were mostly adopted by patients aged between 45 and 64(39.84%); mainly used to treat fracture patients in clinic(17 362 cases, 33.97%); 12 mL(41.81%) was the commonest dosage. And the course of treatment mainly lasted for 1-3 days(28 467 cases, 76.26%), which was basically consistent with the description of package insert. In clinic, traditional Chinese medicines, such as blood activating and stasis removing agents and Bushen Zhuanggu agents, were frequently combined with it(rule support degree of 19.38%). Such western medicine as antibiotics and nutritional drugs were frequently combined with it(rule support 39.9%). The main single combined medicine were vitamin C(13 202 cases, 35%), and Jintiange capsule(7 285 cases, 19.31%). The commonly used combined drug pairs were Hulisan capsule and Jintiange capsule (rule support 4.458%), phenobarbital and ceftazidime azole oxazoline(rule support degree of 10.62%). Cervus and Cucumis Polypeptide injection is mainly adopted by elderly patients in clinic, used to treat fracture patients, and often combined with blood activating and stasis removing agents, Bushen Zhuanggu agents, antibiotics, and nutritional medicine to enhance fracture healing. In clinical application, attention shall be paid to drug safety of elderly patients and types of combined medicines and their interaction, so as to prevent adverse reactions. Copyright© by the Chinese Pharmaceutical Association.

  4. The effect of tra mutations on the synthesis of the F-pilin membrane polypeptide.

    PubMed

    Moore, D; Sowa, B A; Ippen-Ihler, K

    1981-01-01

    We had previously demonstrated that several F specific polypeptide bands could be detected in the membranes of Flac, but not F- strains of Escherichia coli K12, (Moore et al. 1981). One of these polypeptides co-migrated with F-pilin protein on polyacrylamide gels. We have now analyzed 35[S]methionine labelled membrane preparations from a series of strains containing Flac tra mutant plasmids. The F-pilin polypeptide was absent from preparations of strains containing all traA mutants tested, confirming the importance of the traA gene on F-pilin biosynthesis. A polypeptide which migrate in the F-pilin position was still present, however, in membranes prepared from Flac strains carrying mutations in traL, traE, traK, traB, traV, traW, traC, traU, traF, traH or traG despite the inability of these mutants to elaborate F-pili filaments. Thus, all of these gene products may be concerned with F-pilus assembly and outgrowth rather than biosynthesis of the F-pilin subunit. The polar mutation tra-4 did, however, prevent the appearance of pilin polypeptide, indicating that at least one unidentified gene in the region between traE and traG must also be required in F-pilin biosynthesis. Our analysis also permitted the identification of a 100,000 dalton membrane protein as the product of traG. The appearance of an F specific 12,000 dalton protein was prevented by traD amber mutants. As expected, traJ mutants prevented the expression of all the tra operon products detected except the product of traT. The traT product band was reduced only to 50 - 60% of its normal intensity.

  5. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Astrophysics Data System (ADS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-12-01

    We studied the spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay. This led to the unexpected finding that the degrees of polymerization (DP's) of the oligo- and poly-peptides obtained depended on the amounts of polypeptides that were preadsorbed. Plotting average molecular weights obtained against c-spacings of the clay platelet aggregates which widened as a result of polypeptide addition and adsorption before the polymerization, does not permit an obvious explanation of these observations. The best correlation assigns a role to the fractional occupation of the individual intercalation layers of the polypeptides, as the adsorption increases towards a first complete mono-interlayer, then to an incipient and eventually to a complete double layer on to a third interlayer, after which the clay stacking breaks up. Spacings which correspond to an intermediate occupation of any of the three successive interlayers favor amino acids self-addition to polymers. The opposite is true for nearly empty or filled intercalation layers. We hypothesize and describe, how a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances. Moderately filled intercalation spaces may also act as sinks for the newly formed oligomers and facilitate the freeing of reaction sites for the occupation by fresh reagent. The c-spacings required for these mechanisms are the result of the intercalation of the preadsorbed polymer, but similar conditions prevail when polymers are adsorbed as they are generated during polymerization.

  6. Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

    PubMed

    Benzaquen-Geffin, R; Milner, Y; Ginsburg, H

    1987-02-01

    Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level.

  7. pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides

    PubMed Central

    Takei, Toshiaki; Tsumoto, Kouhei; Okonogi, Atsuhito; Kimura, Akiko; Kojima, Shuichi; Yazaki, Kazumori; Takei, Tsunetomo; Ueda, Takuya; Miura, Kin-ichiro

    2015-01-01

    We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation. PMID:25694229

  8. Inhibition of malaria parasite invasion of human erythrocytes by a lymphocyte membrane polypeptide.

    PubMed Central

    Benzaquen-Geffin, R; Milner, Y; Ginsburg, H

    1987-01-01

    Extraction by boiling of the buffy coat of human blood yields a protein solution which inhibits the propagation of the human malaria parasite Plasmodium falciparum in culture with a 50% inhibitory dose of 105 micrograms of protein per ml. The inhibitory activity is associated exclusively with the lymphocytes and affects solely the invasion of erythrocytes by free merozoites. Boiled extracts of isolated lymphocytes had a 50% inhibitory dose of 22 micrograms/ml. Fractionation of surface-labeled or pronase-treated lymphocytes revealed that the antimalarial lymphocyte factor is associated with the intracellular aspect of the membrane fraction and is probably not involved in the host defense system against malaria. Further purification by salt extraction, ion-exchange chromatography, molecular gel filtration, and electroelution from lithium dodecyl sulfate-polyacrylamide gels resulted in 300- to 550-fold purification, i.e., a 50% inhibitory dose of 40 to 70 ng/ml. All inhibitory fractions contained a 48-kilodalton polypeptide which eluted from a gel filtration column as a 400-kilodalton species, implying multimeric association. Some 6,000 molecules of the 48-kilodalton polypeptide bind with high affinity to one merozoite, the free form of the parasite. The Kd of 0.1 to 0.5 nM for the binding of the 48-kilodalton polypeptide correlated well with the 50% inhibitory dose of 0.3 to 0.4 nM obtained with purified active antimalarial lymphocyte factor. We therefore suggest that the 48-kilodalton polypeptide partially purified from lymphocyte membranes is the antimalarial lymphocyte factor and that it exerts its inhibitory activity by binding to merozoites, thereby preventing their invasion into erythrocytes. The antimalarial lymphocyte factor or a polypeptide sequence thereof could serve for further probing of invasion at the molecular level. Images PMID:3542831

  9. Synthesis of gluten-forming polypeptides. 1. Biosynthesis of gliadins and glutenin subunits.

    PubMed

    Abonyi, Tibor; Király, István; Tömösközi, Sándor; Baticz, Orsolya; Guóth, Adrienn; Gergely, Szilveszter; Scholz, Eva; Lásztity, Demeter; Lásztity, Radomir

    2007-05-02

    Five winter wheat cultivars--GK Othalom (HMW-GS composition 2*, 7+8, 5+10), Ukrainka (1, 7+8, 5+10), Palotás (2*, 7+9, 5+10), Ködmön (2*, 7+8, 5+10), and Csongrád (2*, 7+9, 2+12)--grown in Hungary and harvested in the year 2005 were studied. The biosynthesis of gluten-forming polypeptides was followed starting at the 12th day after anthesis to the 53rd. Fresh kernel weight, moisture, and dry matter content of fresh kernels and gliadin and glutenin contents were determined. Gliadin components, total amounts of HMW and LMW polypeptides, and individual HMW polypeptides were determined using a RP-HPLC technique. Although considerable quantitative differences were observed concerning the content of total protein, gliadin, glutenin, and individual gluten-forming polypeptides, the character of accumulation of protein components--determined on the basis protein mass/kernel--was the same for the all of the cultivars studied and could be presented by a sigmoid curve. Small quantities of the gliadin and glutenin monomers may be detected in early stages of kernel development, but the bulk of these proteins is synthesized in later stages of development. It is generally suggested by specialists that the formation and accumulation of glutenin polymers starts later than the synthesis of monomers. Experimental data presented in this paper confirm this suggestion and show that in the first phase of protein synthesis the monomers are in "free" form; polymeric glutenin is detected only later. HMW glutenin subunits are synthesized synchronously, and quantitatively the polypeptides coded by chromosomes D and B dominate.

  10. pH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides.

    PubMed

    Takei, Toshiaki; Tsumoto, Kouhei; Okonogi, Atsuhito; Kimura, Akiko; Kojima, Shuichi; Yazaki, Kazumori; Takei, Tsunetomo; Ueda, Takuya; Miura, Kin-ichiro

    2015-05-01

    We reported previously that our designed polypeptide α3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)3, forms not only an amphipathic α-helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its α-helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of α3 were replaced with Ser. We prepared the following polypeptides: 2Sα3 (LSTLAKA)3, in which all Glu residues were replaced with Ser residues; 6Sα3 (LETLASA)3, in which all Lys residues were replaced with Ser; and 2S6Sα3 (LSTLASA)3; in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2Sα3 formed an α-helix under basic conditions and 6Sα3 formed an α-helix under acid conditions. In 1M KCl, they both formed α-helices under a wide pH range. In addition, 2Sα3 and 6Sα3 formed fibrous assemblies under the same buffer conditions in which they formed α-helices. α-Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6Sα3 formed an α-helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in α-helix formation. © 2015 The Protein Society.

  11. Absence of glycosylation on cyanobacterial phycobilisome linker polypeptides and rhodophytan phycoerythrins.

    PubMed Central

    Fairchild, C D; Jones, I K; Glazer, A N

    1991-01-01

    The 27-, 30-, and 33-kDa rod linker polypeptides and the 75-kDa core linker of phycobilisomes from the cyanobacterium Synechococcus sp. strain PCC 7942 have been reported to be glycoproteins with carbohydrate contents ranging from 3.2 to 18.8% and composed of N-acetylgalactosamine and glucose (H.C. Riethman, T.P. Mawhinney, and L.A. Sherman, J. Bacteriol. 170:2433-2440, 1988). Synechococcus sp. strain PCC 7942 phycobilisomes were purified extensively, and the linker polypeptides were separated from the phycobiliproteins by precipitation in 1 M NaSCN. Upon hydrolysis, the linker fraction yielded 0.037% glucose and 0.015% galactosamine by weight and no other carbohydrate. Phycobilisome polypeptides separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate were subjected to various glycoprotein-specific staining procedures. Linker polypeptides showed very weak concanavalin A binding and no staining by the Schiff-periodate method or by a much more sensitive periodate oxidation-based method. These results indicated that the linker polypeptides are not glycosylated. An earlier report (T. Fujiwara, J. Biochem. 49:361-367, 1961) contended, on the basis of the isolation of sugar-containing peptic chromopeptides from Porphyra tenera R-phycoerythrin, that this red algal phycobiliprotein is a glycoprotein. Analysis of Gastroclonium coulteri R-phycoerythrin and Porphyridium cruentum B-phycoerythrin revealed only traces of carbohydrate in these two proteins, 0.36 and 0.14%, respectively. Results of glycoprotein staining of gels suggested that the carbohydrate in the R-phycoerythrin preparation is due to a glycoprotein contaminant and that neither red algal phycoerythrin is glycosylated. Images PMID:1902214

  12. Final Report

    SciTech Connect

    Stinis, Panos

    2016-08-07

    This is the final report for the work conducted at the University of Minnesota (during the period 12/01/12-09/18/14) by PI Panos Stinis as part of the "Collaboratory on Mathematics for Mesoscopic Modeling of Materials" (CM4). CM4 is a multi-institution DOE-funded project whose aim is to conduct basic and applied research in the emerging field of mesoscopic modeling of materials.

  13. Final Report

    SciTech Connect

    Marchant, Gary E.

    2013-04-23

    This is the final report of a two year project entitled "Governing Nanotechnology Risks and Benefits in the Transition to Regulation: Innovative Public and Private Approaches." This project examined the role of new governance or "soft law" mechanisms such as codes of conduct, voluntary programs and partnership agreements to manage the risks of emerging technologies such as nanotechnology. A series of published or in publication papers and book chapters are attached.

  14. Final Report

    SciTech Connect

    R. Paul Drake

    2001-11-30

    This final report describes work involving 22 investigators from 11 institutions to explore the dynamics present in supernova explosions by means of experiments on the Omega laser. The specific experiments emphasized involved the unstable expansion of a spherical capsule and the coupling of perturbations at a first interface to a second interface by means of a strong shock. Both effects are present in supernovae. The experiments were performed at Omega and the computer simulations were undertaken at several institutions. B139

  15. Polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and polynucleotides encoding same

    DOEpatents

    Morant, Marc

    2017-02-07

    The present invention relates to isolated polypeptides having beta-glucosidase activity, beta-xylosidase activity, or beta-glucosidase and beta-xylosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

  16. Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide

    PubMed Central

    Inchaustegui Gil, Diana P.; Clayton, Christine

    2016-01-01

    The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA. PMID:26808308

  17. Navigating in foldonia: Using accelerated molecular dynamics to explore stability, unfolding and self-healing of the β-solenoid structure formed by a silk-like polypeptide.

    PubMed

    Zhao, Binwu; Cohen Stuart, Martien A; Hall, Carol K

    2017-03-01

    The β roll molecules with sequence (GAGAGAGQ)10 stack via hydrogen bonding to form fibrils which have been themselves been used to make viral capsids of DNA strands, supramolecular nanotapes and pH-responsive gels. Accelerated molecular dynamics (aMD) simulations are used to investigate the unfolding of a stack of two β roll molecules, (GAGAGAGQ)10, to shed light on the folding mechanism by which silk-inspired polypeptides form fibrils and to identify the dominant forces that keep the silk-inspired polypeptide in a β roll configuration. Our study shows that a molecule in a stack of two β roll molecules unfolds in a step-wise fashion mainly from the C terminal. The bottom template is found to play an important role in stabilizing the β roll structure of the molecule on top by strengthening the hydrogen bonds in the layer that it contacts. Vertical hydrogen bonds within the β roll structure are considerably weaker than lateral hydrogen bonds, signifying the importance of lateral hydrogen bonds in stabilizing the β roll structure. Finally, an intermediate structure was found containing a β hairpin and an anti-parallel β sheet consisting of strands from the top and bottom molecules, revealing the self-healing ability of the β roll stack.

  18. Artificial antifreeze polypeptides: alpha-helical peptides with KAAK motifs have antifreeze and ice crystal morphology modifying properties.

    PubMed

    Zhang, W; Laursen, R A

    1999-07-23

    Antifreeze polypeptides from fish are generally thought to inhibit ice crystal growth by specific adsorption onto ice surfaces and preventing addition of water molecules to the ice lattice. Recent studies have suggested that this adsorption results from hydrogen bonding through the side chains of polar amino acids as well as hydrophobic interactions between the non-polar domains on the ice-binding side of antifreeze polypeptides and the clathrate-like surfaces of ice. In order to better understand the activity of one of the antifreeze polypeptide families, namely the alpha-helical type I antifreeze polypeptides, four alpha-helical peptides having sequences not directly analogous to those of known antifreeze polypeptides and containing only positively charged and non-polar side chains were synthesized. Two peptides with regularly spaced lysine residues, GAAKAAKAAAAAAAKAAKAAAAAAAKAAKAAGGY-NH2 and GAALKAAKAAAAAALKAAKAAAAAALKAAKAAGGY-NH2, showed antifreeze activity, albeit weaker than seen in natural antifreeze polypeptides, by the criteria of freezing point depression (thermal hysteresis) and ice crystal modification to a hexagonal trapezohedron. Peptides with irregular spacing of Lys residues were completely inactive. Up to now, lysine residues have not been generally associated with antifreeze activity, though they have been implicated in some antifreeze polypeptides. This work also shows that lysine residues in themselves, when properly positioned on an alpha-helical polyalanine scaffold, have all the requisite properties needed for such an activity.

  19. The location of phosphorylation sites and Ca2+-dependent proteolytic cleavage sites on the major neurofilament polypeptides from Myxicola infundibulum.

    PubMed Central

    Eagles, P A; Gilbert, D S; Maggs, A

    1981-01-01

    1. When axoplasm is incubated with [32P]Pi the main phosphorylated components are the neurofilament polypeptides. 2. Activation with Ca2+ of the proteinase present in axoplasm causes degradation of these neurofilaments and the peptides produced by this reaction have been analysed by fingerprinting. 3. Fingerprinting shows that initially the Ca2+-activated proteinase cleaves the neurofilament polypeptides at three major sites producing polypeptides with mol.wts. 70,000, 50,000 and 47,000. 4. These polypeptides sediment with filaments, originate from the tail-region of the molecule and contain a little radioactive label. 5. As these polypeptides are produced, other polypeptides that come from the head-region of the molecule are liberated as soluble products that contain the bulk of the radioactivity. 6. Fingerprinting therefore shows that at least two regions on the molecule are phosphorylated and that the major one is located towards the head-end of the polypeptides. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:7039615

  20. Synthesis of conformation switchable cationic polypeptides based on poly(S-propargyl-cysteine) for use as siRNA delivery.

    PubMed

    Yi, Ling; Wang, Yisi; Lin, Guanliang; Lin, Danling; Chen, Wenliang; Huang, Yugang; Ye, Guodong

    2017-08-01

    Ring-opening polymerization of S-propargyl-cysteine-N-carboxyanhydride has been used to synthesize conformation switchable poly(S-propargyl-cysteine) starting with l-cysteine, dl- and d-cysteine. Then cationic polypeptides with different backbone chirality are obtained by nearly 100% side-chain grafting of cysteamine via thiol-yne click chemistry. The cationic polypeptides containing mixed conformations of β-sheets, β-turns and random coils are stable against pH, salt and temperature variations. The cationic polypeptides can condense siRNA at a low polypeptide/siRNA weight ratio to form nanoparticles with size depending on the backbone chirality. The cationic polypeptides derived from poly(S-propargyl-l or d-cysteine) are non-cytotoxic to HeLa and HepG2 cells, but interrupting the backbone chirality enhances the cytotoxicity sharply. The cationic polypeptides used for siRNA delivery show good transfection efficiency, but cell internalization process depends on the backbone chirality. The cationic polypeptide derived from the poly(S-propargyl-l-cysteine) is an appropriate siRNA vector with advantages of non-cytotoxicity and high transfection efficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Different populations of vasoactive intestinal polypeptide-immunoreactive interneurons are specialized to control pyramidal cells or interneurons in the hippocampus.

    PubMed

    Acsády, L; Görcs, T J; Freund, T F

    1996-07-01

    The postsynaptic targets of three vasoactive intestinal polypeptide-containing GABAergic interneuron types were examined in the rat hippocampus. Two of them showed remarkable target selectivity for other GABAergic neurons, while the third contacted the somata and proximal dendrites of pyramidal cells. Vasoactive intestinal polypeptide-positive interneurons innervating the stratum oriens/alveus border in the CA1 region were shown to establish multiple contacts with horizontal GABAergic interneurons immunoreactive for type 1 metabotropic glutamate receptor. Similarly, identified axons of vasoactive intestinal polypeptide-positive interneurons projecting to stratum radiatum were found to establish symmetrical synapses largely on GABAergic dendrites. The majority of these postsynaptic GABAergic neurons were shown to contain calbindin or vasoactive intestinal polypeptide. In contrast to the first two vasoactive intestinal polypeptide-containing cell populations, vasoactive intestinal polypeptide-positive interneurons arborizing in stratum pyramidale formed baskets around pyramidal cells. These results revealed a new element in cortical microcircuits, interneurons which are specialized to innervate other GABAergic interneurons. The role of this new component may be the synchronization of dendritic inhibition, or an input-specific disinhibition of pyramidal cells in various dendritic domains. In contrast, vasoactive intestinal polypeptide-containing basket cells are likely to be involved in perisomatic inhibition of pyramidal neurons, and represents a new basket cell type different from that containing parvalbumin.

  2. Redox-responsive, reversibly-crosslinked thiolated cationic helical polypeptides for efficient siRNA encapsulation and delivery.

    PubMed

    Zheng, Nan; Song, Ziyuan; Liu, Yang; Zhang, Rujing; Zhang, Ruoyan; Yao, Catherine; Uckun, Fatih M; Yin, Lichen; Cheng, Jianjun

    2015-05-10

    Cationic helical polypeptides, although highly efficient for inducing membrane penetration, cannot stably condense siRNA molecules via electrostatic interactions, which greatly limit the gene knockdown efficiency. By developing and crosslinking the thiolated polypeptide via formation of disulfide bonds post formation of the polypeptide/siRNA complexes, we were able to obtain stable complexes without compromising the helical secondary structure as well as the membrane activity of the polypeptide. As such, the stable polypeptide/siRNA complex was able to notably protect the siRNA cargo from nuclease digestion in the extracellular environment, while the functions of the polypeptide/siRNA complex for effective cellular internalization and endosomal escape are still largely preserved. Because the disulfide is susceptible to cleavage in response to intracellular redox triggers, siRNA release from the complex is expected upon redox triggering by glutathione (GSH) intracellularly and was actually observed upon redox triggers mediated by glutathione (GSH). With the collective contribution of the potent membrane activity and redox-responsive cargo release profiles, the crosslinked complexes enable efficient gene silencing without appreciable cytotoxicity, thus providing a potential strategy for polypeptide-based intracellular siRNA delivery. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Role of positions e and g in the fibrous assembly formation of an amphipathic α-helix-forming polypeptide.

    PubMed

    Takei, Toshiaki; Tsumoto, Kouhei; Yoshino, Masakuni; Kojima, Shuichi; Yazaki, Kazumori; Ueda, Takuya; Takei, Tsunetomo; Arisaka, Fumio; Miura, Kin-ichiro

    2014-05-01

    We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α-helices. Upon comparison of the amino acid sequences of α3 with other α-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α-helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed β-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies. © 2014 Wiley Periodicals, Inc.

  4. Bioresorbable polypeptide-based comb-polymers efficiently improves the stability and pharmacokinetics of proteins in vivo.

    PubMed

    Turabee, Md Hasan; Thambi, Thavasyappan; Lym, Jae Seung; Lee, Doo Sung

    2017-03-13

    Stimuli-responsive polypeptides are a promising class of biomaterials due to their tunable physicochemical and biological properties. Herein, a series of novel pH- and thermo-responsive block copolymers based on polypeptides were synthesized by ring-opening polymerization of γ-benzyl-l-glutamate-N-carboxyanhydride in the presence of poly(ethylene glycol)-diamine macroinitiator followed by aminolysis. The resulting polypeptide-based triblock copolymer, poly[(2-(dibutylamino)ethyl-l-glutamate)-co-(γ-benzyl-l-glutamate)]-poly(ethylene glycol)-b-poly[(2-(dibutylamino)ethyl-l-glutamate)-co-(γ-benzyl-l-glutamate)] (PNLG-co-PBLG-b-PEG-b-PBLG-co-PNLG), exists as a low viscous sol at low pH and temperature (≤pH 6.4, 25 °C) but it transforms to a soft gel under physiological conditions (pH 7.4 and 37 °C). The physical properties of the polypeptide gel can be tuned by controlling the ratio between hydrophobic PBLG and pH-sensitive PNLG blocks. The polypeptide-based copolymer did not show any noticeable cytotoxicity to fibroblast cells in vitro. It was found that subcutaneous injection of the polypeptide copolymer solution into the dorsal region of Sprague-Dawley (SD) rats formed a gel instantly without major inflammation. The gels were completely biodegraded in six weeks and found to be bioresorbable. Human growth hormone (hGH)-loaded polypeptide-based biodegradable copolymer sols readily formed a viscoelastic gel that inhibited an initial burst and prolonged the hGH release for one week. Overall, due to their bioresorbable and sustained release protein characteristics, polypeptide hydrogels may serve as viable platforms for therapeutic protein delivery and the surface tunable properties of polypeptide hydrogels can be exploited for other potential therapeutic proteins.

  5. Modeling Organic Anion-Transporting Polypeptide 1B1 Inhibition to Elucidate Interaction Risks in Early Drug Design.

    PubMed

    Zamora, Ismael; Winiwarter, Susanne

    2016-10-01

    The importance of transporter proteins for the disposition of drugs has become increasingly apparent during the past decade. A noted drug-drug interaction risk is the inhibition of organic anion-transporting polypeptides (OATPs), key transporters for the liver uptake of the widely used statins. We show here the development of a ligand-based in silico model for interaction with OATP1B1, an important representative of the OATP family. The model is based on a structural overlay of 6 known OATP1B1 inhibitors. A data set of about 150 compounds with published OATP1B1 inhibition data was compared to the resulting "transportophor," and a similarity threshold was defined to distinguish between active and inactive molecules. In addition, using a statistical model based on physicochemical properties of the compounds as prefilter was found to enhance the overall predictivity of the model (final accuracy 0.73, specificity 074, and sensitivity 0.71, based on 126 compounds). The combined model was validated using an in-house data set (accuracy, specificity, and sensitivity were 0.63, 0.59, and 0.78, respectively; 62 compounds). The model gives also a structural overlay to the most similar template enabling visualization of where a change in a given structure might reduce the interaction with the transporter.

  6. Separation of living and dead polymers in synthetic polypeptide mixtures by nonaqueous capillary electrophoresis using differences in ionization states.

    PubMed

    Souaïd, Eddy; Cottet, Hervé

    2005-09-01

    The complexity in the mechanisms of polymerization of N-carboxyanhydrides requires the development of new analytical techniques able to separate mixtures of synthetic polypeptides. This work focuses on the separation of poly(N(epsilon)-trifluoroacetyl-L-lysine) (PTLL) mixtures by nonaqueous capillary electrophoresis (CE). The main goal of this work was to find electrophoretic conditions that permit the separation and the quantification of the dead polymer families that were previously identified in the samples. The influence of the pH of the electrolyte on the selectivity of the separation was carefully investigated. The mechanisms of separation of the PTTLs are discussed as a function of their ionization state. The separations obtained on a noncovalently coated capillary were compared with those obtained on a fused-silica capillary. Finally, using two different electrolytes, it is possible to quantify the three families of PTLLs, namely, the living PTLLs, the dead PTLLs with N-formyl end group and the dead PTLLs with a carboxylic end group. These results confirm the importance of CE for the separation of synthetic organic polymers in nonaqueous electrolytes.

  7. Final Report

    SciTech Connect

    R Paul Drake

    2004-01-12

    OAK-B135 This is the final report from the project Hydrodynamics by High-Energy-Density Plasma Flow and Hydrodynamics and Radiation Hydrodynamics with Astrophysical Applications. This project supported a group at the University of Michigan in the invention, design, performance, and analysis of experiments using high-energy-density research facilities. The experiments explored compressible nonlinear hydrodynamics, in particular at decelerating interfaces, and the radiation hydrodynamics of strong shock waves. It has application to supernovae, astrophysical jets, shock-cloud interactions, and radiative shock waves.

  8. Effect of polypeptides from sea anemone Heteractis crispa on the rodent blood pressure, heart rate, and hemostasis.

    PubMed

    Skobtsova, L A; Dyachenko, I A; Andreev, Ya A; Logashina, Yu A; Murashev, A N; Grishin, E V

    2016-09-01

    АРНС1-3 peptides, modulators of TRPV1 receptors, have been administered to SD rats to study their influence on the animal hemostatic system, heart rate, and blood pressure. None of АЗРС1-3 polypeptides have any effect on the hemostatic system. Both АРНС1 and АРНС2 polypeptides increased significantly the heart rate, but they did not affect blood pressure, which was probably caused by an ability of these polypeptides to modify animal thermoregulation.

  9. Effects of pituitary adenylate cyclase activating polypeptide-27 (PACAP) and vasoactive intestinal polypeptide (VIP) on chloride in HT29 cells studied by X-ray microanalysis.

    PubMed

    Zhang, W; Roomans, G M

    1999-01-01

    The colon cancer cell line HT29 is a useful model to study intestinal chloride secretion. These cells have both cAMP-activated and calcium-activated chloride channels. Changes in elemental content of the cells after stimulation with agonists were determined by X-ray microanalysis in the scanning or scanning transmission electron microscope. Exposure of HT29 cells to pituitary adenylate cyclase activating polypeptide-27 (PACAP) caused a transient decrease in the cellular Cl and K concentrations, indicating (net) efflux of chloride. The effect of PACAP is inhibited by somatostatin, which is known to inhibit cAMP-activated as well as calcium-activated chloride secretion and by U-73122, an inhibitor of phospholipase C. Alloxan, an inhibitor of adenylate cyclase, did not significantly affect the PACAP-induced loss of chloride. The calcium-chelating agent EGTA inhibited the PACAP-induced loss of chloride, indicating the need for extracellular calcium ions. Also vasointestinal polypeptide (VIP) caused a decrease of the cellular chloride concentration in HT29 cells. VIP-induced loss of chloride could be inhibited by pre-treating the cells with somatostatin or UK14,304, an alpha-2 adrenergic agonist that has been shown previously to inhibit purinergically activated chloride efflux. Our results indicate that there is cross-talk between the cAMP- and the calcium-activated pathways for chloride secretion in HT29 cells.

  10. The vasorelaxant effect of pituitary adenylate cyclase activating polypeptide and vasoactive intestinal polypeptide in isolated rat basilar arteries is partially mediated by activation of nitrergic neurons.

    PubMed

    Seebeck, Jörg; Löwe, Marcus; Kruse, Marie Luise; Schmidt, Wolfgang E; Mehdorn, H Maximilian; Ziegler, Albrecht; Hempelmann, Ralf G

    2002-07-15

    The structurally related neuropeptides pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are recognised by two G protein-coupled receptors, termed VPAC(1)-R and VPAC(2)-R, with equal affinity. PACAP and VIP have previously been shown to relax cerebral arteries in an endothelium-independent manner. The aim of the present study was to test if intramural neurons are involved in the mediation of PACAP/VIP-induced vasodilatory responses. Therefore, the vascular tone of isolated rat basilar arteries was measured by means of a myograph. The vasorelaxing effect of PACAP was assessed in arteries precontracted by serotonin in the absence or presence of different test compounds known to selectively inhibit certain signaling proteins. The vasorelaxant effect of PACAP could be significantly reduced by the inhibitor of neuronal N-type calcium channels omega-conotoxin GVIA (omega-CgTx), as well as by 3-bromo-7-nitroindazole (3Br-7-Ni), an inhibitor of the neuronal nitric oxide-synthase (nNOS). The localization of N-type calcium channels and VPAC-Rs within the rat basilar artery was investigated by confocal laser scanning microscopy using omega-CgTx- and VIP-analogs labelled with fluorescent dyes. These findings suggest that activation of intramural neurons may represent an important effector mechanism for mediation of the vasorelaxant PACAP-response.

  11. Design of photocaged puromycin for nascent polypeptide release and spatiotemporal monitoring of translation.

    PubMed

    Buhr, Florian; Kohl-Landgraf, Jörg; tom Dieck, Susanne; Hanus, Cyril; Chatterjee, Deep; Hegelein, Andreas; Schuman, Erin M; Wachtveitl, Josef; Schwalbe, Harald

    2015-03-16

    The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC-puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome-bound polypeptide chains was detected in vitro. Upon illumination, cytotoxic activity, in vitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC-puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2% and the NVOC-puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two-photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Thermodynamic Approach to Enhanced Dispersion and Physical Properties in a Carbon Nanotube/Polypeptide Nanocomposite

    NASA Technical Reports Server (NTRS)

    Lovell, Conrad S.; Wise, Kristopher E.; Kim, Jae-Woo; Lillehei, Peter T.; Harrison, Joycelyn S.; Park, Cheol

    2009-01-01

    A high molecular weight synthetic polypeptide has been designed which exhibits favorable interactions with single wall carbon nanotubes (SWCNTs). The enthalpic and entropic penalties of mixing between these two molecules are reduced due to the polypeptide's aromatic sidechains and helical secondary structure, respectively. These enhanced interactions result in a well dispersed SWCNT/Poly (L-Leucine-ran-L-Phenylalanine) nanocomposite with enhanced mechanical and electrical properties using only shear mixing and sonication. At 0.5 wt% loading of SWCNT filler, the nanocomposite exhibits simultaneous increases in the Young's modulus, failure strain, and toughness of 8%, 120%, and 144%, respectively. At one kHz, the same nanotube loading level also enhances the dielectric constant from 2.95 to 22.81, while increasing the conductivity by four orders of magnitude.

  13. Force measurements of the disruption of the nascent polypeptide chain from the ribosome by optical tweezers.

    PubMed

    Katranidis, Alexandros; Grange, Wilfried; Schlesinger, Ramona; Choli-Papadopoulou, Theodora; Brüggemann, Dorothea; Hegner, Martin; Büldt, Georg

    2011-06-23

    We show that optical tweezers are a valuable tool to study the co-translational folding of a nascent polypeptide chain at the ribosome in real-time. The aim of this study was to demonstrate that a stable and intact population of ribosomes can be tethered to polystyrene beads and that specific hook-ups to the nascent polypeptide chain by dsDNA handles, immobilized on a second bead, can be detected. A rupture force of the nascent chain in the range of 10-50 pN was measured, which demonstrates that the system is anchored to the surface in a stable and specific way. This will allow in numerous future applications to follow protein folding using much lower forces. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  14. Isolation and amino acid sequences of opossum vasoactive intestinal polypeptide and cholecystokinin octapeptide.

    PubMed Central

    Eng, J; Yu, J; Rattan, S; Yalow, R S

    1992-01-01

    Evolutionary history suggests that the marsupials entered South America from North America about 75 million years ago and subsequently dispersed into Australia before the separation between South America and Antarctica-Australia. A question of interest is whether marsupial peptides resemble the corresponding peptides of Old or New World mammals. Previous studies had shown that "little" gastrin of the North American marsupial, the opossum, is identical in length to that of the New World mammals, the guinea pig and chinchilla. In this report, we demonstrate that opossum cholecystokinin octapeptide, like that of the Australian marsupials, the Eastern quoll and the Tamar wallaby, is identical to the cholecystokinin octapeptide of Old World mammals and differs from that of the guinea pig and chinchilla. However, opossum vasoactive intestinal polypeptide differs from the usual Old World mammalian vasoactive intestinal polypeptide in five sites: [sequence; see text]. PMID:1542675

  15. Dual effect of crowders on fibrillation kinetics of polypeptide chains revealed by lattice models

    NASA Astrophysics Data System (ADS)

    Co, Nguyen Truong; Hu, Chin-Kun; Li, Mai Suan

    2013-05-01

    We have developed the lattice model for describing polypeptide chains in the presence of crowders. The influence of crowding confinement on the fibrillation kinetics of polypeptide chains is studied using this model. We observed the non-trivial behavior of the fibril formation time τfib that it decreases with the concentration of crowders if crowder sizes are large enough, but the growth is observed for crowders of small sizes. This allows us to explain the recent experimental observation on the dual effect of crowding particles on fibril growth of proteins that for a fixed crowder concentration the fibrillation kinetics is fastest at intermediate values of total surface of crowders. It becomes slow at either small or large coverages of cosolutes. It is shown that due to competition between the energetics and entropic effects, the dependence of τfib on the size of confined space is described by a parabolic function.

  16. A thermally targeted c-Myc inhibitory polypeptide inhibits breast tumor growth.

    PubMed

    Bidwell, Gene L; Perkins, Eddie; Raucher, Drazen

    2012-06-28

    Although surgical resection with adjuvant chemotherapy and/or radiotherapy are used to treat breast tumors, normal tissue tolerance, development of metastases, and inherent tumor resistance to radiation or chemotherapy can hinder a successful outcome. We have developed a thermally responsive polypeptide, based on the sequence of Elastin-like polypeptide (ELP), that inhibits breast cancer cell proliferation by blocking the activity of the oncogenic protein c-Myc. Following systemic administration, the ELP - delivered c-Myc inhibitory peptide was targeted to tumors using focused hyperthermia, and significantly reduced tumor growth in an orthotopic mouse model of breast cancer. This work provides a new modality for targeted delivery of a specific oncogene inhibitory peptide, and this strategy may be expanded for delivery of other therapeutic peptides or small molecule drugs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  17. UV Spectrophotometric Method for Estimation of Polypeptide-K in Bulk and Tablet Dosage Forms

    NASA Astrophysics Data System (ADS)

    Kaur, P.; Singh, S. Kumar; Gulati, M.; Vaidya, Y.

    2016-01-01

    An analytical method for estimation of polypeptide-k using UV spectrophotometry has been developed and validated for bulk as well as tablet dosage form. The developed method was validated for linearity, precision, accuracy, specificity, robustness, detection, and quantitation limits. The method has shown good linearity over the range from 100.0 to 300.0 μg/ml with a correlation coefficient of 0.9943. The percentage recovery of 99.88% showed that the method was highly accurate. The precision demonstrated relative standard deviation of less than 2.0%. The LOD and LOQ of the method were found to be 4.4 and 13.33, respectively. The study established that the proposed method is reliable, specific, reproducible, and cost-effective for the determination of polypeptide-k.

  18. Self-assembling chimeric polypeptide-doxorubicin conjugate nanoparticles that abolish tumours after a single injection

    NASA Astrophysics Data System (ADS)

    Andrew Mackay, J.; Chen, Mingnan; McDaniel, Jonathan R.; Liu, Wenge; Simnick, Andrew J.; Chilkoti, Ashutosh

    2009-12-01

    New strategies to self-assemble biocompatible materials into nanoscale, drug-loaded packages with improved therapeutic efficacy are needed for nanomedicine. To address this need, we developed artificial recombinant chimeric polypeptides (CPs) that spontaneously self-assemble into sub-100-nm-sized, near-monodisperse nanoparticles on conjugation of diverse hydrophobic molecules, including chemotherapeutics. These CPs consist of a biodegradable polypeptide that is attached to a short Cys-rich segment. Covalent modification of the Cys residues with a structurally diverse set of hydrophobic small molecules, including chemotherapeutics, leads to spontaneous formation of nanoparticles over a range of CP compositions and molecular weights. When used to deliver chemotherapeutics to a murine cancer model, CP nanoparticles have a fourfold higher maximum tolerated dose than free drug, and induce nearly complete tumour regression after a single dose. This simple strategy can promote co-assembly of drugs, imaging agents and targeting moieties into multifunctional nanomedicines.

  19. Membrane-associated polypeptides induced in Chlamydomonas by limiting CO sub 2 concentrations

    SciTech Connect

    Spalding, M.H.; Jeffrey, M. )

    1989-01-01

    Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO{sub 2}, little O{sub 2} inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO{sub 2}-concentrating system. The CO{sub 2}-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO{sub 2} concentrations have inorganic carbon transport activity, but cells grown at 5% CO{sub 2} do not. Four membrane-associated polypeptides (M{sub r}, 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO{sub 2} concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO{sub 2}-concentrating system activity in response to CO{sub 2} limitation.

  20. A novel approach for large-scale polypeptide folding based on elastic networks using continuous optimization.

    PubMed

    Rakshit, Sourav; Ananthasuresh, G K

    2010-02-07

    We present a new computationally efficient method for large-scale polypeptide folding using coarse-grained elastic networks and gradient-based continuous optimization techniques. The folding is governed by minimization of energy based on Miyazawa-Jernigan contact potentials. Using this method we are able to substantially reduce the computation time on ordinary desktop computers for simulation of polypeptide folding starting from a fully unfolded state. We compare our results with available native state structures from Protein Data Bank (PDB) for a few de-novo proteins and two natural proteins, Ubiquitin and Lysozyme. Based on our simulations we are able to draw the energy landscape for a small de-novo protein, Chignolin. We also use two well known protein structure prediction software, MODELLER and GROMACS to compare our results. In the end, we show how a modification of normal elastic network model can lead to higher accuracy and lower time required for simulation.

  1. Catalytic and reactive polypeptides and methods for their preparation and use

    DOEpatents

    Schultz, Peter

    1994-01-01

    Catalytic and reactive polypeptides include a binding site specific for a reactant or reactive intermediate involved in a chemical reaction of interest. The polypeptides further include at least one active functionality proximate the binding site, where the active functionality is capable of catalyzing or chemically participating in the chemical reaction in such a way that the reaction rate is enhanced. Methods for preparing the catalytic peptides include chemical synthesis, site-directed mutagenesis of antibody and enzyme genes, covalent attachment of the functionalities through particular amino acid side chains, and the like. This invention was made with Government support under Grant Contract No. AI-24695, awarded by the Department of health and Human Services, and under Grant Contract No. N 00014-87-K-0256, awarded by the Office of Naval Research. The Government has certain rights in this invention.

  2. A model for the enantiomeric enrichment of polypeptides on the primitive earth

    NASA Technical Reports Server (NTRS)

    Blair, N. E.; Bonner, W. A.

    1981-01-01

    A potential model is presented for the origin of optical activity in polypeptides on the primitive earth due to enantiomeric enrichment in succeeding polymerization-hydrolysis cycles. The model was developed in experiments with the polymerization of a DL-leucine N-carboxyanhydride mixture with a 31.2% enantiomeric excess of the L isomer with sodium methoxide initiator to yield a polyleucine product which was in turn partially hydrolyzed by acid. The polymerization-hydrolysis was found to produce a net 23.8% increase in the enantiomeric excess of the remaining unhydrolyzed polypeptide (14.2% from the polymerization and 9.6% from the partial hydrolysis). On the basis of these results, it is suggested that a slight excess produced by an appropriate chiral physical process may be enhanced by cycles of stereoselective polymerization and hydrolysis driven by fluctuating wet and dry environmental cycles on the primitive earth.

  3. Hierarchical Bionanotubes Formed By the Self Assembly of Microtubules With Cationic Membranes Or Polypeptides

    SciTech Connect

    Raviv, U.; Needleman, D.J.; Ewert, K.K.; Safinya, C.R.

    2009-06-05

    At present there is a surge in interest in biophysical research aimed at elucidating collective interactions between cellular proteins and associated biomolecules leading to supramolecular structures, with the ultimate goal of relating structure to function. The nerve cell cytoskeleton provides a rich example of highly ordered bundles and networks of interacting neurofilaments, microtubules and filamentous actin, where the nature of the interactions, structures and structure-function correlations remain poorly understood. We present synchrotron X-ray diffraction and electron microscopy data, in reconstituted protein systems from the bovine central nervous system, which reveal unexpected structures not predicted by current electrostatic theories. By mixing preassembled microtubules with charged membranes or polypeptides we found hierarchical bionanotubes made of microtubules coated by lipid bilayers or polypeptides, which in turn are coated with a third layer of tubulin oligomers forming rings or spirals.

  4. A simple biochemical method in the search for bioactive polypeptides in a sea anemone (Anemonia sulcata).

    PubMed

    Sanchez, J; Bruhn, T; Aneiros, A; Wachter, E; Béress, L

    1996-01-01

    The sea anemone Anemonia sulcata is a well-known natural source of supply of biologically active polypeptides. So far, five toxins, ATX I, II, III, IV and AS V, several polyvalent protease inhibitors, an elastase inhibitor, two blood pressure-depressive polypeptides and very recently peptides that inhibit competitively the binding of 125I-dendrotoxin to rat brain membranes and block the voltage-sensitive K+ channels, have been isolated from it. The sea anemone toxins (especially toxin II of A. sulcata, ATX II) are very important tools in neurophysiological and pharmacological research, and their structure-function relationship has been investigated. Because of the great scientific value of the sea anemone toxins a simplification of their purification procedure was elaborated.

  5. Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis.

    PubMed Central

    Ito, K; Ebihara, K; Uno, M; Nakamura, Y

    1996-01-01

    Translation termination requires two codon-specific polypeptide release factors in prokaryotes and one omnipotent factor in eukaryotes. Sequences of 17 different polypeptide release factors from prokaryotes and eukaryotes were compared. The prokaryotic release factors share residues split into seven motifs. Conservation of many discrete, perhaps critical, amino acids is observed in eukaryotic release factors, as well as in the C-terminal portion of elongation factor (EF) G. Given that the C-terminal domains of EF-G interacts with ribosomes by mimicry of a tRNA structure, the pattern of conservation of residues in release factors may reflect requirements for a tRNA-mimicry for binding to the A site of the ribosome. This mimicry would explain why release factors recognize stop codons and suggests that all prokaryotic and eukaryotic release factors evolved from the progenitor of EF-G. Images Fig. 2 Fig. 3 PMID:8643594

  6. Aggregation of islet amyloid polypeptide: from physical chemistry to cell biology.

    PubMed

    Cao, Ping; Abedini, Andisheh; Raleigh, Daniel P

    2013-02-01

    Amyloid formation in the pancreas by islet amyloid polypeptide (IAPP) leads to β-cell death and dysfunction, contributing to islet transplant failure and to type-2 diabetes. IAPP is stored in the β-cell insulin secretory granules and cosecreted with insulin in response to β-cell secretagogues. IAPP is believed to play a role in the control of food intake, in controlling gastric emptying and in glucose homeostasis. The polypeptide is natively unfolded in its monomeric state, but is one of the most amyloidogenic sequences known. The mechanisms of IAPP amyloid formation in vivo and in vitro are not understood; the mechanisms of IAPP induced cell death are unclear; and the nature of the toxic species is not completely defined. Recent work is shedding light on these important issues. Copyright © 2012. Published by Elsevier Ltd.

  7. Fingerprint analysis of anti-tumor active polypeptides from Arca subcrenata by HPLC.

    PubMed

    Ren, Sheng-fang; Song, Li-yan; Yan, Chun-yan; Li, Ting-fei; Zhao, Yu; Yu, Rong-min

    2008-08-01

    RP-HPLC was applied to analyze active polypeptides in Arca subcrenata, and the optimal condition for separation was also set up: temperature: 30 degrees C; wavelength: 280 nm; flow rate: 1.0 ml/min; Solvent A consisted of 80% acetonitrile and 0.1% trifluoroacetic acid (TFA) and solvent B contained 0.1% TFA. In this condition, ten samples' fingerprints were gained, in one of which the genuine fraction exhibited fourteen "common peaks" representing the characteristics of the constituents.

  8. Effects of surface hydrophobicity on the conformational changes of polypeptides of different length

    NASA Astrophysics Data System (ADS)

    Mu, Yan

    2011-09-01

    We studied the effects of surface hydrophobicity on the conformational changes of different length polypeptides by calculating the free energy difference between peptide structures using the bias-potential Monte Carlo technique and the probability ratio method. It was found that the hydrophobic surface plays an important role in the stability of secondary structures of the polypeptides with hydrophobic side chains. For short GAAAAG peptides, the hydrophobic surface destabilizes the α helix but stabilizes the β hairpin in the entire temperature region considered in our study. Interestingly, when the surface hydrophobic strength ɛhpsf≥ɛhp, the most stable structure in the low temperature region changes from α helix to β hairpin, and the corresponding phase transition temperature increases slightly. For longer GAAAAAAAAAAG peptides, the effects of the relatively weak hydrophobic surface (ɛhpsf < ɛhp) on α-helical structures may be neglected, while the relatively strongly hydrophobic surface (ɛhpsf≥ɛhp) leads to the obvious partial helicity loss. In contrast, the stability of β structures can be enhanced significantly by the hydrophobic surface, especially by the strongly hydrophobic surface, at low and intermediate temperatures. At high temperatures, in addition to thermal fluctuations, the strongly hydrophobic surface (ɛhpsf>ɛhp) may further disturb the formation of both α-helical and β structures. Moreover, the phase transition temperature between α-helical structures and random coils significantly decreases due to the helicity loss when ɛhpsf>ɛhp. Our findings provide a basic and quantitative picture for understanding the effects of a hydrophobic surface on the conformational changes of the polypeptides with hydrophobic side chains. From an application viewpoint, the present study is helpful in developing alternative strategies of producing high-quality biological fibrillar materials and functional nanoscale devices by the self-assembly of

  9. [Escherichia coli L-asparaginase induces phosphorylation of endogenous polypeptides in human immune cells].

    PubMed

    Mercado, L; Arenas, G

    1999-12-01

    To detect patterns of endogenous polypeptide phosphorylation in monocyte, lymphocyte, and polymorphonuclear leukocyte populations, induced by the products of the catalytic action of L-asparaginase (EcA). Monocytes, polymorphonuclear cells and lymphocytes were isolated from heparinized blood from healthy, voluntary donors. The samples were incubated in 0.4 mCi/ml of [gamma-32P]H3PO4, with: 1 microgram/microliter of EcA, EcA and the substrate or with the products of EcA's catalytic activity: NH4+ and aspartate. The cells were lysated and electrophoresed using denaturing polyacrylamide gels that were then exposed on radiographic plates. The levels of polypeptide phosphorylation were quantified by computer densitometric analysis. The autoradiographs and the densitometric quantification of the electrophoretic profiles of monocytes, polymorphonuclear leukocytes, and lymphocytes revealed an increase in polypeptide phosphorylation when the cells were incubated with the enzyme and its substrate, ammonium and aspartate, or ammonium, which demonstrates that the NH4+ triggers intracellular phosphotransferase activity. A 58 kDa phosphoprotein outstood, it being common to the three cell populations studied. There were also specific phosphorylable polypeptides in monocytes, polymorphonuclear leukocytes, and lymphocytes. Escherichia coli L-asparaginase, binds the plasma membrane in normal human immune cells, catalyzing the L-asparagine substrate. The products of its activity: aspartate and NH4+ modify the extracellular environment, particularly the latter since it could diffuse into the cytosol and modify the pH, which would activate signal transduction pathways associated with the phosphorylation of substrates.

  10. Folding and self-assembly of polypeptides: Dynamics and thermodynamics from molecular simulation

    NASA Astrophysics Data System (ADS)

    Fluitt, Aaron Michael

    Empowered by their exquisite three-dimensional structures, or "folds," proteins carry out biological tasks with high specificity, efficiency, and fidelity. The fold that optimizes biological function represents a stable configuration of the constituent polypeptide molecule(s) under physiological conditions. Proteins and polypeptides are not static, however: battered by thermal motion, they explore a distribution of folds that is determined by the sequence of amino acids, the presence and identity of other molecules, and the thermodynamic conditions. In this dissertation, we apply molecular simulation techniques to the study of two polypeptides that have unusually diffuse distributions of folds under physiological conditions: polyglutamine (polyQ) and islet amyloid polypeptide (IAPP). Neither polyQ nor IAPP adopts a predominant fold in dilute aqueous solution, but at sufficient concentrations, both are prone to self-assemble into stable, periodic, and highly regular aggregate structures known as amyloid. The appearance of amyloid deposits of polyQ in the brain, and of IAPP in the pancreas, are associated with Huntington's disease and type 2 diabetes, respectively. A molecular view of the mechanism(s) by which polyQ and IAPP fold and self-assemble will enhance our understanding of disease pathogenesis, and it has the potential to accelerate the development of therapeutics that target early-stage aggregates. Using molecular simulations with spatial and temporal resolution on the atomic scale, we present analyses of the structural distributions of polyQ and IAPP under various conditions, both in and out of equilibrium. In particular, we examine amyloid fibers of polyQ, the IAPP dimer in solution, and single IAPP fragments at a lipid bilayer. We also benchmark the molecular models, or "force fields," available for such studies, and we introduce a novel simulation algorithm.

  11. Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer.

    PubMed

    Matsuoka, T; Hirakawa, K; Chung, Y S; Yashiro, M; Nishimura, S; Sawada, T; Saiki, I; Sowa, M

    1998-05-01

    We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both alpha2beta1 and alpha3beta1 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequence intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhesion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients.

  12. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  13. Specific photoaffinity labeling of two plasma membrane polypeptides with an azido auxin

    NASA Technical Reports Server (NTRS)

    Hicks, G. R.; Rayle, D. L.; Jones, A. M.; Lomax, T. L.

    1989-01-01

    Plasma membrane vesicles were isolated from zucchini (Cucurbita pepo) hypocotyl tissue by aqueous phase partitioning and assessed for homogeneity by the use of membrane-specific enzyme assays. The highly pure (ca. 95%) plasma membrane vesicles maintained a pH differential across the membrane and accumulated a tritiated azido analogue of 3-indoleacetic acid (IAA), 5-azido-[7-3H]IAA ([3H]N3IAA), in a manner similar to the accumulation of [3H]IAA. The association of the [3H]N3IAA with membrane vesicles was saturable and subject to competition by IAA and auxin analogues. Auxin-binding proteins were photoaffinity labeled by addition of [3H]N3IAA to plasma membrane vesicles prior to exposure to UV light (15 sec; 300 nm) and detected by subsequent NaDodSO4/PAGE and fluorography. When the reaction temperature was lowered to -196 degrees C, high-specific-activity labeling of a 40-kDa and a 42-kDa polypeptide was observed. Triton X-100 (0.1%) increased the specific activity of labeling and reduced the background, which suggests that the labeled polypeptides are intrinsic membrane proteins. The labeled polypeptides are of low abundance, as expected for auxin receptors. Further, the addition of IAA and auxin analogues to the photoaffinity reaction mixture resulted in reduced labeling that was qualitatively similar to their effects on the accumulation of radiolabeled IAA in membrane vesicles. Collectively, these results suggest that the radiolabeled polypeptides are auxin receptors. The covalent nature of the label should facilitate purification and further characterization of the receptors.

  14. Chiral polyamines from reduction of polypeptides: asymmetric pyridoxamine-mediated transaminations.

    PubMed

    Zhou, Wenjun; Yerkes, Nancy; Chruma, Jason J; Liu, Lei; Breslow, Ronald

    2005-03-01

    BH3.THF can reduce polypeptides to polyamines with retention of chirality. The resulting polyamines are intriguing general platforms for asymmetric catalysis, given the diverse structures available and their relative ease of synthesis. We have constructed a number of chiral pyridoxamine catalysts based on reduced peptides. These compounds transaminate alpha-ketoacids with moderate to good enantioselectivity, while their peptidyl counterparts show almost no chiral induction.

  15. Wall-associated kinase-like polypeptide mediates nutritional status perception and response

    DOEpatents

    Yang, Zhenbiao; Karr, Stephen

    2014-02-11

    The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

  16. Hydrogen-bond driven loop-closure kinetics in unfolded polypeptide chains

    SciTech Connect

    Daidone, Isabella; Neuweiler, H; Doose, S; Sauer, M; Smith, Jeremy C

    2010-12-01

    Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient beta-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events.

  17. An altered Q sub B polypeptide as the basis for atrazine resistance in photoautotrophic potato cells

    SciTech Connect

    Smeda, R.J.; Hasegawa, P.M.; Weller, S.C. )

    1990-05-01

    A photoautotrophic potato cell line (variant) was isolated and is capable of sustained growth in media containing the herbicide atrazine at concentrations up to 100-fold greater than the lethal concentration (1.0 {mu}M) for the unselected (wild type) cell line. The basis for atrazine resistance could not be identified by differential uptake or metabolism. Photosynthetic electron transport rates for both intact cell and isolated thylakoid membranes from chloroplasts were unaffected in variant cells at atrazine concentrations up to 100-fold greater than for wild type cells. Photoaffinity labeling of isolated thylakoid membranes from both cell lines with {sup 14}C-azidoatrazine revealed an altered Q{sub B} polypeptide in variant cells resulting in low or no affinity for atrazine. A portion of the chloroplast psbA gene, encoding the Q{sub B} polypeptide, was sequenced for both cell lines. The basis for atrazine resistance in variant cells was identified as a single base change resulting in the alteration of serine to threonine at position 264 of the Q{sub B} polypeptide. In addition to atrazine resistance, variant cells exhibit enhanced tolerance to the herbicides DCMU and metribuzin, but greater sensitivity to bentazon. No reductions in variant cell growth and photosynthetic efficiency in the absence of atrazine were observed.

  18. Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi

    PubMed Central

    Chen, Xiaoshuang; Xu, Huanli; Li, Bo; Wang, Feng; Chen, Xiaoliang; Kong, Dexin; Lin, Xiukun

    2016-01-01

    CS5931 is a novel anticancer agent isolated from the sea squirt Ciona savignyi. However, its content in the species is very low, and developing a novel approach for production of the polypeptide is promising. In the present study, we expressed and purified the polypeptide from E. coli, and the fermentation conditions were studied using response surface methodology. The yield of CS5931 was increased from 2.0 to 7.5 mg/L. The denaturing and renaturation conditions were also studied. Using the optimized renaturation condition, the anticancer activity of refolding CS5931 was increased significantly; the value of IC50 was decreased from 23.2 to 11.6 μM. In vivo study using xenograft nude mice bearing HCT116 cancer cells revealed that CS5931 was able to inhibit the growth of tumor significantly. The study provides a useful approach for obtaining enough amount of CS5931 for further study. This study is also important for developing the polypeptide as a novel anticancer agent. PMID:27007382

  19. Cotranslational and Posttranslational N-Glycosylation of Polypeptides by Distinct Mammalian OST Isoforms

    PubMed Central

    Ruiz-Canada, Catalina; Kelleher, Daniel J.; Gilmore, Reid

    2010-01-01

    Summary Asparagine-linked glycosylation of polypeptides in the lumen of the endoplasmic reticulum is catalyzed by the hetero-oligomeric oligosaccharyltransferase (OST). OST isoforms with different catalytic subunits (STT3A versus STT3B) and distinct enzymatic properties are coexpressed in mammalian cells. Using siRNA to achieve isoform-specific knockdowns, we show that the OST isoforms cooperate and act sequentially to mediate protein N-glycosylation. The STT3A OST isoform is primarily responsible for cotranslational glycosylation of the nascent polypeptide as it enters the lumen of the endoplasmic reticulum. The STT3B isoform is required for efficient cotranslational glycosylation of an acceptor site adjacent to the N-terminal signal sequence of a secreted protein. Unlike STT3A, STT3B efficiently mediates posttranslational glycosylation of a carboxyl-terminal glycosylation site in an unfolded protein. These distinct and complementary roles for the OST isoforms allow sequential scanning of polypeptides for acceptor sites to insure the maximal efficiency of N-glycosylation. PMID:19167329

  20. Rh polypeptide is a major fatty acid-acylated erythrocyte membrane protein

    SciTech Connect

    de Vetten, M.P.; Agre, P.

    1988-12-05

    The erythrocyte Rh antigens contain an Mr = 32,000 integral protein which is thought to contribute in some way to the organization of surrounding phospholipid. To search for possible fatty acid acylation of the Rh polypeptide, intact human erythrocytes were incubated with (3H)palmitic acid prior to preparation of membranes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Several membrane proteins were labeled, but none corresponded to the glycophorins or membrane proteins 1-8. An Mr = 32,000 band was prominently labeled on Rh (D)-negative and -positive erythrocytes and could be precipitated from the latter with anti-D. No similar protein was labeled on membranes from Rhmod erythrocytes, a rare phenotype lacking Rh antigens. Labeling of the Rh polypeptide most likely represents palmitic acid acylation through thioester linkages. The 3H label was not extracted with chloroform/methanol, but was quantitatively eluted with hydroxylamine and co-chromatographed with palmitohydroxamate and free palmitate by thin layer chromatography. The fatty acid acylations occurred independent of protein synthesis and were completely reversed by chase with unlabeled palmitate. It is concluded that the Rh polypeptide is fatty acid-acylated, being a major substrate of an acylation-deacylation mechanism associated with the erythrocyte membrane.