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Sample records for recombinant major outer

  1. Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola.

    PubMed Central

    Fenno, J C; Müller, K H; McBride, B C

    1996-01-01

    The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli. Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M. Haapasalo, K. H. Müller, V. J. Uitto, W. K. Leung, and B. C. McBride, Infect. Immun. 60:2058-2065,1992). The 5' end of msp was obtained by PCR amplification from a T. denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences. The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da. The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1. Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon. The entire msp sequence was amplified from T. denticola genomic DNA and cloned in E. coli by using a tightly regulated T7 RNA polymerase vector system. Expression of Msp was toxic to E. coli when the entire msp gene was present. High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence. Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin. Attachment of recombinant Msp was decreased in the presence of soluble substrate. Attachment of T. denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp. These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T. denticola. PMID

  2. Evaluation of a multisubunit recombinant polymorphic membrane protein and major outer membrane protein T cell vaccine against Chlamydia muridarum genital infection in three strains of mice1

    PubMed Central

    Yu, Hong; Karunakaran, Karuna P.; Jiang, Xiaozhou; Brunham, Robert C.

    2014-01-01

    An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of C. muridarum genital infection, multifunctional mucosal CD4 T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4 T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population. PMID:24992718

  3. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants

    PubMed Central

    Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses. PMID:27219467

  4. Antibody and Cytokine Responses of Koalas (Phascolarctos cinereus) Vaccinated with Recombinant Chlamydial Major Outer Membrane Protein (MOMP) with Two Different Adjuvants.

    PubMed

    Khan, Shahneaz Ali; Desclozeaux, Marion; Waugh, Courtney; Hanger, Jon; Loader, Jo; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2016-01-01

    Developing a vaccine against Chlamydia is key to combating widespread mortalities and morbidities associated with this infection in koalas (Phascolarctos cinereus). In previous studies, we have shown that two or three doses of a Recombinant Major Outer Membrane Protein (rMOMP) antigen-based vaccine, combined with immune stimulating complex (ISC) adjuvant, results in strong cellular and humoral immune responses in koalas. We have also separately evaluated a single dose vaccine, utilising a tri-adjuvant formula that comprises polyphosphazine based poly I: C and host defense peptides, with the same antigen. This formulation also produced strong cellular and humoral immune responses in captive koalas. In this current study, we directly compared the host immune responses of two sub-groups of wild Chlamydia negative koalas in one population vaccinated with the rMOMP protein antigen and adjuvanted with either the ISC or tri-adjuvant formula. Overall, both adjuvants produced strong Chlamydia-specific cellular (IFN-γ and IL-17A) responses in circulating PBMCs as well as MOMP-specific and functional, in vitro neutralising antibodies. While the immune responses were similar, there were adjuvant-specific immune differences between the two adjuvants, particularly in relation to the specificity of the MOMP epitope antibody responses. PMID:27219467

  5. Humoral immune responses in koalas (Phascolarctos cinereus) either naturally infected with Chlamydia pecorum or following administration of a recombinant chlamydial major outer membrane protein vaccine.

    PubMed

    Khan, Shahneaz Ali; Polkinghorne, Adam; Waugh, Courtney; Hanger, Jon; Loader, Jo; Beagley, Kenneth; Timms, Peter

    2016-02-01

    The development of a vaccine is a key strategy to combat the widespread and debilitating effects of chlamydial infection in koalas. One such vaccine in development uses recombinant chlamydial major outer membrane protein (rMOMP) as an antigen and has shown promising results in several koala trials. Previous chlamydial vaccine studies, primarily in the mouse model, suggest that both cell-mediated and antibody responses will be required for adequate protection. Recently, the important protective role of antibodies has been highlighted. In our current study, we conducted a detailed analysis of the antibody-mediated immune response in koalas that are either (a) naturally-infected, and/or (b) had received an rMOMP vaccine. Firstly, we observed that naturally-infected koalas had very low levels of Chlamydia pecorum-specific neutralising antibodies. A strong correlation between low IgG total titers/neutralising antibody levels, and higher C. pecorum infection load was also observed in these naturally-infected animals. In vaccinated koalas, we showed that the vaccine was able to boost the humoral immune response by inducing strong levels of C. pecorum-specific neutralising antibodies. A detailed characterisation of the MOMP epitope response was also performed in naturally-infected and vaccinated koalas using a PepScan epitope approach. This analysis identified unique sets of MOMP epitope antibodies between naturally-infected non-protected and diseased koalas, versus vaccinated koalas, with the latter group of animals producing a unique set of specific epitope-directed antibodies that we demonstrated were responsible for the in vitro neutralisation activity. Together, these results show the importance of antibodies in chlamydial infection and immunity following vaccination in the koala. PMID:26747718

  6. Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C, a host defense peptide and polyphosphazine, elicits strong and long lasting cellular and humoral immune responses.

    PubMed

    Khan, Shahneaz Ali; Waugh, Courtney; Rawlinson, Galit; Brumm, Jacqui; Nilsson, Karen; Gerdts, Volker; Potter, Andrew; Polkinghorne, Adam; Beagley, Kenneth; Timms, Peter

    2014-10-01

    Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime. PMID:25196393

  7. Leptospirosis serodiagnosis by ELISA based on recombinant outer membrane protein.

    PubMed

    Chalayon, Piyanart; Chanket, Phanita; Boonchawalit, Toungporn; Chattanadee, Siriporn; Srimanote, Potjanee; Kalambaheti, Thareerat

    2011-05-01

    The outer membrane protein LipL21, LipL32, LipL41 and Loa22 of Leptospira interrogans serovar Copenhageni were previously revealed by immunoproteomic analysis, using sera from acute phase infection in a guinea pig. The full-length DNA of each protein was then cloned from the same serovar and expressed in pRSET vector. The obtained molecular weight (MW) of recombinant proteins rLipL21, rLipL32 and rLoa22 were slightly higher than the MW predicted from nucleotide sequences of each inserted gene, while only the N-terminal half of rLipL41 was obtained. Mice antiserum raised against each purified recombinant protein could react with the whole cell lysate of leptospiral serovars, implying that leptospiral native proteins shared a common epitope with recombinant protein. Serodiagnosis using recombinant protein antigen based on indirect ELISA procedure was developed in this study. The optimization of the ELISA components lead to determination of optical density (OD) from a single serum-dilution of 1:1000 in the leptospirosis patients group and normal healthy control group. The cut off OD values for both IgG and IgM class were investigated, and based on this fixed dilution only the IgG class could be used for differential diagnosis of patients and normal individuals. Compared with the MAT assay, ELISA assay utilizing both rLipL32 and rLoa22 as antigen, gave high accuracy and could thus be useful as a confirmative serology test. PMID:21353274

  8. Subdominant Outer Membrane Antigens in Anaplasma marginale: Conservation, Antigenicity, and Protective Capacity Using Recombinant Protein

    PubMed Central

    Ducken, Deirdre R.; Brown, Wendy C.; Alperin, Debra C.; Brayton, Kelly A.; Reif, Kathryn E.; Turse, Joshua E.; Palmer, Guy H.; Noh, Susan M.

    2015-01-01

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a defined surface protein complex reproducibly induce protective immunity. However, there are several knowledge gaps limiting progress in vaccine development. First, are these OMPs conserved among the diversity of A. marginale strains circulating in endemic regions? Second, are the most highly conserved outer membrane proteins in the immunogens recognized by immunized and protected animals? Lastly, can this subset of OMPs recognized by antibody from protected vaccinates and conserved among strains recapitulate the protection of outer membrane vaccines? To address the first goal, genes encoding OMPs AM202, AM368, AM854, AM936, AM1041, and AM1096, major subdominant components of the outer membrane, were cloned and sequenced from geographically diverse strains and isolates. AM202, AM936, AM854, and AM1096 share 99.9 to 100% amino acid identity. AM1041 has 97.1 to 100% and AM368 has 98.3 to 99.9% amino acid identity. While all four of the most highly conserved OMPs were recognized by IgG from animals immunized with outer membranes, linked surface protein complexes, or unlinked surface protein complexes and shown to be protected from challenge, the highest titers and consistent recognition among vaccinates were to AM854 and AM936. Consequently, animals were immunized with recombinant AM854 and AM936 and challenged. Recombinant vaccinates and purified outer membrane vaccinates had similar IgG and IgG2 responses to both proteins. However, the recombinant vaccinates developed higher bacteremia after challenge as compared to adjuvant-only controls and outer membrane vaccinates. These results provide the first evidence that vaccination with specific antigens may exacerbate disease. Progressing from the protective capacity of outer membrane formulations to recombinant vaccines

  9. Isolation of the outer membrane and characterization of the major outer membrane protein from Spirochaeta aurantia.

    PubMed Central

    Kropinski, A M; Parr, T R; Angus, B L; Hancock, R E; Ghiorse, W C; Greenberg, E P

    1987-01-01

    The outer membrane of Spirochaeta aurantia was isolated after cells were extracted with sodium lauryl sarcosinate and was subsequently purified by differential centrifugation and KBr isopycnic gradient centrifugation. The purified outer membrane was obtained in the form of carotenoid-containing vesicles. Four protein species with apparent molecular weights of 26,000 (26K), 36.5K, 41K, and 48.5K were readily observed as components of the vesicles. The 36.5K protein was the major polypeptide and constituted approximately 90% of the outer membrane protein observed on sodium dodecyl sulfate-polyacrylamide gels. Under mild denaturing conditions the 36.5K major protein exhibited an apparent molecular weight of approximately 90,000. This, together with the results of protein cross-linking studies, indicates that the 36.5K polypeptide has an oligomeric conformation in the native state. Reconstitution of solubilized S. aurantia outer membrane into lipid bilayer membranes revealed the presence of a porin, presumably the 36.5K protein, with an estimated channel diameter of 2.3 nm based on the measured single channel conductance of 7.7 nS in 1 M KCl. Images PMID:3025168

  10. Characterization of the major outer membrane antigens of Treponema hyodysenteriae.

    PubMed Central

    Wannemuehler, M J; Hubbard, R D; Greer, J M

    1988-01-01

    Outer membrane extracts of Treponema hyodysenteriae were used to evaluate the antibody responses in immunized or convalescent pigs. Western blot (immunoblot) analysis identified antibodies in sera reactive with 14- to 19-kilodalton (kDa) antigens. Reactivity against these antigens could be removed only by absorption of sera with butanol-water-extracted endotoxin from the homologous strain of T. hyodysenteriae. Treatment of the outer membrane extracts with 0.1 M sodium meta-periodate, but not with proteinase K, abolished reactivity with both outer membrane and endotoxin antigens (14 and 19 kDa). These results indicate that swine vaccinated with the outer membrane extract of T. hyodysenteriae develop antibody responses to outer membrane antigens qualitatively similar to those of swine convalescing from active infection, especially antibodies against low-molecular-mass antigens. The nature of the 14- to 19-kDa antigens appears consistent with that of treponemal endotoxin and lipopolysaccharide. Images PMID:2460406

  11. Recombinant Outer Capsid Glycoprotein (VP7) of Rotavirus Expressed in Insect Cells Induces Neutralizing Antibodies in Rabbits

    PubMed Central

    Khodabandehloo, M; Shahrabadi, M Shamsi; Keyvani, H; Bambai, B; Sadigh, ZA

    2012-01-01

    Background: Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection. Many efforts have been done to produce recombinant VP7 that maintain native characteristics. We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Methods: Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Results: Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants. Injection of recombinant VP7 in rabbits elicited the production of serum antibodies, which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture. Conclusion: Recombinant outer capsid glycoprotein (VP7) of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine. PMID:23113180

  12. Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC).

    PubMed

    Stenkova, Anna M; Bystritskaya, Evgeniya P; Guzev, Konstantin V; Rakin, Alexander V; Isaeva, Marina P

    2016-01-01

    The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system. PMID:27578962

  13. Molecular Evolution of the Yersinia Major Outer Membrane Protein C (OmpC)

    PubMed Central

    Stenkova, Anna M.; Bystritskaya, Evgeniya P.; Guzev, Konstantin V.; Rakin, Alexander V.; Isaeva, Marina P.

    2016-01-01

    The genus Yersinia includes species with a wide range of eukaryotic hosts (from fish, insects, and plants to mammals and humans). One of the major outer membrane proteins, the porin OmpC, is preferentially expressed in the host gut, where osmotic pressure, temperature, and the concentrations of nutrients and toxic products are relatively high. We consider here the molecular evolution and phylogeny of Yersinia ompC. The maximum likelihood gene tree reflects the macroevolution processes occurring within the genus Yersinia. Positive selection and horizontal gene transfer are the key factors of ompC diversification, and intraspecies recombination was revealed in two Yersinia species. The impact of recombination on ompC evolution was different from that of another major porin gene, ompF, possibly due to the emergence of additional functions and conservation of the basic transport function. The predicted antigenic determinants of OmpC were located in rapidly evolving regions, which may indicate the evolutionary mechanisms of Yersinia adaptation to the host immune system. PMID:27578962

  14. Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies.

    PubMed

    Yadav, Sunita Kumari; Meena, Jitendra Kumar; Sharma, Mahima; Dixit, Aparna

    2016-08-01

    Aeromonas hydrophila is a gram-negative fish pathogenic bacterium, also responsible for causing opportunistic pathological conditions in humans. It causes a number of diseases in fish due to which the fish industry incurs huge economic losses annually. Due to problems of antibiotic resistance, and the rapidity with which the infection spreads among fishes, vaccination remains the most effective strategy to combat this infection in fish populations. Among various virulence factors associated with bacterial virulence, outer membrane proteins have been widely evaluated for their vaccine potential owing to their surface exposure and related role in pathogenicity. In the present study, we have investigated the immunogenic potential of a non-specific porin, outer membrane protein C (OmpC) whose expression is regulated by the two-component regulatory system and plays a major role in the survival of A. hydrophila under different osmolaric conditions. The full-length gene (~1 kb) encoding OmpC of A. hydrophila was cloned, characterized and expressed in E. coli. High yield (~112 mg/L at shake flask level) of the recombinant OmpC (rOmpC) (~40 kDa) of A. hydrophila was obtained upon purification from inclusion bodies using Ni(2+)-NTA affinity chromatography. Immunization with purified rOmpC in murine model generated high endpoint (>1:40,000) titers. IgG isotyping, ELISA and ELISPOT assay indicated mixed immune response with a TH2 bias. Also, the anti-rOmpC antibodies were able to agglutinate A. hydrophila in vitro and exhibited specific cross-reactivity with different Aeromonas strains, which will facilitate easy detection of different Aeromonas isolates in infected samples. Taken together, these data clearly indicate that rOmpC could serve as an effective vaccine against different strains of Aeromonas, a highly heterogenous group of bacteria. PMID:27328672

  15. The Major Outer Sheath Protein (Msp) of Treponema denticola Has a Bipartite Domain Architecture and Exists as Periplasmic and Outer Membrane-Spanning Conformers

    PubMed Central

    Anand, Arvind; Luthra, Amit; Edmond, Maxwell E.; Ledoyt, Morgan; Caimano, Melissa J.

    2013-01-01

    The major outer sheath protein (Msp) is a primary virulence determinant in Treponema denticola, as well as the parental ortholog for the Treponema pallidum repeat (Tpr) family in the syphilis spirochete. The Conserved Domain Database (CDD) server revealed that Msp contains two conserved domains, major outer sheath proteinN (MOSPN) and MOSPC, spanning residues 77 to 286 and 332 to 543, respectively, within the N- and C-terminal regions of the protein. Circular dichroism (CD) spectroscopy, Triton X-114 (TX-114) phase partitioning, and liposome incorporation demonstrated that full-length, recombinant Msp (MspFl) and a recombinant protein containing MOSPC, but not MOSPN, form amphiphilic, β-sheet-rich structures with channel-forming activity. Immunofluorescence analysis of intact T. denticola revealed that only MOSPC contains surface-exposed epitopes. Data obtained using proteinase K accessibility, TX-114 phase partitioning, and cell fractionation revealed that Msp exists as distinct OM-integrated and periplasmic trimers. MspFl folded in Tris buffer contained slightly less β-sheet structure than detergent-folded MspFl; both forms, however, partitioned into the TX-114 detergent-enriched phase. CDD analysis of the nine Tpr paralogs predicted to be outer membrane proteins (OMPs) revealed that seven have an Msp-like bipartite structure; phylogenetic analysis revealed that the MOSPN and MOSPC domains of Msp are most closely related to those of TprK. Based upon our collective results, we propose a model whereby a newly exported, partially folded intermediate can be either processed for OM insertion by the β-barrel assembly machinery (BAM) or remain periplasmic, ultimately forming a stable, water-soluble trimer. Extrapolated to T. pallidum, our model enables us to explain how individual Tprs can localize to either the periplasmic (e.g., TprK) or OM (e.g., TprC) compartments. PMID:23457251

  16. Warp of the ionized gas layer in the outer Galaxy, traced by recombination line observations

    NASA Astrophysics Data System (ADS)

    Azcárate, I. N.; Cersosimo, J. C.

    We report results of H166α recombination line observations from the outer Galaxy in both the Northern and Southern Galactic Plane. The Southern observations were made with the 30 m antenna of the Instituto Argentino de Radioastronomía in Villa Elisa, Buenos Aires, Argentina, and the Northern ones ( more sensitive, high quality observations, performed with an ``state of the art'' receiver) with the 43 m antenna of the National Radio Astronomy Observatory, in Green Bank, West Virginia, USA. >From the two sets of observations we obtain evidence of the warp of the low-density ionized gas layer, traced by the H166α emission in the outer Milky Way, towards positive galactic latitudes in the Northern and towards negative latitudes in the Southern Galaxy. The warp of this tracer qualitatively agrees with that of the HI.

  17. Porin activity of the native and recombinant outer membrane protein Oms28 of Borrelia burgdorferi.

    PubMed Central

    Skare, J T; Champion, C I; Mirzabekov, T A; Shang, E S; Blanco, D R; Erdjument-Bromage, H; Tempst, P; Kagan, B L; Miller, J N; Lovett, M A

    1996-01-01

    The outer membrane-spanning (Oms) proteins of Borrelia burgdorferi have been visualized by freeze-fracture analysis but, until recently, not further characterized. We developed a method for the isolation of B. burgdorferi outer membrane vesicles and described porin activities with single-channel conductances of 0.6 and 12.6 nS in 1 M KCI. By using both nondenaturing isoelectric focusing gel electrophoresis and fast-performance liquid chromatography separation after detergent solubilization, we found that the 0.6-nS porin activity resided in a 28-kDa protein, designated Oms28. The oms28 gene was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence of Oms28 predicted a 257-amino-acid precursor protein with a putative 24-amino-acid leader peptidase I signal sequence. Processed Oms28 yielded a mature protein with a predicted molecular mass of 25,363 Da. When overproduced in Escherichia coli, the Oms28 porin fractionated in part to the outer membrane. Sodium dodecyl sulfate-polyacrylamide gel-purified recombinant Oms28 from E. coli retained functional activity as demonstrated by an average single-channel conductance of 1.1 nS in the planar lipid bilayer assay. These findings confirmed that Oms28 is a B. burgdorferi porin, the first to be described. As such, it is potential relevance to the pathogenesis of Lyme borreliosis and to the physiology of the spirochete. PMID:8759855

  18. Expression of the major outer membrane protein of Chlamydia trachomatis in Escherichia coli.

    PubMed Central

    Manning, D S; Stewart, S J

    1993-01-01

    The major outer membrane protein (MOMP) of Chlamydia trachomatis was expressed in Escherichia coli. To assess whether it assembled into a conformationally correct structure at the cell surface, we characterized the recombinant MOMP (rMOMP) by Western immunoblot analysis, indirect immunofluorescence, and immunoprecipitation with monoclonal antibodies (MAbs) that recognize contiguous and conformational MOMP epitopes. Western blot analysis showed that most of the rMOMP comigrated with authentic monomer MOMP, indicating that its signal peptide was recognized and cleaved by E. coli. The rMOMP could not be detected on the cell surface of viable or formalin-killed E. coli organisms by indirect immunofluorescence staining with a MAb specific for a MOMP contiguous epitope. In contrast, the same MAb readily stained rMOMP-expressing E. coli cells that had been permeabilized by methanol fixation. A MAb that recognizes a conformational MOMP epitope and reacted strongly with formalin- or methanol-fixed elementary bodies failed to stain formalin- or methanol-fixed E. coli expressing rMOMP. Moreover, this MAb did not immunoprecipitate rMOMP from expressing E. coli cells even though it precipitated the authentic protein from lysates of C. trachomatis elementary bodies. Therefore we concluded that rMOMP was not localized to the E. coli cell surface and was not recognizable by a conformation-dependent antibody. These results indicate that rMOMP expressed by E. coli is unlikely to serve as an accurate model of MOMP structure and function. They also question the utility of rMOMP as a source of immunogen for eliciting neutralizing antibodies against conformational antigenic sites of the protein. Images PMID:8406797

  19. Construction and Immunogenicity of Recombinant Swinepox Virus Expressing Outer Membrane Protein L of Salmonella.

    PubMed

    Fang, Yizhen; Lin, Huixing; Ma, Zhe; Fan, Hongjie

    2016-07-28

    Salmonella spp. are gram-negative flagellated bacteria that cause a variety of diseases in humans and animals, ranging from mild gastroenteritis to severe systemic infection. To explore development of a potent vaccine against Salmonella infections, the gene encoding outer membrane protein L (ompL) was inserted into the swinepox virus (SPV) genome by homologous recombination. PCR, western blot, and immunofluorescence assays were used to verify the recombinant swinepox virus rSPV-OmpL. The immune responses and protection efficacy of rSPV-OmpL were assessed in a mouse model. Forty mice were assigned to four groups, which were immunized with rSPV-OmpL, inactive Salmonella (positive control), wildtype SPV (wtSPV; negative control), or PBS (challenge control), respectively. The OmpLspecific antibody in the rSPV-OmpL-immunized group increased dramatically and continuously over time post-vaccination, and was present at a significantly higher level than in the positive control group (p < 0.05). The concentrations of IFN-γ and IL-4, which represent Th1-type and Th2-type cytokine responses, were significantly higher (p < 0.05) in the rSPVOmpL- vaccinated group than in the other three groups. After intraperitoneal challenge with a lethal dose of Salmonella typhimurium CVCC542, eight out of ten mice in the rSPV-OmpLvaccinated group were protected, whereas all the mice in the negative control and challenge control groups died within 3 days. Passive immune protection assays showed that hyperimmune sera against OmpL could provide mice with effective protection against challenge from S. typhimurium. The recombinant swinepox virus rSPV-OmpL might serve as a promising vaccine against Salmonella infection. PMID:27012234

  20. A poliovirus hybrid expressing a neutralization epitope from the major outer membrane protein of Chlamydia trachomatis is highly immunogenic.

    PubMed Central

    Murdin, A D; Su, H; Manning, D S; Klein, M H; Parnell, M J; Caldwell, H D

    1993-01-01

    Trachoma and sexually transmitted diseases caused by Chlamydia trachomatis are major health problems worldwide. Epitopes on the major outer membrane protein (MOMP) of C. trachomatis have been identified as important targets for the development of vaccines. In order to examine the immunogenicity of a recombinant vector expressing a chlamydial epitope, a poliovirus hybrid was constructed in which part of neutralization antigenic site I of poliovirus type 1 Mahoney (PV1-M) was replaced by a sequence from variable domain I of the MOMP of C. trachomatis serovar A. The chlamydial sequence included the neutralization epitope VAGLEK. This hybrid was viable, grew very well compared with PV1-M, and expressed both poliovirus and chlamydial antigenic determinants. When inoculated into rabbits, this hybrid was highly immunogenic, inducing a strong response against both PV1-M and C. trachomatis serovar A. Antichlamydia titers were 10- to 100-fold higher than the titers induced by equimolar amounts of either purified MOMP or a synthetic peptide expressing the VAGLEK epitope. Furthermore, rabbit antisera raised against this hybrid neutralized chlamydial infectivity both in vitro, for hamster kidney cells, and passively in vivo, for conjunctival epithelia of cynomolgus monkeys. Because poliovirus infection induces a strong mucosal immune response in primates and humans, these results indicate that poliovirus-chlamydia hybrids could become powerful tools for the study of mucosal immunity to chlamydial infection and for the development of recombinant chlamydial vaccines. Images PMID:7691749

  1. Purification and partial characterization of the major outer membrane protein of Chlamydia trachomatis

    SciTech Connect

    Caldwell, H.D.; Kromhout, J.; Schachter, J.

    1981-03-01

    Elementary bodies (EB) of Chlamydia trachomatis serotypes C, E, and L2 were extrinsically radioiodinated, and whole-cell lysates of these serotypes were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Autoradiography of the polypeptide profiles identified a major surface protein with an apparent subunit molecular weight of 39,500 that was common to each C. trachomatis serotype. The abilities of nonionic (Triton X-100), dipolar ionic (Zwittergent TM-314), mild (sodium deoxycholate and sodium N-lauroyl sarcosine), and strongly anionic (SDS) detergents to extract this protein from intact EB of the L2 serotype were investigated by SDS-PAGE analysis of the soluble and insoluble fractions obtained after each detergent treatment. Only SDS readily extracted this protein from intact EB. Sarkosyl treatment selectively solubilized the majority of other EB proteins, leaving the 39,500-dalton protein associated with the Sarkosyl-insoluble fraction. Ultrastructural studies of the Sarkosyl-insoluble EB pellet showed it to consist of empty EB particles possessing an apparently intact outer membrane. No structural evidence for a peptidoglycan-like cell wall was found. Morphologically these chlamydial outer membrane complexes (COMC) resembled intact chlamydial EB outer membranes. The 39,500-dalton outer membrane protein was quantitatively extracted from COMC by treating them with 2% SDS at 60 degrees C. This protein accounted for 61% of the total COMC-associated protein, and its extraction resulted in a concomitant loss of the COMC membrane structure and morphology. The 39,500-dalton major outer membrane protein is a serogroup antigen of C. trachomatis organisms.

  2. Protective Immunity to Vaccinia Virus Induced by Vaccination with Multiple Recombinant Outer Membrane Proteins of Intracellular and Extracellular Virions

    PubMed Central

    Fogg, Christiana; Lustig, Shlomo; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Moss, Bernard

    2004-01-01

    Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine. PMID:15367588

  3. Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae.

    PubMed Central

    Carbonetti, N H; Sparling, P F

    1987-01-01

    Protein I (P.I) is the major outer membrane protein of Neisseria gonorrhoeae and serves as a porin. By using oligonucleotide probes derived from the known amino-terminal sequence of the mature protein, we have cloned the gene encoding the P.I of gonococcal strain FA19 in three overlapping fragments and determined the DNA sequence. The gene sequence predicts a protein with characteristics typical of the porins of other Gram-negative bacteria. A clone expressing P.I in Escherichia coli was obtained by removing a portion of the P.I gene promoter and reconstructing the entire P.I gene in a position just downstream from a phage T7 promoter. Expression of P.I was then achieved by introducing this recombinant plasmid into an E. coli strain containing an inducible T7 polymerase gene. The clone produced a protein that was identical in size to native P.I and reacted with anti-P.I monoclonal antibodies. Prolonged expression of the protein apparently was lethal for E. coli, possibly explaining failures to clone an intact P.I gene with its own promoter. Images PMID:3122212

  4. Outer membrane protein DsrA is the major fibronectin-binding determinant of Haemophilus ducreyi.

    PubMed

    Leduc, Isabelle; White, C Dinitra; Nepluev, Igor; Throm, Robert E; Spinola, Stanley M; Elkins, Christopher

    2008-04-01

    The ability to bind extracellular matrix proteins is a critical virulence determinant for skin pathogens. Haemophilus ducreyi, the etiological agent of the genital ulcer disease chancroid, binds extracellular matrix components, including fibronectin (FN). We investigated H. ducreyi FN binding and report several important findings about this interaction. First, FN binding by H. ducreyi was greatly increased in bacteria grown on heme and almost completely inhibited by hemoglobin. Second, wild-type strain 35000HP bound significantly more FN than did a dsrA mutant in two different FN binding assays. Third, the expression of dsrA in the dsrA mutant restored FN binding and conferred the ability to bind FN to a non-FN-binding Haemophilus influenzae strain. Fourth, an anti-DsrA monoclonal antibody partially blocked FN binding by H. ducreyi. The hemoglobin receptor, the collagen-binding protein, the H. ducreyi lectin, the fine-tangle pili, and the outer membrane protein OmpA2 were not involved in H. ducreyi FN binding, since single mutants bound FN as well as the parent strain did. However, the major outer membrane protein may have a minor role in FN binding by H. ducreyi, since a double dsrA momp mutant bound less FN than did the single dsrA mutant. Finally, despite major sequence differences, DsrA proteins from both class I and class II H. ducreyi strains mediated FN and vitronectin binding. We concluded that DsrA is the major factor involved in FN binding by both classes of H. ducreyi strains. PMID:18212073

  5. Long-Term Stability of a Vaccine Formulated with the Amphipol-Trapped Major Outer Membrane Protein from Chlamydia trachomatis

    PubMed Central

    Feinstein, H. Eric; Tifrea, Delia; Sun, Guifeng; Popot, Jean-Luc; de la Maza, Luis M.

    2014-01-01

    Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3–14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3–14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of 15N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3–14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations. PMID:24942817

  6. High-Resolution Patterns of Meiotic Recombination across the Human Major Histocompatibility Complex

    PubMed Central

    Cullen, Michael; Perfetto, Stephen P.; Klitz, William; Nelson, George; Carrington, Mary

    2002-01-01

    Definitive characteristics of meiotic recombination events over large (i.e., >1 Mb) segments of the human genome remain obscure, yet they are essential for establishing the haplotypic structure of the genome and for efficient mapping of complex traits. We present a high-resolution map of recombination at the kilobase level across a 3.3-Mb interval encompassing the major histocompatibility complex (MHC). Genotyping of 20,031 single sperm from 12 individuals resulted in the identification and fine mapping of 325 recombinant chromosomes within genomic intervals as small as 7 kb. Several principal characteristics of recombination in this region were observed: (1) rates of recombination can differ significantly between individuals; (2) intense hot spots of recombination occur at least every 0.8 Mb but are not necessarily evenly spaced; (3) distribution in the location of recombination events can differ significantly among individuals; (4) between hot spots, low levels of recombination occur fairly evenly across 100-kb segments, suggesting the presence of warm spots of recombination; and (5) specific sequence motifs associate significantly with recombination distribution. These data provide a plausible model for recombination patterns of the human genome overall. PMID:12297984

  7. Pore-forming ability of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Egli, C; Hancock, R E; Holt, S C

    1992-01-01

    Three major outer membrane proteins with apparent molecular masses of 43, 45, and 51 kDa were purified from Wolinella recta ATCC 33238, and their pore-forming abilities were determined by the black lipid bilayer method. The non-heat-modifiable 45-kDa protein (Omp 45) showed no pore-forming activity even at high KCl concentrations. The single-channel conductances in 1 M KCl of the heat-modifiable proteins with apparent molecular masses of 43 kDa (Omp 43) and 51 kDa (Omp 51) were 0.49 and 0.60 nS, respectively. The proteins formed nonselective channels and, as determined by experiments of ion selectivity and zero-current potential, were weakly anion selective. Images PMID:1370429

  8. Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

    NASA Astrophysics Data System (ADS)

    Yuan, Ye; Wang, Xiuli; Guo, Sheping; Qiu, Xuemei

    2011-06-01

    Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene (ompW) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by V. parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

  9. Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).

    PubMed Central

    Champion, C I; Blanco, D R; Exner, M M; Erdjument-Bromage, H; Hancock, R E; Tempst, P; Miller, J N; Lovett, M A

    1997-01-01

    In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum. PMID:9023206

  10. Subdominant outer membrane antigens in anaplasma marginale: conservation, antigenicity, and protective capacity using recombinant protein

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Anaplasma marginale is a tick-borne rickettsial pathogen of cattle with a worldwide distribution. Currently a safe and efficacious vaccine is unavailable. Outer membrane protein (OMP) extracts or a well- defined surface protein complex reproducibly induce protective immunity. However, there are seve...

  11. Extraction, purification, and characterization of major outer membrane proteins from Wolinella recta ATCC 33238.

    PubMed Central

    Kennell, W L; Holt, S C

    1991-01-01

    The outer membrane of Wolinella recta ATCC 33238 was isolated by French pressure cell disruption and differential centrifugation. Outer membrane proteins (OMPs) were solubilized by Zwittergent 3.14 extraction and separated by DEAE-Sephacel ion-exchange chromatography. The major OMPs that were found in W. recta ATCC 33238 and in several other Wolinella spp. consisted of proteins with apparent molecular masses of 51, 45, and 43 kDa. These three conserved proteins were purified to essential homogeneity by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized chemically. Heating at between 75 and 100 degrees C revealed both the 43- and 51-kDa proteins to be heat modified from apparent molecular masses of 32 and 38 kDa, respectively. The 45-kDa protein was unmodified at all temperatures tested. Two-dimensional isoelectric focusing-SDS-PAGE revealed the 51-kDa protein to be composed of multiple pIs between a pH of 5.0 and greater than 8.0 while the 43- and 45-kDa proteins had a pI of approximately 6.0. N'-terminal amino acid sequence analysis of the first 30 to 40 amino acids and search of the Protein Identification Resource data base for similar proteins only revealed the 43-kDa protein to be similar to the P.69 OMP of Bordetella pertussis; however, the homology was weak (33%). Amino acid analysis revealed the 43-kDa protein to be noncharged and the 45- and 51-kDa proteins to be hydrophilic, containing between 38 to 42% polar residues but no cysteine. This study reports the purification and partial characterization of three conserved proteins in W. recta ATCC 33238. Images PMID:1894372

  12. Biochemical and biophysical characterization of the major outer surface protein, OSP-A from North American and European isolates of Borrelia burgdorferi

    SciTech Connect

    McGrath, B.C.; Dunn, J.J.; France, L.L.; Jaing, W.; Polin, D.; Gorgone, G.; Luft, B.; Dykhuizen, D.

    1995-12-31

    Lyme borreliosis, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne disease in North America and Western Europe. As the major delayed immune response in humans, a better understanding of the major outer surface lipoproteins OspA and OspB are of much interest. These proteins have been shown to exhibit three distinct phylogenetic genotypes based on their DNA sequences. This paper describes the cloning of genomic DNA for each variant and amplification of PCR. DNA sequence data was used to derive computer driven phylogenetic analysis and deduced amino acid sequences. Overproduction of variant OspAs was carried out in E. coli using a T7-based expression system. Circular dichroism and fluorescence studies was carried out on the recombinant B31 PspA yielding evidence supporting a B31 protein containing 11% alpha-helix, 34% antiparallel beta-sheet, 12% parallel beta sheet.

  13. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

    PubMed Central

    Gabay, J E; Blake, M; Niles, W D; Horwitz, M A

    1985-01-01

    We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins. Images PMID:2579942

  14. Recombinant outer membrane protein F-B subunit of LT protein as a prophylactic measure against Pseudomonas aeruginosa burn infection in mice

    PubMed Central

    Farsani, Hassan Heydari; Rasooli, Iraj; Gargari, Seyed Latif Mousavi; Nazarian, Shahram; Astaneh, Shakiba Darwish Alipour

    2015-01-01

    AIM: To study immunogenicity of outer membrane protein F (OprF) fused with B subunit of LT (LTB), against Pseudomonas aeruginosa (P. aeruginosa). METHODS: The OprF, a major surface exposed outer membrane protein that is antigenically conserved in various strains of P. aeruginosa, is a promising immunogen against P. aeruginosa. In the present study recombinant OprF and OprF-LTB fusion gene was cloned, expressed and purified. BALB/c mice and rabbits were immunized using recombinant OprF and OprF-LTB and challenged at the burn site with P. aeruginosa lethal dose of 104 CFU. The protective efficacy of rabbit anti OprF IgG against P. aeruginosa burn infection was investigated by passive immunization. RESULTS: It has been well established that the LTB is a powerful immunomodulator with strong adjuvant activity. LTB as a bacterial adjuvant enhanced immunogenicity of OprF and anti OprF IgG titer in serum was increased. Experimental findings showed significantly higher average survival rate in burned mice immunized with OprF-LTB than immunized with OprF or the control group. Rabbits anti OprF IgG brought about 75% survival of mice following challenge with P. aeruginosa. Post challenge hepatic and splenic tissues of mice group immunized with OprF-LTB had significantly lower bacterial load than those immunized with OprF or the control groups. CONCLUSION: These results demonstrate that LTB-fused OprF might be a potential candidate protein for a prophylactic measure against P. aeruginosa in burn infection. PMID:26713284

  15. Vaccination of ducks with recombinant outer membrane protein (OmpA) and a 41 kDa partial protein (P45N') of Riemerella anatipestifer.

    PubMed

    Huang, Bin; Subramaniam, Sumathi; Frey, Joachim; Loh, Hilda; Tan, Hai-Meng; Fernandez, Charlene J; Kwang, Jimmy; Chua, Kim-Lee

    2002-01-23

    The generation of protective immunity against Riemerella anatipestifer infection in ducks were investigated by immunizations with recombinant glutathione sulfatransferase (GST) fusion's proteins of OmpA, a 42kDa major outer membrane protein, and P45N', a 41kDa N-terminal fragment of a newly identified 45kDa potential surface protein from R. anatipestifer. The DNA encoding OmpA and P45N' were isolated from R. anatipestifer serotype 15 (field strain 110/89) and serotype 19 (reference strain 30/90), respectively. Immunoblotting and ELISA results showed that the purified recombinant proteins induced the production of antibodies in immunized ducks. However, neither was protective against subsequent challenge with the virulent serotype 15 strain, 34/90. All the five ducks immunized with formalinized R. anatipestifer strain 34/90 survived the challenge with the homologous strain whereas six out of seven ducks in the non-immunized control group died within a week following the challenge. PMID:11731174

  16. Overexpression, refolding, and purification of the major immunodominant outer membrane porin OmpC from Salmonella typhi: characterization of refolded OmpC.

    PubMed

    Kumar, P D; Krishnaswamy, S

    2005-03-01

    The major immunodominant integral outer membrane protein C (OmpC) from Salmonella typhi Ty21a was overexpressed, without the signal peptide, in Escherichia coli. The protein aggregates as inclusion bodies (IBs) in the cytoplasm. OmpC from IBs was solubilized with 4 M urea and refolded. This involved rapid dilution of unfolded OmpC into a refolding buffer containing polyoxyethylene-9-lauryl ether (C(12)E(9)) and glycerol. The refolded OmpC (rfOmpC) was shown to be structurally similar to the native OmpC by SDS-PAGE, Western blotting, tryptic digestion, ultrafiltration, circular dichroism, and fluorescence spectroscopic techniques. Crystals of rfOmpC were obtained in preliminary crystallization trials. The rfOmpC also sets a stage for rational design by recombinant DNA technology for vaccine design and high resolution structure determination. PMID:15721780

  17. The recent recombinant evolution of a major crop pathogen, potato virus Y.

    PubMed

    Visser, Johan Christiaan; Bellstedt, Dirk Uwe; Pirie, Michael David

    2012-01-01

    Potato virus Y (PVY) is a major agricultural disease that reduces crop yields worldwide. Different strains of PVY are associated with differing degrees of pathogenicity, of which the most common and economically important are known to be recombinant. We need to know the evolutionary origins of pathogens to prevent further escalations of diseases, but putatively reticulate genealogies are challenging to reconstruct with standard phylogenetic approaches. Currently available phylogenetic hypotheses for PVY are either limited to non-recombinant strains, represent only parts of the genome, and/or incorrectly assume a strictly bifurcating phylogenetic tree. Despite attempts to date potyviruses in general, no attempt has been made to date the origins of pathogenic PVY. We test whether diversification of the major strains of PVY and recombination between them occurred within the time frame of the domestication and modern cultivation of potatoes. In so doing, we demonstrate a novel extension of a phylogenetic approach for reconstructing reticulate evolutionary scenarios. We infer a well resolved phylogeny of 44 whole genome sequences of PVY viruses, representative of all known strains, using recombination detection and phylogenetic inference techniques. Using Bayesian molecular dating we show that the parental strains of PVY diverged around the time potatoes were first introduced to Europe, that recombination between them only occurred in the last century, and that the multiple recombination events that led to highly pathogenic PVY(NTN) occurred within the last 50 years. Disease causing agents are often transported across the globe by humans, with disastrous effects for us, our livestock and crops. Our analytical approach is particularly pertinent for the often small recombinant genomes involved (e.g. HIV/influenza A). In the case of PVY, increased transport of diseased material is likely to blame for uniting the parents of recombinant pathogenic strains: this process needs

  18. Functional properties of the major outer membrane protein in Stenotrophomonas maltophilia.

    PubMed

    Chen, Yih-Yuan; Wu, Han-Chiang; Lin, Juey-Wen; Weng, Shu-Fen

    2015-08-01

    Stenotrophomonas maltophilia is an opportunistic pathogen that is closely associated with high morbidity and mortality in debilitated and immunocompromised individuals. Therefore, to investigate the pathogenesis mechanism is urgently required. However, there are very few studies to evaluate the functional properties of outer membrane protein, which may contribute to the pathogenesis in S. maltophilia. In this study, three abundant proteins in the outer membrane fraction of S. maltophilia were identified by liquid chromatography-tandem mass spectrometry as OmpW1, MopB, and a hypothetical protein. MopB, a member of the OmpA family, was firstly chosen for functional investigation in this study because many OmpA-family proteins are known to be involved in pathogenesis and offer potential as vaccines. Membrane fractionation analyses demonstrated that MopB was indeed the most abundant outer membrane protein (OMP) in S. maltophilia. For functional studies, the mopB mutant of S. maltophilia (SmMopB) was constructed by insertional mutation. MopB deficiency resulted in a change in the protein composition of OMPs and altered the architecture of the outer membrane. The SmMopB strain exhibited reduced cytotoxicity toward L929 fibroblasts and was more sensitive to numerous stresses, including human serum, sodium dodecyl sulfate, and hydrogen peroxide compared with wildtype S. maltophilia. These results suggest that MopB may be a good candidate for the design of vaccines or anti-MopB drugs for controlling serious nosocomial infections of multidrug-resistant S. maltophilia, especially in immunosuppressed patients. PMID:26224456

  19. Renaturation of Recombinant Treponema pallidum Rare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation

    PubMed Central

    Zhang, Hongwei H.; Blanco, David R.; Exner, Maurice M.; Shang, Ellen S.; Champion, Cheryl I.; Phillips, Martin L.; Miller, James N.; Lovett, Michael A.

    1999-01-01

    We have previously observed that while native Treponema pallidum rare outer membrane protein 1 (Tromp1) is hydrophobic and has porin activity, recombinant forms of Tromp1 do not possess these properties. In this study we show that these properties are determined by conformation and can be replicated by proper renaturation of recombinant Tromp1. Native Tromp1, but not the 47-kDa lipoprotein, extracted from whole organisms by using Triton X-114, was found to lose hydrophobicity after treatment in 8 M urea, indicating that Tromp1’s hydrophobicity is conformation dependent. Native Tromp1 was purified from 0.1% Triton X-100 extracts of whole organisms by fast-performance liquid chromatography (FPLC) and shown to have porin activity in planar lipid bilayers. Cross-linking studies of purified native Tromp1 with an 11 Å cross-linking agent showed oligomeric forms consistent with dimers and trimers. For renaturation studies of recombinant Tromp1 (rTromp1), a 31,109-Da signal-less construct was expressed in Escherichia coli and purified by FPLC. FPLC-purified rTromp1 was denatured in 8 M urea and then renatured in the presence of 0.5% Zwittergent 3,14 during dialysis to remove the urea. Renatured rTromp1 was passed through a Sephacryl S-300 gel exclusion column previously calibrated with known molecular weight standards. While all nonrenatured rTromp1 eluted from the column at approximately the position of the carbonic anhydrase protein standard (29 kDa), all renatured rTromp1 eluted at the position of the phosphorylase b protein standard (97 kDa), suggesting a trimeric conformation. Trimerization was confirmed by using an 11 Å cross-linking agent which showed both dimers and trimers similar to that of native Tromp1. Triton X-114 phase separations showed that all of renatured rTromp1, but none of nonrenatured rTromp1, phase separated exclusively into the hydrophobic detergent phase, similar to native Tromp1. Circular dichroism of nonrenatured and renatured rTromp1

  20. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

    PubMed

    Monaris, D; Sbrogio-Almeida, M E; Dib, C C; Canhamero, T A; Souza, G O; Vasconcellos, S A; Ferreira, L C S; Abreu, P A E

    2015-08-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  1. Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

    PubMed Central

    Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

    2015-01-01

    Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

  2. Ultraviolet-irradiated vaccinia virus recombinants, exposing HIV-envelope on their outer membrane, induce antibodies against this antigen in rabbits.

    PubMed

    Loewinger, M; Katz, E

    2002-01-01

    The construction and isolation of recombinants of vaccinia virus (IHD-J strain), bearing on their outer membrane a chimeric protein consisting of the cytoplasmic and transmembrane domains of vaccinia B5R protein and the external domain of HIV envelope, has been previously described by us. The present study aimed to investigate the potential use of such recombinants as a vaccine, following inactivation of their infectivity by ultraviolet (UV) irradiation. The minimal dose of UV irradiation, required for the complete inactivation of the infectivity of these recombinants, was determined. Injections of rabbits with the irradiated noninfectious recombinant viruses successfully induced specific antibodies against the HIV envelope antigen, in addition to those against the poxvirus. PMID:12479396

  3. Monoclonal antibodies against LipL32, the major outer membrane protein of pathogenic Leptospira: production, characterization, and testing in diagnostic applications.

    PubMed

    Fernandes, Cláudia P H; Seixas, Fabiana K; Coutinho, Mariana L; Vasconcellos, Flávia A; Seyffert, Núbia; Croda, Julio; McBride, Alan J; Ko, Albert I; Dellagostin, Odir A; Aleixo, José A G

    2007-02-01

    Pathogenic serovars of Leptospira have a wide antigenic diversity attributed mainly to the lipopolysaccharide present in the outer membrane. In contrast, antigens conserved among pathogenic serovars are mainly represented by outer membrane proteins. Surface exposure of a major and highly conserved outer membrane lipoprotein (LipL32) was recently demonstrated on pathogenic Leptospira. LipL32 in its recombinant form (rLipL32) was used to immunize BALB/c mice to develop murine monoclonal antibodies (MAbs). Three MAbs against rLipL32 were produced, isotyped, and evaluated for further use in diagnostic tests of leptospirosis using different approaches. MAbs were conjugated to peroxidase and evaluated in a native protein enzyme-linked immunosorbent assay (ELISA) with intact and heat-treated leptospiral cells, conjugated to fluorescein isothiocyanate (FITC) for indirect immunofluorescence with intact and methanol fixed cells and were used for LipL32 immunoprecipitation from leptospiral cells. rLipL32 MAbs conjugated to peroxidase or used as primary antibody bound to intact and heat-treated cells in ELISA, proving that they could be used in enzyme immunoassays for detection of the native protein. In immunofluorescence assay, MAbs labeled bacterial cells either intact or methanol fixed. Two MAbs were able to immunoprecipitate the native protein from live and motile leptospiral cells and, adsorbed onto magnetic beads, captured intact bacteria from artificially contaminated human sera for detection by polymerase chain reaction (PCR) amplification. Results of this study suggest that the MAbs produced can be useful for the development of diagnostic tests based on detection of LipL32 leptospiral antigen in biological fluids. PMID:17316084

  4. Major substructure in the M31 Outer Halo: the East Cloud

    NASA Astrophysics Data System (ADS)

    McMonigal, B.; Bate, N. F.; Conn, A. R.; Mackey, A. D.; Lewis, G. F.; Irwin, M. J.; Martin, N. F.; McConnachie, A. W.; Ferguson, A. M. N.; Ibata, R. A.; Huxor, A. P.

    2016-02-01

    We present the first detailed analysis of the East Cloud, a highly disrupted diffuse stellar substructure in the outer halo of M31. The core of the substructure lies at a projected distance of ˜100 kpc from the centre of M31 in the outer halo, with possible extensions reaching right into the inner halo. Using Pan-Andromeda Archaeological Survey photometry of red giant branch stars, we measure the distance, metallicity and brightness of the cloud. Using Hubble Space Telescope data, we independently measure the distance and metallicity to the two globular clusters coincident with the East Cloud core, PA-57 and PA-58, and find their distances to be consistent with the cloud. Four further globular clusters coincident with the substructure extensions are identified as potentially associated. Combining the analyses, we determine a distance to the cloud of 814^{+20}_{-9} kpc, a metallicity of [Fe/H] = -1.2 ± 0.1, and a brightness of MV = -10.7 ± 0.4 mag. Even allowing for the inclusion of the potential extensions, this accounts for less than 20 per cent of the progenitor luminosity implied by the luminosity-metallicity relation. Using the updated techniques developed for this analysis, we also refine our estimates of the distance and brightness of the South-West Cloud, a separate substructure analysed in the previous work in this series.

  5. Major substructure in the M31 outer halo: the South-West Cloud

    NASA Astrophysics Data System (ADS)

    Bate, N. F.; Conn, A. R.; McMonigal, B.; Lewis, G. F.; Martin, N. F.; McConnachie, A. W.; Veljanoski, J.; Mackey, A. D.; Ferguson, A. M. N.; Ibata, R. A.; Irwin, M. J.; Fardal, M.; Huxor, A. P.; Babul, A.

    2014-02-01

    We undertake the first detailed analysis of the stellar population and spatial properties of a diffuse substructure in the outer halo of M31. The South-West Cloud lies at a projected distance of ˜100 kpc from the centre of M31 and extends for at least ˜50 kpc in projection. We use Pan-Andromeda Archaeological Survey photometry of red giant branch stars to determine a distance to the South-West Cloud of 793^{+45}_{-45} kpc. The metallicity of the cloud is found to be [Fe/H] = -1.3 ± 0.1. This is consistent with the coincident globular clusters PAndAS-7 and PAndAS-8, which have metallicities determined using an independent technique of [Fe/H] = -1.35 ± 0.15. We measure a brightness for the Cloud of MV = -12.1 mag; this is ˜75 per cent of the luminosity implied by the luminosity-metallicity relation. Under the assumption that the South-West Cloud is the visible remnant of an accreted dwarf satellite, this suggests that the progenitor object was amongst M31's brightest dwarf galaxies prior to disruption.

  6. Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein.

    PubMed

    Pohlner, J; Meyer, T F; Jalajakumari, M B; Manning, P A

    1986-12-01

    The nucleotide sequence of the ompV gene of Vibrio cholerae was determined. The product of the gene is a 28,000 dalton protein which, after the removal of a 19 amino acid signal sequence, produces a mature outer membrane protein of 26,000 daltons. The cleavage site was determined by amino-terminal amino acid sequencing of the purified mature protein. The DNA upstream of the gene shows the presence of a typical promoter region as judged from the Escherichia coli consensus information; however, the Shine-Dalgarno sequence is associated with a region capable of forming a secondary structure in the mRNA. The formation of this structure would inhibit binding of the mRNA to the ribosome and reduce translation. It is proposed that this structure is recognized by a positive activator in V. cholerae and because of its absence in E. coli ompV is poorly expressed. The distribution of rare codons within ompV suggests that they may serve to slow down the translation of particular domains such that the nascent polypeptide has an opportunity to take up its conformation without interference from the later formed regions. Such a mechanism could aid localization of the protein if export were by a contranslational secretion system. PMID:3031428

  7. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    PubMed

    Hoehn, G T; Clark, V L

    1990-12-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. PMID:2123827

  8. Distribution of a protein antigenically related to the major anaerobically induced gonococcal outer membrane protein among other Neisseria species.

    PubMed Central

    Hoehn, G T; Clark, V L

    1990-01-01

    The Pan 1 protein of Neisseria gonorrhoeae is a novel 54-kDa outer membrane protein expressed only when gonococci are grown in the absence of oxygen. It is a major antigen recognized by sera from patients with gonococcal infection. We raised mouse monospecific polyclonal antiserum to gel-purified Pan 1 from gonococcal strain F62. The antiserum was broadly cross-reactive among gonococcal strains; all strains tested reacted in immunoblot analysis proportionate to the amount of Pan 1 visible in silver-stained sodium dodecyl sulfate (SDS)-polyacrylamide gels. In immunoblot experiments, N. lactamica and N. cinerea reacted very strongly to the anti-Pan 1 antiserum, whereas N. sicca, N. flava, and N. mucosa did not react at all. The other commensals tested, N. subflava and N. perflava, exhibited only a minor reaction. These results correlated with the apparent amount of Pan 1 seen on SDS-polyacrylamide gels of outer membranes. SDS-polyacrylamide gel analysis of six meningococcal strains revealed no visible anaerobically induced outer membrane proteins, and the subsequent immunoblots showed only slight or no reaction to the anti-Pan 1 antibody. In the four meningococcal strains that did react slightly with the antiserum, a Pan 1-like protein was seen only in anaerobically grown cells. Thus, meningococci did not express Pan 1 at levels comparable to that found in gonococci; however, when Pan 1 was expressed in meningococcal strains, it was oxygen regulated. This is the first example of a protein found in the gonococcal outer membrane that, under identical growth conditions, is not expressed at similar levels in the meningococcus. Images PMID:2123827

  9. THE INVISIBLE MAJORITY? EVOLUTION AND DETECTION OF OUTER PLANETARY SYSTEMS WITHOUT GAS GIANTS

    SciTech Connect

    Mann, Andrew W.; Gaidos, Eric; Gaudi, B. Scott

    2010-08-20

    due to the presence of additional outer planets. Future microlensing surveys will detect {approx}130 analogs over a 5 yr survey, including a handful of multiple-planet systems. Finally, the Space Interferometry Mission (SIM-Lite) should be capable of detecting 96% of the innermost icy planets over the course of a 5 yr mission.

  10. Major substructure in the M31 outer halo: distances and metallicities along the giant stellar stream

    NASA Astrophysics Data System (ADS)

    Conn, A. R.; McMonigal, B.; Bate, N. F.; Lewis, G. F.; Ibata, R. A.; Martin, N. F.; McConnachie, A. W.; Ferguson, A. M. N.; Irwin, M. J.; Elahi, P. J.; Venn, K. A.; Mackey, A. D.

    2016-05-01

    We present a renewed look at M31's giant stellar stream along with the nearby structures streams C and D, exploiting a new algorithm capable of fitting to the red giant branch (RGB) of a structure in both colour and magnitude space. Using this algorithm, we are able to generate probability distributions in distance, metallicity and RGB width for a series of subfields spanning these structures. Specifically, we confirm a distance gradient of approximately 20 kpc per degree along a 6 deg extension of the giant stellar stream, with the farthest subfields from M31 lying ˜120 kpc more distant than the innermost subfields. Further, we find a metallicity that steadily increases from -0.7^{+0.1}_{-0.1} to -0.2^{+0.2}_{-0.1} dex along the inner half of the stream before steadily dropping to a value of -1.0^{+0.2}_{-0.2} dex at the farthest reaches of our coverage. The RGB width is found to increase rapidly from 0.4^{+0.1}_{-0.1} to 1.1^{+0.2}_{-0.1} dex in the inner portion of the stream before plateauing and decreasing marginally in the outer subfields of the stream. In addition, we estimate stream C to lie at a distance between 794 and 862 kpc and stream D between 758 and 868 kpc. We estimate the median metallicity of stream C to lie in the range -0.7 to -1.6 dex and a metallicity of -1.1^{+0.3}_{-0.2} dex for stream D. RGB widths for the two structures are estimated to lie in the range 0.4-1.2 dex and 0.3-0.7 dex, respectively. In total, measurements are obtained for 19 subfields along the giant stellar stream, four along stream C, five along stream D and three general M31 spheroid fields for comparison. We thus provide a higher resolution coverage of the structures in these parameters than has previously been available in the literature.

  11. Bordetella pertussis major outer membrane porin protein forms small, anion-selective channels in lipid bilayer membranes.

    PubMed Central

    Armstrong, S K; Parr, T R; Parker, C D; Hancock, R E

    1986-01-01

    The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures. The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCl was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cl- and a maximum possible conductance through the channel of 1.28 nS. Images PMID:2420780

  12. Recombinant Major Antigenic Protein 2 of Ehrlichia canis: a Potential Diagnostic Tool

    PubMed Central

    Alleman, A. Rick; McSherry, Leo J.; Barbet, Anthony F.; Breitschwerdt, Edward B.; Sorenson, Heather L.; Bowie, Michael V.; Bélanger, Myriam

    2001-01-01

    The major antigenic protein 2 (MAP2) of Ehrlichia canis was cloned and expressed. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of canine monocytic ehrlichiosis. The recombinant protein, which contained a C-terminal polyhistidine tag, had a molecular mass of approximately 26 kDa. The antigen was clearly identified by Western immunoblotting using antihistidine antibody and immune serum from an experimentally infected dog. The recombinant MAP2 (rMAP2) was tested in an ELISA format using 141 serum samples from E. canis immunofluorescent antibody (IFA)-positive and IFA-negative dogs. Fifty-five of the serum samples were from dogs experimentally or naturally infected with E. canis and were previously demonstrated to contain antibodies reactive with E. canis by indirect immunofluorescence assays. The remaining 86 samples, 33 of which were from dogs infected with microorganisms other than E. canis, were seronegative. All of the samples from experimentally infected animals and 36 of the 37 samples from naturally infected animals were found to contain antibodies against rMAP2 of E. canis in the ELISA. Only 3 of 53 IFA-negative samples tested positive on the rMAP2 ELISA. There was 100% agreement among IFA-positive samples from experimentally infected animals, 97.3% agreement among IFA-positive samples from naturally infected animals, and 94.3% agreement among IFA-negative samples, resulting in a 97.2% overall agreement between the two assays. These data suggest that rMAP2 of E. canis could be used as a recombinant test antigen for the serodiagnosis of canine monocytic ehrlichiosis. PMID:11427559

  13. Temperature Dependence of the Rate Constant for the CH3 Recombination Reaction: A Loss Process in Outer Planet Atmospheres

    NASA Technical Reports Server (NTRS)

    Cody, R. J.; Payne, W. A.; Thorn, R. P., Jr.; Romani, P. N.; Stief, L. J.; Nesbitt, F. L.; Iannone, M. A.; Tardy, D. C.

    2002-01-01

    The methyl free radical (CH3) has been observed in the atmospheres of Saturn and Neptune by the ISO satellite. There are discrepancies between the column densities for the CH3 radical derived from the ISO observations and the column densities derived from atmospheric photochemical models. For Neptune the model column density is 1.5 times that derived from ISO. For Saturn the model is 6 times that from ISO. The recombination of methyl radicals is the major loss process for methyl in these atmospheres. The serious disagreement between observed and calculated levels of CH3 has led to suggestions that the atmospheric models greatly underestimated the loss of CH3 due to poor knowledge of the rate of the reaction (1) CH3 + CH3 + M goes to C2H6 + M at the low temperatures and pressures of these atmospheric systems. Although the reaction CH3 + CH3 + M goes to C2H6 + M has been extensively studied both theoretically and experimentally, the laboratory conditions have been, with only a few exceptions, higher temperatures (T greater than 298K), higher pressures (P greater than or equal to 10 Torr - 13.3 mbar) or M=Ar rather than H2 or He as the bath gas.

  14. Warp of the ionized gas layer in the outer Galaxy, traced by Recombination Line Observations in both the First and Fourth Quadrants

    NASA Astrophysics Data System (ADS)

    Azcarate, I. N.; Cersosimo, J. C.

    2000-05-01

    Results of H166α recombination line observations from the Outer Galaxy in both the Northern and Southern Galactic Plane, are reported. The Southern observations were made with the 30m antenna of the Instituto Argentino de Radioastronomia in Villa Elisa, Buenos Aires, Argentina and the Northern ones ( more sensitive, high quality observations, performed with an "state of the art" receiver) with the 43m antenna of the National Radio Astronomy Observatory, in Green Bank, West Virginia, USA. From the two sets of observations, we obtain evidence of the warp of the low density ionized gas layer, traced by the H166α emission in the outer MIlky Way, towards positive galactic latitudes in the Northern and towards negative latitudes in the Southern Galaxy. The warp of this tracer qualitatively agrees with that of the HI.

  15. Lipopolysaccharide Modifications of a Cholera Vaccine Candidate Based on Outer Membrane Vesicles Reduce Endotoxicity and Reveal the Major Protective Antigen

    PubMed Central

    Leitner, Deborah R.; Feichter, Sandra; Schild-Prüfert, Kristina; Rechberger, Gerald N.; Reidl, Joachim

    2013-01-01

    The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay. PMID:23630951

  16. Enzyme-linked immunosorbent assay with major outer membrane proteins of Brucella melitensis to measure immune response to Brucella species.

    PubMed Central

    Hunter, S B; Bibb, W F; Shih, C N; Kaufmann, A F; Mitchell, J R; McKinney, R M

    1986-01-01

    We developed an enzyme-linked immunosorbent assay (ELISA) system to measure human immunoglobulin G (IgG) and IgM response to the major outer membrane proteins of Brucella melitensis. The ELISA was more sensitive in detecting antibody than a standard microagglutination (MA) test with B. abortus antigen. Of 101 sera from persons with suspected brucellosis, 79 (78.2%) gave ELISA IgM titers greater than or equal to the B. abortus MA titer without 2-mercaptoethanol (2ME), which measures both IgM and IgG. Of the 101 sera, 97% gave ELISA IgG titers greater than or equal to the MA with 2ME titer. A total of 58 sera, drawn from 11 human patients from 1 to 29 weeks after onset of brucellosis, gave higher geometric mean titers for the ELISA IgG test than for the MA with 2ME test. These 58 sera also gave ELISA IgM geometric mean titers that were greater than or within one doubling dilution of the geometric mean titers of MA without 2ME. In addition to detecting antibody response to B. abortus, B. melitensis, and B. suis, the ELISA was sensitive to antibody response to human and canine infections with B. canis. The B. canis antibody response is not detected by the MA test with B. abortus antigen. The ELISA, with a standard preparation of major outer membrane proteins of B. melitensis as antigen, appears to be useful in measuring antibody response in humans to infections by all species of Brucella known to infect humans. PMID:3095364

  17. Recombination and selection in the major histocompatibility complex of the endangered forest musk deer (Moschus berezovskii).

    PubMed

    Cai, Ruibo; Shafer, Aaron B A; Laguardia, Alice; Lin, Zhenzhen; Liu, Shuqiang; Hu, Defu

    2015-01-01

    The forest musk deer (Moschus berezovskii) is a high elevation species distributed across western China and northern Vietnam. Once abundant, habitat loss and poaching has led to a dramatic decrease in population numbers prompting the IUCN to list the species as endangered. Here, we characterized the genetic diversity of a Major Histocompatibility Complex (MHC) locus and teased apart driving factors shaping its variation. Seven DRB exon 2 alleles were identified among a group of randomly sampled forest musk deer from a captive population in the Sichuan province of China. Compared to other endangered or captive ungulates, forest musk deer have relatively low levels of MHC genetic diversity. Non-synonymous substitutions primarily occurred in the putative peptide-binding region (PBR), with analyses suggesting that recombination and selection has shaped the genetic diversity across the locus. Specifically, inter-allelic recombination generated novel allelic combinations, with evidence for both positive selection acting on the PBR and negative selection on the non-PBR. An improved understanding of functional genetic variability of the MHC will facilitate better design and management of captive breeding programs for this endangered species. PMID:26603338

  18. Recombination and selection in the major histocompatibility complex of the endangered forest musk deer (Moschus berezovskii)

    PubMed Central

    Cai, Ruibo; Shafer, Aaron B.A.; Laguardia, Alice; Lin, Zhenzhen; Liu, Shuqiang; Hu, Defu

    2015-01-01

    The forest musk deer (Moschus berezovskii) is a high elevation species distributed across western China and northern Vietnam. Once abundant, habitat loss and poaching has led to a dramatic decrease in population numbers prompting the IUCN to list the species as endangered. Here, we characterized the genetic diversity of a Major Histocompatibility Complex (MHC) locus and teased apart driving factors shaping its variation. Seven DRB exon 2 alleles were identified among a group of randomly sampled forest musk deer from a captive population in the Sichuan province of China. Compared to other endangered or captive ungulates, forest musk deer have relatively low levels of MHC genetic diversity. Non-synonymous substitutions primarily occurred in the putative peptide-binding region (PBR), with analyses suggesting that recombination and selection has shaped the genetic diversity across the locus. Specifically, inter-allelic recombination generated novel allelic combinations, with evidence for both positive selection acting on the PBR and negative selection on the non-PBR. An improved understanding of functional genetic variability of the MHC will facilitate better design and management of captive breeding programs for this endangered species. PMID:26603338

  19. Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge

    PubMed Central

    Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

    2014-01-01

    African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR −/−) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. PMID:24837765

  20. Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein.

    PubMed Central

    Hoffman, P S; Seyer, J H; Butler, C A

    1992-01-01

    The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via

  1. Mechanistic Insight into the TH1-Biased Immune Response to Recombinant Subunit Vaccines Delivered by Probiotic Bacteria-Derived Outer Membrane Vesicles

    PubMed Central

    Rosenthal, Joseph A.; Huang, Chung-Jr.; Doody, Anne M.; Leung, Tiffany; Mineta, Kaho; Feng, Danielle D.; Wayne, Elizabeth C.; Nishimura, Nozomi; Leifer, Cynthia; DeLisa, Matthew P.; Mendez, Susana; Putnam, David

    2014-01-01

    Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV’s ability to induce self-adjuvanted robust and protective humoral and TH1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered “pathogen-like particles” derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion. PMID:25426709

  2. Mechanistic insight into the TH1-biased immune response to recombinant subunit vaccines delivered by probiotic bacteria-derived outer membrane vesicles.

    PubMed

    Rosenthal, Joseph A; Huang, Chung-Jr; Doody, Anne M; Leung, Tiffany; Mineta, Kaho; Feng, Danielle D; Wayne, Elizabeth C; Nishimura, Nozomi; Leifer, Cynthia; DeLisa, Matthew P; Mendez, Susana; Putnam, David

    2014-01-01

    Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion. PMID:25426709

  3. Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

    PubMed Central

    von Specht, B U; Knapp, B; Muth, G; Bröker, M; Hungerer, K D; Diehl, K D; Massarrat, K; Seemann, A; Domdey, H

    1995-01-01

    Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa. PMID:7729895

  4. Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.

    PubMed Central

    Blanco, D R; Champion, C I; Exner, M M; Shang, E S; Skare, J T; Hancock, R E; Miller, J N; Lovett, M A

    1996-01-01

    We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure. PMID:8955283

  5. Evidence of Localized Prophage-Host Recombination in the lytA Gene, Encoding the Major Pneumococcal Autolysin ▿

    PubMed Central

    Morales, María; García, Pedro; de la Campa, Adela G.; Liñares, Josefina; Ardanuy, Carmen; García, Ernesto

    2010-01-01

    According to a highly polymorphic region in the lytA gene, encoding the major autolysin of Streptococcus pneumoniae, two different families of alleles can be differentiated by PCR and restriction digestion. Here, we provide evidence that this polymorphic region arose from recombination events with homologous genes of pneumococcal temperate phages. PMID:20304992

  6. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  7. Development and Comparative Evaluation of a Plate Enzyme-Linked Immunosorbent Assay Based on Recombinant Outer Membrane Antigens Omp28 and Omp31 for Diagnosis of Human Brucellosis

    PubMed Central

    Tiwari, Sapana; Kumar, Ashu; Mangalgi, Smita; Rathod, Vedika; Prakash, Archana; Barua, Anita; Arora, Sonia; Sathyaseelan, Kannusamy

    2013-01-01

    Brucellosis is an important zoonotic infectious disease of humans and livestock with worldwide distribution and is caused by bacteria of the genus Brucella. The diagnosis of brucellosis always requires laboratory confirmation by either isolation of pathogens or detection of specific antibodies. The conventional serological tests available for the diagnosis of brucellosis are less specific and show cross-reactivity with other closely related organisms. These tests also necessitate the handling of Brucella species for antigen preparation. Therefore, there is a need to develop reliable, rapid, and user-friendly systems for disease diagnosis and alternatives to vaccine approaches. Keeping in mind the importance of brucellosis as an emerging infection and the prevalence in India, we carried out the present study to compare the recombinant antigens with the native antigens (cell envelope and sonicated antigen) of Brucella for diagnosis of human brucellosis by an indirect plate enzyme-linked immunosorbent assay (ELISA). Recombinant outer membrane protein 28 (rOmp28) and rOmp31 antigens were cloned, expressed, and purified in the bacterial expression system, and the purified proteins were used as antigens. Indirect plate ELISAs were then performed and standardized for comparison of the reactivities of recombinant and native antigens against the 433 clinical samples submitted for brucellosis testing, 15 culture-positive samples, and 20 healthy donor samples. The samples were separated into four groups based on their positivity to rose bengal plate agglutination tests (RBPTs), standard tube agglutination tests (STATs), and 2-mercaptoethanol (2ME) tests. The sensitivities and specificities of all the antigens were calculated, and the rOmp28 antigen was found to be more suitable for the clinical diagnosis of brucellosis than the rOmp31 antigen and native antigens. The rOmp28-based ELISA showed a very high degree of agreement with the conventional agglutination tests and

  8. Pore-forming properties of the major 53-kilodalton surface antigen from the outer sheath of Treponema denticola.

    PubMed Central

    Egli, C; Leung, W K; Müller, K H; Hancock, R E; McBride, B C

    1993-01-01

    A 53-kDa protein from the outer sheath of the oral spirochete Treponema denticola was purified to homogeneity and shown to reconstitute channels in black lipid bilayer model membranes. The channel had a single-channel conductance of 1.8 nS in 0.1 M KCl, making this the largest porin channel observed to date (estimated diameter, 3.4 nm). Electron micrographs of 53-kDa-protein-containing outer sheaths of T. denticola showed a regular hexagonal array of darker staining pits. Images PMID:7682993

  9. Identification and Characterization of a Novel Porin Family Highlights a Major Difference in the Outer Membrane of Chlamydial Symbionts and Pathogens

    PubMed Central

    Aistleitner, Karin; Heinz, Christian; Hörmann, Alexandra; Heinz, Eva; Montanaro, Jacqueline; Schulz, Frederik; Maier, Elke; Pichler, Peter; Benz, Roland; Horn, Matthias

    2013-01-01

    The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydia outer membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts. PMID:23383036

  10. Comparative analysis of the structures of the outer membrane protein P1 genes from major clones of Haemophilus influenzae type b.

    PubMed Central

    Munson, R; Grass, S; Einhorn, M; Bailey, C; Newell, C

    1989-01-01

    P1 outer membrane proteins from Haemophilus influenzae type b are heterogeneous antigenically and with respect to apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For determination of the molecular basis for the differences in the P1 proteins, the genes for the P1 proteins from strain 1613, representative of outer membrane protein subtype 3L, and strain 8358, representative of outer membrane protein subtype 6U, were cloned, sequenced, and compared with the previously reported gene for the P1 protein from strain MinnA, a strain with the outer membrane protein subtype 1H. These prototype strains are representatives of the three major clonal families of H. influenzae type b responsible for invasive disease in diverse areas of the world. The nucleotide sequences of the P1 genes from strains 1613 and 8358 were 94 and 90% identical to the MinnA sequence, respectively. The derived amino acid sequences were 91 and 86% identical, respectively. Heterogeneity between the MinnA and 1613 proteins was largely localized to two short variable regions; the protein from strain 8538 contained a third variable region not observed in the other P1 proteins. Thus, the outer membrane protein P1 genes are highly conserved; the variable regions may code for the previously demonstrated strain-specific antigenic determinants. Images PMID:2572549

  11. The tolC locus of Escherichia coli affects the expression of three major outer membrane proteins.

    PubMed Central

    Morona, R; Reeves, P

    1982-01-01

    tolC mutants, which are resistant to colicin E1 and also highly sensitive to detergents and dyes, were shown to lack the OmpF outer membrane protein. There was little effect on transcription as judged by the use of an ompF-lac operon fusion strain, and the tolC effect was probably due to a post-transcriptional effect. The NmpC protein and protein 2 were also tolC dependent. Images PMID:6281230

  12. Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein.

    PubMed

    Arockiasamy, A; Krishnaswamy, S

    2000-07-15

    Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins. PMID:10929809

  13. Comparison of the Effect of Two Purification Methods on the Immunogenicity of Recombinant Outer Membrane Protein H of Pasteurella multocida Serovar A:1

    PubMed Central

    Thanasarasakulpong, Arunee; Poolperm, Pichayanut; Tangjitjaroen, Weerapongse; Varinrak, Thanya; Sawada, Takuo; Pfeiffer, Dirk; Sthitmatee, Nattawooti

    2016-01-01

    Recombinant outer membrane protein H (rOmpH) of Pasteurella multocida strain X-73 can be purified using affinity chromatography but this adversely affects its immunogenicity. The current study presents the results from an intervention study comparing the immunogenicity of rOmpH purified using electroelution with rOmpH purified using affinity chromatography and native OmpH purified using electroelution and a nonimmunized control group. Chickens immunized with rOmpH purified using electroelution produced the highest ELISA antibody levels against P. multocida strains. Chickens in each of the 5 treatment groups were split into two subgroups for challenge with two different P. multocida strains. The average number of adhesions to CEF cells was statistically significantly lower in sera from chickens immunized with rOmpH or native OmpH purified using electroelution than in those of the three other treatment groups. The survival amongst chickens immunized with rOmpH or native OmpH purified using electroelution indicated high levels of protection. In contrast, survival probability was zero or low in the groups immunized with rOmpH purified using affinity chromatography and in the nonimmunized group. These findings show that the rOmpH purified using electroelution retains its immunogenicity and stimulates high levels of protection in chickens against P. multocida infection. PMID:26885439

  14. Isolation and nucleotide sequence of the gene (aniA) encoding the major anaerobically induced outer membrane protein of Neisseria gonorrhoeae.

    PubMed

    Hoehn, G T; Clark, V L

    1992-11-01

    When grown under anaerobic conditions, Neisseria gonorrhoeae, the etiologic agent of the sexually transmitted disease gonorrhea, expresses several novel outer membrane proteins. One of these, Pan 1, has an apparent molecular mass of 54 kDa in electrophoresis and is recognized by serum samples from patients with gonococcal infection. The presence of antibodies to this protein in patient sera suggests that Pan 1 is expressed during gonococcal infection and, more importantly, that N. gonorrhoeae grows anaerobically in vivo. We have cloned the Pan 1 structural gene, aniA, by screening a gonococcal lambda gt11 expression library with monospecific, polyclonal anti-Pan 1 antiserum. Three distinct immunoreactive recombinants, containing overlapping fragments of DNA, were isolated and confirmed to be coding for Pan 1 protein sequences. Northern (RNA blot) hybridization of an insert from an aniA recombinant to total gonococcal cellular RNA revealed the presence of a 1.5-kb transcript that was specific to RNA from anaerobically grown gonococci, indicating that the aniA gene is regulated at the transcriptional level and is monocistronic. To characterize the aniA gene, we have sequenced the entire 2-kb region spanned by the overlapping recombinants. We have also performed primer extension analysis on RNA isolated from aerobically and anaerobically grown gonococci in order to define the aniA promoter region. Two putative primer extension products specific to organisms grown anaerobically were identified by homology to known Escherichia coli promoter sequences, suggesting that the regulation of aniA expression involves multiple promoter regions. PMID:1383156

  15. Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

    PubMed Central

    Gong, Wenping; Wang, Pengcheng; Xiong, Xiaolu; Jiao, Jun; Yang, Xiaomei; Wen, Bohai

    2015-01-01

    The obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii. PMID:25909586

  16. A novel recombinant bivalent outer membrane protein of Vibrio vulnificus and Aeromonas hydrophila as a vaccine antigen of American eel (Anguilla rostrata).

    PubMed

    SongLin, Guo; PanPan, Lu; JianJun, Feng; JinPing, Zhao; Peng, Lin; LiHua, Duan

    2015-04-01

    The immogenicity of a novel vaccine antigen was evaluated after immunized American eels (Anguilla rostrata) with a recombinant bivalent expressed outer membrane protein (OMP) of Vibrio vulnificus and Aeromonas hydrophila. Three groups of eels were intraperitoneal (i.p) injected with phosphate-buffered saline (PBS group), formaline-killed-whole-cell (FKC) of A. hydrophila and V. vulnificus (FKC group) or the bivalent OMP (OMP group). On 14, 21, 28 and 42 days post-vaccination respectively, proliferation of the whole blood cells, titers of specific antibody and lysozyme activities of experimental eels were detected. On 28 day post-vaccination, eels from three groups were challenged by i.p injection of live A. hydrophila or V. vulnificus. The results showed that, compared with the PBS group, proliferation of whole blood cells in OMP group was significant enhanced on 28 days, and the serum titers of anti-A.hydrophila and anti-V. vulnificus antibody in eels of FKC and OMP group were significant increased on 14, 21 and 28d. Lysozyme Activities in serum, skin mucus, liver and kidney were significant changed between the three groups. Relative Percent Survival (RPS) after challenged A. hydrophila in KFC vs. PBS group and OMP vs. PBS group were 62.5% and 50% respectively, and the RPS challenged V. vulnificus in FKC and OMP vs. PBS group were 37.5% and 50% respectively. These results suggest that American eels immunized with the bivalent OMP would positively affect specific as well as non-specific immune parameters and protect against infection by the two pathogens in fresh water farming. PMID:25655329

  17. A Multiplex PCR Approach for Detecting Dual Infections and Recombinants Involving Major HIV Variants.

    PubMed

    Cappy, Pierre; De Oliveira, Fabienne; Gueudin, Marie; Alessandri-Gradt, Elodie; Plantier, Jean-Christophe

    2016-05-01

    The cocirculation of different HIV types and groups can lead to dual infections and recombinants, which hinder diagnosis and therapeutic management. We designed two multiplex PCRs (mPCRs) coupled with capillary electrophoresis to facilitate the detection of such infections. The first, MMO2, targets three variants (HIV-1/M, HIV-1/O, and HIV-2), and the second, MMO, targets HIV-1/M and HIV-1/O. These mPCRs were validated on DNA and RNA extracts from 19 HIV-1/M, 12 HIV-1/O, and 13 HIV-2 cultures and from mixtures simulating dual infections. They were then assessed with DNA and RNA extracts from samples of 47 clinical monoinfections and HIV-1/M+O dual infections or infections with HIV-1/MO recombinants. Both mPCRs had excellent specificity. Sensitivities ranged from 80 to 100% for in vitro samples and from 58 to 100% for clinical samples, with the results obtained depending on the material used and the region of the genome concerned. Sensitivity was generally lower for DNA than for RNA and for amplifications of the integrase and matrix regions. In terms of global detection (at least one target gene for each strain), both mPCRs yielded a detection rate of 100% for in vitro samples. MMO2 detected 100% of the clinical strains from DNA and 97% from RNA, whereas MMO detected 100% of the strains from both materials. Thus, for in vitro and clinical samples, MMO2 was a useful tool for detecting dual infections with HIV-1 and HIV-2 (referred to as HIV-1+HIV-2) and HIV-1/M+O, and MMO was useful for detecting both MO dual infections and MO mosaic patterns. PMID:26912747

  18. A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity.

    PubMed Central

    Mage, M G; Lee, L; Ribaudo, R K; Corr, M; Kozlowski, S; McHugh, L; Margulies, D H

    1992-01-01

    Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines. Images PMID:1438262

  19. Protein typing of major outer membrane lipoproteins from Chinese pathogenic Leptospira spp. and characterization of their immunogenicity.

    PubMed

    Luo, Dongjiao; Xue, Feng; Ojcius, David M; Zhao, Jinfang; Mao, Yafei; Li, Liwei; Lin, Xuai; Yan, Jie

    2009-12-10

    Leptospirosis, caused by different Leptospira species, is one of the most widespread zoonotic infections worldwide. Here we expressed three major leptospiral lipoproteins and examined their immunogenicity. All the pathogenic Leptospira strains tested possess the lipL21, lipL32 and lipL41 genes, but the latter two can be further divided into different gene types (lipL32-1, lipL32-2, lipL41-1, lipL41-2). Microscopic agglutination test revealed that rLipLs antisera had extensive cross-immunoagglutination among the 178 leptospiral strains in which rLipL32-1 contributed the highest agglutination titer. The rLipLs-based ELISAs established in this study demonstrated that in the sera of 385 leptospirosis patients infected with different serovars of Leptospira interrogans, rLipL32-1 had the highest positive rates for IgG and IgM (89.4-98.7%), followed by the IgG/IgM positive rates of rLipL21 (87.0-96.1%) and rLipL32-2 (86.5-96.9%), while the two rLipL41s presented the lowest IgG/IgM positive rates (69.9-83.9%). The immunoprotective levels in guinea pigs of rLipL32-1 (58.3% and 66.7%) were the highest, compared to those of the other rLipLs (25.0-58.3%). Multiple different rLipLs would increase immunoprotective levels (from 58.3% and 66.7% to 83.3% and 91.7%). The data suggest that all the rLipLs are the genus-specific superficial antigens of pathogenic Leptospira species and should be considered in designing universal vaccines against leptospirosis. PMID:19796723

  20. Purification and recombinant expression of major peanut allergen Ara h 1.

    PubMed

    Wu, Zhihua; Yan, Fei; Wei, Xiaonan; Li, Xin; Tong, Ping; Yang, Anshu; Tang, Ronghua; Chen, Hongbing

    2015-01-01

    Reaction to peanut, as one of the major food allergens, has become an increasingly common life-threatening disorder. Although peanut allergens have been extensively identified, Ara h 1 is still too expensive to be applied in food safety or clinical utility. In this study, the purification, expression, and immunological analyses of Ara h 1 are investigated. It was shown that a high purity (>95%) of Ara h 1 could be prepared by either purification or expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, and mass spectroscopy were used to identify the Ara h 1, and it was found that natural Ara h 1 (nAra h 1) and expressed Ara h 1 (rAra h 1) have the same properties, including amino acid sequence. In particular, rAra h 1 reacted positively with anti-nAra h 1 serum, showing their similar immunological property. Thus, by either purification or expression, Ara h 1 could be prepared with low cost, as performed in the present work. SDS-PAGE, mag trix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS), and immunological analysis confirmed that both forms of Ara h 1 had the same properties. PMID:25036339

  1. cis- and trans-acting elements involved in regulation of aniA, the gene encoding the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae.

    PubMed

    Householder, T C; Belli, W A; Lissenden, S; Cole, J A; Clark, V L

    1999-01-01

    AniA (formerly Pan1) is the major anaerobically induced outer membrane protein in Neisseria gonorrhoeae. AniA has been shown to be a major antigen in patients with gonococcal disease, and we have been studying its regulation in order to understand the gonococcal response to anaerobiosis and its potential role in virulence. This study presents a genetic analysis of aniA regulation. Through deletion analysis of the upstream region, we have determined the minimal promoter region necessary for aniA expression. This 130-bp region contains a sigma 70-type promoter and an FNR (fumarate and nitrate reductase regulator protein) binding site, both of which are absolutely required for anaerobic expression. Also located in the minimal promoter region are three T-rich direct repeats and several potential NarP binding sites. This 80-bp region is required for induction by nitrite. By site-directed mutagenesis of promoter sequences, we have determined that the transcription of aniA is initiated only from the sigma 70-type promoter. The gearbox promoter, previously believed to be the major promoter, does not appear to be active during anaerobiosis. The gonococcal FNR and NarP homologs are involved in the regulation of aniA, and we demonstrate that placing aniA under the control of the tac promoter compensates for the inability of a gonococcal fnr mutant to grow anaerobically. PMID:9882668

  2. Molecular cloning, sequencing, and expression of omp-40, the gene coding for the major outer membrane protein from the acidophilic bacterium Thiobacillus ferrooxidans.

    PubMed

    Guiliani, N; Jerez, C A

    2000-06-01

    Thiobacillus ferrooxidans is one of the chemolithoautotrophic bacteria important in industrial biomining operations. Some of the surface components of this microorganism are probably involved in adaptation to their acidic environment and in bacterium-mineral interactions. We have isolated and characterized omp40, the gene coding for the major outer membrane protein from T. ferrooxidans. The deduced amino acid sequence of the Omp40 protein has 382 amino acids and a calculated molecular weight of 40,095.7. Omp40 forms an oligomeric structure of about 120 kDa that dissociates into the monomer (40 kDa) by heating in the presence of sodium dodecyl sulfate. The degree of identity of Omp40 amino acid sequence to porins from enterobacteria was only 22%. Nevertheless, multiple alignments of this sequence with those from several OmpC porins showed several important features conserved in the T. ferrooxidans surface protein, such as the approximate locations of 16 transmembrane beta strands, eight loops, including a large external L3 loop, and eight turns which allowed us to propose a putative 16-stranded beta-barrel porin structure for the protein. These results together with the previously known capacity of Omp40 to form ion channels in planar lipid bilayers strongly support its role as a porin in this chemolithoautotrophic acidophilic microorganism. Some characteristics of the Omp40 protein, such as the presence of a putative L3 loop with an estimated isoelectric point of 7.21 allow us to speculate that this can be the result of an adaptation of the acidophilic T. ferrooxidans to prevent free movement of protons across its outer membrane. PMID:10831405

  3. A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization

    PubMed Central

    Mahdavi, Jafar; Pirinccioglu, Necmettin; Oldfield, Neil J.; Carlsohn, Elisabet; Stoof, Jeroen; Aslam, Akhmed; Self, Tim; Cawthraw, Shaun A.; Petrovska, Liljana; Colborne, Natalie; Sihlbom, Carina; Borén, Thomas; Wooldridge, Karl G.; Ala'Aldeen, Dlawer A. A.

    2014-01-01

    Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr268; previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr268 led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(β1–3)-GalNAc(β1–4)-GalNAc(β1–4)-GalNAcα1-Thr268; modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr268 promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis. PMID:24451549

  4. Hexahydrated magnesium ions bind in the deep major groove and at the outer mouth of A-form nucleic acid duplexes.

    SciTech Connect

    Robinson, H.; Gao, Y.-G.; Sanishvili, R.; Joachimiak, A.; Wang, A. H.-J.; Univ. of Illinois; Northwestern Univ.

    2000-01-01

    Magnesium ions play important roles in the structure and function of nucleic acids. Whereas the tertiary folding of RNA often requires magnesium ions binding to tight places where phosphates are clustered, the molecular basis of the interactions of magnesium ions with RNA helical regions is less well understood. We have refined the crystal structures of four decamer oligonucleotides, d(ACCGGCCGGT), r(GCG)d(TATACGC), r(GC)d(GTATACGC) and r(G)d(GCGTATACGC) with bound hexahydrated magnesium ions at high resolution. The structures reveal that A-form nucleic acid has characteristic [Mg(H2O)6]2+ binding modes. One mode has the ion binding in the deep major groove of a GpN step at the O6/N7 sites of guanine bases via hydrogen bonds. Our crystallographic observations are consistent with the recent NMR observations that in solution [Co(NH3)6]3+, a model ion of [Mg(H2O)6]2+, binds in an identical manner. The other mode involves the binding of the ion to phosphates, bridging across the outer mouth of the narrow major groove. These [Mg(H2O)6]2+ ions are found at the most negative electrostatic potential regions of A-form duplexes. We propose that these two binding modes are important in the global charge neutralization, and therefore stability, of A-form duplexes.

  5. Transfer of fatty acids from the 1-position of phosphatidyl-ethanolamine to the major outer membrane lipoprotein of E coli

    SciTech Connect

    Jackowski, S.; Rock, C.O.

    1986-05-01

    The fatty acids esterified to Braun's lipoprotein are derived from the phospholipid pool in E. coli. Mutants lacking acyl-CoA synthetase activity (fadD) incorporated extracellular fatty acids specifically into the 1-position of phosphatidylethanolamine (PtdEtn). This pathway was blocked by chloramphenicol and was depressed by preventing the acylation of the amino terminus of the lipoprotein with globomycin. Transfer of fatty acids to lipoprotein was investigated in fadD mutants harboring hybrid plasmids containing either the lipoprotein gene or a lipoprotein-..beta..-lactamase gene fusion under control of the lactose promoter. Labeling of the 1-position of the PtdEtn pool prior to induction of lipoprotein biosynthesis resulted in the transfer of fatty acids from PtdEtn to the lipoproteins. Induction of lipoprotein synthesis in the presence of exogenous (1-/sup 14/C)palmitate increased the amount of radioactivity entering the PtdEtn pool and efficiently labeled lipoprotein acyl moieties. Lipoprotein fatty acids derived from the 1-position of PtdEtn were resistant to hydroxylamine hydrolysis, and globomycin reduced the incorporation of exogenous (1-/sup 14/C)palmitic acid into lipoproteins by 80% suggesting that the fatty acid is attached to the amino terminus. These data illustrate the metabolic relationship between turnover of fatty acids in the 1-position of PtdEtn and the maturation of the major outer membrane lipoprotein.

  6. A spatial correlation of the flow distribution on the outer continental shelf of Louisiana during the major hurricanes in the Gulf of Mexico during the 2005 season

    NASA Astrophysics Data System (ADS)

    Coor, J. L.; Li, C. Y.; Rouse, L. J.

    2007-12-01

    The 2005 hurricane season was unusually active, producing 31 named storms in the Atlantic, Caribbean, and Gulf of Mexico. Of these 31 storms, 11 entered the Gulf of Mexico, the most notable of which were Hurricanes Cindy, Dennis, Katrina, and Rita. Data were collected during these storms by acoustic Doppler current profilers (ADCPs) based on 58 oil and gas platforms scattered across the outer continental shelf (OCS) region of the northern Gulf of Mexico. Nine to 31 ADCPs were active and recording data during each major storm passage through the Gulf of Mexico. Data were recorded from depths of 60 to 70m down to 1100 to 1200m, with a few extending to depths around 2000m. From these data, the flow distribution of the OCS region was studied with the use of time series and spectrum analysis. Preliminary analysis has shown temporal variations in the vertical structure, increased diurnal oscillation current velocities (by a factor of approximately two), near-inertial oscillations, and variations in the overall direction of the flow before, during, and after the passage of the hurricanes. Methods of harmonic analysis and rotary spectra were implemented in this study. These data and results provide an estimate of the spatial extent to which a hurricane influences subsurface currents.

  7. Fate of the major outer membrane protein P.IA in early and late events of gonococcal infection of epithelial cells.

    PubMed

    Weel, J F; van Putten, J P

    1991-01-01

    We investigated the fate of the major outer membrane protein of Neisseria gonorrhoeae, P.IA, during gonococcal infection of Chang conjunctiva epithelial cells by using immunoelectron microscopy. Probing of P.IA epitopes with mono- and polyclonal antibodies revealed variable, fixation-dependent P.IA epitope exposure in the gonococci used as an inoculum in the infection experiments. Detection of invariable exposed P.IA epitopes in cryosections of infected epithelial cells with a polyclonal antiserum revealed unaltered P.IA exposure on the bacterial membranes during early attachment of the bacteria to the eukaryotic cells. Upon entry of the bacteria into the host cells, however, labelling was extended to membraneous structures that intercalated between the bacteria and the host cell surface, and, occasionally, to the host cell plasma membrane. The latter observation is consistent with the suggested active role of P.I. in the uptake process (as shown in 1985 by E.C. Gotschlich). Once inside the epithelial cells, both morphologically intact and disintegrating bacteria could be distinguished. The disintegration of the bacteria was accompanied by a loss of P.IA immunoreactivity. PMID:1725221

  8. Structure analysis of OmpC, one of the major proteins in the outer membrane of E. coli, by high resolution electron microscopy

    SciTech Connect

    Chang, C.F.

    1983-07-01

    This dissertation is concerned with the structure analysis of a pore-forming membrane protein, OmpC, which is one of the major proteins in the outer membrane of Escherichia coli. In order to obtain structural information it was necessary to develop a suitable technique for preparing two-dimensional crystalline arrays of this membrane protein in an unfixed, unstained and hydrated condition. Electron micrographs were recorded at exposures of less than 5 electrons/A/sup 2/ in order to avoid severe radiation damage. The resulting images were crystallographically averaged, in order to overcome the statistical limitations associated with the low electron exposures. The resulting images, which extend to a resolution of approx. 13.5 A, lend themselves to a natural interpretation that is consistent with the mass density of protein, water and lipid, prior data from 2-D and 3-D structure studies of negatively stained specimens at approx. = 20 A resolution, and published spectroscopic data on the peptide chain secondary structure.

  9. Major outer membrane protein variants of Chlamydia trachomatis are associated with severe upper genital tract infections and histopathology in San Francisco.

    PubMed

    Dean, D; Oudens, E; Bolan, G; Padian, N; Schachter, J

    1995-10-01

    Cervical and endometrial samples from 33 women with lower genital tract infection (LGTI) or pelvic inflammatory disease (PID) were evaluated for Chlamydia trachomatis major outer membrane protein gene (omp1) polymorphism. Polymorphism was correlated with symptoms, clinical findings, and histopathology. F, E, I, D, H, K, and G genotypes were represented. Thirty-seven genotyped samples (66%) displayed omp1 mutations compared with prototype sequences. Significantly, 7 of 7 women with variant F infections had PID compared with 6 non-variant F infections in women with LGTI (P = .003). PID was defined by clinical findings or plasma cells on endometrial biopsy. Of interest, F variants were associated with histopathology. Eleven women (92%) with E genotypes were asymptomatic. Our data suggest that F variants are associated with symptomatic, severe endometrial disease, whereas E genotypes are associated with asymptomatic, milder infections. Detection of virulent genotypes may provide a prognostic indicator for serious sequelae. Larger studies are required to evaluate the molecular, immunologic, and epidemiologic basis for these findings. PMID:7561174

  10. Immunogenicity and protective efficacy of recombinant major envelope protein (rH3L) of buffalopox virus in animal models.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Shivachandra, Sathish Bhadravati

    2016-02-01

    Buffalopox virus, a zoonotic Indian vaccinia-like virus, is responsible for contagious disease affecting mainly buffaloes, cattle and humans. H3L gene, encoding for an immunodominant major envelope protein of intracellular mature virion of orthopoxviruses, is highly conserved and found to elicit neutralizing antibodies. Therefore in the present study, the immunogenicity and protective efficacy of the recombinant H3L protein of buffalopox virus in laboratory animal models has been evaluated. A partial H3L gene encoding for the C-terminal truncated ectodomain of H3L protein (1M to I280) of BPXV-Vij/96 strain was cloned, over-expressed and purified as histidine-tagged fusion protein (50 kDa) from Escherichia coli using Ni-NTA affinity chromatography. The purified rH3L protein was further used for active immunization of guinea pig (250 μg/dose) and adult mice (10 μg and 50 μg/dose) with or without adjuvants (alum, Freund's Complete Adjuvant and CpG). Subsequently, a gradual increase in antigen specific serum IgG as well as neutralizing antibody titres measured by using indirect-ELISA and serum neutralization test respectively, was noted in both guinea pigs and mouse models. Suckling mice immunized passively with anti-H3L serum showed 80% pre-exposure prophylaxis upon challenge with virulent buffalopox virus strain. An indirect-ELISA based on rH3L protein showed no cross-reactivity with hyperimmune sera against sheeppox virus (SPPV), goatpox virus (GTPV), orf virus (ORFV), foot- and- mouth disease virus (FMDV), peste des petits ruminants virus (PPRV) and bluetongue virus (BTV) during the course of study. The study highlights the potential utility of rH3L protein as a safer prophylactic and diagnostic reagent for buffalopox. PMID:26723250

  11. Confirmation of immuno-reactivity of the recombinant major birch pollen allergen Bet v 1a by affinity-CIEF.

    PubMed

    Dullnig, Verena; Weiss, Richard; Amon, Sabine; Rizzi, Andreas; Stutz, Hanno

    2009-07-01

    Affinity-CIEF has been applied to characterize a recombinant product of the major birch pollen allergen Betula verrucosa isoform 1a (Bet v 1a) immuno-chemically. For this purpose mAbs of the IgG-type have been produced in-lab from two murine hybridoma lines, specified as clones 2 and 5.1. Both IgG clones were characterized by SDS-PAGE, MALDI-TOF-MS and CIEF. The purified IgG solutions had to be dialysed against 10 mmol/L phosphate (pH 7.4) to prevent IgG precipitation and to ensure appropriate CIEF separation. Both tested monoclonal IgGs (mIgGs) comprised four constituents covering pI ranges of 6.98-7.09 and 6.78-7.03 for clones 2 and 5.1 with major peaks at pI 7.09 and 7.03, respectively. When increasing amounts of Bet v 1a (pI 4.95) were incubated with 2.0 mumol/L mIgG, novel peaks were progressively induced in a pI range slightly more acidic than the focusing region of mIgGs. These peaks grew on the expense of original mIgG peaks. All pI values were calculated using two pI marker compounds with a repeatability of better than 0.03 units. New peaks represent complexes between Bet v 1a and mIgG either of 1:1 or of 2:1 binding stoichiometry. At a molar ratio of 2:1, saturation of both IgG paratopes with allergen (Ag) molecules was achieved as indicated by unbound Bet v 1a. The current CIEF approach addresses the proof of single epitope integrity in the course of immuno-chemical characterization of Bet v 1a. Contrary to traditional immunoassays, affinity CIEF allows for a distinction and relative quantification of mAbs, Ag-antibody complexes and Ag variants coexisting in one sample. PMID:19621361

  12. Human T-lymphotropic virus tax activates human cytomegalovirus major-immediate early promoter and improves production of recombinant proteins in HEK293 cells.

    PubMed

    Lwa, Teng Rhui; Lee, Jialing; Ng, Chew Har; Lew, Qiao Jing; Hia, Hui Ching; Chao, Sheng-Hao

    2011-01-01

    The human cytomegalovirus (CMV) major immediate-early (MIE) promoter is widely used in mammalian cells for production of recombinant proteins. It is of great interest to further enhance protein production driven by the CMV promoter. Here, we report that the Tax protein of human T-lymphotropic virus stimulates the transgene expression under the control of CMV MIE promoter in HEK293 cells. At least threefold increases in transient production of recombinant proteins, including luciferase and two biopharmaceutical proteins (erythropoietin and interferon-γ), were detected. Furthermore, cyclic adenosine monophosphate (AMP)-response element binding protein 2 (CREB2) was identified as a cellular cofactor, which might be responsible for Tax transactivation of the CMV MIE promoter. Our results not only demonstrate the potential use of this novel expression strategy for improvement of recombinant protein production in HEK293 cells but also provide the molecular mechanism for Tax-mediated activation of CMV MIE promoter. PMID:21425252

  13. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection

    PubMed Central

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-01-01

    Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed. PMID:27525325

  14. Expression of recombinant T-cell epitopes of major Japanese cedar pollen allergens fused with cholera toxin B subunit in Escherichia coli.

    PubMed

    Hoang, Vinh Van; Zou, Yanshuang; Kurata, Kentaro; Enomoto, Keiichi

    2015-05-01

    Peptides containing T-cell epitopes from allergens, which are not reactive to allergen-specific IgE, are appropriate candidates as antigens for specific immunotherapy against allergies. To develop a vaccine that can be used in practical application to prevent and treat Japanese cedar pollen allergy, four major T-cell epitopes from the Cry j 1 antigen and six from the Cry j 2 antigen were selected to design cry j 1 epi and cry j 2 epi, DNA constructs encoding artificial polypeptides of the selected epitopes. To apply cholera toxin B subunit (CTB) as an adjuvant, cry j 1 epi and cry j 2 epi were linked and then fused to the CTB gene in tandem to construct a fusion gene, ctb-linker-cry j 1 epi- cry j 2 epi-flag. The fusion gene was introduced into a pET-28a(+) vector and expressed in Escherichia coli BL21(DE3). The expressed recombinant protein was purified by a His-tag affinity column and confirmed by western blot analysis using anti-CTB and anti-FLAG antibodies. The purified recombinant protein also proved to be antigenic against anti-Cry j 1 and anti-Cry j 2 antibodies. Expression of the recombinant protein induced with 1mM IPTG reached a maximum in 3-5h, and recovery of the affinity-purified recombinant protein was approximately 120mg/L of culture medium. The present study indicates that production of sufficient amounts of recombinant protein with antigenic epitopes may be possible by recombinant techniques using E. coli or other bacterial strains for protein expression. PMID:25665505

  15. Enhancement of uptake of lipopolysaccharide in macrophages by the major outer membrane protein OmpA of gram-negative bacteria.

    PubMed Central

    Korn, A; Rajabi, Z; Wassum, B; Ruiner, W; Nixdorff, K

    1995-01-01

    Monoclonal antibodies (MAb) to lipopolysaccharide (LPS) and to the major outer membrane protein OmpA from Proteus mirabilis were generated and used to monitor the kinetics of uptake in macrophages of LPS as well as LPS bound to OmpA. Uptake was measured by a modified enzyme-linked immunosorbent assay (ELISA) in a microtiter culture system. The MAb were of various immunoglobulin G subclasses and showed strong reactivities with their antigens. Four hybridoma clones recognizing LPS and three recognizing OmpA from P. mirabilis 19 were selected for the present study on the basis of reactions in ELISA and Western blot (immunoblot) analyses. In the uptake assay, it was possible to differentiate between antigen on the cell surface and antigen which had been internalized. Uptake of LPS by macrophages was relatively rapid during the first 4 h of culture and then progressed more slowly over the remaining 24-h observation period. The level of detection of LPS in this assay system was in the nanogram range. When macrophages were pulsed with LPS for 30 min and subsequently washed to remove antigen not bound to the cells, the amount of LPS detectable on the macrophage surface decreased progressively for 3 h after the pulse, which indicated internalization of the antigen. Thereafter, LPS rose to an increased level on the cell surface. The rate of uptake of LPS was more rapid when it was in complex with OmpA. When the fate of OmpA was monitored in the same LPS-protein complexes by use of MAb to OmpA in a pulse experiment, the level of protein measured on the cell surface decreased after an initial rise, which again indicated internalization, but the protein did not reappear on the cell surface in a form detectable with the MAb. Compared with the LPS monitoring system, detection of OmpA associated with macrophages was weak, although the MAb to OmpA reacted strongly with the protein in the ELISA and Western blot analyses. PMID:7790087

  16. ST2249-MRSA-III: a second major recombinant methicillin-resistant Staphylococcus aureus clone causing healthcare infection in the 1970s.

    PubMed

    Nimmo, G R; Steen, J A; Monecke, S; Ehricht, R; Slickers, P; Thomas, J C; Appleton, S; Goering, R V; Robinson, D A; Coombs, G W

    2015-05-01

    Typing of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) from Australia in the 1970s revealed a novel clone, ST2249-MRSA-III (CC45), present from 1973 to 1979. This clone was present before the Australian epidemic caused by the recombinant clone, ST239-MRSA-III. This study aimed to characterize the genome of ST2249-MRSA-III to establish its relationship to other MRSA clones. DNA microarray analysis was conducted and a draft genome sequence of ST2249 was obtained. The recombinant structure of the ST2249 genome was revealed by comparisons to publicly available ST239 and ST45 genomes. Microarray analysis of genomic DNA of 13 ST2249 isolates showed gross similarities with the ST239 chromosome in a segment around the origin of replication and with ST45 for the remainder of the chromosome. Recombination breakpoints were precisely determined by the changing pattern of nucleotide polymorphisms in the genome sequence of ST2249 isolate SK1585 compared with ST239 and ST45. One breakpoint was identified to the right of oriC, between sites 1014 and 1065 of the gene D484_00045. Another was identified to the left of oriC, between sites 1185 and 1248 of D484_01632. These results indicate that ST2249 inherited approximately 35.3% of its chromosome from an ST239-like parent and 64.7% from an ST45-like parent. ST2249-MRSA-III resulted from a major recombination between parents that resemble ST239 and ST45. Although only limited Australian archival material is available, the oldest extant isolate of ST2249 predates the oldest Australian isolate of ST239 by 3 years. It is therefore plausible that these two recombinant clones were introduced into Australia separately. PMID:25708549

  17. ST2249-MRSA-III: a second major recombinant methicillin-resistant Staphylococcus aureus clone causing healthcare infection in the 1970s

    PubMed Central

    Nimmo, G. R.; Steen, J. A.; Monecke, S.; Ehricht, R.; Slickers, P.; Thomas, J. C.; Appleton, S.; Goering, R. V.; Robinson, D. A.; Coombs, G. W.

    2015-01-01

    Typing of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) from Australia in the 1970s revealed a novel clone, ST2249-MRSA-III (CC45), present from 1973 to 1979. This clone was present before the Australian epidemic caused by the recombinant clone, ST239-MRSA-III. This study aimed to characterize the genome of ST2249-MRSA-III to establish its relationship to other MRSA clones. DNA microarray analysis was conducted and a draft genome sequence of ST2249 was obtained. The recombinant structure of the ST2249 genome was revealed by comparisons to publicly available ST239 and ST45 genomes. Microarray analysis of genomic DNA of 13 ST2249 isolates showed gross similarities with the ST239 chromosome in a segment around the origin of replication and with ST45 for the remainder of the chromosome. Recombination breakpoints were precisely determined by the changing pattern of nucleotide polymorphisms in the genome sequence of ST2249 isolate SK1585 compared with ST239 and ST45. One breakpoint was identified to the right of oriC, between sites 1014 and 1065 of the gene D484_00045. Another was identified to the left of oriC, between sites 1185 and 1248 of D484_01632. These results indicate that ST2249 inherited approximately 35.3% of its chromosome from an ST239-like parent and 64.7% from an ST45-like parent. ST2249-MRSA-III resulted from a major recombination between parents that resemble ST239 and ST45. Although only limited Australian archival material is available, the oldest extant isolate of ST2249 predates the oldest Australian isolate of ST239 by 3 years. It is therefore plausible that these two recombinant clones were introduced into Australia separately. PMID:25708549

  18. Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV.

    PubMed

    Liu, Hsin-I; Chiou, Pinwen Peter; Gong, Hong-Yi; Chou, Hsin-Yiu

    2015-01-01

    Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines. PMID:26633384

  19. Cloning of the Major Capsid Protein (MCP) of Grouper Iridovirus of Taiwan (TGIV) and Preliminary Evaluation of a Recombinant MCP Vaccine against TGIV

    PubMed Central

    Liu, Hsin-I; Chiou, Pinwen Peter; Gong, Hong-Yi; Chou, Hsin-Yiu

    2015-01-01

    Fish iridoviruses cause systemic diseases with high mortality in various species of wild and farm-raised fish, resulting in severe economic losses. In 1998, we isolated a new epizootic iridovirus in cultured grouper (Epinephelus sp.) in Taiwan, thus named as grouper iridovirus of Taiwan (TGIV). We report here the cloning of TGIV major capsid protein (MCP). Phylogenetic analysis of the iridoviral MCPs confirmed the classification of TGIV into the Megalocytivirus genus. Recombinant TGIV MCP and GIV MCP were then generated to produce polyclonal antibodies. Western blot analysis revealed that the two antisera were species-specific, indicating no common epitope shared by the MCPs of the two viruses. We further assayed the potency of a subunit vaccine containing recombinant TGIV MCP. The vaccine effectively protected grouper from TGIV infection. The result demonstrated that MCP is a suitable antigen for anti-TGIV vaccines. PMID:26633384

  20. Comparative Analyses Between Lolium/Festuca Introgression Lines and Rice Reveal the Major Fraction of Functionally Annotated Gene Models Is Located in Recombination-Poor/Very Recombination-Poor Regions of the Genome

    PubMed Central

    King, Julie; Armstead, Ian P.; Donnison, S. Iain; Roberts, Luned A.; Harper, John A.; Skøt, Kirsten; Elborough, Kieran; King, Ian P.

    2007-01-01

    Publication of the rice genome sequence has allowed an in-depth analysis of genome organization in a model monocot plant species. This has provided a powerful tool for genome analysis in large-genome unsequenced agriculturally important monocot species such as wheat, barley, rye, Lolium, etc. Previous data have indicated that the majority of genes in large-genome monocots are located toward the ends of chromosomes in gene-rich regions that undergo high frequencies of recombination. Here we demonstrate that a substantial component of the coding sequences in monocots is localized proximally in regions of very low and even negligible recombination frequencies. The implications of our findings are that during domestication of monocot plant species selection has concentrated on genes located in the terminal regions of chromosomes within areas of high recombination frequency. Thus a large proportion of the genetic variation available for selection of superior plant genotypes has not been exploited. In addition our findings raise the possibility of the evolutionary development of large supergene complexes that confer a selective advantage to the individual. PMID:17603095

  1. Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

    SciTech Connect

    Kane, W.H.; Devore-Carter, D.; Ortel, T.L. )

    1990-07-24

    Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 {plus minus} 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 {plus minus} 0.012 to 0.124 {plus minus} 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 {plus minus} 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1,800 {plus minus} 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1,700-2,000 units/mg. Immunoprecipitation of ({sup 35}S)methionine-labeled rHFV revealed a single high molecular mass component. Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. The authors have also expressed a mutant, rHFV-des-B{sub 811-1441}, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. This mutant constitutively expressed 38 {plus minus} 7% of the activity of the RVV-V-activated protein. These results suggest that one of the functions of the large connecting region in factor V is to inhibit constitutive procoagulant activity.

  2. Outer membrane protein purification.

    PubMed

    Arigita, C; Jiskoot, W; Graaf, M R; Kersten, G F

    2001-01-01

    The major outer membrane proteins (OMPs) from Neisseria meningitidis, which are expressed at high levels, are subdivided in five classes based on molecular weight (1,2) (see Table 1). Table 1 Major Meningococcal Outer-Membrane Proteins Outer-membrane proteins Name Molecular maass Function/characteristics Class 1 PorA 44-47 kDa Porin Class 2/3 PorB 37-42 kDa Porin Class 4 Rmp Reductionmodifiableprotein, unknown Class 5 Opa 26-30 kDa Adhesion,opacity protein Opc 25 kDa Invasion, opacity protein Iron-regulated proteins Mirp 37 kDa Iron acquisition (?);majoriron-regulatedprotein FrpB 70 kDa Ferric enterobactin receptor (also FetA) Adapted from ref. (1). PMID:21336748

  3. Both V(D)J coding ends but neither signal end can recombine at the bcl-2 major breakpoint region, and the rejoining is ligase IV dependent.

    PubMed

    Raghavan, Sathees C; Hsieh, Chih-Lin; Lieber, Michael R

    2005-08-01

    The t(14;18) chromosomal translocation is the most common translocation in human cancer, and it occurs in all follicular lymphomas. The 150-bp bcl-2 major breakpoint region (Mbr) on chromosome 18 is a fragile site, because it adopts a non-B DNA conformation that can be cleaved by the RAG complex. The non-B DNA structure and the chromosomal translocation can be recapitulated on intracellular human minichromosomes where immunoglobulin 12- and 23-signals are positioned downstream of the bcl-2 Mbr. Here we show that either of the two coding ends in these V(D)J recombination reactions can recombine with either of the two broken ends of the bcl-2 Mbr but that neither signal end can recombine with the Mbr. Moreover, we show that the rejoining is fully dependent on DNA ligase IV, indicating that the rejoining phase relies on the nonhomologous DNA end-joining pathway. These results permit us to formulate a complete model for the order and types of cleavage and rejoining events in the t(14;18) translocation. PMID:16024785

  4. Growth promotion of red sea bream, Pagrosomus major, by oral administration of recombinant eel and salmon growth hormone

    NASA Astrophysics Data System (ADS)

    Xu, Bin; Mai, Kang-Sen; Xu, Ying-Li; Miao, Hong-Zhi; Liu, Zhen-Hui; Dong, Yong; Lan, Shan; Wang, Rao; Zhang, Pei-Jun

    2001-06-01

    Recombinant eel GH and yeast containing chinook salmon growth hormone (reGH and rcsGH) were incorporated into gelatin and sodium alginate (reGH-GS and rcsGH-GS) or polymer matrix (reGH-HP55) to protect the hormone from proteolytic cleavage in the stomach. The diets containing reGH-GS, rcsGH-GS, reGH-HP55 and free-reGH or uncoated-rcsGH were administered to red sea bream. Feeding of reGH-GS, reGH-HP55 and rcsGH-GS diets resulted in significant increases in body weight and fork length over those of controls. These results strongly suggest that gelatin and sodium alginate as well as polymer matrix protected the hormone from proteolytic enzymes in the gastrointestinal tract to allow the bioactive hormone to enter the circulation and eventually stimulate fish growth.

  5. Recombinant duck enteritis viruses expressing major structural proteins of the infectious bronchitis virus provide protection against infectious bronchitis in chickens.

    PubMed

    Li, Huixin; Wang, Yulong; Han, Zongxi; Wang, Yu; Liang, Shulin; Jiang, Lu; Hu, Yonghao; Kong, Xiangang; Liu, Shengwang

    2016-06-01

    To design an alternative vaccine for control of infectious bronchitis in chickens, three recombinant duck enteritis viruses (rDEVs) expressing the N, S, or S1 protein of infectious bronchitis virus (IBV) were constructed using conventional homologous recombination methods, and were designated as rDEV-N, rDEV-S, and rDEV-S1, respectively. Chickens were divided into five vaccinated groups, which were each immunized with one of the rDEVs, covalent vaccination with rDEV-N & rDEV-S, or covalent vaccination with rDEV-N & rDEV-S1, and a control group. An antibody response against IBV was detectable and the ratio of CD4(+)/CD8(+) T-lymphocytes decreased at 7 days post-vaccination in each vaccinated group, suggesting that humoral and cellular responses were elicited in each group as early as 7 days post-immunization. After challenge with a homologous virulent IBV strain at 21 days post-immunization, vaccinated groups showed significant differences in the percentage of birds with clinical signs, as compared to the control group (p < 0.01), as the two covalent-vaccination groups and the rDEV-S group provided better protection than the rDEV-N- or rDEV-S1-vaccinated group. There was less viral shedding in the rDEV-N & rDEV-S- (2/10) and rDEV-N & rDEV-S1- (2/10) vaccinated groups than the other three vaccinated groups. Based on the clinical signs, viral shedding, and mortality rates, rDEV-N & rDEV-S1 covalent vaccination conferred better protection than use of any of the single rDEVs. PMID:26946113

  6. Specific Antibodies Reactive with the 22-Kilodalton Major Outer Surface Protein of Borrelia anserina Ni-NL Protect Chicks from Infection

    PubMed Central

    Sambri, Vittorio; Marangoni, Antonella; Olmo, Andrea; Storni, Elisa; Montagnani, Marco; Fabbi, Massimo; Cevenini, Roberto

    1999-01-01

    An outer surface lipoprotein of 22 kDa was identified in the avian pathogen Borrelia anserina Ni-NL by using antibody preparations reactive with bacterial surface-exposed proteins. Amino acid sequence analysis of the 22-kDa protein demonstrated 90% identity with VmpA of B. turicatae, suggesting that the protein belongs to the family of 20-kDa outer surface proteins of the genus Borrelia. All of the 60 chicks intramuscularly treated with antibodies specifically reacting with the 22-kDa protein and infected with strain Ni-NL were completely protected from infection, since no spirochetemia was detected, and from death. Control chicks were treated with immune sera raised against apathogenic strain B. anserina Es, which expresses a prominent 20-kDa polypeptide that is also a member of the Vmp family but does not cross-react immunologically with the 22-kDa protein of the Ni-NL strain. These animals, infected with B. anserina Ni-NL, showed a high degree of spirochetemia 10 days after infection, and all died between 14 and 21 days after infection. The results showed that the 22-kDa surface protein of B. anserina Ni-NL is a determinant of the pathogenic potential of the strain and also confirmed that only strain-specific antibodies are protective against B. anserina infection. PMID:10225933

  7. MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein

    PubMed Central

    Guo, Monica S.; Updegrove, Taylor B.; Gogol, Emily B.; Shabalina, Svetlana A.; Gross, Carol A.; Storz, Gisela

    2014-01-01

    In enteric bacteria, the transcription factor σE maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σE-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σE activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σE regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σE to repress the synthesis of all abundant outer membrane proteins in response to stress. PMID:25030700

  8. Recombination suppression in the vicinity of the breakpoints of a balanced 1:11 autosomal translocation associated with schizophrenia and other forms of major mental illness

    SciTech Connect

    He, L.; Blackwood, D.H.R.; Maclean, A.W.

    1994-09-01

    The frequency and extent of pairing failure around human translocations is unknown. We have examined the pattern of recombination around the breakpoints of a balanced autosomal translocation t(1:11)(q43:q21) associated with major mental illness. We have postulated that the association with mental illness in the family has not arisen by chance, but rather that functional disruption of a gene at or near a breakpoint site is responsible. Efforts to isolate the breakpoints for molecular analysis of the region are now at an advanced stage. On the other hand if pairing failure is occurring in the family in the region of the breakpoints, a susceptibility allele for mental illness, acting independently of the translocation, may be located some distance away. DNA was available from seventeen carriers and ten non-translocation carriers, giving a total of thirty-one informative meioses spanning 4 generations. The derivative one and eleven chromosomes were also isolated in somatic cell hybrids and were used to confirm allele phase. We genotyped the pedigree members using nine markers covering 30 cMs on either side of both the chromosome one and eleven breakpoints. No recombinants were found with markers within 3 cMs of either breakpoint. Four markers at an average of 7 cMs respectively on either side of the two breakpoints gave a total of three crossovers from thirty-one meioses versus an expected 9, demonstrating (p<0.05) significant recombination suppression. By contrast, examination of chromosome regions at greater distances from the breakpoints showed recombination rates similar to those expected from CEPH data with no evidence of suppression. We conclude that crossover suppression occurs in this family but is restricted to a region within 7 cMs of the breakpoints.

  9. Saturn's outer magnetosphere

    NASA Technical Reports Server (NTRS)

    Schardt, A. W.; Behannon, K. W.; Carbary, J. F.; Eviatar, A.; Lepping, R. P.; Siscoe, G. L.

    1983-01-01

    Similarities between the Saturnian and terrestrial outer magnetosphere are examined. Saturn, like Earth, has a fully developed magnetic tail, 80 to 100 RS in diameter. One major difference between the two outer magnetospheres is the hydrogen and nitrogen torus produced by Titan. This plasma is, in general, convected in the corotation direction at nearly the rigid corotation speed. Energies of magnetospheric particles extend to above 500 keV. In contrast, interplanetary protons and ions above 2 MeV have free access to the outer magnetosphere to distances well below the Stormer cutoff. This access presumably occurs through the magnetotail. In addition to the H+, H2+, and H3+ ions primarily of local origin, energetic He, C, N, and O ions are found with solar composition. Their flux can be substantially enhanced over that of interplanetary ions at energies of 0.2 to 0.4 MeV/nuc.

  10. A recombinant antibody with the antigen-specific, major histocompatibility complex-restricted specificity of T cells.

    PubMed Central

    Andersen, P S; Stryhn, A; Hansen, B E; Fugger, L; Engberg, J; Buus, S

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might lead to novel approaches in immunotherapy. However, it has proven difficult to generate antibodies with the specificity of T cells by conventional hybridoma techniques. Here we report that the phage display technology is a feasible alternative to generate antibodies recognizing specific, predetermined peptide/MHC complexes. Images Fig. 2 PMID:8700842

  11. Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium

    PubMed Central

    Ithurbide, Solenne; Bentchikou, Esma; Coste, Geneviève; Bost, Bruno; Servant, Pascale; Sommer, Suzanne

    2015-01-01

    The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA + as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA + cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA + bacteria exposed to ionizing radiation. PMID:26517555

  12. Single Strand Annealing Plays a Major Role in RecA-Independent Recombination between Repeated Sequences in the Radioresistant Deinococcus radiodurans Bacterium.

    PubMed

    Ithurbide, Solenne; Bentchikou, Esma; Coste, Geneviève; Bost, Bruno; Servant, Pascale; Sommer, Suzanne

    2015-10-01

    The bacterium Deinococcus radiodurans is one of the most radioresistant organisms known. It is able to reconstruct a functional genome from hundreds of radiation-induced chromosomal fragments. Our work aims to highlight the genes involved in recombination between 438 bp direct repeats separated by intervening sequences of various lengths ranging from 1,479 bp to 10,500 bp to restore a functional tetA gene in the presence or absence of radiation-induced DNA double strand breaks. The frequency of spontaneous deletion events between the chromosomal direct repeats were the same in recA+ and in ΔrecA, ΔrecF, and ΔrecO bacteria, whereas recombination between chromosomal and plasmid DNA was shown to be strictly dependent on the RecA and RecF proteins. The presence of mutations in one of the repeated sequence reduced, in a MutS-dependent manner, the frequency of the deletion events. The distance between the repeats did not influence the frequencies of deletion events in recA+ as well in ΔrecA bacteria. The absence of the UvrD protein stimulated the recombination between the direct repeats whereas the absence of the DdrB protein, previously shown to be involved in DNA double strand break repair through a single strand annealing (SSA) pathway, strongly reduces the frequency of RecA- (and RecO-) independent deletions events. The absence of the DdrB protein also increased the lethal sectoring of cells devoid of RecA or RecO protein. γ-irradiation of recA+ cells increased about 10-fold the frequencies of the deletion events, but at a lesser extend in cells devoid of the DdrB protein. Altogether, our results suggest a major role of single strand annealing in DNA repeat deletion events in bacteria devoid of the RecA protein, and also in recA+ bacteria exposed to ionizing radiation. PMID:26517555

  13. The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form.

    PubMed Central

    Reverbel-Leroy, C; Pages, S; Belaich, A; Belaich, J P; Tardif, C

    1997-01-01

    The recombinant form of the cellulase CelF of Clostridium cellulolyticum, tagged by a C-terminal histine tail, was overproduced in Escherichia coli. The fusion protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column. The intact form of CelF (Mr, 79,000) was rapidly degraded at the C terminus, giving a shorter stable form, called truncated CelF (Mr, 71,000). Both the entire and the truncated purified forms degraded amorphous cellulose (kcat = 42 and 30 min(-1), respectively) and microcrystalline cellulose (kcat = 13 and 10 min(-1), respectively). The high ratio of soluble reducing ends to insoluble reducing ends released by truncated CelF from amorphous cellulose showed that CelF is a processive enzyme. Nevertheless, the diversity of the cellodextrins released by truncated CelF from phosphoric acid-swollen cellulose at the beginning of the reaction indicated that the enzyme might randomly hydrolyze beta-1,4 bonds. This hypothesis was supported by viscosimetric measurements and by the finding that CelF and the endoglucanase CelA are able to degrade some of the same cellulose sites. CelF was therefore called a processive endocellulase. The results of immunoblotting analysis showed that CelF was associated with the cellulosome of C. cellulolyticum. It was identified as one of the three major components of cellulosomes. The ability of the entire form of CelF to interact with CipC, the cellulosome integrating protein, or mini-CipC1, a recombinant truncated form of CipC, was monitored by interaction Western blotting (immunoblotting) and by binding assays using a BIAcore biosensor-based analytical system. PMID:8981979

  14. Structural analysis and immunogenicity of recombinant major envelope protein (rA27L) of buffalopox virus, a zoonotic Indian vaccinia-like virus.

    PubMed

    Kumar, Amit; Yogisharadhya, Revanaiah; Bhanuprakash, Veerakyathappa; Venkatesan, Gnanavel; Shivachandra, Sathish Bhadravati

    2015-10-01

    Buffalopox virus (BPXV), an Indian variant of vaccinia virus (VACV), is a zoonotic agent and affects buffaloes, cattle and humans. A27L is one of the conserved major immuno-dominant envelope proteins of orthopox viruses (OPVs) involved in viral entry/maturation and elicits neutralizing antibodies. In this study, the A27L gene of BPXV-Vij/96 strain encoding recombinant mature A27L (21S to E110) and C-terminal truncated A27L-LZD (21S to N84aa) proteins were cloned and over-expressed in Escherichia coli as fusion proteins. Structurally, A27L of BPXV was similar to that of VACV and found to contain four regions including a potential coiled-coil motif (CCM) in the centre (43 to 84aa). Oligomerization of recombinant A27L fusion protein (∼30 kDa) leads to the formation of dimer/trimers/tetramers under non-reducing conditions. Further, the purified rA27L protein was used for active immunization of rabbit (250 μg/rabbit) and adult mice (10 μg and 50 μg/mice) with or without adjuvants (FCA, alum and CpG). Immune response measured by using indirect-ELISA and SNT revealed a gradual increase in antigen specific serum IgG as well as neutralization antibody titers. Upon challenge with virulent BPXV strain, a protection of 60% was observed in suckling mice passively administered with anti-rA27L sera. No cross-reactivity of rA27L protein with hyperimmune sera against ORFV, GTPV, SPPV, PPRV, FMDV and BTV was noticed in indirect-ELISA. The study indicated that the rA27L protein is a safe and potential prophylactic as well as diagnostic antigen for buffalopox. PMID:26319070

  15. Modulation of effects of lipopolysaccharide on macrophages by a major outer membrane protein of Proteus mirabilis as measured in a chemiluminescence assay.

    PubMed Central

    Weber, G; Heck, D; Bartlett, R R; Nixdorff, K

    1992-01-01

    Our previous studies have shown that a major protein isolated from purified cell walls of Proteus mirabilis (39-kDa protein) is a strong modulator of the specific immune responses to lipopolysaccharide (LPS) from this bacterium. When the protein is mixed with LPS before immunization of mice, the responses of antibody-producing cells specific for LPS are greatly enhanced and converted predominantly to the immunoglobulin G isotype. In the present study, the immunomodulating effects of the 39-kDa protein were tested at the level of interaction of LPS with macrophages. Activation of macrophages was determined by measuring the production of oxygen radicals in a chemiluminescence assay with lucigenin as the amplifier. LPS from P. mirabilis induced strong oxidative metabolism in both peritoneal and bone marrow-derived murine macrophages. These responses were inhibited in a dose-dependent manner by mixing LPS with increasing amounts of the protein. In contrast, bovine serum albumin and methylated bovine serum albumin enhanced the response of macrophages dramatically when complexed with LPS. The inhibiting activity of the 39-kDa protein was also observed with LPS from Escherichia coli K-12. PMID:1541521

  16. Cosmological Recombination

    NASA Astrophysics Data System (ADS)

    Wong, Wan Yan

    2008-11-01

    In this thesis we focus on studying the physics of cosmological recombination and how the details of recombination affect the Cosmic Microwave Background (CMB) anisotropies. We present a detailed calculation of the spectral line distortions on the CMB spectrum arising from the Lyman-alpha and the lowest two-photon transitions in the recombination of hydrogen (H), and the corresponding lines from helium (He). The peak of these distortions mainly comes from the Lyman-alpha transition and occurs at about 170 microns, which is the Wien part of the CMB. The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. With this motivation, we perform a multi-level calculation of the recombination of H and He with the addition of the spin-forbidden transition for neutral helium (He I), plus the higher order two-photon transitions for H and among singlet states of He I. We find that the inclusion of the spin-forbidden transition results in more than a percent change in the ionization fraction, while the other transitions give much smaller effects. Last we modify RECFAST by introducing one more parameter to reproduce recent numerical results for the speed-up of helium recombination. Together with the existing hydrogen `fudge factor', we vary these two parameters to account for the remaining dominant uncertainties in cosmological recombination. By using a Markov Chain Monte Carlo method with Planck forecast data, we find that we need to determine the parameters to better than 10% for He I and 1% for H, in order to obtain negligible effects on the cosmological parameters.

  17. Evaluation of recombinant Leishmania poly-protein plus GLA-SE vaccines against sand fly-transmitted Leishmania major in C57Bl/6 mice1

    PubMed Central

    Peters, Nathan C.; Bertholet, Sylvie; Lawyer, Phillip G.; Charmoy, Melanie; Romano, Audrey; Ribeiro-Gomes, Flavia L.; Stamper, Lisa W.; Sacks, David L.

    2012-01-01

    Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania poly-proteins delivered in a stable emulsion (SE) with the TLR 4 agonists monophosphoryl lipid A (MPL) or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease following sand fly transmission of L. major in susceptible BALB/c mice. Similar poly-protein adjuvant combinations are the vaccine candidates most likely to see clinical evaluation. We assessed immunity generated by KSAC or L110f vaccination with GLA-SE following challenge with L. major by needle or infected sand fly bite in resistant C57BL/6 mice. Poly-protein vaccinated mice had a 60-fold increase in CD4+IFN-γ+ T cell numbers versus control animals at 2 weeks post needle inoculation of L. major and this correlated with a 100-fold reduction in parasite load. Immunity did not, however, reach levels observed in mice with a healed primary infection. Following challenge by infected sand fly bite, poly-protein vaccinated animals had comparable parasite loads, greater numbers of neutrophils at the challenge site, and reduced CD4+ IFN-γ+:IL-17+ ratios versus non-vaccinated controls. In contrast, healed animals had significantly reduced parasite loads and higher CD4+ IFN-γ+:IL-17+ ratios. These observations demonstrate that vaccine-induced protection against needle challenge does not necessarily translate to protection following challenge by infected sand fly bite. PMID:23045616

  18. Introduction of protein or DNA delivered via recombinant Salmonella typhimurium into the major histocompatibility complex class I presentation pathway of macrophages.

    PubMed

    Catic, A; Dietrich, G; Gentschev, I; Goebel, W; Kaufmann, S H; Hess, J

    1999-02-01

    Recombinant (r) Salmonella typhimurium aroA strains which display the hen egg ovalbumin OVA(257-264) peptide SIINFEKL in secreted form were constructed. In addition, attenuated rS. typhimurium pcDNA-OVA constructs harbouring a eukaryotic expression plasmid encoding complete OVA were used to introduce the immunodominant OVA(257-264) epitope into the major histocompatibility complex (MHC) class I presentation pathway. Both modes of antigen delivery (DNA and protein) by Salmonella vaccine carriers stimulated OVA(257-264)-specific CD8 T-cell hybridomas. An in vitro infection system was established that allowed both rSalmonella carrier devices to facilitate MHC class I delivery of OVA(257-264) by coexpression of listeriolysin (Hly) or by coinfection with rS. typhimurium Hlys (Hess J., Gentschev I., Miko D., Welzel M., Ladel C., Goebel W., Kaufmann S.H.E., Proc. Natl. Acad. Sci. USA 93 (1996) 1458-1463). Coexpression of Hly and coinfection with rS. typhimurium Hlys slightly improved MHC class I processing of OVA. Our data provide further evidence for the feasibility of attenuated, Hly-expressing rS. typhimurium carriers secreting heterologous antigens or harbouring heterologous DNA as effective vaccines for stimulating CD8 T cells in addition to CD4 T cells. PMID:10594975

  19. In Vitro Infectivity Assessment by Drug Susceptibility Comparison of Recombinant Leishmania major Expressing Enhanced Green Fluorescent Protein or EGFP-Luciferase Fused Genes with Wild-Type Parasite

    PubMed Central

    Sadeghi, Somayeh; Seyed, Negar; Etemadzadeh, Mohammad-Hossein; Abediankenari, Saeid; Rafati, Sima; Taheri, Tahereh

    2015-01-01

    Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-γ/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency. PMID:26323836

  20. Immunoglobulin Heavy Chain Variable Region and Major Histocompatibility Region Genes Are Linked to Induced Graves' Disease in Females From Two Very Large Families of Recombinant Inbred Mice

    PubMed Central

    Aliesky, Holly; Banuelos, Bianca; Magana, Jessica; Williams, Robert W.; Rapoport, Basil

    2014-01-01

    Graves' hyperthyroidism is caused by antibodies to the TSH receptor (TSHR) that mimic thyroid stimulation by TSH. Stimulating TSHR antibodies and hyperthyroidism can be induced by immunizing mice with adenovirus expressing the human TSHR A-subunit. Prior analysis of induced Graves' disease in small families of recombinant inbred (RI) female mice demonstrated strong genetic control but did not resolve trait loci for TSHR antibodies or elevated serum T4. We investigated the genetic basis for induced Graves' disease in female mice of two large RI families and combined data with earlier findings to provide phenotypes for 178 genotypes. TSHR antibodies measured by inhibition of TSH binding to its receptor were highly significantly linked in the BXD set to the major histocompatibility region (chromosome 17), consistent with observations in 3 other RI families. In the LXS family, we detected linkage between T4 levels after TSHR-adenovirus immunization and the Ig heavy chain variable region (Igvh, chromosome 12). This observation is a key finding because components of the antigen binding region of Igs determine antibody specificity and have been previously linked to induced thyroid-stimulating antibodies. Data from the LXS family provide the first evidence in mice of a direct link between induced hyperthyroidism and Igvh genes. A role for major histocompatibility genes has now been established for genetic susceptibility to Graves' disease in both humans and mice. Future studies using arrays incorporating variation in the complex human Ig gene locus will be necessary to determine whether Igvh genes are also linked to Graves' disease in humans. PMID:25051451

  1. Recombinant Salmonella typhimurium outer membrane protein A is recognized by synovial fluid CD8 cells and stimulates synovial fluid mononuclear cells to produce interleukin (IL)-17/IL-23 in patients with reactive arthritis and undifferentiated spondyloarthropathy.

    PubMed

    Chaurasia, S; Shasany, A K; Aggarwal, A; Misra, R

    2016-08-01

    In developing countries, one-third of patients with reactive arthritis (ReA) and undifferentiated spondyloarthropathy (uSpA) are triggered by Salmonella typhimurium. Synovial fluid mononuclear cells (SFMCs) of patients with ReA and uSpA proliferate to low molecular weight fractions (lmwf) of outer membrane proteins (Omp) of S. typhimurium. To characterize further the immunity of Omp of Salmonella, cellular immune response to two recombinant proteins of lmwf, OmpA and OmpD of S. typhimurium (rOmpA/D-sal) was assessed in 30 patients with ReA/uSpA. Using flow cytometry, 17 of 30 patients' SF CD8(+) T cells showed significant intracellular interferon (IFN)-γ to Omp crude lysate of S. typhimurium. Of these 17, 11 showed significantly more CD8(+) CD69(+) IFN-γ T cells to rOmpA-sal, whereas only four showed reactivity to rOmpD-sal. The mean stimulation index was significantly greater in rOmpA-sal than rOmpD-sal [3·0 (1·5-6·5) versus 1·5 (1·0-2·75), P < 0·005]. Similarly, using enzyme-linked immunospot (ELISPOT) in these 17 patients, the mean spots of IFN-γ-producing SFMCs were significantly greater in rOmpA-sal than rOmpD-sal [44·9 (3·5-130·7) versus 19·25 (6-41), P < 0·05]. SFMCs stimulated by rOmpA-sal produced significantly more proinflammatory cytokines than rOmpD-sal: IFN-γ [1·44 (0·39-20·42) versus 0·72 (0·048-9·15) ng/ml, P < 0·05], interleukin (IL)-17 [28·60 (6·15-510·86) versus 11·84 (6·83-252·62) pg/ml, P < 0·05], IL-23 [70·19 (15-1161·16) versus 28·25 (> 15-241·52) pg/ml, P < 0·05] and IL-6 [59·78 (2·03-273·36) versus 10·17 (0·004-190·19) ng/ml, P < 0·05]. The rOmpA-sal-specific CD8(+) T cell response correlated with duration of current synovitis (r = 0·53, P < 0·05). Thus, OmpA of S. typhimurium is a target of SF CD8(+) T cells and drives SFMC to produce increased cytokines of the IL-17/IL-23 axis which contribute to the pathogenesis of Salmonella-triggered ReA. PMID:27060348

  2. Thermoelectric Outer Planets Spacecraft (TOPS)

    NASA Technical Reports Server (NTRS)

    1973-01-01

    The research and advanced development work is reported on a ballistic-mode, outer planet spacecraft using radioisotope thermoelectric generator (RTG) power. The Thermoelectric Outer Planet Spacecraft (TOPS) project was established to provide the advanced systems technology that would allow the realistic estimates of performance, cost, reliability, and scheduling that are required for an actual flight mission. A system design of the complete RTG-powered outer planet spacecraft was made; major technical innovations of certain hardware elements were designed, developed, and tested; and reliability and quality assurance concepts were developed for long-life requirements. At the conclusion of its active phase, the TOPS Project reached its principal objectives: a development and experience base was established for project definition, and for estimating cost, performance, and reliability; an understanding of system and subsystem capabilities for successful outer planets missions was achieved. The system design answered long-life requirements with massive redundancy, controlled by on-board analysis of spacecraft performance data.

  3. Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

    PubMed Central

    Miller, M M; Goto, R M; Taylor, R L; Zoorob, R; Auffray, C; Briles, R W; Briles, W E; Bloom, S E

    1996-01-01

    Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8632997

  4. Engineering, purification and applications of His-tagged recombinant antibody fragments with specificity for the major birch pollen allergen, bet v1.

    PubMed

    Flicker, S; Laffer, S; Steinberger, P; Alhani, B; Zhu, Y; Laukkanen, M L; Keinänen, K; Kraft, D; Valenta, R

    2000-01-01

    Type I allergy, an immunodisorder affecting almost 20% of the population worldwide, is based on the production of IgE antibodies against per se harmless allergens. We report the expression of hexahistidine-tagged antibody fragments (Fabs) with specificity for Bet v1, the major birch pollen allergen, in Escherichia coli. The cDNA coding for the heavy chain fragment of a mouse monoclonal anti-Bet v1 antibody, Bip 1, was engineered by PCR to contain a hexahistidine-encoding 3' end. The modified Bip1 heavy chain cDNA was co-expressed in E. coli XL-1 Blue with the Bip 1 light chain cDNA using the combinatorial plasmid pComb3H. His-tagged recombinant (r) Bip 1 Fabs were isolated by nickel affinity chromatography and rBip 1 Fabs without His-tag were purified via affinity to rBet v1. rBip 1 Fabs with and without His-tag bound specifically to rBet v1 and, like Bet v1 -specific human serum IgE and rabbit-anti rBet v1 antibodies, cross-reacted with Bet v1-related allergens in other plant-species (alder, oak, hazelnut). We demonstrate the usefulness of His-tagged rBip 1 Fabs (1) for the identification of pollen samples containing Bet v 1 by particle blotting, (2) forthe detection of Bet v1-specific IgE antibodies in human serum samples by sandwich ELISA and (3) for the quantification of Bet v1 in solution. Based on these examples we suggest to use rBip 1 Fabs for the detection of Bet v1 and Bet v1-related allergens in natural allergen sources for allergy prevention, as well as for the standardization of natural allergen extracts produced for diagnosis and immunotherapy of birch pollen allergy. PMID:10722049

  5. The effects of recombinant eel growth hormone, methyltestosterone and L-thyroxine on the growth of red sea bream, Pagrosomus major

    NASA Astrophysics Data System (ADS)

    Xu, Bin; Ge, Guo-Chang; Miao, Hong-Zhi

    1995-03-01

    the effects of recombinant eel growth hormone (reGH), methyltestosterone (MT) and L-thyroxine (T4) on the growth of red sea bream, Pagrosomus major, were investigated. Administration of reGH to fry by immersion at 2 mg/1 for 2 h every 5 days resulted in significant increase in both weight and length, but the condition factor (CF) diminished relative to that of similarly treated controls over the 37 day treatment period. Immersion in 0.1 mg/l T4 also resulted in significant increase in both weight and length and higher survival rate of test fry compared to the controls. Immersion in MT had less effect on growth and high-dose resulted in high mortality. In the second study, injection of 2 μg reGH/(g·wk) caused a significant increase in the specific growth rate (SGR) of test red sea bream fingerlings relative to that of the controls during the 4—week treatment period and maintained the increasing trend over the post-treatment period (weeks 4 6). Injection of MT at a dosage of 1μg/(g·wk) resulted in a significant increase in SGR during the 4—week treatment period. Intramuscular injection of 0.1 μg T4/(g·wk) also resulted in a significant increase in weight. Injection of high-doses of MT and T4 inhibited growth and resulted in darkening skin, bulging eyes and thinning body. In muscle chemical composition, the treated groups had no significant differences compared to the controls, but the high-dose MT and T4 groups showed significant increases in lipid content. Potential practical methods for hormone application in fish culture are discussed.

  6. Outer atmospheric research

    NASA Technical Reports Server (NTRS)

    Anderson, John L.

    1988-01-01

    The region above the earth from about 90 km to 150 km is a major part of the upper or outer atmosphere. It is relatively unexplored, being too high for balloons or aircraft and too low for persistent orbiting spacecraft. However, the concept of a tethered subsatellite, deployed downward from an orbiting, more massive craft such as the Space Shuttle, opens the possibility of a research capability that could provide global mapping of this region. The need for research in this thick spherical shell above the earth falls into two major categories: (1) scientific data for understanding and modeling the global atmosphere and thereby determining its role in the earth system, and (2) engineering data for the design of future aerospace vehicles that will operate there. This paper presents an overview and synthesis of the currently perceived research needs and the state-of-the-art of the proposed tethered research capability.

  7. λ Recombination and Recombineering.

    PubMed

    Murphy, Kenan C

    2016-05-01

    The bacteriophage λ Red homologous recombination system has been studied over the past 50 years as a model system to define the mechanistic details of how organisms exchange DNA segments that share extended regions of homology. The λ Red system proved useful as a system to study because recombinants could be easily generated by co-infection of genetically marked phages. What emerged from these studies was the recognition that replication of phage DNA was required for substantial Red-promoted recombination in vivo, and the critical role that double-stranded DNA ends play in allowing the Red proteins access to the phage DNA chromosomes. In the past 16 years, however, the λ Red recombination system has gained a new notoriety. When expressed independently of other λ functions, the Red system is able to promote recombination of linear DNA containing limited regions of homology (∼50 bp) with the Escherichia coli chromosome, a process known as recombineering. This review explains how the Red system works during a phage infection, and how it is utilized to make chromosomal modifications of E. coli with such efficiency that it changed the nature and number of genetic manipulations possible, leading to advances in bacterial genomics, metabolic engineering, and eukaryotic genetics. PMID:27223821

  8. Origin of Outer Solar System

    NASA Technical Reports Server (NTRS)

    Holman, Matthew J.; Boyce, J. (Technical Monitor)

    2003-01-01

    We feel that at the present moment the available theoretical models of the Kuiper belt are still in advance of the data, and thus our main task has been to conduct observational work guided by theoretical motivations. Our efforts over the past year can be divided into four categories: A) Wide-field Searches for Kuiper Belt Objects; B) Pencil-beam Searches for Kuiper Belt Objects; C) Wide-field Searches for Moons of the Outer Planets; D) Pencil-beam Searches for Faint Uranian and Neptunian Moons; E) Recovery Observations. As of April 2002, we have conducted several searches for Kuiper belt objects using large-format mosaic CCD camera on 4-meter class telescopes. In May 1999, we used the Kitt Peak 4-meter with the NOAO Mosaic camera we attempted a search for KBOs at a range of ecliptic latitudes. In addition to our wide-field searches, we have conducted three 'pencil-beam' searches in the past year. In a pencil-beam search we take repeated integrations of the same field throughout a night. After preprocessing the resulting images we shift and recombine them along a range of rates and directions consistent with the motion of KBOs. Stationary objects then smear out, while objects moving at near the shift rate appear as point sources. In addition to our searches for Kuiper belt objects, we are completing the inventory of the outer solar system by search for faint satellites of the outer planets. In August 2001 we conducted pencil beam searches for faint Uranian and Neptunian satellites at CFHT and CTIO. These searches resulted in the discover of two Neptunian and four Uranian satellite candidates. The discovery of Kuiper belt objects and outer planet satellites is of little use if the discoveries are not followed by systematic, repeated astrometric observations that permit reliable estimates of their orbits.

  9. Methanotroph outer membrane preparation.

    PubMed

    Karlsen, Odd A; Berven, Frode S; Jensen, Harald B; Fjellbirkeland, Anne

    2011-01-01

    All presently known methanotrophs are gram-negative bacteria suggesting that they are surrounded by a two-layered membrane: an inner or cytoplasmic membrane and an outer membrane. In the methanotroph Methylococcus capsulatus (Bath), separation of the two membranes has allowed studies on protein and lipid composition of the outer membrane. Its outer membrane can be isolated from purified cell envelopes by selective solubilization of the inner membranes with the detergent Triton X-100. The proteins associated with the outer membrane can further be fractionated into integral and tightly associated proteins and peripheral loosely associated proteins. We present here protocols for this fractionation and show how the proteins associated with the outer leaflet of the outer membrane can be isolated and identified by whole-cell biotin surface labeling. PMID:21419921

  10. Immunization with the Chlamydia trachomatis mouse pneumonitis major outer membrane protein by use of CpG oligodeoxynucleotides as an adjuvant induces a protective immune response against an intranasal chlamydial challenge.

    PubMed

    Pal, Sukumar; Davis, Heather L; Peterson, Ellena M; de la Maza, Luis M

    2002-09-01

    Recently, we have shown that a vaccine consisting of a purified preparation of the Chlamydia trachomatis mouse pneumonitis (MoPn) major outer membrane protein (MOMP) and Freund's adjuvant can protect mice against a genital challenge. Here, we wanted to determine if CpG motifs could be used as an immune modulator to the MOMP to induce protection in mice against an intranasal (i.n.) challenge. One-week-old BALB/c mice were immunized intramuscularly and subcutaneously either once or three times at 2-week intervals with MOMP and CpG suspended in aluminum hydroxide (alum). Negative controls received ovalbumin, CpG, and alum. Positive controls were immunized i.n. with C. trachomatis MoPn elementary bodies (EB). Six weeks after the last immunization, mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of the C. trachomatis MoPn serovar. Mice that received MOMP, CpG, and alum had a strong immune response, as shown by a high titer of serum antibodies to Chlamydia and significant lymphoproliferation of T-cells following stimulation with C. trachomatis EB. After the i.n. challenge mice immunized with MOMP, CpG, and alum showed significantly less body weight loss than the corresponding control mice immunized with ovalbumin, CpG, and alum. Ten days after the challenge the animals were euthanized, their lungs were weighed, and the numbers of IFU in the lungs were determined. The average weight of the lungs of the mice immunized with MOMP, CpG, and alum was significantly less than average weight of the lungs of the mice immunized with ovalbumin, CpG, and alum. Also, the average number of IFU recovered per mouse immunized with MOMP, CpG, and alum was significantly less than the average number of IFU per mouse detected in the mice inoculated with ovalbumin, CpG, and alum. In conclusion, our data show that CpG sequences can be used as an effective adjuvant with the C. trachomatis MoPn MOMP to elicit a protective immune response in mice against a chlamydial respiratory

  11. Physics of the outer heliosphere

    SciTech Connect

    Gazis, P.R. )

    1991-01-01

    Major advances in the physics of the outer heliosphere are reviewed for the 1987-1990 time frame. Emphasis is placed on five broad topics: the detailed structure of the solar wind at large heliocentric distances, the global structure of the interplanetary field, latidudinal variations and meridional flows, radial and temporal variations, and the interaction of the solar wind with the local interstellar medium. 122 refs.

  12. The Outer Limits: English.

    ERIC Educational Resources Information Center

    Tyler, Barbara R.; Biesekerski, Joan

    The Quinmester course "The Outer Limits" involves an exploration of unknown worlds, mental and physical, through fiction and nonfiction. Its purpose is to focus attention on the ongoing conquest of the frontiers of the mind, the physical world, and outer space. The subject matter includes identification and investigation of unknown worlds in the…

  13. Outer Solar System Nomenclature

    NASA Technical Reports Server (NTRS)

    Owen, Tobias C.; Grant, John (Technical Monitor)

    2003-01-01

    This grant has supported work by T. Owen and B. A. Smith on planetary and satellite nomenclature, carried out under the general auspices of the International Astronomical Union (IAU). The IAU maintains a Working Group on Planetary and Satellite Nomenclature (WGPSN) whose current chair is Prof.Kaare Aksnes of the Rosseland Institute for Theoretical Astrophysics in Oslo, Norway. Both Owen and Smith are members of the WGPSN; Owen as chair of the Outer Solar System Task Group, and Smith as chair of the Mars Task Group. The major activity during the last grant period (2002) was the approval of several new names for features on Mars by Smith's group and features on Jovian satellites plus new names for satellites of Jupiter, Saturn and Uranus by Owen's group. Much of this work was accomplished by e-mail exchanges, but the new nomenclature was formally discussed and approved at a meeting of the WGPSN held in conjunction with the Division for Planetary Sciences meeting in Birmingham, Alabama in October 2002.

  14. Genomic homologous recombination in planta.

    PubMed Central

    Gal, S; Pisan, B; Hohn, T; Grimsley, N; Hohn, B

    1991-01-01

    A system for monitoring intrachromosomal homologous recombination in whole plants is described. A multimer of cauliflower mosaic virus (CaMV) sequences, arranged such that CaMV could only be produced by recombination, was integrated into Brassica napus nuclear DNA. This set-up allowed scoring of recombination events by the appearance of viral symptoms. The repeated homologous regions were derived from two different strains of CaMV so that different recombinant viruses (i.e. different recombination events) could be distinguished. In most of the transgenic plants, a single major virus species was detected. About half of the transgenic plants contained viruses of the same type, suggesting a hotspot for recombination. The remainder of the plants contained viruses with cross-over sites distributed throughout the rest of the homologous sequence. Sequence analysis of two recombinant molecules suggest that mismatch repair is linked to the recombination process. Images PMID:2026150

  15. Immunization with Recombinant Brucella Species Outer Membrane Protein Omp16 or Omp19 in Adjuvant Induces Specific CD4+ and CD8+ T Cells as Well as Systemic and Oral Protection against Brucella abortus Infection▿

    PubMed Central

    Pasquevich, Karina A.; Estein, Silvia M.; Samartino, Clara García; Zwerdling, Astrid; Coria, Lorena M.; Barrionuevo, Paula; Fossati, Carlos A.; Giambartolomei, Guillermo H.; Cassataro, Juliana

    2009-01-01

    Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane proteins (OMPs) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated Omp16 (L-Omp16) or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection against B. abortus infection. Vaccination with unlipidated Omp16 (U-Omp16) or U-Omp19 in IFA induced a higher degree of protection than the respective lipidated versions. Moreover, the level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to that elicited by live B. abortus S19 immunization. Flow cytometric analysis showed that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4+ as well as CD8+ T cells producing gamma interferon. In vivo depletion of CD4+ or CD8+ T cells in mice immunized with U-Omp16 or U-Omp19 plus IFA resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in aluminum hydroxide formulation, and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51, respectively). Overall these results indicate that U-Omp16 or U-Omp19 would be a useful candidate for a subunit vaccine against human and animal brucellosis. PMID:18981242

  16. Immunization with recombinant Brucella species outer membrane protein Omp16 or Omp19 in adjuvant induces specific CD4+ and CD8+ T cells as well as systemic and oral protection against Brucella abortus infection.

    PubMed

    Pasquevich, Karina A; Estein, Silvia M; García Samartino, Clara; Samartino, Clara García; Zwerdling, Astrid; Coria, Lorena M; Barrionuevo, Paula; Fossati, Carlos A; Giambartolomei, Guillermo H; Cassataro, Juliana

    2009-01-01

    Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane proteins (OMPs) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated Omp16 (L-Omp16) or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection against B. abortus infection. Vaccination with unlipidated Omp16 (U-Omp16) or U-Omp19 in IFA induced a higher degree of protection than the respective lipidated versions. Moreover, the level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to that elicited by live B. abortus S19 immunization. Flow cytometric analysis showed that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4(+) as well as CD8(+) T cells producing gamma interferon. In vivo depletion of CD4(+) or CD8(+) T cells in mice immunized with U-Omp16 or U-Omp19 plus IFA resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in aluminum hydroxide formulation, and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51, respectively). Overall these results indicate that U-Omp16 or U-Omp19 would be a useful candidate for a subunit vaccine against human and animal brucellosis. PMID:18981242

  17. Validation of Recombinant Salivary Protein PpSP32 as a Suitable Marker of Human Exposure to Phlebotomus papatasi, the Vector of Leishmania major in Tunisia

    PubMed Central

    Bettaieb, Jihene; Abdeladhim, Maha; Hadj Kacem, Saoussen; Abdelkader, Rania; Gritli, Sami; Chemkhi, Jomaa; Aslan, Hamide; Kamhawi, Shaden; Ben Salah, Afif; Louzir, Hechmi; Valenzuela, Jesus G.; Ben Ahmed, Melika

    2015-01-01

    Background During a blood meal, female sand flies, vectors of Leishmania parasites, inject saliva into the host skin. Sand fly saliva is composed of a large variety of components that exert different pharmacological activities facilitating the acquisition of blood by the insect. Importantly, proteins present in saliva are able to elicit the production of specific anti-saliva antibodies, which can be used as markers for exposure to vector bites. Serological tests using total sand fly salivary gland extracts are challenging due to the difficulty of obtaining reproducible salivary gland preparations. Previously, we demonstrated that PpSP32 is the immunodominant salivary antigen in humans exposed to Phlebotomus papatasi bites and established that humans exposed to P. perniciosus bites do not recognize it. Methodology/Principal Findings Herein, we have validated, in a large cohort of 522 individuals, the use of the Phlebotomus papatasi recombinant salivary protein PpSP32 (rPpSP32) as an alternative method for testing exposure to the bite of this sand fly. We also demonstrated that screening for total anti-rPpSP32 IgG antibodies is sufficient, being comparable in efficacy to the screening for IgG2, IgG4 and IgE antibodies against rPpSP32. Additionally, sera obtained from dogs immunized with saliva of P. perniciosus, a sympatric and widely distributed sand fly in Tunisia, did not recognize rPpSP32 demonstrating its suitability as a marker of exposure to P. papatasi saliva. Conclusions/Significance Our data indicate that rPpSP32 constitutes a useful epidemiological tool to monitor the spatial distribution of P. papatasi in a particular region, to direct control measures against zoonotic cutaneous leishmaniasis, to assess the efficiency of vector control interventions and perhaps to assess the risk of contracting the disease. PMID:26368935

  18. Characterization of recombinant plant cinnamate 4-hydroxylase produced in yeast. Kinetic and spectral properties of the major plant P450 of the phenylpropanoid pathway.

    PubMed

    Urban, P; Werck-Reichhart, D; Teutsch, H G; Durst, F; Regnier, S; Kazmaier, M; Pompon, D

    1994-06-15

    Helianthus tuberosus cinnamate 4-hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p-coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR-amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose-inducible artificial promoter. Engineered S. cerevisiae strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced iron/carbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4-hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low-activity form. The spectral and kinetic characterizations of the yeast-produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH-dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data. PMID:8026495

  19. Outer planet satellites

    SciTech Connect

    Schenk, P.M. )

    1991-01-01

    Recent findings on the outer-planet satellites are presented, with special consideration given to data on the rheologic properties of ice on icy satellites, the satellite surfaces and exogenic processes, cratering on dead cratered satellites, volcanism, and the interiors of outer-planet satellites. Particular attention is given to the state of Titan's surface and the properties of Triton, Pluto, and Charon. 210 refs.

  20. Incorporation of squalene into rod outer segments

    SciTech Connect

    Keller, R.K.; Fliesler, S.J. )

    1990-08-15

    We have reported previously that squalene is the major radiolabeled nonsaponifiable lipid product derived from ({sup 3}H)acetate in short term incubations of frog retinas. In the present study, we demonstrate that newly synthesized squalene is incorporated into rod outer segments under similar in vitro conditions. We show further that squalene is an endogenous constituent of frog rod outer segment membranes; its concentration is approximately 9.5 nmol/mumol of phospholipid or about 9% of the level of cholesterol. Pulse-chase experiments with radiolabeled precursors revealed no metabolism of outer segment squalene to sterols in up to 20 h of chase. Taken together with our previous absolute rate studies, these results suggest that most, if not all, of the squalene synthesized by the frog retina is transported to rod outer segments. Synthesis of protein is not required for squalene transport since puromycin had no effect on squalene incorporation into outer segments. Conversely, inhibition of isoprenoid synthesis with mevinolin had no effect on the incorporation of opsin into the outer segment. These latter results support the conclusion that the de novo synthesis and subsequent intracellular trafficking of opsin and isoprenoid lipids destined for the outer segment occur via independent mechanisms.

  1. Genetic Recombination

    ERIC Educational Resources Information Center

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  2. Comparison of /sup 125/I-labeled and /sup 14/C-Labeled peptides of the major outer membrane protein of Chlamydia Trachomatis Strain L2/434 separated by high-performance liquid chromatography

    SciTech Connect

    Judd, R.C.; Caldwell, H.D.

    1985-01-01

    The objective of this study was to determine if in-gel chloramine-T radioiodination adequately labels OM proteins to allow for accurate and precise structural comparison of these molecules. Therefore, intrinsically /sup 14/C-amino acid labeled proteins and /sup 125/I-labeled proteins were cleaved with two endopeptidic reagents and the peptide fragments separated by HPLC. A comparison of retention times of the fragments, as determined by differential radiation counting, thus indicated whether /sup 125/Ilabeling identified of all the peptide peaks seen in the /sup 14/Clabeled proteins. Results demonstrated that radioiodination yields complete and accurate information about the primary structure of outer membrane proteins. In addition, it permits the use of extremely small amounts of protein allowing for method optimization and multiple separations to insure reproducibility.

  3. The Major Phase-Variable Outer Membrane Protein of Escherichia coli Structurally Resembles the Immunoglobulin A1 Protease Class of Exported Protein and Is Regulated by a Novel Mechanism Involving Dam and OxyR

    PubMed Central

    Henderson, Ian R.; Owen, Peter

    1999-01-01

    Here we report the characterization of an Escherichia coli gene (agn43) which encodes the principal phase-variable outer membrane protein termed antigen 43 (Ag43). The agn43 gene encodes a precursor protein of 107 kDa containing a 52-amino-acid signal sequence. Posttranslational processing generates an α43 subunit (predicted Mr of 49,789) and a C-terminal domain (β43) with features typical of a bacterial integral outer membrane protein (predicted Mr of 51,642). Secondary structure analysis predicts that β43 exists as an 18-stranded β barrel and that Ag43 shows structural organization closely resembling that of immunoglobulin A1 protease type of exoprotein produced by pathogenic Neisseria and Haemophilus spp. The correct processing of the polyprotein to α43 and β43 in OmpT, OmpP, and DegP protease-deficient E. coli strains points to an autocatalytic cleavage mechanism, a hypothesis supported by the occurrence of an aspartyl protease active site within α43. Ag43, a species-specific antigen, possesses two RGD motifs of the type implicated in binding to human integrins. The mechanism of reversible phase variation was studied by immunochemical analysis of a panel of well-defined regulatory mutants and by analysis of DNA sequences upstream of agn43. Evidence strongly suggests that phase variation is regulated by both deoxyadenosine methylase (Dam) and by OxyR. Thus, oxyR mutants are locked on for Ag43 expression, whereas dam mutants are locked off for Ag43 expression. We propose a novel mechanism for the regulation of phase switching in which OxyR competes with Dam for unmethylated GATC sites in the regulatory region of the agn43 gene. PMID:10094691

  4. Outer Planet Flagship Mission

    NASA Astrophysics Data System (ADS)

    Cutts, James; Niebur, C.; Dudzinski, L.; Coradini, M.; Lebreton, J.

    2008-09-01

    Studies for Outer Planet Missions have been ongoing for many years, but in 2007 NASA commissioned four specific studies to be considered for further examination; the Europa Explorer, Titan Explorer, Enceladus Mission and Jupiter Science Orbiter. During the same time frame ESA invited Outer Planet proposals under the Cosmic Vision call. Two were submitted, TandEm and LaPlace, which focused on Titan/Enceladus and Jupiter System science respectively. In 2008, NASA selected two of the missions, Europa Explorer and Titan Explorer, and ESA selected the two outer planet proposals for further study. This poster describes the process by which NASA and ESA are collaborating on the current studies which are now named the Titan/Saturn (TSSM) and Europa/Jupiter Missions (EJSM). We provide an update on the background, organization and schedule for these two mission studies.

  5. Outer Planet Flagship Missions

    NASA Astrophysics Data System (ADS)

    Niebur, C.; Dudzinski, L.; Coradini, M.; Lebreton, J.; Cutts, J. A.

    2008-05-01

    Studies for Outer Planet Missions have been ongoing for many years, but in 2007 NASA commissioned four specific studies to be considered for further examination; the Europa Explorer, Titan Explorer, Enceladus Mission and Jupiter Science Orbiter. During the same time frame ESA invited Outer Planet proposals under the Cosmic Vision call. Two were submitted, TandEM and LaPlace, which focused on Titan/Enceladus and Jupiter System science respectively. In 2008, NASA selected two of the missions, Europa Explorer and Titan Explorer, and ESA selected the two outer planet proposals for further study. This poster describes the process by which NASA and ESA are collaborating on the current studies which are now named the Titan/Saturn and Europa/Jupiter Missions. We provide the background, organization and schedule that are presently envisaged for these two mission studies.

  6. Outer Planets Flagship Mission

    NASA Astrophysics Data System (ADS)

    Niebur, C.; Dudzinski, L.; Coradini, M.; Lebreton, J. P.; Cutts, J. A.

    2008-09-01

    Studies for Outer Planet Missions have been ongoing for many years, but in 2007 NASA commissioned four specific studies to be considered for further examination; the Europa Explorer, Titan Explorer, Enceladus Mission and Jupiter Science Orbiter. During the same time frame ESA invited Outer Planet proposals under the Cosmic Vision call. Two were submitted, TandEm and LaPlace, which focused on Titan/Enceladus and Jupiter System science respectively. In 2008, NASA selected two of the missions, Europa Explorer and Titan Explorer, and ESA selected the two outer planet proposals for further study. This poster describes the process by which NASA and ESA are collaborating on the current studies which are now named the Titan/Saturn (TSSM) and Europa/Jupiter Missions (EJSM). We provide an update on the background, organization and schedule for these two mission studies.

  7. The Leptospiral Outer Membrane

    PubMed Central

    Haake, David A; Zückert, Wolfram R

    2015-01-01

    The outer membrane (OM) is the front line of leptospiral interactions with their environment and the mammalian host. Unlike most invasive spirochetes, pathogenic leptospires must be able survive in both free-living and host-adapted states. As organisms move from one set of environmental conditions to another, the OM must cope with a series of conflicting challenges. For example, the OM must be porous enough to allow nutrient uptake, yet robust enough to defend the cell against noxious substances. In the host, the OM presents a surface decorated with adhesins and receptors for attaching to, and acquiring, desirable host molecules such as the complement regulator, Factor H. On the other hand, the OM must enable leptospires to evade detection by the host’s immune system on their way from sites of invasion through the bloodstream to the protected niche of the proximal tubule. The picture that is emerging of the leptospiral OM is that, while it shares many of the characteristics of the OMs of spirochetes and Gram-negative bacteria, it is also unique and different in ways that make it of general interest to microbiologists. For example, unlike most other pathogenic spirochetes, the leptospiral OM is rich in lipopolysaccharide (LPS). Leptospiral LPS is similar to that of Gram-negative bacteria but has a number of unique structural features that may explain why it is not recognized by the LPS-specific Toll-like receptor 4 of humans. As in other spirochetes, lipoproteins are major components of the leptospiral OM, though their roles are poorly understood. The functions of transmembrane OMPs in many cases are better understood thanks to homologies with their Gram-negative counterparts and the emergence of improved genetic techniques. This chapter will review recent discoveries involving the leptospiral OM and its role in leptospiral physiology and pathogenesis. Readers are referred to earlier, excellent summaries related to this subject (Adler and de la Peña Moctezuma

  8. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    PubMed Central

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

    2013-01-01

    Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  9. The isolation of novel phage display-derived human recombinant antibodies against CCR5, the major co-receptor of HIV.

    PubMed

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-08-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  10. Intranasal Delivery of Recombinant AAV Containing BDNF Fused with HA2TAT: a Potential Promising Therapy Strategy for Major Depressive Disorder

    PubMed Central

    Ma, Xian-cang; Liu, Peng; Zhang, Xiao-ling; Jiang, Wen-hui; Jia, Min; Wang, Cai-xia; Dong, Ying-ying; Dang, Yong-hui; Gao, Cheng-ge

    2016-01-01

    Depression is a disturbing psychiatric disease with unsatisfied therapy. Not all patients are sensitive to anti-depressants currently in use, side-effects are unavoidable during therapy, and the cases with effectiveness are always accompanied with delayed onset of clinical efficacy. Delivering brain-derived neurotrophic factor (BDNF) to brain seems to be a promising therapy. However, a better approach to delivery is still rudimentary. The purpose of our present work is to look for a rapid-onset and long-lasting therapeutic strategy for major depressive disorder (MDD) by effectively delivering BDNF to brain. BDNF, fused with cell-penetrating peptides (TAT and HA2), was packaged in adenovirus associated virus (AAV) to construct the BDNF-HA2TAT/AAV for intranasally delivering BDNF to central nervous system (CNS) via nose-brain pathway. Intranasal administration of BDNF-HA2TAT/AAV to normal mice displayed anti-depression effect in forced swimming test when the delivery lasted relatively longer. The AAV applied to mice subjected to chronic mild stress (CMS) through intranasal administration for 10 days also alleviated depression-like behaviors. Western-blotting analysis revealed that BDNF-HA2TAT/AAV nasal administration enhanced hippocampal BDNF content. These results indicate intranasal administration of constructed BDNF-HA2TAT/AAV exerts anti-depression effect in CMS mice by increasing hippocampal BDNF, suggesting that this strategy holds a promising therapeutic potential for MDD. PMID:26935651

  11. Intranasal Delivery of Recombinant AAV Containing BDNF Fused with HA2TAT: a Potential Promising Therapy Strategy for Major Depressive Disorder.

    PubMed

    Ma, Xian-cang; Liu, Peng; Zhang, Xiao-ling; Jiang, Wen-hui; Jia, Min; Wang, Cai-xia; Dong, Ying-ying; Dang, Yong-hui; Gao, Cheng-ge

    2016-01-01

    Depression is a disturbing psychiatric disease with unsatisfied therapy. Not all patients are sensitive to anti-depressants currently in use, side-effects are unavoidable during therapy, and the cases with effectiveness are always accompanied with delayed onset of clinical efficacy. Delivering brain-derived neurotrophic factor (BDNF) to brain seems to be a promising therapy. However, a better approach to delivery is still rudimentary. The purpose of our present work is to look for a rapid-onset and long-lasting therapeutic strategy for major depressive disorder (MDD) by effectively delivering BDNF to brain. BDNF, fused with cell-penetrating peptides (TAT and HA2), was packaged in adenovirus associated virus (AAV) to construct the BDNF-HA2TAT/AAV for intranasally delivering BDNF to central nervous system (CNS) via nose-brain pathway. Intranasal administration of BDNF-HA2TAT/AAV to normal mice displayed anti-depression effect in forced swimming test when the delivery lasted relatively longer. The AAV applied to mice subjected to chronic mild stress (CMS) through intranasal administration for 10 days also alleviated depression-like behaviors. Western-blotting analysis revealed that BDNF-HA2TAT/AAV nasal administration enhanced hippocampal BDNF content. These results indicate intranasal administration of constructed BDNF-HA2TAT/AAV exerts anti-depression effect in CMS mice by increasing hippocampal BDNF, suggesting that this strategy holds a promising therapeutic potential for MDD. PMID:26935651

  12. Application of quality by design principles to the development and technology transfer of a major process improvement for the manufacture of a recombinant protein.

    PubMed

    Looby, Mairead; Ibarra, Neysi; Pierce, James J; Buckley, Kevin; O'Donovan, Eimear; Heenan, Mary; Moran, Enda; Farid, Suzanne S; Baganz, Frank

    2011-01-01

    This study describes the application of quality by design (QbD) principles to the development and implementation of a major manufacturing process improvement for a commercially distributed therapeutic protein produced in Chinese hamster ovary cell culture. The intent of this article is to focus on QbD concepts, and provide guidance and understanding on how the various components combine together to deliver a robust process in keeping with the principles of QbD. A fed-batch production culture and a virus inactivation step are described as representative examples of upstream and downstream unit operations that were characterized. A systematic approach incorporating QbD principles was applied to both unit operations, involving risk assessment of potential process failure points, small-scale model qualification, design and execution of experiments, definition of operating parameter ranges and process validation acceptance criteria followed by manufacturing-scale implementation and process validation. Statistical experimental designs were applied to the execution of process characterization studies evaluating the impact of operating parameters on product quality attributes and process performance parameters. Data from process characterization experiments were used to define the proven acceptable range and classification of operating parameters for each unit operation. Analysis of variance and Monte Carlo simulation methods were used to assess the appropriateness of process design spaces. Successful implementation and validation of the process in the manufacturing facility and the subsequent manufacture of hundreds of batches of this therapeutic protein verifies the approaches taken as a suitable model for the development, scale-up and operation of any biopharmaceutical manufacturing process. PMID:21948302

  13. Law in Outer Space.

    ERIC Educational Resources Information Center

    Schmidt, William G.

    1997-01-01

    Provides an overview of the current practice and fascinating future of legal issues involved in outer space exploration and colonization. Current space law, by necessity, addresses broad principles rather than specific incidents. Nonetheless, it covers a variety of issues including commercial development, rescue agreements, object registration,…

  14. Control of tick infestations and pathogen prevalence in cattle and sheep farms vaccinated with the recombinant Subolesin-Major Surface Protein 1a chimeric antigen

    PubMed Central

    2014-01-01

    Background Despite the use of chemical acaricides, tick infestations continue to affect animal health and production worldwide. Tick vaccines have been proposed as a cost-effective and environmentally friendly alternative for tick control. Vaccination with the candidate tick protective antigen, Subolesin (SUB), has been shown experimentally to be effective in controlling vector infestations and pathogen infection. Furthermore, Escherichia coli membranes containing the chimeric antigen composed of SUB fused to Anaplasma marginale Major Surface Protein 1a (MSP1a) (SUB-MSP1a) were produced using a simple low-cost process and proved to be effective for the control of cattle tick, Rhipicephalus (Boophilus) microplus and R. annulatus infestations in pen trials. In this research, field trials were conducted to characterize the effect of vaccination with SUB-MSP1a on tick infestations and the prevalence of tick-borne pathogens in a randomized controlled prospective study. Methods Two cattle and two sheep farms with similar geographical locations and production characteristics were randomly assigned to control and vaccinated groups. Ticks were collected, counted, weighed and classified and the prevalence of tick-borne pathogens at the DNA and serological levels were followed for one year prior to and 9 months after vaccination. Results Both cattle and sheep developed antibodies against SUB in response to vaccination. The main effect of the vaccine in cattle was the 8-fold reduction in the percent of infested animals while vaccination in sheep reduced tick infestations by 63%. Female tick weight was 32-55% lower in ticks collected from both vaccinated cattle and sheep when compared to controls. The seroprevalence of Babesia bigemina was lower by 30% in vaccinated cattle, suggesting a possible role for the vaccine in decreasing the prevalence of this tick-borne pathogen. The effect of the vaccine in reducing the frequency of one A. marginale msp4 genotype probably reflected

  15. A Pseudorabies Virus Recombinant Simultaneously Lacking the Major Tegument Proteins Encoded by the UL46, UL47, UL48, and UL49 Genes Is Viable in Cultured Cells

    PubMed Central

    Fuchs, Walter; Granzow, Harald; Mettenleiter, Thomas C.

    2003-01-01

    The UL46, UL47, UL48, and UL49 genes, which encode major tegument proteins, are conserved in most alphaherpesvirus genomes. However, the relative importance of each of these proteins for replication of individual alphaherpesviruses appears to be different. Recently, we demonstrated that single deletions of UL47 or UL48 impair maturation and egress of pseudorabies virus (PrV) particles to different extents, whereas deletions of UL46 or UL49 have no significant effects on virus replication in cell culture (W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp, and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002; M. Kopp, B. G. Klupp, H. Granzow, W. Fuchs, and T. C. Mettenleiter, J. Virol. 76:8820-8833, 2002). To test for possible functional redundancy between the four tegument proteins, a quadruple gene deletion mutant (PrV-ΔUL46-49) was generated and characterized in vitro. Although plaque formation by this mutant was almost abolished and maximum titers were reduced more than 100-fold compared to those of parental wild-type virus, PrV-ΔUL46-49 could be propagated and serially passaged in noncomplementing porcine and rabbit kidney cells. Electron-microscopic studies revealed that nucleocapsid formation and egress of PrV-ΔUL46-49 from the host cell nucleus were not affected, but secondary envelopment of nucleocapsids in the cytoplasm was only rarely observed. The replication defect of PrV-ΔUL46-49 could be fully corrected by reinsertion of the UL46-to-UL49 gene cluster. Plaque sizes and virus titers were only slightly increased after restoration of only UL47 expression, whereas repair of only UL48 resulted in a significant increase in replication capacity to the level of a UL47 deletion mutant. In conclusion, we show that none of the UL46 to UL49 tegument proteins is absolutely required for productive replication of PrV. Moreover, our data indicate that the UL47 and UL48 proteins function independently during cell-to-cell spread and virus egress. PMID:14610211

  16. The recombination of genetic material

    SciTech Connect

    Low, K.B.

    1988-01-01

    Genetic recombination is the major mechanism by which new arrangements of genetic elements are produced in all living organisms, from the simplest bacterial viruses to humans. This volume presents an overview of the types of recombination found in prokaryotes and eukaryotes.

  17. Bacterial Recombineering: Genome Engineering via Phage-Based Homologous Recombination.

    PubMed

    Pines, Gur; Freed, Emily F; Winkler, James D; Gill, Ryan T

    2015-11-20

    The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing. PMID:25856528

  18. Recombinant phytochrome A in yeast differs by its spectroscopic and photochemical properties from the major phyA' and is close to the minor phyA": evidence for posttranslational modification of the pigment in plants.

    PubMed

    Sineshchekov, V; Hennig, L; Lamparter, T; Hughes, J; Gärtner, W; Schäfer, E

    2001-06-01

    Previously, two pools of phytochrome A (phyA' and phyA") have been detected by in situ low-temperature fluorescence spectroscopy and photochemistry; it was suggested that they might differ in the nature of their posttranslational modification. In order to verify this possibility Arabidopsis and rice (Oryza) phyA were expressed in yeast and the pigments were assembled in vivo with phycocyanobilin (PCB) and phytochromobilin (P phi B). The resulting recombinant phytochromes in the red-light-absorbing form (Pr) were characterized in the yeast cell by (1) the fluorescence emission spectra; (2) the temperature dependence of Pr fluorescence intensity and activation energy of fluorescence decay; and (3) the extent of photoconversion of Pr into photoproduct lumi-R (gamma 1) or far-red-light absorbing form (Pfr) (gamma 2). Both Arabidopsis phyA/PCB and Oryza phyA/P phi B had low gamma 1 of ca 0.05, allowing their attribution to the Pr" phenomenological type of phytochrome comprising phyA", phyB and cryptogam phytochromes. The spectroscopic properties of Oryza phyA/P phi B were also very close to phyA". However, both investigated holoproteins differed from phyA", both with respect to the character of temperature dependence of the fluorescence yield and activation energy. Thus, recombinant Oryza phyA/P phi B is similar but not identical to phyA". The data demonstrate that the low-abundance-fraction plant phyA (phyA") comes from the same gene as the major (phyA') fraction. Because both endogenous phyA fractions differ from the phytochrome expressed in yeast, they appear to be posttranslationally modified and/or bound to partner proteins or cellular substructures. However, the character of the presumed chemical modification is different in phyA' and phyA" and its extent is more profound in the case of the former. PMID:11421077

  19. Outer Solar System Nomenclature

    NASA Technical Reports Server (NTRS)

    Owen, Tobias C.

    1998-01-01

    The Principal Investigator's responsibilities on this grant fell into two categories according to his participation. In the nomenclature work of the International Astronomical Union (IAU). Owen is chair of the Task Group for the Outer Solar System. He is also a member of the IAU's Working Group on Planetary and Satellite Nomenclature (WGPSN) which is composed of the chairs of the several Task Groups plus the presidents of two IAU Commissions and several outside consultants. The WGPSN is presided over by its President, Professor Kaare Aksnes from the Rosseland Institute for Theoretical Astrophysics in Oslo, Norway.

  20. Jupiter's outer atmosphere.

    NASA Technical Reports Server (NTRS)

    Brice, N. M.

    1973-01-01

    The current state of the theory of Jupiter's outer atmosphere is briefly reviewed. The similarities and dissimilarities between the terrestrial and Jovian upper atmospheres are discussed, including the interaction of the solar wind with the planetary magnetic fields. Estimates of Jovian parameters are given, including magnetosphere and auroral zone sizes, ionospheric conductivity, energy inputs, and solar wind parameters at Jupiter. The influence of the large centrifugal force on the cold plasma distribution is considered. The Jovian Van Allen belt is attributed to solar wind particles diffused in toward the planet by dynamo electric fields from ionospheric neutral winds, and the consequences of this theory are indicated.

  1. Outer Hair Cell Electromotility and Otoacoustic Emissions*

    PubMed Central

    Brownell, William E.

    2009-01-01

    Outer hair cell electromotility is a rapid, force generating, length change in response to electrical stimulation. DC electrical pulses either elongate or shorten the cell and sinusoidal electrical stimulation results in mechanical oscillations at acoustic frequencies. The mechanism underlying outer hair cell electromotility is thought to be the origin of spontaneous otoacoustic emissions. The ability of the cell to change its length requires that it be mechanically flexible. At the same time the structural integrity of the organ of Corti requires that the cell possess considerable compressive rigidity along its major axis. Evolution appears to have arrived at novel solutions to the mechanical requirements imposed on the outer hair cell. Segregation of cytoskeletal elements in specific intracellular domains facilitates the rapid movements. Compressive strength is provided by a unique hydraulic skeleton in which a positive hydrostatic pressure in the cytoplasm stabilizes a flexible elastic cortex with circumferential tensile strength. Cell turgor is required in order that the pressure gradients associated with the electromotile response can be communicated to the ends of the cell. A loss in turgor leads to loss of outer hair cell electromotility. Concentrations of salicylate equivalent to those that abolish spontaneous otoacoustic emissions in patients weaken the outer hair cell’s hydraulic skeleton. There is a significant diminution in the electromotile response associated with the loss in cell turgor. Aspirin’s effect on outer hair cell electromotility attests to the role of the outer hair cell in generating otoacoustic emissions and demonstrates how their physiology can influence the propagation of otoacoustic emissions. PMID:2187727

  2. Spectrum Recombination.

    ERIC Educational Resources Information Center

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  3. Spontaneous Regeneration of Human Photoreceptor Outer Segments

    PubMed Central

    Horton, Jonathan C.; Parker, Alicia B.; Botelho, James V.; Duncan, Jacque L.

    2015-01-01

    Photoreceptors are damaged in many common eye diseases, such as macular degeneration, retinal detachment, and retinitis pigmentosa. The development of methods to promote the repair or replacement of affected photoreceptors is a major goal of vision research. In this context, it would be useful to know whether photoreceptors are capable of undergoing some degree of spontaneous regeneration after injury. We report a subject who lost retinal function in a wide zone around the optic disc, giving rise to massive enlargement of the physiological blind spot. Imaging with an adaptive optics scanning laser ophthalmoscope (AOSLO) showed depletion of cone outer segments in the affected retina. A year later visual function had improved, with shrinkage of the enlarged blind spot. AOSLO imaging showed repopulation of cone outer segments, although their density remained below normal. There was a one-to-one match between sites of formerly missing outer segments and new outer segments that had appeared over the course of the year’s recovery. This correspondence provided direct morphological evidence that damaged cones are capable, under some circumstances, of generating new outer segments. PMID:26213154

  4. Outer planets satellites

    NASA Technical Reports Server (NTRS)

    Morrison, D.

    1983-01-01

    The present investigation takes into account the published literature on outer planet satellites for 1979-1982. It is pointed out that all but three (the moon and the two Martian satellites) of the known planetary satellites are found in the outer solar system. Most of these are associated with the three regular satellite systems of Jupiter, Saturn, and Uranus. The largest satellites are Titan in the Saturn system and Ganymede and Callisto in the Jupiter system. Intermediate in size between Mercury and Mars, each has a diameter of about 5000 km. Presumably each has an internal composition about 60 percent rock and 40 ice, and each is differentiated with a dense core extending out about 75 percent of the distance to the surface, with a mantle of high-pressure ice and a crust of ordinary ice perhaps 100 km thick. Attention is also given to Io, Europa, the icy satellites of Saturn, the satellites of Uranus, the small satellites of Jupiter and Saturn, Triton and the Pluto system, and plans for future studies.

  5. Inner and outer beauty.

    PubMed

    Raymond, Kenneth N; Brown, Casey J

    2012-01-01

    Symmetry and pattern are precious forms of beauty that can be appreciated on both the macroscopic and molecular scales. Crystallographers have long appreciated the intimate connections between symmetry and molecular structure, reflected in their appreciation for the artwork of Escher. This admiration has been applied in the design of highly symmetrical coordination compounds. Two classes of materials are discussed: extended coordination arrays and discrete supramolecular assemblies. Extended coordination polymers have been implemented in gas separation and storage due to the remarkably porosity of these materials, aided by the ability to design ever-larger inner spaces within these frameworks. In the case of discrete symmetrical structures, defined inner and outer space present a unique aesthetic and chemical environment. The consequent host-guest chemistry and applications in catalysis are discussed. PMID:22076081

  6. Strategy for outer planets exploration

    NASA Technical Reports Server (NTRS)

    1975-01-01

    NASA's Planetary Programs Office formed a number of scientific working groups to study in depth the potential scientific return from the various candidate missions to the outer solar system. The results of these working group studies were brought together in a series of symposia to evaluate the potential outer planet missions and to discuss strategies for exploration of the outer solar system that were consistent with fiscal constraints and with anticipated spacecraft and launch vehicle capabilities. A logical, scientifically sound, and cost effective approach to exploration of the outer solar system is presented.

  7. Polyclonal activation of naïve T cells by urease deficient-recombinant BCG that produced protein complex composed of heat shock protein 70, CysO and major membrane protein-II

    PubMed Central

    2014-01-01

    Background Mycobacterium bovis bacillus Calmette-Guérin (BCG) is known to be only partially effective in inhibiting M. tuberculosis (MTB) multiplication in human. A new recombinant (r) urease-deficient BCG (BCG-dHCM) that secretes protein composed of heat shock protein (HSP)70, MTB-derived CysO and major membrane protein (MMP)-II was produced for the efficient production of interferon gamma (IFN-γ) which is an essential element for mycobacteriocidal action and inhibition of neutrophil accumulation in lungs. Methods Human monocyte-derived dendritic cells (DC) and macrophages were differentiated from human monocytes, infected with BCG and autologous T cells-stimulating activity of different constructs of BCG was assessed. C57BL/6 mice were used to test the effectiveness of BCG for the production of T cells responsive to MTB-derived antigens (Ags). Results BCG-dHCM intracellularly secreted HSP70-CysO-MMP-II fusion protein, and activated DC by up-regulating Major Histcompatibility Complex (MHC), CD86 and CD83 molecules and enhanced various cytokines production from DC and macrophages. BCG-dHCM activated naïve T cells of both CD4 and CD8 subsets through DC, and memory type CD4+ T cells through macrophages in a manner dependent on MHC and CD86 molecules. These T cell activations were inhibited by the pre-treatment of Ag-presenting cells (APCs) with chloroquine. The single and primary BCG-dHCM-inoculation produced long lasting T cells responsive to in vitro secondarily stimulation with HSP70, CysO, MMP-II and H37Rv-derived cytosolic protein, and partially inhibited the replication of aerosol-challenged MTB. Conclusions The results indicate that introduction of different type of immunogenic molecules into a urease-deficient rBCG is useful for providing polyclonal T cell activating ability to BCG and for production of T cells responsive to secondary stimulation. PMID:24690183

  8. Biogenesis of outer membranes in Gram-negative bacteria.

    PubMed

    Tokuda, Hajime

    2009-03-23

    The outer membrane, an essential organelle of Gram-negative bacteria, is composed of four major components: lipopolysaccharide, phospholipids, beta-barrel proteins, and lipoproteins. The mechanisms underlying the transport of these components to outer membranes are currently under extensive examination. Among them, the sorting of lipoproteins to the outer membrane of Escherichia coli has been clarified in detail. The Lol system, composed of five proteins, catalyzes outer membrane sorting of lipoproteins. Various Lpt proteins have recently been identified as factors involved in the transport of lipopolysaccharide to the outer membrane, although the mechanism remains largely unknown. Proteins with alpha-helical membrane spanning segments are found in the inner membrane, whereas amphipathic beta-barrel proteins span the outer membrane. These beta-barrel proteins are inserted into the outer membranes through a central core protein BamA (YaeT) with the help of four outer membrane lipoproteins. In contrast, little is known about how phospholipids are transported to the outer membrane. PMID:19270402

  9. Molecular identity of human outer radial glia during cortical development.

    PubMed

    Pollen, Alex A; Nowakowski, Tomasz J; Chen, Jiadong; Retallack, Hanna; Sandoval-Espinosa, Carmen; Nicholas, Cory R; Shuga, Joe; Liu, Siyuan John; Oldham, Michael C; Diaz, Aaron; Lim, Daniel A; Leyrat, Anne A; West, Jay A; Kriegstein, Arnold R

    2015-09-24

    Radial glia, the neural stem cells of the neocortex, are located in two niches: the ventricular zone and outer subventricular zone. Although outer subventricular zone radial glia may generate the majority of human cortical neurons, their molecular features remain elusive. By analyzing gene expression across single cells, we find that outer radial glia preferentially express genes related to extracellular matrix formation, migration, and stemness, including TNC, PTPRZ1, FAM107A, HOPX, and LIFR. Using dynamic imaging, immunostaining, and clonal analysis, we relate these molecular features to distinctive behaviors of outer radial glia, demonstrate the necessity of STAT3 signaling for their cell cycle progression, and establish their extensive proliferative potential. These results suggest that outer radial glia directly support the subventricular niche through local production of growth factors, potentiation of growth factor signals by extracellular matrix proteins, and activation of self-renewal pathways, thereby enabling the developmental and evolutionary expansion of the human neocortex. PMID:26406371

  10. Controlled Release from Recombinant Polymers

    PubMed Central

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-01-01

    Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

  11. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli.

    PubMed

    Kay, Emily J; Yates, Laura E; Terra, Vanessa S; Cuccui, Jon; Wren, Brendan W

    2016-04-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  12. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli

    PubMed Central

    Kay, Emily J.; Yates, Laura E.; Terra, Vanessa S.; Cuccui, Jon; Wren, Brendan W.

    2016-01-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  13. Improved diagnostic performance of a commercial anaplasma antibody competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5–glutathione S-transferase fusion protein as antigen

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study tested the hypothesis that removal of maltose binding protein from recombinant antigen used for plate coating would improve the specificity of Anaplasma antibody competitive ELISA. Three hundred and eight sera with significant MBP antibody binding (=30%I) in Anaplasma negative herds was 1...

  14. Turbine airfoil with outer wall thickness indicators

    DOEpatents

    Marra, John J; James, Allister W; Merrill, Gary B

    2013-08-06

    A turbine airfoil usable in a turbine engine and including a depth indicator for determining outer wall blade thickness. The airfoil may include an outer wall having a plurality of grooves in the outer surface of the outer wall. The grooves may have a depth that represents a desired outer surface and wall thickness of the outer wall. The material forming an outer surface of the outer wall may be removed to be flush with an innermost point in each groove, thereby reducing the wall thickness and increasing efficiency. The plurality of grooves may be positioned in a radially outer region of the airfoil proximate to the tip.

  15. Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

    PubMed Central

    Plaza, H; Whelchel, T R; Garczynski, S F; Howerth, E W; Gherardini, F C

    1997-01-01

    We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%. PMID:9286995

  16. Nondisjunction of chromosome 15: Origin and recombination

    SciTech Connect

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. ); Langlois, S. ); Morris, M.A.; Malcolm, S.

    1993-09-01

    Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N=27) and Angelman syndrome patients (N-5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, more paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. 33 refs., 1 fig., 7 tabs.

  17. Radio wave scattering in the outer heliosphere

    NASA Technical Reports Server (NTRS)

    Cairns, Iver H.

    1995-01-01

    Current models for the 2-3 kHz emissions observed by the Voyager spacecraft in the outer heliosphere involve 2f(p) radiation generated near the termination shock or the heliopause. Radio wave scattering by solar wind density irregularities strongly affects observed sources of f(p) and 2f(p) emission in the inner heliosphere and the characteristics of astrophysical sources. In particular, the angular size, brightness temperature, and time variability of the source are strongly affected by scattering, thereby having major implications for the inferred size, energy budget, time variability, location, and nature of the source if scattering is ignored. This paper addresses whether scattering is important for interpreting the Voyager 2-3 kHz emissions. Quantitative calculations (with and without diffraction) are performed for the angular broadening of an outer heliospheric source as a function of path length, radiation frequency relative to f(p) and the spectrum of density irregularities. The effects of scattering in both the solar wind and the heliosheath are considered. Predictions for radial gradients in the source's apparent angular size and in the source's modulation index are presented. The calculations are compared with observations and the results discussed. First estimates suggest that scattering plausibly dominates the observed source size. The observed trend in modulation index with heliocentric distance is consistent with scattering being important and the source being in the outer heliosphere. Additional arguments for scattering being important are summarized.

  18. Delivery of foreign antigens by engineered outer membrane vesicle vaccines.

    PubMed

    Chen, David J; Osterrieder, Nikolaus; Metzger, Stephan M; Buckles, Elizabeth; Doody, Anne M; DeLisa, Matthew P; Putnam, David

    2010-02-16

    As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines. PMID:20133740

  19. η Carinae - The outer ejecta

    NASA Astrophysics Data System (ADS)

    Weis, K.

    2009-03-01

    η Carinae is a unique object among the most massive evolved stars in the LBV phase. The central object(s) is(are) surrounded by a complex circumstellar nebula ejected during more than one eruption in the 19th century. Beyond the well-defined edges of its famous bipolar nebula, the Homunculus, are additional nebulous features referred to as the outer ejecta. The outer ejecta contains a large variety of structures of very different sizes and morphologies distributed in a region 0.67 pc in diameter. Individual features in the outer ejecta are moving extremely fast, up to 3200 km/s, in general the expansion velocities are between 400-900 km/s. A consequence of these high velocities is that structures in the outer ejecta interact with the surrounding medium and with each other. The strong shocks that arise from these interactions give rise to soft X-ray emission. The global expansion pattern of the outer ejecta reveals an overall bipolar distribution, giving a symmetric structure to its morphologically more irregular appearance. The long, highly collimated filaments, called strings, are particularly unusual. The material in the strings follows a Hubble-flow and appears to originate at the central object.

  20. Dissociation of immune determinants of outer membrane proteins of Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

    PubMed Central

    Westbay, T D; Dascher, C C; Hsia, R C; Bavoil, P M; Zauderer, M

    1994-01-01

    Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines. Images PMID:7525489

  1. Origin of Outer Solar System

    NASA Technical Reports Server (NTRS)

    Holman, Matthew J.; Lindstrom, David (Technical Monitor)

    2005-01-01

    Our ongoing research program combines extensive deep and wide-field observations using a variety of observational platforms with numerical studies of the dynamics of small bodies in the outer solar system in order to advance the main scientific goals of the community studying the Kuiper belt and the outer solar system. These include: (1) determining the relative populations of the known classes of KBOs as well as other possible classes; ( 2 ) determining the size distributions or luminosity function of the individual populations or the Kuiper belt as a whole; (3) determining the inclinations distributions of these populations; (4) establishing the radial extent of the Kuiper belt; ( 5 ) measuring and relating the physical properties of different types of KBOs to those of other solar system bodies; and, (6) completing our systematic inventory of the satellites of the outer planets.

  2. Outer Planet Assessment Group (OPAG) Recommended Exploration Strategy for the Outer Planets 2013-2022

    NASA Astrophysics Data System (ADS)

    McKinnon, William B.; Steering Committee, Opag; Planets Community, Outer

    2010-05-01

    The Outer Solar System provides critical clues to how solar systems form and evolve, how planetary systems become habitable, and how life has evolved in our solar system. NASA's Outer Planets Assessment Group (OPAG) was established to identify scientific priorities and pathways for Outer Solar System exploration. Fundamental new discoveries are best made with a mixture of mission sizes that includes large (flagship) missions, and medium-sized and smaller-sized (as practical) missions, along with vigorous support for basic research, data analysis, and technology development — a balanced strategy most efficiently implemented as an Outer Planets Exploration Program. Missions to the Outer Solar System are major undertakings, requiring large and expensive launch vehicles, long mission durations, highly reliable (frequently radiation hard) and autonomous spacecraft, and radioisotope power sources in most cases. OPAG has recommended to the US National Research Council Planetary Science Decadal Survey to explore the possibilities for ‘small flagship' class missions to be considered, providing a greater range of choice and capabilities in the mix to balance program size and science return. With the Galileo mission concluded, the Cassini equinox mission in progress, and Juno in development, OPAG has strongly endorsed the competitive selection by NASA of the Jupiter Europa Orbiter (JEO) as the next Outer Planets Flagship and as part of the Europa Jupiter System Mission (EJSM) with ESA, a collaboration that includes a Ganymede orbiter and an increased focus on Jupiter science; OPAG has strongly recommended support of JEO and EJSM in the Decadal Survey. In addition, OPAG has strongly endorsed approval by NASA of the Cassini Solstice Mission, including the Juno-like end-of-mission scenario, given the likely phenomenal return on investment. OPAG also advocates the need for a focused technology program for the next Outer Planet Flagship Mission after EJSM, in order to be ready

  3. PROGENITORS OF RECOMBINING SUPERNOVA REMNANTS

    SciTech Connect

    Moriya, Takashi J.

    2012-05-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with ionization temperature higher than the electron temperature, has been recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the supernova explosion. If the circumstellar medium is dense enough, collisional ionization equilibrium can be established in the early stage of the evolution of the supernova remnant and subsequent adiabatic cooling, which occurs after the shock wave gets out of the dense circumstellar medium, makes the electron temperature lower than the ionization temperature. We study the circumstellar medium around several supernova progenitors and show which supernova progenitors can have a circumstellar medium dense enough to establish collisional ionization equilibrium soon after the explosion. We find that the circumstellar medium around red supergiants (especially massive ones) and the circumstellar medium dense enough to make Type IIn supernovae can establish collisional ionization equilibrium soon after the explosion and can evolve to become recombining supernova remnants. Wolf-Rayet stars and white dwarfs have the possibility to be recombining supernova remnants but the fraction is expected to be very small. As the occurrence rate of the explosions of red supergiants is much higher than that of Type IIn supernovae, the major progenitors of recombining supernova remnants are likely to be red supergiants.

  4. Turbine airfoil with a compliant outer wall

    DOEpatents

    Campbell, Christian X.; Morrison, Jay A.

    2012-04-03

    A turbine airfoil usable in a turbine engine with a cooling system and a compliant dual wall configuration configured to enable thermal expansion between inner and outer layers while eliminating stress formation in the outer layer is disclosed. The compliant dual wall configuration may be formed a dual wall formed from inner and outer layers separated by a support structure. The outer layer may be a compliant layer configured such that the outer layer may thermally expand and thereby reduce the stress within the outer layer. The outer layer may be formed from a nonplanar surface configured to thermally expand. In another embodiment, the outer layer may be planar and include a plurality of slots enabling unrestricted thermal expansion in a direction aligned with the outer layer.

  5. Recombinant protein production technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recombinant protein production is an important technology for antibody production, biochemical activity study, and structural determination during the post-genomic era. Limiting factors in recombinant protein production include low-level protein expression, protein precipitation, and loss of protein...

  6. Gas hydrates of outer continental margins

    SciTech Connect

    Kvenvolden, K.A. )

    1990-05-01

    Gas hydrates are crystalline substances in which a rigid framework of water molecules traps molecules of gas, mainly methane. Gas-hydrate deposits are common in continental margin sediment in all major oceans at water depths greater than about 300 m. Thirty-three localities with evidence for gas-hydrate occurrence have been described worldwide. The presence of these gas hydrates has been inferred mainly from anomalous lacoustic reflectors seen on marine seismic records. Naturally occurring marine gas hydrates have been sampled and analyzed at about tensites in several regions including continental slope and rise sediment of the eastern Pacific Ocean and the Gulf of Mexico. Except for some Gulf of Mexico gas hydrate occurrences, the analyzed gas hydrates are composed almost exclusively of microbial methane. Evidence for the microbial origin of methane in gas hydrates includes (1) the inverse relation between methane occurence and sulfate concentration in the sediment, (2) the subparallel depth trends in carbon isotopic compositions of methane and bicarbonate in the interstitial water, and (3) the general range of {sup 13}C depletion ({delta}{sub PDB}{sup 13}C = {minus}90 to {minus}60 {per thousand}) in the methane. Analyses of gas hydrates from the Peruvian outer continental margin in particular illustrate this evidence for microbially generated methane. The total amount of methane in gas hydrates of continental margins is not known, but estimates of about 10{sup 16} m{sup 3} seem reasonable. Although this amount of methane is large, it is not yet clear whether methane hydrates of outer continental margins will ever be a significant energy resource; however, these gas hydrates will probably constitute a drilling hazard when outer continental margins are explored in the future.

  7. 76 FR 54787 - Outer Continental Shelf Official Protraction Diagram, Lease Maps, and Supplemental Official Outer...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-09-02

    ... Bureau of Ocean Energy Management, Regulation and Enforcement Outer Continental Shelf Official Protraction Diagram, Lease Maps, and Supplemental Official Outer Continental Shelf Block Diagrams AGENCY... revised North American Datum of 1927 (NAD 27) Outer Continental Shelf Official Protraction Diagram,...

  8. 76 FR 2919 - Outer Continental Shelf Official Protraction Diagram and Supplemental Official Outer Continental...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-18

    ... Bureau of Ocean Energy Management, Regulation and Enforcement Outer Continental Shelf Official Protraction Diagram and Supplemental Official Outer Continental Shelf Block Diagrams AGENCY: Bureau of Ocean... American Datum of 1983 (NAD 83) Outer Continental Shelf Official Protraction Diagram and...

  9. Therapeutic Recombinant Monoclonal Antibodies

    ERIC Educational Resources Information Center

    Bakhtiar, Ray

    2012-01-01

    During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

  10. Recombineering homologous recombination constructs in Drosophila.

    PubMed

    Carreira-Rosario, Arnaldo; Scoggin, Shane; Shalaby, Nevine A; Williams, Nathan David; Hiesinger, P Robin; Buszczak, Michael

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner. PMID:23893070

  11. Outer membrane proteins of Methylococcus capsulatus (Bath).

    PubMed

    Fjellbirkeland, A; Kleivdal, H; Joergensen, C; Thestrup, H; Jensen, H B

    1997-08-01

    Membranes obtained from whole-cell lysates of Methylococcus capsulatus (Bath) were separated by Triton X-100 extraction. The resulting insoluble fraction was enriched in outer membranes as assessed by electron microscopy and by the content of beta-hydroxy palmitic acid and particulate methane monooxygenase. Major proteins with molecular masses of approximately 27, 40, 46, 59, and 66 kDa were detected by SDS-PAGE of the Triton-X-100-insoluble membranes. MopA, MopB, MopC, MopD, and MopE (Methylococcus outer membrane protein) are proposed to designate these proteins. Several of the Mop proteins exhibited heat-modifiable properties in SDS-PAGE and were influenced by the presence of 2-mercaptoethanol in the sample buffer. The 46- and 59-kDa bands migrated as a single high-molecular-mass 95-kDa oligomer under mild denaturing conditions. When reconstituted into black lipid membranes, this oligomer was shown to serve as a channel with an estimated single-channel conductance of 1.4 nS in 1 M KCl. PMID:9238104

  12. Radio Emissions from the Outer Heliosphere

    NASA Technical Reports Server (NTRS)

    Gurnett, D. A.; Kurth, W. S.

    1996-01-01

    For nearly fifteen years the Voyager 1 and 2 spacecraft have been detecting an unusual radio emission in the outer heliosphere in the frequency range from about 2 to 3 kHz. Two major events have been observed, the first in 1983-84 and the second in 1992-93. In both cases the onset of the radio emission occurred about 400 days after a period of intense solar activity, the first in mid-July 1982, and the second in May-June 1991. These two periods of solar activity produced the two deepest cosmic ray Forbush decreases ever observed. Forbush decreases are indicative of a system of strong shocks and associated disturbances propagating outward through the heliosphere. The radio emission is believed to have been produced when this system of shocks and disturbances interacted with one of the outer boundaries of the heliosphere, most likely in the vicinity of the the heliopause. The emission is believed to be generated by the shock-driven Langmuir-wave mode conversion mechanism, which produces radiation at the plasma frequency (f(sub p)) and at twice the plasma frequency (2f(sub p)). From the 400-day travel time and the known speed of the shocks, the distance to the interaction region can be computed, and is estimated to be in the range from about 110 to 160 AU.

  13. Cloning and expression of the major 47-kilodalton surface immunogen of Treponema pallidum in Escherichia coli.

    PubMed Central

    Norgard, M V; Chamberlain, N R; Swancutt, M A; Goldberg, M S

    1986-01-01

    Monoclonal antibodies directed against the 47-kilodalton (kDa) major outer membrane surface immunogen of virulent Treponema pallidum subsp. pallidum were used to select Escherichia coli recombinant clones expressing the 47-kDa immunogen. The phenotype of the clones was dependent on the presence of recombinant plasmid in the host cell. Southern hybridization revealed that the cloned T. pallidum subsp. pallidum DNA sequence was an accurate representation of the T. pallidum subsp. pallidum genomic DNA arrangement. Purified immunoglobulin G from rabbits experimentally infected with T. pallidum subsp. pallidum and human secondary syphilitic sera specifically reacted with the clones, while normal human serum or immunoglobulin G from normal rabbit serum did not. Results of Southern hybridization indicated that a homologous 47-kDa immunogen gene was absent in at least four species of nonpathogenic treponemes tested, as well as from total rabbit genomic DNA. Rabbit anti-T. phagedenis biotype Reiter (treponemal nonpathogen) antiserum and a monoclonal antibody directed against a common treponemal determinant were unreactive with the clones. Western blotting and radioimmunoprecipitation experiments with specific monoclonal antibodies revealed that the recombinant (E. coli) and native (T. pallidum subsp. pallidum) forms of the antigen had identical electrophoretic mobilities. The availability of recombinant 47-kDa immunogen provides a new opportunity for biochemical analysis of the protein, structure-function studies, examination of its role in microbial pathogenesis, and assessment of its diagnostic and vaccinogenic potentials. Images PMID:3021631

  14. Major depression

    MedlinePlus

    Depression - major; Depression - clinical; Clinical depression; Unipolar depression; Major depressive disorder ... Doctors do not know the exact causes of depression. It is believed that chemical changes in the ...

  15. Geometrical interpretation for the outer SU(3) outer multiplicity label

    NASA Technical Reports Server (NTRS)

    Draayer, Jerry P.; Troltenier, D.

    1995-01-01

    A geometrical interpretation for the outer multiplicity rho that occurs in a reduction of the product of two SU(3) representations, (lambda(sub pi), mu(sub pi)) x (lambda(sub nu), mu(sub nu)) approaches sigma(sub rho)(lambda, mu)(sub rho), is introduced. This coupling of proton (pi) and neutron (nu) representations arises, for example, in both boson and fermion descriptions of heavy deformed nuclei. Attributing a geometry to the coupling raises the possibility of introducing a simple interaction that provides a physically meaningful way for distinguishing multiple occurrences of (lambda, mu) values that can arise in such products.

  16. Photoionization and Recombination

    NASA Technical Reports Server (NTRS)

    Nahar, Sultana N.

    2000-01-01

    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  17. Magnetospheres of the outer planets

    SciTech Connect

    Cheng, A.F.

    1986-12-01

    The magnetospheres of the outer planets have been shown by Voyager explorations to strongly interact with the surfaces and atmospheres of their planetary satellites and rings. In the cases of Jupiter, Saturn and Uranus, the processes of charged particle sputtering, neutral gas cloud formation, and rapid plasma injection from the ionization of the neutral clouds, have important implications both for the magnetospheres as a whole and for the surfaces and atmospheres of their satellites. The general methodology employed in these researches has involved comparisons of the planetary magnetospheres in order to identify common physical processes. 16 references.

  18. Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

    SciTech Connect

    Eren, Elif; Vijayaraghavan, Jagamya; Liu, Jiaming; Cheneke, Belete R.; Touw, Debra S.; Lepore, Bryan W.; Indic, Mridhu; Movileanu, Liviu; van den Berg, Bert; Dutzler, Raimund

    2012-01-17

    Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  19. Recombination of cluster ions

    NASA Technical Reports Server (NTRS)

    Johnsen, Rainer

    1993-01-01

    Some of our recent work on molecular band emissions from recombination of molecular dimer ions (N4(+) and CO(+) CO) is discussed. Much of the experimental work was done by Y. S. Cao; the results on N4(+) recombination have been published. A brief progress report is given on our ongoing measurements of neutral products of recombination using the flowing-afterglow Langmuir-probe technique in conjunction with laser-induced fluorescence.

  20. Recombination in electron coolers

    NASA Astrophysics Data System (ADS)

    Wolf, A.; Gwinner, G.; Linkemann, J.; Saghiri, A. A.; Schmitt, M.; Schwalm, D.; Grieser, M.; Beutelspacher, M.; Bartsch, T.; Brandau, C.; Hoffknecht, A.; Müller, A.; Schippers, S.; Uwira, O.; Savin, D. W.

    2000-02-01

    An introduction to electron-ion recombination processes is given and recent measurements are described as examples, focusing on low collision energies. Discussed in particular are fine-structure-mediated dielectronic recombination of fluorine-like ions, the moderate recombination enhancement by factors of typically 1.5-4 found for most ion species at relative electron-ion energies below about 10 meV, and the much larger enhancement occurring for specific highly charged ions of complex electronic structure, apparently caused by low-energy dielectronic recombination resonances. Recent experiments revealing dielectronic resonances with very large natural width are also described.

  1. Outer membrane proteins preferentially load MHC class II peptides: Implications for as a Chlamydia trachomatis T cell vaccine

    PubMed Central

    Karunakaran, Karuna P.; Yu, Hong; Jiang, Xiaozhou; Chan, Queenie; Moon, Kyung-Mee; Foster, Leonard J.; Brunham, Robert C.

    2015-01-01

    CD4 T cell immune responses such as interferon-γ and tumor necrosis factor-α secretion are necessary for Chlamydia immunity. We used an immunoproteomic approach in which Chlamydia trachomatis and Chlamydia muridarum-derived peptides presented by MHC class II molecules on the surface of infected dendritic cells (DCs) were identified by tandem mass spectrometry using bone marrow derived DCs (BMDCs) from mice of different MHC background. We first compared the C. muridarum immunoproteome in C3H mice to that previously identified in C57BL/6 mice. Fourteen MHC class II binding peptides from 11 Chlamydia proteins were identified from C3H infected BMDCs. Two C. muridarum proteins overlapped between C3H and C57B/6 mice and both were polymorphic membrane proteins (Pmps) which presented distinct class II binding peptides. Next we studied DCs from C57BL/6 mice infected with the human strain, C. trachomatis serovar D. Sixty MHC class II binding peptides derived from 27 C. trachomatis proteins were identified. Nine proteins were orthologous T cell antigens between C. trachomatis and C. muridarum and 2 of the nine were Pmps which generated MHC class II binding epitopes at distinct sequences within the proteins. As determined by antigen specific splenocyte responses outer membrane proteins PmpF, -G and -H and the major outer membrane protein (MOMP) were antigenic in mice previously infected with C. muridarum or C. trachomatis. Furthermore a recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with MOMP formulated with a Th1 polarizing adjuvant significantly accelerated (p < 0.001) clearance in the C57BL/6 mice C. trachomatis transcervical infection model. We conclude that Chlamydia outer membrane proteins are important T cell antigens useful in the development of a C. trachomatis subunit vaccine. PMID:25738816

  2. Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli.

    PubMed

    Cullen, Paul A; Lo, Miranda; Bulach, Dieter M; Cordwell, Stuart J; Adler, Ben

    2003-01-01

    Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS-PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface. PMID:12583997

  3. Outer Appearances Can Be Deceiving

    NASA Technical Reports Server (NTRS)

    2004-01-01

    This graph shows the chemical composition of the rock at Gusev Crater dubbed 'Mazatzal' after it was brushed and ground by the Mars Exploration Rover Spirit's rock abrasion tool. The data, taken by the rover's alpha particle X-ray spectrometer over the last few sols, show that the amount of chlorine and sulfur tri-oxide in Mazatzal first increased after brushing, then diminished after grinding. The interior of the rock appears to have the same chemical make-up as other volcanic or basalt rocks studied in the Gusev Crater area ('Adirondack' and 'Humphrey'). Its outer coating or rind, on the other hand, appears to be of a different constitution. Scientists are still puzzling out the implications of these data.

    The larger symbols on the graph represent inferred rock compositions, while the smaller symbols are actual data points. Observations were made at the target dubbed 'New York' on Mazatzal.

  4. Aft outer rim seal arrangement

    DOEpatents

    Lee, Ching-Pang; Tham, Kok-Mun; Schroeder, Eric; Meeroff, Jamie; Miller, Jr., Samuel R; Marra, John J; Campbell, Christian X

    2015-04-28

    An outer rim seal arrangement (10), including: an annular rim (70) centered about a longitudinal axis (30) of a rotor disc (31), extending fore and having a fore-end (72), an outward-facing surface (74), and an inward-facing surface (76); a lower angel wing (62) extending aft from a base of a turbine blade (22) and having an aft end (64) disposed radially inward of the rim inward-facing surface to define a lower angel wing seal gap (80); an upper angel wing (66) extending aft from the turbine blade base and having an aft end (68) disposed radially outward of the rim outward-facing surface to define a upper angel wing seal gap (80, 82); and guide vanes (100) disposed on the rim inward-facing surface in the lower angel wing seal gap. Pumping fins (102) may be disposed on the upper angel wing seal aft end in the upper angel wing seal gap.

  5. CATALYTIC RECOMBINER FOR A NUCLEAR REACTOR

    DOEpatents

    King, L.D.P.

    1960-07-01

    A hydrogen-oxygen recombiner is described for use with water-boiler type reactors. The catalyst used is the wellknown platinized alumina, and the novelty lies in the structural arrangement used to prevent flashback through the gas input system. The recombiner is cylindrical, the gases at the input end being deflected by a baffle plate through a first flashback shield of steel shot into an annular passage adjacent to and extending the full length of the housing. Below the baffle plate the gases flow first through an outer annular array of alumina pellets which serve as a second flashback shield, a means of distributing the flowing gases evenly and as a means of reducing radiation losses to the walls. Thereafter the gases flow inio the centrally disposed catalyst bed where recombination is effected. The steam and uncombined gases flow into a centrally disposed cylindrical passage inside the catalyst bod and thereafter out through the exit port. A high rate of recombination is effected.

  6. Recombination Pattern Reanalysis of Some HIV-1 Circulating Recombination Forms Suggest the Necessity and Difficulty of Revision

    PubMed Central

    Jia, Lei; Li, Lin; Li, Hanping; Liu, Siyang; Wang, Xiaolin; Bao, Zuoyi; Li, Tianyi; Zhuang, Daomin; Liu, Yongjian; Li, Jingyun

    2014-01-01

    Background Recombination is one of the major mechanisms underlying the generation of HIV-1 variability. Currently 61 circulating recombinant forms of HIV-1 have been identified. With the development of recombination detection techniques and accumulation of HIV-1 reference stains, more accurate mosaic structures of circulating recombinant forms (CRFs), like CRF04 and CRF06, have undergone repeated analysis and upgrades. Such revisions may also be necessary for other CRFs. Unlike previous studies, whose results are based primarily on a single recombination detection program, the current study was based on multiple recombination analysis, which may have produced more impartial results. Methods Representative references of 3 categories of intersubtype recombinants were selected, including BC recombinants (CRF07 and CRF08), BG recombinants (CRF23 and CRF24), and BF recombinants (CRF38 and CRF44). They were reanalyzed in detail using both the jumping profile hidden Markov model and RDP3. Results The results indicate that revisions and upgrades are very necessary and the entire re-analysis suggested 2 types of revision: (i) length of inserted fragments; and (ii) number of inserted fragments. The reanalysis also indicated that determination of small regions of about 200 bases or fewer should be performed with more caution. Conclusion Results indicated that the involvement of multiple recombination detection programs is very necessary. Additionally, results suggested two major challenges, one involving the difficulty of accurately determining the locations of breakpoints and the second involving identification of small regions of about 200 bases or fewer with greater caution. Both indicate the complexity of HIV-1 recombination. The resolution would depend critically on development of a recombination analysis algorithm, accumulation of HIV-1 stains, and a higher sequencing quality. With the changes in recombination pattern, phylogenetic relationships of some CRFs may also

  7. Chemistry of the outer planets

    NASA Technical Reports Server (NTRS)

    Scattergood, Thomas W.

    1992-01-01

    Various aspects were studied of past or present chemistry in the atmospheres of the outer planets and their satellites using lab simulations. Three areas were studied: (1) organic chemistry induced by kinetically hot hydrogen atoms in the region of Jupiter's atmosphere containing the ammonia cirrus clouds; (2) the conversion of NH3 into N2 by plasmas associated with entry of meteors and other objects into the atmosphere of early Titan; and (3) the synthesis of simple hydrocarbons and HCN by lightning in mixtures containing N2, CH4, and NH3 representing the atmospheres of Titan and the outer planets. The results showed that: (1) hot H2 atoms formed from the photodissociation of NH3 in Jupiter's atmosphere could account for some of the atmospheric chemistry in the ammonia cirrus cloud region; (2) the thermalization of hot H2 atoms in atmospheres predominated by molecular H is not as rapid as predicted by elastic collision theory; (3) the net quantum loss of NH3 in the presence of a 200 fold excess of H2 is 0.02, much higher than was expected from the amount of H2 present; (4) the conversion of NH3 into N2 in plasmas associated with infalling meteors is very efficient and rapid, and could account for most of the N2 present on Titan; (5) the yields of C2H2 and HCN from lightning induced chemistry in mixtures of CH4 and N2 is consistent with quenched thermodynamic models of the discharge core; and (6) photolysis induced by the UV light emitted by the gases in the hot plasmas may account for some, if not most, of the excess production of C2H6 and the more complex hydrocarbons.

  8. Outer membrane protein (OMP) based vaccine for Neisseria meningitidis serogroup B.

    PubMed

    Pillai, Subramonia; Howell, Alan; Alexander, Kristin; Bentley, B Erin; Jiang, Han-Qian; Ambrose, Karita; Zhu, Duzhang; Zlotnick, Gary

    2005-03-18

    A family of outer membrane lipoproteins of Neisseria meningitidis, LP2086, has been shown to induce serum bactericidal activity against a broad variety of meningococcal strains. Two sub-families of serologically distinct LP2086 proteins (A and B) have been identified. In the present study, we have shown that polyclonal anti-serum against rLP2086 is protective in vivo in an infant rat passive-protection model. Additionally, the LP2086 protein is displayed on the surface of 91% meningococcal strains as measured in a whole cell ELISA using polyclonal anti-sera raised against these proteins. We also demonstrate based on the reactivity of anti-rLP2086 antibody with recombinantly expressed C- and N-terminal fragments of rLP2086 in a Western blot assay that the C-terminal fragment of LP2086 dictates sub-family specificity and the N-terminal fragment determines the family specificity. A formulation containing family A and B of LP2086 potentially would provide broad protection against a majority of Neisseria meningitidis strains. PMID:15755596

  9. Genetic recombination. [Escherichia coli

    SciTech Connect

    Stahl, F.W.

    1987-02-01

    The molecular pathways of gene recombination are explored and compared in studies of the model organisms, Escherichia coli and phase lambda. In the discussion of data from these studies it seems that recombination varies with the genetic idiosyncrasies of the organism and may also vary within a single organism.

  10. In vitro analysis of a primary, major histocompatibility complex (MHC)-restricted, cytotoxic T-lymphocyte response to avian leukosis virus (ALV), using target cells expressing MHC class I cDNA inserted into a recombinant ALV vector.

    PubMed

    Thacker, E L; Fulton, J E; Hunt, H D

    1995-10-01

    The interaction between the major histocompatibility complex (MHC) and cytotoxic T lymphocytes (CTLs) is an important component of the host's resistance to viral infections and tumor formation. In this study, an avian leukosis virus (ALV) vector system, RCASBP, expressing MHC chicken class I (B-F) cDNA was used to develop target cells expressing the chicken class I glycoproteins complexed with ALV antigens on the cell surface. Peripheral blood from chickens inoculated with ALV was shown to contain antigen-specific, MHC-restricted, CD8+ effector CTLs, using a 51Cr release assay utilizing the RCASBP B-F target cells. The stimulated effector cells were also predominantly alpha beta T-cell receptor-positive (TCR2) T cells. The CTL response varied between two haplotypes of chickens which differed in their response to Rous sarcoma virus (RSV)-induced tumors. Chickens with the B21 haplotype which regress RSV-induced tumors showed maximal cytolytic activity, while chickens with the B13 haplotype which do not regress RSV-induced tumors had minimal to no cytolytic activity. In addition to assessing the CTL response to ALV, the creation of MHC-specific immortal target cell lines will be extremely useful in evaluating CTL responses to other viral disease in chickens. PMID:7666545

  11. Induction of cross-priming of naive CD8+ T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein.

    PubMed

    Mukai, Tetsu; Maeda, Yumi; Tamura, Toshiki; Matsuoka, Masanori; Tsukamoto, Yumiko; Makino, Masahiko

    2009-11-15

    Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8(+) T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8(+) T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8(+) T cells, but also CD4(+) T cells of both types to produce IFN-gamma. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8(+) T cells by BCG-70M through DC. Thus, the CD8(+) T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8(+) T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4(+) T cells, CD62L(low)CD8(+) T cells and perforin-producing CD8(+) T cells were efficiently produced. MMP-II-reactive CD4(+) and CD8(+) memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4(+) T cells, and CD8(+) T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation. PMID:19846882

  12. Outer planets grand tours: Planetary radio astronomy team report

    NASA Technical Reports Server (NTRS)

    Warwick, J. W.

    1972-01-01

    Requirements related to scientific observations of planetary radio emissions during outer planets grand tours are discussed. Observations at low frequencies where non-thermal cooperative plasma phenomena play a major role are considered for determining dynamical processes and magnetic fields near a planet. Magnetic field measurements by spacecraft magnetometers, and by radio receivers in their harmonic modes are proposed for interpretation of planetary radio emission.

  13. Law and politics in outer space: A bibliography.

    NASA Technical Reports Server (NTRS)

    White, I. L.; Wilson, C. E.; Vosburgh, J. A.

    1972-01-01

    The materials are categorized by specific topics and by types of materials. The sources are books, articles, reports, United Nations materials, U.S. Government documents, etc. Books are listed by geographical areas, and articles are divided into what are considered to be the major space topics. Book and article sections are also divided into English and foreign language entries. A bibliographical essay introduces the literature to those unacquainted with law and politics of outer space.

  14. Major Links.

    ERIC Educational Resources Information Center

    Henderson, Tona

    1995-01-01

    Provides electronic mail addresses for resources and discussion groups related to the following academic majors: art, biology, business, chemistry, computer science, economics, health sciences, history, literature, math, music, philosophy, political science, psychology, sociology, and theater. (AEF)

  15. Disulfide-bonded outer membrane proteins in the genus Legionella.

    PubMed Central

    Butler, C A; Street, E D; Hatch, T P; Hoffman, P S

    1985-01-01

    Legionella pneumophila and related species were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis for outer membrane proteins. Of the 10 species examined, 9 contained a 24-kilodalton (kDa) major outer membrane protein (MOMP) that was resolvable only when outer membrane material was heated in the presence of 2-mercaptoethanol. Labeling studies with [35S]cysteine indicated that the protein contained cysteine, and disulfide cross-linking of the unreduced complex was demonstrated by labeling with iodoacetamide. The unreduced outer membrane preparation contained peptidoglycan, and after treatment with lysozyme to remove peptidoglycan, a protein complex of 95 kDa was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the absence of 2-mercaptoethanol. Reduction of the 95-kDa complex yielded 24-kDa monomers, suggesting that the 95-kDa complex was composed of four subunits. The 24-kDa MOMP from L. pneumophila was purified, and antibody produced to this protein cross-reacted with all species of Legionella as determined from an immunoblot of a sodium dodecyl sulfate gel. Only serogroup 1 strains of L. bozemanii lacked the 24-kDa MOMP and showed no cross-reactivity. These results suggest that the 24-kDa MOMP common to most species of Legionella contains a genus-specific epitope. Images PMID:3980079

  16. Molecular Basis of Bacterial Outer Membrane Permeability Revisited

    PubMed Central

    Nikaido, Hiroshi

    2003-01-01

    Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions. PMID:14665678

  17. Interiors and atmospheres of the outer planets

    NASA Technical Reports Server (NTRS)

    Hubbard, W. B.

    1991-01-01

    This theoretical/observational project constrains structure of outer planet atmospheres and interiors through observational data. The primary observation tool is through observations of occultations of stars by outer solar system objects, which yield information about atmospheric temperatures and dynamics, and planetary dimensions and oblateness. The theoretical work relates the data to interior structures in a variety of ways.

  18. Design Of Robots For Outer Space

    NASA Technical Reports Server (NTRS)

    Roston, Gerald P.

    1990-01-01

    Report discusses design of robots for use in zero gravity and vacuum, with attention to differences between requirements imposed on designs by outer space and by terrestrial applications. Terrestrial robots designed for multiple purposes and for minimal cost. Outer-space robots designed specialized to one task where cost has relatively low priority. Design optimal in one environment unlikely optimal in another.

  19. 76 FR 63654 - Outer Continental Shelf Official Protraction Diagram, Lease Maps, and Supplemental Official Outer...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-13

    ... Bureau of Ocean Energy Management Outer Continental Shelf Official Protraction Diagram, Lease Maps, and Supplemental Official Outer Continental Shelf Block Diagrams AGENCY: Bureau of Ocean Energy Management (BOEM), Interior. ACTION: Availability of revised North American Datum of 1927 (NAD 27) Outer Continental...

  20. Recombination hotspots: Models and tools for detection.

    PubMed

    Paul, Prosenjit; Nag, Debjyoti; Chakraborty, Supriyo

    2016-04-01

    Recombination hotspots are the regions within the genome where the rate, and the frequency of recombination are optimum with a size varying from 1 to 2kb. The recombination event is mediated by the double-stranded break formation, guided by the combined enzymatic action of DNA topoisomerase and Spo 11 endonuclease. These regions are distributed non-uniformly throughout the human genome and cause distortions in the genetic map. Numerous lines of evidence suggest that the number of hotspots known in humans has increased manifold in recent years. A few facts about the hotspot evolutions were also put forward, indicating the differences in the hotspot position between chimpanzees and humans. In mice, recombination hot spots were found to be clustered within the major histocompatibility complex (MHC) region. Several models, that help explain meiotic recombination has been proposed. Moreover, scientists also developed some computational tools to locate the hotspot position and estimate their recombination rate in humans is of great interest to population and medical geneticists. Here we reviewed the molecular mechanisms, models and in silico prediction techniques of hot spot residues. PMID:26991854

  1. Astroparticles: Messengers from Outer Space

    NASA Astrophysics Data System (ADS)

    Desiati, Paolo

    2016-07-01

    Since Galileo pointed a spyglass toward the sky, 400 years ago, observations empowered by man-made instrumentation have provided us with an enormous leap in the knowledge of how the Universe functions. More and more powerful optical telescopes made it possible for us to reach the farthest corners of space. At the same time, the advances in microphysics and the discovery of the electromagnetic spectrum, made it possible to directly look at the Universe in a way that our eyes cannot see. The discoveries of the intimate structure of matter, of subatomic particles and of how they interact with each other, have led astronomers to use the smallest objects in Nature to observe the farthest reaches of the otherwise invisible Universe. Not unlike Galileo, today we observe Outer Space with visible light and beyond, across the entire electromagnetic spectrum, from long wavelength radio waves to short wavelength gamma rays. But also with instruments detecting cosmic rays (the atomic nuclei we know on Earth) neutrinos (neutral subatomic particles that interact very weakly with matter) and gravitational waves (perturbations of spacetime predicted by General Relativity). Each cosmic messenger provides us with a unique piece of information about their source and the history of their journey to us. Modern astrophysics has the challenging goal to collect as much information as possible from all those messengers, to reconstruct the story of the Universe and how it became what it is today. This journey started with the unsettling discovery that we are only one minuscule dot in the immensity of the Universe and yet we are able to observe objects that are far in space and time. This journey is yet to complete its course, and the more we advance our knowledge, the more we need to understand. This interdisciplinary talk provides an overview of this journey and the future perspectives.

  2. [Recombinant antibodies against bioweapons].

    PubMed

    Thullier, Philippe; Pelat, Thibaut; Vidal, Dominique

    2009-12-01

    The threat posed by bioweapons (BW) could lead to the re-emergence of such deadly diseases as plague or smallpox, now eradicated from industrialized countries. The development of recombinant antibodies allows tackling this risk because these recombinant molecules are generally well tolerated in human medicine, may be utilized for prophylaxis and treatment, and because antibodies neutralize many BW. Recombinant antibodies neutralizing the lethal toxin of anthrax, botulinum toxins and the smallpox virus have in particular been isolated recently, with different technologies. Our approach, which uses phage-displayed immune libraries built from non-human primates (M. fascicularis) to obtain recombinant antibodies, which may later be super-humanized (germlinized), has allowed us to obtain such BWs-neutralizing antibodies. PMID:20035695

  3. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  4. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  5. Major depression.

    PubMed

    Bentley, Susan M; Pagalilauan, Genevieve L; Simpson, Scott A

    2014-09-01

    Major depression is a common, disabling condition seen frequently in primary care practices. Non-psychiatrist ambulatory providers are increasingly responsible for diagnosing, and primarily managing patients suffering from major depressive disorder (MDD). The goal of this review is to help primary care providers to understand the natural history of MDD, identify practical tools for screening, and a thoughtful approach to management. Clinically challenging topics like co-morbid conditions, treatment resistant depression and pharmacotherapy selection with consideration to side effects and medication interactions, are also covered. PMID:25134869

  6. The Magnetic Field in the Outer Heliosphere

    NASA Technical Reports Server (NTRS)

    Suess, S. T.

    2004-01-01

    One of the great achievements of Parker was the prediction that the solar magnetic field would be drawn into Archimedian spirals as it is carried away from the Sun by the solar wind. This prediction has been amply confirmed by many in situ measurements in the intervening four decades. But, Parker made his prediction for a solar wind that expands into infinite space while we now know that the local interstellar medium (LISM) is far from empty and, in fact, confines the solar wind to a finite volume, known as the heliosphere, that extends to approximately 100 AU in the upstream direction (the solar system is moving through the LISM). Voyagers 1/2, presently at -80 AU, are approaching the upstream boundaries of the heliosphere and returning data on the properties of the magnetic field. This is important for understanding how galactic cosmic rays (GCRs) reach the Earth. Voyagers show that the IMF at 10-80 AU behaves much as Parker predicted - with two important exceptions. This is not surprising since the field is essentially passively advected by the solar wind out to 80 AU. But, new models say that nearer the heliosphere boundaries the field plays a major role in the solar wind-LISM interaction. However, of the many physical ingredients that constitute the outer heliosphere, the magnetic field poses some of the most interesting and difficult numerical modeling problems. Presently, only a few results have been published and much remains to be done. Here I will summarize the expected and measured behavior of the magnetic field at 80 AU. Then I will describe modeling predictions beyond 80 AU: magnetic "tornadoes", polarity envelopes, the Axford-Cranfill effect, inner and outer magnetic walls and more. I will also list what I believe to be important new modeling objectives. Finally, I will speculate on what is happening with the magnetic field near the nose of the heliosphere. My conclusion is that models of GCR modulation rarely incorporate even crudely realistic

  7. Major Andre

    ERIC Educational Resources Information Center

    Henisch, B. A.; Henisch, H. K.

    1976-01-01

    If most Revolutionary era people seem two-dimensional their lives simpler to understand than ours, it may be only that history, with the benefit of hindsight, clarifies. Examines a profile of Major John Andre, the British liaison officer in Benedict Arnold's plan to surrender West Point, as both hero and villain to show the complexity of early…

  8. Composition and Localization of Treponema denticola Outer Membrane Complexes ▿

    PubMed Central

    Godovikova, Valentina; Goetting-Minesky, M. Paula; Fenno, J. Christopher

    2011-01-01

    The Treponema denticola outer membrane lipoprotein-protease complex (dentilisin) contributes to periodontal disease by degrading extracellular matrix components and disrupting intercellular host signaling pathways. We recently demonstrated that prcB, located upstream of and cotranscribed with prcA and prtP, encodes a 22-kDa lipoprotein that interacts with PrtP and is required for its activity. Here we further characterize products of the protease locus and their roles in expression, formation, and localization of outer membrane complexes. PrcB migrates in native gels as part of a >400-kDa complex that includes PrtP and PrcA, as well as the major outer sheath protein Msp. PrcB is detectable as a minor constituent of the purified active protease complex, which was previously reported to consist of only PrtP and auxiliary polypeptides PrcA1 and PrcA2. Though it lacks the canonical ribosome binding site present upstream of both prcA and prtP, PrcB is present at levels similar to those of PrtP in whole-cell extracts. Immunofluorescence microscopy demonstrated cell surface exposure of the mature forms of PrtP, PrcA1, PrcB, and Msp. The 16-kDa N-terminal acylated fragment of PrtP (predicted to be released during activation of PrtP) was present in cell extracts but was detected neither in the purified active protease complex nor on the cell surface. PrcA2, detectable on the surface of Msp-deficient cells but not that of wild-type cells, coimmunoprecipitated with Msp. Our results indicate that PrcB is a component of the outer membrane lipoprotein protease complex and that Msp and PrcA2 interaction may mediate formation of a very-high-molecular-weight outer membrane complex. PMID:21986628

  9. Depletion of the Outer Asteroid Belt

    NASA Technical Reports Server (NTRS)

    Liou, Jer-Chyi; Malhotra, Renu

    1997-01-01

    During the early history of the solar system, it is likely that the outer planets changed their distance from the sun, and hence, their influence on the asteroid belt evolved with time. The gravitational influence of Jupiter and Saturn on the orbital evolution of asteroids in the outer asteroid belt was calculated. The results show that the sweeping of mean motion resonances associated with planetary migration efficiently destabilizes orbits in the outer asteroid belt on a time scale of 10 million years. This mechanism provides an explanation for the observed depletion of asteroids in that region.

  10. Immunogenicity of Pasteurella multocida and Mannheimia haemolytica outer membrane vesicles

    PubMed Central

    Roier, Sandro; Fenninger, Judith C.; Leitner, Deborah R.; Rechberger, Gerald N.; Reidl, Joachim; Schild, Stefan

    2013-01-01

    Pasteurella multocida is able to cause disease in humans and in a wide range of animal hosts, including fowl cholera in birds, atrophic rhinitis in pigs, and snuffles in rabbits. Together with Mannheimia haemolytica, P. multocida also represents a major bacterial causative agent of bovine respiratory disease (BRD), which is one of the most important causes for economic losses for the cattle backgrounding and feedlot industry. Commercially available vaccines only partially prevent infections caused by P. multocida and M. haemolytica. Thus, this study characterized the immunogenicity of P. multocida and M. haemolytica outer membrane vesicles (OMVs) upon intranasal immunization of BALB/c mice. Enzyme-linked immunosorbent assays (ELISA) revealed that OMVs derived from P. multocida or M. haemolytica are able to induce robust humoral and mucosal immune responses against the respective donor strain. In addition, also significant cross-immunogenic potential was observed for both OMV types. Colonization studies showed that a potential protective immune response against P. multocida is not only achieved by immunization with P. multocida OMVs, but also by immunization with OMVs derived from M. haemolytica. Immunoblot and immunoprecipitation analyses demonstrated that M. haemolytica OMVs induce a more complex immune response compared to P. multocida OMVs. The outer membrane proteins OmpA, OmpH, and P6 were identified as the three major immunogenic proteins of P. multocida OMVs. Amongst others, the serotype 1-specific antigen, an uncharacterized outer membrane protein, as well as the outer membrane proteins P2 and OmpA were found to be the most important antigens of M. haemolytica OMVs. These findings are useful for the future development of broad-spectrum OMV based vaccines against BRD and other infections caused by P. multocida or M. haemolytica. PMID:23731905

  11. Regulation of Meiotic Recombination

    SciTech Connect

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  12. Outer planet probe engineering model structural tests

    NASA Technical Reports Server (NTRS)

    Smittkamp, J. A.; Gustin, W. H.; Griffin, M. W.

    1977-01-01

    A series of proof of concept structural tests was performed on an engineering model of the Outer Planets Atmospheric Entry Probe. The tests consisted of pyrotechnic shock, dynamic and static loadings. The tests partially verified the structural concept.

  13. Outer Membrane Permeability and Antibiotic Resistance

    PubMed Central

    Delcour, Anne H.

    2009-01-01

    Summary To date most antibiotics are targeted at intracellular processes, and must be able to penetrate the bacterial cell envelope. In particular, the outer membrane of Gram-negative bacteria provides a formidable barrier that must be overcome. There are essentially two pathways that antibiotics can take through the outer membrane: a lipid-mediated pathway for hydrophobic antibiotics, and general diffusion porins for hydrophilic antibiotics. The lipid and protein compositions of the outer membrane have a strong impact on the sensitivity of bacteria to many types of antibiotics, and drug resistance involving modifications of these macromolecules is common. This review will describe the molecular mechanisms for permeation of antibiotics through the outer membrane, and the strategies that bacteria have deployed to resist antibiotics by modifications of these pathways. PMID:19100346

  14. Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins.

    PubMed

    Siroy, Axel; Mailaender, Claudia; Harder, Daniel; Koerber, Stephanie; Wolschendorf, Frank; Danilchanka, Olga; Wang, Ying; Heinz, Christian; Niederweis, Michael

    2008-06-27

    Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes. PMID:18434314

  15. The outer solar system - Perspectives for exobiology

    NASA Technical Reports Server (NTRS)

    Owen, T.

    1974-01-01

    An attempt is made to summarize the current knowledge about the composition and structures of outer planet atmospheres with special emphasis on Jupiter, Saturn, and Titan. The nature of the substances which are responsible for the yellow coloration observed on both Jupiter and Saturn is discussed. The analysis of planetary conditions conducted shows that the outer solar system offers a variety of environments in which natural experiments in prebiotic organic synthesis must be taking place at the present time.

  16. Recombination and DNA Repair in Helicobacter pylori

    PubMed Central

    Dorer, Marion S.; Sessler, Tate H.; Salama, Nina R.

    2013-01-01

    All organisms have pathways that repair the genome, ensuring their survival and that of their progeny. But these pathways also serve to diversify the genome, causing changes on the level of nucleotide, whole gene, and genome structure. Sequencing of bacteria has revealed wide allelic diversity and differences in gene content within the same species, highlighting the importance of understanding pathways of recombination and DNA repair. The human stomach pathogen Helicobacter pylori is an excellent model system for studying these pathways. H. pylori harbors major recombination and repair pathways and is naturally competent, facilitating its ability to diversify its genome. Elucidation of DNA recombination, repair, and diversification programs in this pathogen will reveal connections between these pathways and their importance to infection. PMID:21682641

  17. Transcription and Recombination: When RNA Meets DNA

    PubMed Central

    Aguilera, Andrés; Gaillard, Hélène

    2014-01-01

    A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

  18. Genome-wide recombination dynamics are associated with phenotypic variation in maize.

    PubMed

    Pan, Qingchun; Li, Lin; Yang, Xiaohong; Tong, Hao; Xu, Shutu; Li, Zhigang; Li, Weiya; Muehlbauer, Gary J; Li, Jiansheng; Yan, Jianbing

    2016-05-01

    Meiotic recombination is a major driver of genetic diversity, species evolution, and agricultural improvement. Thus, an understanding of the genetic recombination landscape across the maize (Zea mays) genome will provide insight and tools for further study of maize evolution and improvement. Here, we used c. 50 000 single nucleotide polymorphisms to precisely map recombination events in 12 artificial maize segregating populations. We observed substantial variation in the recombination frequency and distribution along the ten maize chromosomes among the 12 populations and identified 143 recombination hot regions. Recombination breakpoints were partitioned into intragenic and intergenic events. Interestingly, an increase in the number of genes containing recombination events was accompanied by a decrease in the number of recombination events per gene. This kept the overall number of intragenic recombination events nearly invariable in a given population, suggesting that the recombination variation observed among populations was largely attributed to intergenic recombination. However, significant associations between intragenic recombination events and variation in gene expression and agronomic traits were observed, suggesting potential roles for intragenic recombination in plant phenotypic diversity. Our results provide a comprehensive view of the maize recombination landscape, and show an association between recombination, gene expression and phenotypic variation, which may enhance crop genetic improvement. PMID:26720856

  19. The carbon budget in the outer solar nebula.

    PubMed

    Simonelli, D P; Pollack, J B; McKay, C P; Reynolds, R T; Summers, A L

    1989-01-01

    Detailed models of the internal structures of Pluto and Charon, assuming rock and water ice as the only constituents, indicate that the mean silicate mass fraction of this two-body system is on the order of 0.7; thus the Pluto/Charon system is significantly "rockier" than the satellites of the giant planets (silicate mass fraction approximately 0.55). This compositional contrast reflects different formation mechanisms: it is likely that Pluto and Charon formed directly from the solar nebula, while the circumplanetary nebulae that produced the giant planet satellites were derived from envelopes that surrounded the forming giant planets (envelopes in which icy planetesimals dissolved more readily than rocky planetesimals). Simple cosmic abundance calculations, and the assumption that the Pluto/Charon system formed directly from solar nebula condensates, strongly suggest that the majority of the carbon in the outer solar nebula was in the form of carbon monoxide; these results are consistent with (1) inheritance from the dense molecular clouds in the interstellar medium (where CH4/CO < 10(-2) in the gas phase) and/or (2) of the Lewis and Prinn kinetic inhibition model of solar nebula chemistry. Theoretical predictions of the C/H enhancements in the atmospheres of the giant planets, when compared to the actual observed enhancements, suggest that 10%, or slightly more, of the carbon in the outer solar nebula was in the form of condensed materials (although the amount of condensed C may have dropped slightly with increasing heliocentric distance). Strict compositional limits computed for the Pluto/Charon system using the densities of CH4 and CO ices indicate that these pure ices are at best minor components in the interiors of these bodies, and imply that CH4 and CO ices were not the dominant C-bearing solids in the outer nebula. Clathrate-hydrates could not have appropriated enough CH4 or CO to be the major form of condensed carbon, although such clathrates may be

  20. Immunogenicity studies with a genetically engineered hexavalent PorA and a wild-type meningococcal group B outer membrane vesicle vaccine in infant cynomolgus monkeys.

    PubMed

    Rouppe van der Voort, E; Schuller, M; Holst, J; de Vries, P; van der Ley, P; van den Dobbelsteen, G; Poolman, J

    2000-01-31

    The immunogenicity of two meningococcal outer membrane vesicle (OMV) vaccines, namely the Norwegian wild-type OMV vaccine and the Dutch hexavalent PorA OMV vaccine, were examined in infant cynomolgus monkeys. For the first time, a wild-type- and a recombinant OMV vaccine were compared. Furthermore, the induction of memory and the persistence of circulating antibodies were measured. The Norwegian vaccine contained all four classes of major outer membrane proteins (OMP) and wild-type L3/L8 lipopolysaccharide (LPS). The Dutch vaccine consisted for 90% of class 1 OMPs, had low expression of class 4 and 5 OMP, and GalE LPS. Three infant monkeys were immunised with a human dose at the age of 1.5, 2.5 and 4.5 months. Two monkeys of each group received a fourth dose at the age of 11 months. In ELISA, both OMV vaccines were immunogenic and induced booster responses, particularly after the fourth immunisation. The Norwegian vaccine mostly induced sero-subtype P1.7,16 specific serum bactericidal antibodies (SBA), although some other SBA were induced as well. The antibody responses against P1.7,16, induced by the Norwegian vaccine, were generally higher than for the Dutch vaccine. However, the Dutch vaccine induced PorA specific SBA against all six sero-subtypes included in the vaccine showing differences in the magnitude of SBA responses to the various PorAs. PMID:10618530

  1. Cratering Rates in the Outer Solar System

    NASA Technical Reports Server (NTRS)

    Zahnle, Kevin

    2003-01-01

    We have constructed a self-consistent study of cratering rates in the outer solar system. Two papers were written, one on cratering asymmetries on synchronously rotating satellites and the other on the cratering rates themselves. The first addresses the well-founded expectation that the leading hemisphere of a synchronously rotating satellite should be more heavily cratered than the trailing hemisphere, and how our solar system has avoided showing much sign of this. We conclude that Ganymede has in the past rotated nonsynchronously, which may imply that it once harboured a thicker inner ocean than it does now. The other study began as an attempt to determine the age of the surface of Europa at a time when Europa was regarded as a major Exobiological target. In keeping with changing times the study expanded to the point that it now recommends cratering rates for worlds as diverse as Charon and Pluto, and includes the contributions of several invaluable co-authors, none of whom would agree with all of my conclusions. The nexus of the work is the size-frequency distribution of comets striking Jupiter (Figure). This was determined using the historically observed record of comets striking or nearly striking Jupiter; the size-frequency distributions of craters on lightly cratered surfaces of Europa, Ganymede, and Triton; and the size-frequncy distribution of Kuiper Belt objects. Extreme reductionists will be happy to know that the surface of Europa probably has an age of around 50 million years. Perhaps more intriguing is that Neptune's moon Triton, by origin a giant comet and by capture and orbital evolution a once fully melted giant comet, has a surface that is probably no older than Europa's.

  2. Meiotic recombination mechanisms.

    PubMed

    Grelon, Mathilde

    2016-01-01

    Meiosis is a specialized cell division at the origin of the haploid cells that eventually develop into the gametes. It therefore lies at the heart of Mendelian heredity. Recombination and redistribution of the homologous chromosomes arising during meiosis constitute an important source of genetic diversity, conferring to meiosis a particularly important place in the evolution and the diversification of the species. Our understanding of the molecular mechanisms governing meiotic recombination has considerably progressed these last decades, benefiting from complementary approaches led on various model species. An overview of these mechanisms will be provided as well as a discussion on the implications of these recent discoveries. PMID:27180110

  3. 75 FR 61512 - Outer Continental Shelf Official Protraction Diagrams

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-05

    ... Bureau of Ocean Energy Management, Regulation and Enforcement Outer Continental Shelf Official... Outer Continental Shelf Official Protraction Diagrams (OPDs) located within Atlantic Ocean areas, with... informational purposes only. Outer Continental Shelf Official Protraction Diagrams in the North Atlantic,...

  4. Recombination in Eukaryotic Single Stranded DNA Viruses

    PubMed Central

    Martin, Darren P.; Biagini, Philippe; Lefeuvre, Pierre; Golden, Michael; Roumagnac, Philippe; Varsani, Arvind

    2011-01-01

    Although single stranded (ss) DNA viruses that infect humans and their domesticated animals do not generally cause major diseases, the arthropod borne ssDNA viruses of plants do, and as a result seriously constrain food production in most temperate regions of the world. Besides the well known plant and animal-infecting ssDNA viruses, it has recently become apparent through metagenomic surveys of ssDNA molecules that there also exist large numbers of other diverse ssDNA viruses within almost all terrestrial and aquatic environments. The host ranges of these viruses probably span the tree of life and they are likely to be important components of global ecosystems. Various lines of evidence suggest that a pivotal evolutionary process during the generation of this global ssDNA virus diversity has probably been genetic recombination. High rates of homologous recombination, non-homologous recombination and genome component reassortment are known to occur within and between various different ssDNA virus species and we look here at the various roles that these different types of recombination may play, both in the day-to-day biology, and in the longer term evolution, of these viruses. We specifically focus on the ecological, biochemical and selective factors underlying patterns of genetic exchange detectable amongst the ssDNA viruses and discuss how these should all be considered when assessing the adaptive value of recombination during ssDNA virus evolution. PMID:21994803

  5. Syrtis Major

    NASA Technical Reports Server (NTRS)

    2002-01-01

    (Released 1 May 2002) The Science This image is from the region of Syrtis Major, which is dominated by a low-relief shield volcano. This area is believed to be an area of vigorous aeolian activity with strong winds in the east-west direction. The effects of these winds are observed as relatively bright streaks across the image, extending from topographic features such as craters. The brighter surface material probably indicates a smaller relative particle size in these areas, as finer particles have a higher albedo. The bright streaks seen off of craters are believed to have formed during dust storms. A raised crater rim can cause a reduction in the wind velocity directly behind it, which results in finer particles being preferentially deposited in this location. In the top half of the image, there is a large bright streak that crosses the entire image. There is no obvious topographic obstacle, therefore it is unclear whether it was formed in the same manner as described above. This image is located northwest of Nili Patera, a large caldera in Syrtis Major. Different flows from the caldera eruptions can be recognized as raised ridges, representing the edge of a flow lobe. The Story In the 17th century, Holland was in its Golden Age, a time of cultural greatness and immense political and economic influence in the world. In that time, lived a inquisitive person named Christian Huygens. As a boy, he loved to draw and to figure out problems in mathematics. As a man, he used these talents to make the first detailed drawings of the Martian surface - - only 50 years or so after Galileo first turned his telescope on Mars. Mars suddenly became something other than a small red dot in the sky. One of the drawings Huygens made was of a dark marking on the red planet's surface named Syrtis Major. Almost 350 years later, here we are with an orbiter that can show us this place in detail. Exploration lives! It's great we can study this area up close. In earlier periods of history

  6. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis

    PubMed Central

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  7. Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis.

    PubMed

    Ma, Qiao-Li; Liu, Ai-Cui; Ma, Xiao-Juan; Wang, Yan-Bai; Hou, Yu-Ting; Wang, Zhen-Hai

    2015-01-01

    Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection. PMID:26770344

  8. Molecular cloning and expression of Neisseria meningitidis class 1 outer membrane protein in Escherichia coli K-12.

    PubMed Central

    Barlow, A K; Heckels, J E; Clarke, I N

    1987-01-01

    A genomic library of meningococcal DNA from a clinical isolate of Neisseria meningitidis was constructed in the expression vector lambda gt11. Outer membrane complex was prepared from the same strain and used to immunize rabbits to raise polyclonal anti-outer membrane complex serum. The amplified library was probed with this polyclonal serum, and seven expressing recombinants were isolated; further investigations indicated these to be identical. The expressed meningococcal gene in these recombinants was fused to vector beta-galactosidase and shown to encode epitopes present on the 42-kilodalton class 1 outer membrane protein. Estimation of the size of the recombinant fusion protein suggests that up to 40 kilodaltons of protein-coding sequence is present. The lambda gt11 recombinant contains a 3.4-kilobase DNA insert, which has been recloned into a plasmid and characterized by restriction endonuclease analysis. A restriction fragment from the insert, representing the protein-coding region hybridizes to a single 2.2-kilobase XbaI fragment from the homologous strain and to similar-sized XbaI fragments in other strains of meningococci, expressing antigenically distinct class 1 proteins. Images PMID:3117690

  9. Dissociative recombination in planetary ionospheres

    NASA Technical Reports Server (NTRS)

    Fox, J. L.

    1993-01-01

    Ionization in planetary atmospheres can be produced by solar photoionization, photoelectron impact ionization, and, in auroral regions, by impact of precipitating particles. This ionization is lost mainly in dissociative recombination (DR) of molecular ions. Although atomic ions cannot undergo DR, they can be transformed locally through ion-molecule reactions into molecular ions, or they may be transported vertically or horizontally to regions of the atmosphere where such transformations are possible. Because DR reactions tend to be very exothermic, they can be an important source of kinetically or internally excited fragments. In interplanetary thermospheres, the neutral densities decrease exponentially with altitude. Below the homopause (or turbopause), the atmosphere is assumed to be throughly mixed by convection and/or turbulence. Above the homopause, diffusion is the major transport mechanism, and each species is distributed according to its mass, with the logarithmic derivative of the density with repect to altitude given approximately by -1/H, where H = kT/mg is the scale height. In this expression, T is the neutral temperature, g is the local acceleratiion of gravity, and m is the mass of the species. Thus lighter species become relatively more abundant, and heavier species less abundant, as the altitude increases. This variation of the neutral composition can lead to changes in the ion composition; furthermore, as the neutral densities decrease, dissociative recombination becomes more important relative to ion-neutral reactions as a loss mechanism for molecular ions.

  10. Recombination in viruses: mechanisms, methods of study, and evolutionary consequences.

    PubMed

    Pérez-Losada, Marcos; Arenas, Miguel; Galán, Juan Carlos; Palero, Ferran; González-Candelas, Fernando

    2015-03-01

    Recombination is a pervasive process generating diversity in most viruses. It joins variants that arise independently within the same molecule, creating new opportunities for viruses to overcome selective pressures and to adapt to new environments and hosts. Consequently, the analysis of viral recombination attracts the interest of clinicians, epidemiologists, molecular biologists and evolutionary biologists. In this review we present an overview of three major areas related to viral recombination: (i) the molecular mechanisms that underlie recombination in model viruses, including DNA-viruses (Herpesvirus) and RNA-viruses (Human Influenza Virus and Human Immunodeficiency Virus), (ii) the analytical procedures to detect recombination in viral sequences and to determine the recombination breakpoints, along with the conceptual and methodological tools currently used and a brief overview of the impact of new sequencing technologies on the detection of recombination, and (iii) the major areas in the evolutionary analysis of viral populations on which recombination has an impact. These include the evaluation of selective pressures acting on viral populations, the application of evolutionary reconstructions in the characterization of centralized genes for vaccine design, and the evaluation of linkage disequilibrium and population structure. PMID:25541518