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Sample records for recombinant major outer

  1. Vaccination of koalas with a recombinant Chlamydia pecorum major outer membrane protein induces antibodies of different specificity compared to those following a natural live infection.

    PubMed

    Kollipara, Avinash; Polkinghorne, Adam; Beagley, Kenneth W; Timms, Peter

    2013-01-01

    Chlamydial infection in koalas is common across the east coast of Australia and causes significant morbidity, infertility and mortality. An effective vaccine to prevent the adverse consequences of chlamydial infections in koalas (particularly blindness and infertility in females) would provide an important management tool to prevent further population decline of this species. An important step towards developing a vaccine in koalas is to understand the host immune response to chlamydial infection. In this study, we used the Pepscan methodology to identify B cell epitopes across the Major Outer Membrane Protein (MOMP) of four C. pecorum strains/genotypes that are recognized, either following (a) natural live infection or (b) administration of a recombinant MOMP vaccine. Plasma antibodies from the koalas naturally infected with a C. pecorum G genotype strain recognised the epitopes located in the variable domain (VD) four of MOMP G and also VD4 of MOMP H. By comparison, plasma antibodies from an animal infected with a C. pecorum F genotype strain recognised epitopes in VD1, 2 and 4 of MOMP F, but not from other genotype MOMPs. When Chlamydia-free koalas were immunised with recombinant MOMP protein they produced antibodies not only against epitopes in the VDs but also in conserved domains of MOMP. Naturally infected koalas immunised with recombinant MOMP protein also produced antibodies against epitopes in the conserved domains. This work paves the way for further refinement of a MOMP-based Chlamydia vaccine that will offer wide cross-protection against the variety of chlamydial infections circulating in wild koala populations.

  2. Cloning and characterization of multigenes encoding the immunodominant 30-kilodalton major outer membrane proteins of Ehrlichia canis and application of the recombinant protein for serodiagnosis.

    PubMed

    Ohashi, N; Unver, A; Zhi, N; Rikihisa, Y

    1998-09-01

    A 30-kDa major outer membrane protein of Ehrlichia canis, the agent of canine ehrlichiosis, is the major antigen recognized by both naturally and experimentally infected dog sera. The protein cross-reacts with a serum against a recombinant 28-kDa protein (rP28), one of the outer membrane proteins of a gene (omp-1) family of Ehrlichia chaffeensis. Two DNA fragments of E. canis were amplified by PCR with two primer pairs based on the sequences of E. chaffeensis omp-1 genes, cloned, and sequenced. Each fragment contained a partial 30-kDa protein gene of E. canis. Genomic Southern blot analysis with the partial gene probes revealed the presence of multiple copies of these genes in the E. canis genome. Three copies of the entire gene (p30, p30-1, and p30a) were cloned and sequenced from the E. canis genomic DNA. The open reading frames of the two copies (p30 and p30-1) were tandemly arranged with an intergenic space. The three copies were similar but not identical and contained a semivariable region and three hypervariable regions in the protein molecules. The following genes homologous to three E. canis 30-kDa protein genes and the E. chaffeensis omp-1 family were identified in the closely related rickettsiae: wsp from Wolbachia sp. , p44 from the agent of human granulocytic ehrlichiosis, msp-2 and msp-4 from Anaplasma marginale, and map-1 from Cowdria ruminantium. Phylogenetic analysis among the three E. canis 30-kDa proteins and the major surface proteins of the rickettsiae revealed that these proteins are divided into four clusters and the two E. canis 30-kDa proteins are closely related but that the third 30-kDa protein is not. The p30 gene was expressed as a fusion protein, and the antibody to the recombinant protein (rP30) was raised in a mouse. The antibody reacted with rP30 and a 30-kDa protein of purified E. canis. Twenty-nine indirect fluorescent antibody (IFA)-positive dog plasma specimens strongly recognized the rP30 of E. canis. To evaluate whether the r

  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. Genetic diversity of Chlamydia pecorum strains in wild koala locations across Australia and the implications for a recombinant C. pecorum major outer membrane protein based vaccine.

    PubMed

    Kollipara, Avinash; Polkinghorne, Adam; Wan, Charles; Kanyoka, Pride; Hanger, Jon; Loader, Joanne; Callaghan, John; Bell, Alicia; Ellis, William; Fitzgibbon, Sean; Melzer, Alistar; Beagley, Kenneth; Timms, Peter

    2013-12-27

    The long term survival of the koala (Phascolarctos cinereus) is at risk due to a range of threatening processes. A major contributing factor is disease caused by infection with Chlamydia pecorum, which has been detected in most mainland koala populations and is associated with ocular and genital tract infections. A critical aspect for the development of vaccines against koala chlamydial infections is a thorough understanding of the prevalence and strain diversity of C. pecorum infections across wild populations. In this study, we describe the largest survey (403 koalas from eight wild populations and three wildlife hospitals) examining the diversity of C. pecorum infections. 181 of the 403 koalas tested (45%) positive for C. pecorum by species-specific quantitative PCR with infection rates ranging from 20% to 61% in the eight wild populations sampled. The ompA gene, which encodes the chlamydial major outer membrane protein (MOMP), has been the major target of several chlamydial vaccines. Based on our analysis of the diversity of MOMP amino types in the infected koalas, we conclude that, (a) there exists significant diversity of C. pecorum strains in koalas, with 10 distinct, full length C. pecorum MOMP amino types identified in the 11 koala locations sampled, (b) despite this diversity, there are predicted T and B cell epitopes in both conserved as well as variable domains of MOMP which suggest cross-amino type immune responses, and (c) a recombinant MOMP-based vaccine consisting of MOMP "F" could potentially induce heterotypic protection against a range of C. pecorum strains.

  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. 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.

  7. A vaccine formulated with a combination of TLR-2 and TLR-9 adjuvants and the recombinant major outer membrane protein elicits a robust immune response and significant protection against a Chlamydia muridarum challenge.

    PubMed

    Cheng, Chunmei; Pal, Sukumar; Tifrea, Delia; Jia, Zhenyu; de la Maza, Luis M

    2014-03-01

    Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen in the World and there is a need for a vaccine. To enhance the immunogenicity of a vaccine formulated with the Chlamydia muridarum (Cm) mouse pneumonitis recombinant major outer membrane protein (MOMP), we used combinations of Pam2CSK4 + CpG-1826 and Montanide ISA 720 VG + CpG-1826 as adjuvants. Neisseria gonorrhoeae recombinant porin B (Ng-PorB) was used as the antigen control with the same adjuvants. Female BALB/c mice were immunized twice in the nares (i.n.) or in the colon (cl.) and were boosted twice by the intramuscular plus subcutaneous (i.m. + s.c.) routes. Based on the IgG2a/IgG1 ratio in sera, mice immunized with MOMP + Pam2CSK4 + CpG-1826 showed a strong Th2 response while animals vaccinated with MOMP + Montanide ISA 720 VG + CpG-1826 had a Th1 response. Both groups of mice also developed robust Cm-specific T cell proliferation and high levels of IFN-γ. Four weeks after the last immunization, the mice were challenged i.n. with 10(4) inclusion-forming units (IFU) of Cm. Using changes in body weight and number of IFU recovered from the lungs at 10 days post-challenge mice immunized i.n. + i.m./s.c. with MOMP + Pam2CSK4 + CpG-1826 were better protected than other groups. In conclusion, MOMP adjuvanted with Pam2CSK4 + CpG-1826, elicits strong humoral and cellular immune responses and induces significant protection against Chlamydia.

  8. 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.

  9. 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

  10. 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

  11. 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

  12. The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena.

    PubMed

    Edamatsu, Masaki

    2014-05-16

    Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.

  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. 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

  15. 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

  16. Aeromonas salmonicida possesses two genes encoding homologs of the major outer membrane protein, OmpA.

    PubMed Central

    Costello, G M; Vipond, R; MacIntyre, S

    1996-01-01

    Two homologs of the outer membrane protein OmpA were identified in Aeromonas salmonicida by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and amino-terminal sequence analyses. An A. salmonicida genomic DNA library was constructed by using lambda GEM-11 and recombinant phage carrying both genes ompAI and ompAII) selected by immunoscreening. A 5.0-kb BamHI fragment containing the two genes in tandem was subcloned in pBluescript and used for further subcloning and sequencing of the genes. The encoded proteins (Mr = 33,564 and 32,536 for mature OmpAI and OmpAII, respectively) had only 64% identity with each other and otherwise had the highest level of homology to OmpA proteins from the members of the family Enterobacteriaceae. Based on the Escherichia coli OmpA model, an eight-stranded amphipathic beta-barrel model for the membrane assembly of the N-terminal half of OmpAI and OmpAII was predicted. Most variation between the two proteins was localized to the predicted surface loops and periplasmic turns, while the transmembrane strands and C-terminals domains were highly conserved. Expression of ompAI and ompAII separately in E. coli indicated that both genes could be independently transcribed from their own promoters and that both gene products were assembled into the E. coli outer membrane. A survey of different Aeromonas spp. by PCR revealed that possession of two tandem ompA genes was widespread among this genus. This is the first report of any bacterial species possessing two genes for homologs of this major outer membrane protein. PMID:8626290

  17. Major outer membrane proteins in moderately halophilic eubacteria of genera Chromohalobacter and Halomonas.

    PubMed

    Tokunaga, Hiroko; Mitsuo, Kenjiro; Kamekura, Masahiro; Tokunaga, Masao

    2004-01-01

    Outer and inner membrane fractions of Chromohalobacter marismortui and Halomonas elongata were isolated by differential detergent solubilization, and profiles of membrane proteins, especially major outer membrane proteins, were analyzed. These type strains possessed one extremely abundant outer membrane protein which showed similarity in amino-terminal amino acid sequence with the outer membrane porin proteins in other Gram-negative bacteria. Three halophilic eubacterial strains isolated from saline environments were also characterized. Strains 160 and 43 were found to be Chromohalobacter spp. and strain 40 to be a Halomonas sp. by sequence analysis of their 16 S ribosomal RNA genes. Extremely abundant porin proteins with an apparent molecular mass of 49 kDa were found in Chromohalobacter sp.160 and Halomonas sp. 40, but no major outer membrane protein was detected in Chromohalobacter sp. 43, suggesting strain 43 was most likely a naturally defective porin mutant. Porin proteins from Chromohalobacter spp. and Halomonas spp. showed the same migration on SDS-polyacrylamide gel electrophoresis with or without heat-treatment, indicating that these porin proteins did not form a SDS-resistant trimeric structure, which was detected in most of the Gram-negative bacterial porin proteins.

  18. 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.

  19. Native surface association of a recombinant 38-kilodalton Treponema pallidum antigen isolated from the Escherichia coli outer membrane.

    PubMed Central

    Fehniger, T E; Radolf, J D; Walfield, A M; Cunningham, T M; Miller, J N; Lovett, M A

    1986-01-01

    A recombinant plasmid designated pAW305, containing a 6-kilobase insert of Treponema pallidum DNA, directed the expression of a 38-kilodalton (kDa) treponemal antigen in Escherichia coli. The 38-kDa antigen copurified with the outer membrane fraction of the E. coli cell envelope after treatment with nonionic detergents or sucrose density gradient centrifugation. Rabbits immunized with the recombinant 38-kDa antigen developed antibodies which reacted specifically with a 38-kDa T. pallidum antigen on immunoblots, and 38-kDa antisera specifically immobilized T. pallidum in a complement-dependent manner in the T. pallidum immobilization test. Antisera to the 38-kDa recombinant antigen were also used to demonstrate its native surface association on T. pallidum by immunoelectron microscopy. Images PMID:3516880

  20. Major psychological factors affecting acceptance of gene-recombination technology.

    PubMed

    Tanaka, Yutaka

    2004-12-01

    The purpose of this study was to verify the validity of a causal model that was made to predict the acceptance of gene-recombination technology. A structural equation model was used as a causal model. First of all, based on preceding studies, the factors of perceived risk, perceived benefit, and trust were set up as important psychological factors determining acceptance of gene-recombination technology in the structural equation model. An additional factor, "sense of bioethics," which I consider to be important for acceptance of biotechnology, was added to the model. Based on previous studies, trust was set up to have an indirect influence on the acceptance of gene-recombination technology through perceived risk and perceived benefit in the model. Participants were 231 undergraduate students in Japan who answered a questionnaire with a 5-point bipolar scale. The results indicated that the proposed model fits the data well, and showed that acceptance of gene-recombination technology is explained largely by four factors, that is, perceived risk, perceived benefit, trust, and sense of bioethics, whether the technology is applied to plants, animals, or human beings. However, the relative importance of the four factors was found to vary depending on whether the gene-recombination technology was applied to plants, animals, or human beings. Specifically, the factor of sense of bioethics is the most important factor in acceptance of plant gene-recombination technology and animal gene-recombination technology, and the factors of trust and perceived risk are the most important factors in acceptance of human being gene-recombination technology.

  1. Homogeneity of the major outer membrane protein gene of feline Chlamydia psittaci.

    PubMed

    Sayada, C; Andersen, A; Rodriguez, P; Eb, F; Milon, A; Elion, J; Denamur, E

    1994-01-01

    Thirteen feline Chlamydia psittaci strains isolated in various countries over a 50-year period were examined by restriction mapping of the major outer membrane protein (MOMP) gene obtained after DNA amplification by the polymerase chain reaction and compared with avian, ovine and guinea pig inclusion conjunctivitis (GPIC) C psittaci strains. All the feline isolates produced a unique AluI pattern distinct from the other C psittaci strains and were characterised by a typical fragment doublet of 185/180 base pairs. Such a homogeneity argues for their genetic relatedness and suggests their clonal origin. These data demonstrate the usefulness of MOMP-gene restriction mapping in C psittaci typing.

  2. Multiple tandem promoters of the major outer membrane protein gene (omp1) of Chlamydia psittaci.

    PubMed

    Yuan, Y; Zhang, Y X; Manning, D S; Caldwell, H D

    1990-09-01

    The transcription of omp1, the gene encoding the major outer membrane protein, was studied for two strains of Chlamydia psittaci, guinea pig inclusion conjunctivitis (GPIC) and mouse pneumonitis (Mn). The transcriptional initiation sites for the omp1 of each strain were mapped by S1 nuclease and primer extension analyses. Three different sizes of omp1 transcripts were observed for GPIC and four were observed for Mn. The production of these transcripts appeared to be the consequence of multiple tandem promoters. The order in which the omp1 RNA transcripts appeared during the growth cycle of the C. psittaci strains was found to differ from that of C. trachomatis.

  3. Protective efficacy of recombinant Yersinia outer proteins against bubonic plague caused by encapsulated and nonencapsulated Yersinia pestis.

    PubMed

    Andrews, G P; Strachan, S T; Benner, G E; Sample, A K; Anderson, G W; Adamovicz, J J; Welkos, S L; Pullen, J K; Friedlander, A M

    1999-03-01

    To evaluate the role of Yersinia outer proteins (Yops) in conferring protective immunity against plague, six yop loci from Yersinia pestis were individually amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant proteins were purified and injected into mice. Most Yop-vaccinated animals succumbed to infection with either wild-type encapsulated Y. pestis or a virulent, nonencapsulated isogenic variant. Vaccination with YpkA significantly prolonged mean survival time but did not increase overall survival of mice infected with the nonencapsulated strain. The only significant protection against death was observed in YopD-vaccinated mice challenged with the nonencapsulated strain. PMID:10024607

  4. Structure-function studies of the Neisseria gonorrhoeae major outer membrane porin.

    PubMed

    Chen, Adrienne; Seifert, H Steven

    2013-12-01

    The major outer membrane porin (PorB) expressed by Neisseria gonorrhoeae plays multiple roles during infection, in addition to its function as an outer membrane pore. We have generated a panel of mutants of N. gonorrhoeae strain FA1090 expressing a variety of mutant porB genes that all function as porins. We identified multiple regions of porin that are involved in its binding to the complement regulatory factors C4b-binding protein and factor H and confirmed that the ability to bind at least one factor is required for FA1090 to survive the bactericidal effects of human serum. We tested the ability of these mutants to inhibit both apoptosis and the oxidative burst in polymorphonuclear leukocytes but were unable to identify the porin domains required for either function. This study has identified nonessential porin domains and some potentially essential portions of the protein and has further expanded our understanding of the contribution of the porin domains required for complement regulation.

  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. 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.

  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. Cloning and sequence analysis of the major outer membrane protein genes of two Chlamydia psittaci strains.

    PubMed

    Zhang, Y X; Morrison, S G; Caldwell, H D; Baehr, W

    1989-05-01

    We cloned and sequenced the gene encoding the major outer membrane protein (MOMP) of two Chlamydia psittaci strains, guinea pig inclusion conjunctivitis (GPIC) strain 1, and meningopneumonitis (Mn) strain Cal-10. Intraspecies alignment of the two C. psittaci MOMP genes revealed 80.6% similarity, and interspecies comparison of C. trachomatis and C. psittaci MOMP genes yielded about 68% similarity. As found previously for C. trachomatis MOMP sequences, stretches of predominantly conserved sequences of GPIC and Mn MOMPs were interrupted by four variable domains whose locations were identical to those of C. trachomatis MOMPs. Seven of eight cysteine residues were found at precisely the same positions in GPIC, Mn, and C. trachomatis MOMPs, emphasizing their importance in structure and function of the protein. Collectively, these results indicate that C. psittaci and C. trachomatis MOMP genes diverged from a common ancestor.

  9. 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

  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. Production of a monoclonal antibody specific for the major outer membrane protein of Campylobacter jejuni and characterization of the epitope.

    PubMed

    Qian, Hongliang; Pang, Ervinna; Du, Qingyun; Chang, Jason; Dong, Jin; Toh, Say Ling; Ng, Fook Kheong; Tan, Ai Ling; Kwang, Jimmy

    2008-02-01

    Campylobacter species are important enteric pathogens causing disease in humans and animals. There is a lack of a good immunological test that can be used routinely to separate Campylobacter jejuni from other Campylobacter species. We produced monoclonal antibodies (MAbs) directed against the major outer membrane protein (MOMP) of C. jejuni using recombinant MOMP as the antigen. One MAb, designated MAb5C4 and of the immunoglobulin G1 isotype, was found to be potentially specific for C. jejuni. Dot blots demonstrated that MAb5C4 reacted with all 29 isolates of C. jejuni tested but did not react with 2 C. jejuni isolates, 26 other Campylobacter spp. isolates, and 19 non-Campylobacter isolates. Western blotting showed that MAb5C4 bound to a single protein band approximately 43 kDa in size, corresponding to the expected size of C. jejuni MOMP. The detection limit of MAb5C4 in a dot blot assay was determined to be about 5 x 10(3) bacteria. The epitope on the MOMP was mapped to a region six amino acids in length with the sequence 216GGQFNP221, which is 97% conserved among C. jejuni strains but divergent in other Campylobacter spp.; a GenBank search indicated that 95% of C. jejuni isolates will be able to be detected from non-Campylobacter spp. based on the highly specific and conserved region of the GGQFNP polypeptide. The epitope is predicted to be located in a region that is exposed to the periplasm. MAb5C4 is a potentially specific and sensitive MAb that can be used for the specific detection and identification of C. jejuni.

  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. Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli.

    PubMed

    Dascher, C; Roll, D; Bavoil, P M

    1993-12-01

    The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli. A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.

  14. Cloning and sequence analysis of the major outer membrane protein gene of Chlamydia psittaci 6BC.

    PubMed

    Everett, K D; Andersen, A A; Plaunt, M; Hatch, T P

    1991-08-01

    The gene encoding the major outer membrane protein (MOMP) of the psittacine Chlamydia psittaci strain 6BC was cloned and sequenced. N-terminal protein sequencing of the mature MOMP indicated that it is posttranslationally processed at a site identical to the site previously identified in the MOMP of Chlamydia trachomatis L2. The nucleotide sequence of the C. psittaci 6BC MOMP gene was found to be 67 to 68% identical to those of human C. trachomatis strains, 73% identical to that of Chlamydia pneumoniae IOL-207, 79% identical to that of the C. psittaci guinea pig inclusion conjunctivitis strain, GPIC, and 83% identical to that of the C. psittaci ovine abortion strain S26/3. In contrast, the 6BC sequence was found to be greater than 99% identical to the sequences reported for two strains of C. psittaci, A22/M and Cal-10 meningopneumonitis, believed to be of nonpsittacine avian origin. Monoclonal antibody analysis confirmed the nonpsittacine avian origin of A22/M but identified the Cal-10 strain from which the MOMP gene was previously sequenced as a psittacine strain. These results confirm that psittacine and nonpsittacine avian strains of C. psittaci are closely related and distinct from the mammalian guinea pig inclusion conjunctivitis and ovine abortion strains of C. psittaci.

  15. 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

  16. Vaccination with major outer membrane protein proteosomes elicits protection in mice against a Chlamydia respiratory challenge.

    PubMed

    Tifrea, Delia F; Pal, Sukumar; Toussi, Deana N; Massari, Paola; de la Maza, Luis M

    2013-11-01

    Vaccines formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) have so far been shown to elicit the most robust protection against this pathogen. nMOMP is a membrane protein and therefore, detergents are used to keep it in solution. Detergents however, have toxic effects. To address this limitation, we tested a nMOMP proteosome vaccine and compared its ability to elicit protection against nMOMP solubilized in the detergent Z3-14. The two preparations were formulated with or without CpG + Montanide (C/M). As a control antigen we used ovalbumin. Mice vaccinated with nMOMP developed strong humoral and cell mediated Chlamydia-specific immune responses. Based on the IgG2a/IgG1 levels in serum and amounts of IFN-γ in splenocytes supernatants the immune responses were predominantly Th1-biased. The animals were subsequently challenged intranasally with 2 × 10(3)Chlamydia inclusion forming units (IFU) and the course of the infection was followed for 10 days when the mice were euthanized. Based on changes in body weight, weight of the lungs and number of IFU recovered from the lungs, mice immunized with nMOMP-Ps and nMOMP + Z3-14 adjuvanted with C/M showed the most robust protection. In summary, nMOMP-Ps should be considered as Chlamydia vaccine candidates.

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

    PubMed

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

    1985-04-01

    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.

  18. Monoclonal antibody binding to the major outer membrane protein of Campylobacter coli.

    PubMed

    Qian, Hongliang; Pang, Ervinna; Chang, Jason; Toh, Say Ling; Ng, Fook Kheong; Tan, Ai Ling; Kwang, Jimmy

    2008-11-30

    Campylobacter species are major enteric pathogens causing diarrhea illness in humans and animals. Immunological tests are needed for accurate and rapid identification of C. coli, in conjunction with the use of standard biochemical tests. We initiated the creation of monoclonal antibodies (MAbs) using whole C. coli cells as antigen. Four positive clones were identified, namely MAb2G6, MAb3B9, MAb4A10 and MAb5B9. Dot-blot assay and ELISA revealed that only MAb2G6 did not cross react with C. jejuni and other Campylobacter isolates. As demonstrated by dot-blot assay, MAb2G6 reacted with all 23 C. coli isolates tested but did not react with 29 isolates of C. jejuni, 3 other Campylobacter spp. isolates and 19 non-Campylobacter isolates, with the lowest detection limit was in the range of 10(3) to 10(4) bacteria. Western blots and dot blots showed that the antigen of MAb2G6 was a native protein, with immunoprecipitation assay showed that MAb2G6 bound to a protein band of approximately 43 kDa in size, corresponding to major outer membrane protein (MOMP) of C. coli revealed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Immunofluorescence assay (IFA) showed that MOMP of C. coli was indeed the antigen of MAb2G6, with immunogold-electron microscopy demonstrated that MAb2G6 conjugated with immunogold particles bound to all over the surface of C. coli cells. MAb2G6 also showed potential usage in direct detection of C. coli in faecal samples.

  19. Recombinant outer membrane vesicles carrying Chlamydia muridarum HtrA induce antibodies that neutralize chlamydial infection in vitro

    PubMed Central

    Bartolini, Erika; Ianni, Elvira; Frigimelica, Elisabetta; Petracca, Roberto; Galli, Giuliano; Berlanda Scorza, Francesco; Norais, Nathalie; Laera, Donatello; Giusti, Fabiola; Pierleoni, Andrea; Donati, Manuela; Cevenini, Roberto; Finco, Oretta; Grandi, Guido; Grifantini, Renata

    2013-01-01

    Background Outer membrane vesicles (OMVs) are spheroid particles released by all Gram-negative bacteria as a result of the budding out of the outer membrane. Since they carry many of the bacterial surface-associated proteins and feature a potent built-in adjuvanticity, OMVs are being utilized as vaccines, some of which commercially available. Recently, methods for manipulating the protein content of OMVs have been proposed, thus making OMVs a promising platform for recombinant, multivalent vaccines development. Methods Chlamydia muridarum DO serine protease HtrA, an antigen which stimulates strong humoral and cellular responses in mice and humans, was expressed in Escherichia coli fused to the OmpA leader sequence to deliver it to the OMV compartment. Purified OMVs carrying HtrA (CM rHtrA-OMV) were analyzed for their capacity to induce antibodies capable of neutralizing Chlamydia infection of LLC-MK2 cells in vitro. Results CM rHtrA-OMV immunization in mice induced antibodies that neutralize Chlamydial invasion as judged by an in vitro infectivity assay. This was remarkably different from what observed with an enzymatically functional recombinant HtrA expressed in, and purified from the E. coli cytoplasm (CM rHtrA). The difference in functionality between anti-CM rHtrA and anti-CM rHtrA-OMV antibodies was associated to a different pattern of protein epitopes recognition. The epitope recognition profile of anti-CM HtrA-OMV antibodies was similar to that induced in mice during Chlamydial infection. Conclusions When expressed in OMVs HtrA appears to assume a conformation similar to the native one and this results in the elicitation of functional immune responses. These data further support the potentiality of OMVs as vaccine platform. PMID:24009891

  20. Immunogenicity of Mannheimia haemolytica Recombinant Outer Membrane Proteins Serotype 1-Specific Antigen, OmpA, OmpP2, and OmpD15▿

    PubMed Central

    Ayalew, Sahlu; Shrestha, Binu; Montelongo, Marie; Wilson, Amanda E.; Confer, Anthony W.

    2011-01-01

    We previously identified Mannheimia haemolytica outer membrane proteins (OMPs) that may be important immunogens by using immunoproteomic analyses. Genes for serotype 1-specific antigen (SSA-1), OmpA, OmpP2, and OmpD15 were cloned and expressed, and recombinant proteins were purified. Objective 1 of this study was to demonstrate immunogenicity of the four recombinant OMPs in mice and cattle. Objective 2 was to determine if the addition of individual recombinant OMPs or combinations of them would modify immune responsiveness of mice to the recombinant chimeric protein SAC89, containing the main epitope from M. haemolytica outer membrane lipoprotein PlpE and the neutralizing epitope of M. haemolytica leukotoxin. Mice vaccinated with recombinant OmpA (rOmpA), rSSA-1, rOmpD15, and rOmpP2 developed significant antibody responses to M. haemolytica outer membranes and to the homologous recombinant OMP. Cattle vaccinated with rOmpA and rSSA-1 developed significant antibodies to M. haemolytica outer membranes by day 28, whereas cattle vaccinated with rOmpD15 and rOmpP2 developed only minimal responses. Sera from cattle vaccinated with each of the recombinant proteins stimulated complement-mediated killing of the bacterium. Concurrent vaccination with SAC89 plus any of the four rOMPs singly resulted in increased endpoint anti-SAC89 titers, and for the SAC89/rSSA-1 vaccinees, the response was increased significantly. In contrast, the SAC89/P2/SSA-1 and SAC89/OmpA/P2/D15/SSA-1 combination vaccines resulted in significant decreases in anti-SAC89 antibodies compared to SAC89 vaccination alone. In conclusion, under the conditions of these experiments, vaccination of mice and cattle with rOmpA and rSSA-1 stimulated high antibody responses and may have protective vaccine potential. PMID:21976226

  1. 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

  2. Recombination and spontaneous mutation at the major cluster of resistance genes in lettuce (Lactuca sativa).

    PubMed Central

    Chin, D B; Arroyo-Garcia, R; Ochoa, O E; Kesseli, R V; Lavelle, D O; Michelmore, R W

    2001-01-01

    Two sets of overlapping experiments were conducted to examine recombination and spontaneous mutation events within clusters of resistance genes in lettuce. Multiple generations were screened for recombinants using PCR-based markers flanking Dm3. The Dm3 region is not highly recombinagenic, exhibiting a recombination frequency 18-fold lower than the genome average. Recombinants were identified only rarely within the cluster of Dm3 homologs and no crossovers within genes were detected. Three populations were screened for spontaneous mutations in downy mildew resistance. Sixteen Dm mutants were identified corresponding to spontaneous mutation rates of 10(-3) to 10(-4) per generation for Dm1, Dm3, and Dm7. All mutants carried single locus, recessive mutations at the corresponding Dm locus. Eleven of the 12 Dm3 mutations were associated with large chromosome deletions. When recombination could be analyzed, deletion events were associated with exchange of flanking markers, consistent with unequal crossing over; however, although the number of Dm3 paralogs was changed, no novel chimeric genes were detected. One mutant was the result of a gene conversion event between Dm3 and a closely related homolog, generating a novel chimeric gene. In two families, spontaneous deletions were correlated with elevated levels of recombination. Therefore, the short-term evolution of the major cluster of resistance genes in lettuce involves several genetic mechanisms including unequal crossing over and gene conversion. PMID:11157000

  3. 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

  4. 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

  5. Expression of the major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis in Escherichia coli using an arabinose-inducible plasmid vector.

    PubMed

    Hoelzle, L E; Hoelzle, K; Wittenbrink, M M

    2003-10-01

    The ompA genes encoding the 40 kDa major outer membrane protein (MOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were cloned into the arabinose-inducible plasmid vector pBADMycHis, and recombinant MOMPs (rMOMP) from the three chlamydial species were expressed at high levels in Escherichia (E.) coli. The proteins lacking the 22 aa N-terminal signal peptide were expressed as insoluble cytoplasmic inclusion bodies which were readily purified using immobilized metal-affinity chromatography. The rMOMPs including the N-terminal signal peptide were expressed and translocated as a surface-exposed immunoaccessible protein into the outer membrane of E. coli. Transformants expressing this full-length rMOMP were significantly reduced in viability. Purified native elementary bodies (EB) and rMOMPs of the three chlamydial species purified from the E. coli cytoplasm were used for immunization of rabbits. The resulting sera were analysed for their ability to recognize homologous and heterologous rMOMP and native EB. When testing rMOMP antisera against rMOMP and EB antigens, marked cross-reactivities were detected between the three species. Using EB antisera and rMOMPs as antigens, a significant species-specific reactivity was measured.

  6. 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

  7. Two bacteriophages which utilize a new Escherichia coli major outer membrane protein as part of their receptor.

    PubMed

    Chai, T J; Foulds, J

    1978-07-01

    Escherichia coli strain JF694 contains a new major outer membrane protein which we have called protein E (J. Foulds, and T. Chai, J. Bacteriol. 133:1478-1483). Two new bacteriophages, TC45 and TC23, were isolated that require the presence of protein E in the outer membrane of host cells for growth. Both of these bacteriophages have a morphology similar to T-even bacteriophages but are distinct in properties such as plaque morphology, buoyant density, and burst size. Although strain JF694, containing protein E, adsorbs bacteriophage TC45 efficiently, cells killed with heat or chloroform are unable to inactivate this bacteriophage. Purified protein E either in the presence or absence of additional probable cofactors such as lipopolysaccharide was also unable to inactivate bacteriophage TC45. Both bacteriophages probably use protein E as at least part of their receptor but require, in addition, other outer membrane components or a specific orientation or organization of this protein in the outer membrane.

  8. 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

  9. Evaluation of recombinant outer membrane protein C based indirect enzyme-linked immunoassay for the detection of Salmonella antibodies in poultry

    PubMed Central

    Manoj, Jinu; Agarwal, Rajesh K.; Sailo, Blessa; Wani, Mudasir Ahmed; Singh, Manoj Kumar

    2015-01-01

    Aim: To evaluate the efficacy of recombinant outer membrane proteinC (rOmpC) based enzyme-linked immunoassay (ELISA) for the diagnosis of salmonellosis in poultry. Materials and Methods: Three antigens were prepared, and the indirect ELISA was standardized using the antigens and the antiserum raised in chicken against Omp and rOmpC. Sera were collected from a total of 255 apparently healthy field chickens and screened for the presence of Salmonella antibodies by this ELISA. Results: The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of Omp revealed major polypeptides at 36, 42 and 52 kDa, and the rOmpC was evident by a single protein band of 43 kDa. The Omp and rOmpC antigen revealed an optimum concentration of 78 and 156 ng, respectively, in the assay, while the whole cell antigen gave an optimum reaction at a concentration of 106 organisms/ml. The test was found to be specific as it did not react with any of the antisera of seven other organisms. The developed ELISA detected Salmonella antibodies from 22 (8.62%) samples with rOmpC antigen, while 24 (9.41%) samples gave a positive reaction with both Omp and whole cell antigens. Conclusion: We suggest rOmpC based indirect ELISA as a suitable screening tool for serological monitoring of poultry flocks. PMID:27047189

  10. On the internal structure of the major satellites of the outer planets

    NASA Technical Reports Server (NTRS)

    Reynolds, R. T.; Cassen, P. M.

    1979-01-01

    The structures and thermal evolutions of the large icy-satellites of the outer solar system are considered. It is shown (for bodies comparable in size and mass to the Galilean satellites, having sizeable mass fraction of H2O, and with meteoritic abundances of radioactive materials contained within their silicate fractions) that the crust of solid ice over a liquid mantle predicted by conductive heat-transfer calculations is unstable to large-scale solid-state convection. For appropriate material parameters, convective heat-transfer rates are sufficient to freeze a large liquid mantle on a time scale that is short compared to the lifetime of the body. It is also concluded that the ice layer is convecting at the present time. A reevaluation of previous work, using improved values for material parameters and boundary conditions, reverses earlier conclusions and implies a rigid outer crust with resulting long-term stability of surface features to creep deformation. The combination of a rigid crust with active internal convection presents the additional possibility of surface features that are produced and maintained by dynamic internal processes.

  11. Recombinant M2e outer membrane vesicle vaccines protect against lethal influenza A challenge in BALB/c mice.

    PubMed

    Rappazzo, C Garrett; Watkins, Hannah C; Guarino, Cassandra M; Chau, Annie; Lopez, Jody L; DeLisa, Matthew P; Leifer, Cynthia A; Whittaker, Gary R; Putnam, David

    2016-03-01

    Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature. PMID:26827663

  12. Temperate Bacteriophage Which Causes the Production of a New Major Outer Membrane Protein by Escherichia coli

    PubMed Central

    Schnaitman, Carl; Smith, Deborah; de Salsas, Montserrat Forn

    1975-01-01

    Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as “protein 1” or “matrix protein”. In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens. Images PMID:16789148

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium

    PubMed Central

    Cangi, Nídia; Gordon, Jonathan L.; Bournez, Laure; Pinarello, Valérie; Aprelon, Rosalie; Huber, Karine; Lefrançois, Thierry; Neves, Luís; Meyer, Damien F.; Vachiéry, Nathalie

    2016-01-01

    The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago. PMID:27747194

  19. Serum antibody response to recombinant major inner capsid protein following human infection with group B rotavirus.

    PubMed Central

    Eiden, J J; Mouzinho, A; Lindsay, D A; Glass, R I; Fang, Z Y; Taylor, J L

    1994-01-01

    Recombinant major inner capsid protein (VP6) of the IDIR strain of group B rotavirus (GBR) was incorporated in a solid-phase immunoassay to access antibody response to infection in humans. Expression of VP6 in insect cells permitted design of a highly sensitive assay that avoided the contaminants present in GBR antigens obtained from fecal specimens. Among patients infected with the ADRV strain of GBR in China, increased reactivity with recombinant VP6 was observed in convalescent-phase sera in comparison with sera obtained shortly after infection (P = 0.0084). Anti-VP6 antibodies were detectable as soon as 7 days after onset of gastrointestinal symptoms, and serum reactivity persisted in specimens drawn more than 1 year after infection. Solid-phase immunoassay with recombinant VP6 was next employed in order to assess anti-GBR antibody in 513 serum specimens obtained from 423 Maryland residents (ages, 7 months to 96 years; median age, 42 years). Four individuals (< 1%) exhibited serum antibodies directed against the recombinant VP6 (ages, 54 to 95 years; mean age, 77 years). Examination of 129 additional serum specimens including some from other geographic regions of the United States failed to reveal the presence of anti-GBR antibody. Anti-GBR antibody was also not detected in any of 131 serum specimens from 60 staff and residents of a nursing home in Switzerland. While infection of humans with GBR has been uncommon in these locations outside of China, the detection of serum antibodies in older individuals in the United States either indicated an unknown, age-related risk factor or may have indicated infection in the more distant past. The availability of these reagents should allow surveys for GBR infection among additional populations that have not previously been investigated. PMID:8077413

  20. Structural analysis and cross-protective efficacy of recombinant 87 kDa outer membrane protein (Omp87) of Pasteurella multocida serogroup B:2.

    PubMed

    Kumar, Abhinendra; Yogisharadhya, Revanaiah; Ramakrishnan, Muthannan A; Viswas, K N; Shivachandra, Sathish B

    2013-12-01

    Pasteurella multocida serogroup B:2, a causative agent of haemorrhagic septicaemia (HS) in cattle and buffalo especially in tropical regions of Asian and African countries, is known to possess several outer membrane proteins (OMPs) as immunogenic antigens. In the present study, omp87 gene encoding for 87 kDa OMP (Omp87) protein of P. multocida serogroup B:2 strain P52, has been amplified (∼2304 bp), cloned in to pET32a vector and over-expressed in recombinant Escherichia coli as fusion protein. The recombinant Omp87 protein (∼102 kDa) including N-terminus hexa-histidine tag was purified under denaturing condition. Immunization of mice with rOmp87 resulted in increased antigen specific IgG titres in serum and provided protection of 66.6 and 83.3% following homologous (B:2) and heterologous (A:1) challenge, respectively. A homology model of Omp87 revealed the presence of two distinct domains; N-terminal domain with four POTRA repeats in the periplasmic space and a pore forming C-terminal β-barrel domain (β1- β16) in the outer membrane of P. multocida, which belong to Omp85-TpsB transporter superfamily of OMPs. The study indicated the potential possibilities to use rOmp87 protein along with suitable adjuvant in developing subunit vaccine for haemorrhagic septicaemia and pasteurellosis in livestock.

  1. Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.

    PubMed Central

    Roy, S; Das, A B; Ghosh, A N; Biswas, T

    1994-01-01

    The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteriae type 1. The purification method involved selective extraction of MOMP with sodium dodecyl sulfate in the presence of 0.4 M sodium chloride followed by size exclusion chromatography with Sephacryl S-200 HR. MOMP was found to form hydrophilic diffusion pores by incorporation into artificial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibited significant porin activity. However, the liposomes containing heat-denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl sulfate-10% polyacrylamide gel it moved as an oligomer of 78,000 molecular weight. Upon boiling, fully dissociated monomers of 38,000 molecular weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice immunized with MOMP of S. dysenteriae type 1 reacted strongly with purified MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent assay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surface by immunoelectron microscopy with anti-MOMP antibody. S. dysenteriae type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitrocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S. boydii, and S. sonnei, indicating that MOMPs were antigenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella species an immunologically significant macromolecule for study. Images PMID:7927692

  2. 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

  3. 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

  4. 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

  5. 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

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

    2014-06-17

    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.

  6. 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

  7. 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

  8. Structural and Functional Characterization of Recombinant Interleukin-10 from Indian Major Carp Labeo rohita

    PubMed Central

    Karan, Sweta; Dash, Pujarini; Kaushik, Himani; Sahoo, Pramoda K.; Garg, Lalit C.

    2016-01-01

    Interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. Though it is one of the major cytokines, IL-10 from the Indian major carp, Labeo rohita, has not yet been characterized. In the present study, we report large scale production and purification of biologically active recombinant IL-10 of L. rohita (rLrIL-10) using a heterologous expression system and its biophysical and functional characterization. High yield (~70 mg/L) of soluble rLrIL-10 was obtained at shake flask level. The rLrIL-10 was found to exist as a dimer. Far-UV CD spectroscopy showed presence of predominantly alpha helices. The tertiary structure of the purified rLrIL-10 was verified by fluorescence spectroscopy. Two-dimensional gel analysis revealed the presence of six isoforms of the rLrIL-10. The rLrIL-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1β, TNFα, and IL-8, and augmented the NKEF transcript levels in spleen of L. rohita. Anti-inflammatory role of the rLrIL-10 was further established by inhibition of phagocytosis using NBT reduction assay in vitro. The data indicate that the dimeric alpha helical structure and function of IL-10 of L. rohita as a key regulator of anti-inflammatory response have remained conserved during evolution. PMID:27689097

  9. Structural and Functional Characterization of Recombinant Interleukin-10 from Indian Major Carp Labeo rohita

    PubMed Central

    Karan, Sweta; Dash, Pujarini; Kaushik, Himani; Sahoo, Pramoda K.; Garg, Lalit C.

    2016-01-01

    Interleukin-10, an important regulator of both the innate and adaptive immune systems, is a multifunctional major cytokine. Though it is one of the major cytokines, IL-10 from the Indian major carp, Labeo rohita, has not yet been characterized. In the present study, we report large scale production and purification of biologically active recombinant IL-10 of L. rohita (rLrIL-10) using a heterologous expression system and its biophysical and functional characterization. High yield (~70 mg/L) of soluble rLrIL-10 was obtained at shake flask level. The rLrIL-10 was found to exist as a dimer. Far-UV CD spectroscopy showed presence of predominantly alpha helices. The tertiary structure of the purified rLrIL-10 was verified by fluorescence spectroscopy. Two-dimensional gel analysis revealed the presence of six isoforms of the rLrIL-10. The rLrIL-10 was biologically active and its administration significantly reduced serum proinflammatory cytokines, namely, interleukin 1β, TNFα, and IL-8, and augmented the NKEF transcript levels in spleen of L. rohita. Anti-inflammatory role of the rLrIL-10 was further established by inhibition of phagocytosis using NBT reduction assay in vitro. The data indicate that the dimeric alpha helical structure and function of IL-10 of L. rohita as a key regulator of anti-inflammatory response have remained conserved during evolution.

  10. Saturating mutagenesis of an essential gene: a majority of the Neisseria gonorrhoeae major outer membrane porin (PorB) is mutable.

    PubMed

    Chen, Adrienne; Seifert, H Steven

    2014-02-01

    The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin's multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.

  11. Point mutations in the major outer membrane protein drive hypervirulence of a rapidly expanding clone of Campylobacter jejuni.

    PubMed

    Wu, Zuowei; Periaswamy, Balamurugan; Sahin, Orhan; Yaeger, Michael; Plummer, Paul; Zhai, Weiwei; Shen, Zhangqi; Dai, Lei; Chen, Swaine L; Zhang, Qijing

    2016-09-20

    Infections due to clonal expansion of highly virulent bacterial strains are clear and present threats to human and animal health. Association of genetic changes with disease is now a routine, but identification of causative mutations that enable disease remains difficult. Campylobacter jejuni is an important zoonotic pathogen transmitted to humans mainly via the foodborne route. C. jejuni typically colonizes the gut, but a hypervirulent and rapidly expanding clone of C. jejuni recently emerged, which is able to translocate across the intestinal tract, causing systemic infection and abortion in pregnant animals. The genetic basis responsible for this hypervirulence is unknown. Here, we developed a strategy, termed "directed genome evolution," by using hybridization between abortifacient and nonabortifacient strains followed by selection in an animal disease model and whole-genome sequence analysis. This strategy successfully identified SNPs in porA, encoding the major outer membrane protein, are responsible for the hypervirulence. Defined mutagenesis verified that these mutations were both necessary and sufficient for causing abortion. Furthermore, sequence analysis identified porA as the gene with the top genome-wide signal of adaptive evolution using Fu's Fs, a population genetic metric for recent population size changes, which is consistent with the recent expansion of clone "sheep abortion." These results identify a key virulence factor in Campylobacter and a potential target for the control of this zoonotic pathogen. Furthermore, this study provides general, unbiased experimental and computational approaches that are broadly applicable for efficient elucidation of disease-causing mutations in bacterial pathogens. PMID:27601641

  12. Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

    PubMed

    Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

    2009-01-01

    Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

  13. Lack of allelic polymorphism for the major outer membrane protein gene of the agent of guinea pig inclusion conjunctivitis (Chlamydia psittaci).

    PubMed

    Zhao, Q; Schachter, J; Stephens, R S

    1993-07-01

    The major outer membrane protein gene (omp1) was sequenced for each of six Chlamydia psittaci (guinea pig inclusion conjunctivitis [GPIC]) strains isolated from guinea pigs. Five of the isolates were obtained in the United States during the 1960s and 1970s, including the prototype strain isolated by Murray in 1962. The other isolate was obtained from a guinea pig in England. The nucleotide sequence of the omp1 gene for each strain was identical. The lack of omp1 allelic polymorphism among GPIC isolates suggests that, unlike C. trachomatis, the GPIC agent lacks antigenic variation in the major outer membrane protein.

  14. Major epiplasmic proteins of ciliates are articulins: cloning, recombinant expression, and structural characterization.

    PubMed

    Huttenlauch, I; Geisler, N; Plessmann, U; Peck, R K; Weber, K; Stick, R

    1995-09-01

    The cytoskeleton of certain protists comprises an extensive membrane skeleton, the epiplasm, which contributes to the cell shape and patterning of the species-specific cortical architecture. The isolated epiplasm of the ciliated protist Pseudomicrothorax dubius consists of two major groups of proteins with molecular masses of 78-80 kD and 11-13 kD, respectively. To characterize the structure of these proteins, peptide sequences of two major polypeptides (78-80 kD) as well as a cDNA representing the entire coding sequence of a minor and hitherto unidentified component (60 kD; p60) of the epiplasm have been determined. All three polypeptides share sequence similarities. They contain repeated valine- and proline-rich motifs of 12 residues with the consensus VPVP--V-V-V-. In p60 the central core domain consists of 24 tandemly repeated VPV motifs. Within the repeat motifs positively and negatively charged residues, when present, show an alternating pattern in register with the V and P positions. Recombinant p60 was purified in 8 M urea and dialyzed against buffer. Infrared spectroscopic measurements indicate 30% beta-sheet. Electron microscopy reveals short filamentous polymers with a rather homogenous diameter (approximately 15-20 nm), but variable lengths. The small polymers form thicker filaments, ribbons, and larger sheets or tubes. A core domain similar to that of P. dubius p60 is also found in the recently described epiplasmic proteins of the flagellate Euglena, the so-called articulins. Our results show that the members of this protein family are not restricted to flagellates, but are also present in the distantly related ciliates where they are major constituents of the epiplasm. Comparison of flagellate and ciliate articulins highlights common features of this novel family of cytoskeletal proteins.

  15. Evaluation of protective potential of Yersinia pestis outer membrane protein antigens as possible candidates for a new-generation recombinant plague vaccine.

    PubMed

    Erova, Tatiana E; Rosenzweig, Jason A; Sha, Jian; Suarez, Giovanni; Sierra, Johanna C; Kirtley, Michelle L; van Lier, Christina J; Telepnev, Maxim V; Motin, Vladimir L; Chopra, Ashok K

    2013-02-01

    Plague caused by Yersinia pestis manifests itself in bubonic, septicemic, and pneumonic forms. Although the U.S. Food and Drug Administration recently approved levofloxacin, there is no approved human vaccine against plague. The capsular antigen F1 and the low-calcium-response V antigen (LcrV) of Y. pestis represent excellent vaccine candidates; however, the inability of the immune responses to F1 and LcrV to provide protection against Y. pestis F1(-) strains or those which harbor variants of LcrV is a significant concern. Here, we show that the passive transfer of hyperimmune sera from rats infected with the plague bacterium and rescued by levofloxacin protected naive animals against pneumonic plague. Furthermore, 10 to 12 protein bands from wild-type (WT) Y. pestis CO92 reacted with the aforementioned hyperimmune sera upon Western blot analysis. Based on mass spectrometric analysis, four of these proteins were identified as attachment invasion locus (Ail/OmpX), plasminogen-activating protease (Pla), outer membrane protein A (OmpA), and F1. The genes encoding these proteins were cloned, and the recombinant proteins purified from Escherichia coli for immunization purposes before challenging mice and rats with either the F1(-) mutant or WT CO92 in bubonic and pneumonic plague models. Although antibodies to Ail and OmpA protected mice against bubonic plague when challenged with the F1(-) CO92 strain, Pla antibodies were protective against pneumonic plague. In the rat model, antibodies to Ail provided protection only against pneumonic plague after WT CO92 challenge. Together, the addition of Y. pestis outer membrane proteins to a new-generation recombinant vaccine could provide protection against a wide variety of Y. pestis strains.

  16. Point mutations in the major outer membrane protein drive hypervirulence of a rapidly expanding clone of Campylobacter jejuni

    PubMed Central

    Wu, Zuowei; Periaswamy, Balamurugan; Sahin, Orhan; Yaeger, Michael; Plummer, Paul; Zhai, Weiwei; Shen, Zhangqi; Dai, Lei; Zhang, Qijing

    2016-01-01

    Infections due to clonal expansion of highly virulent bacterial strains are clear and present threats to human and animal health. Association of genetic changes with disease is now a routine, but identification of causative mutations that enable disease remains difficult. Campylobacter jejuni is an important zoonotic pathogen transmitted to humans mainly via the foodborne route. C. jejuni typically colonizes the gut, but a hypervirulent and rapidly expanding clone of C. jejuni recently emerged, which is able to translocate across the intestinal tract, causing systemic infection and abortion in pregnant animals. The genetic basis responsible for this hypervirulence is unknown. Here, we developed a strategy, termed “directed genome evolution,” by using hybridization between abortifacient and nonabortifacient strains followed by selection in an animal disease model and whole-genome sequence analysis. This strategy successfully identified SNPs in porA, encoding the major outer membrane protein, are responsible for the hypervirulence. Defined mutagenesis verified that these mutations were both necessary and sufficient for causing abortion. Furthermore, sequence analysis identified porA as the gene with the top genome-wide signal of adaptive evolution using Fu’s Fs, a population genetic metric for recent population size changes, which is consistent with the recent expansion of clone “sheep abortion.” These results identify a key virulence factor in Campylobacter and a potential target for the control of this zoonotic pathogen. Furthermore, this study provides general, unbiased experimental and computational approaches that are broadly applicable for efficient elucidation of disease-causing mutations in bacterial pathogens. PMID:27601641

  17. In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae.

    PubMed

    Mihara, K; Blobel, G; Sato, R

    1982-12-01

    We have isolated an outer mitochondrial membrane (OMM) fraction from baker's yeast. Saccharomyces cerevisiae, that possesses porin activity and contains a major polypeptide of 29,000 daltons. By analogy to similar data for an OMM fraction from rat liver and mung bean [Zalman, L. S., Nikaido, N. & Kagawa, Y. (1980) J. Biol. Chem. 255, 1771-1774], the 29,000-dalton polypeptide of the isolated yeast OMM fraction has been tentatively identified as porin. Evidence to substantiate this identification was provided by the finding that both the porin activity and the 29,000-dalton polypeptide were entirely resistant when the OMM fraction was exposed to trypsin digestion, with the 29,000-dalton polypeptide being virtually the only polypeptide in the OMM fraction to be unaffected by trypsin digestion. There was no protection when trypsin digestion was carried out in the presence of detergent. Using monospecific antibodies, we have shown that yeast porin is apparently not synthesized as a larger precursor in a cell-free translation system. In vitro-synthesized porin could not be integrated into dog pancreas microsomal vesicles or into an isolated OMM fraction from yeast, either co- or posttranslationally. In vitro-synthesized porin, however, could be integrated posttranslationally into whole isolated mitochondria. This membrane specificity suggests that integration does not proceed by unassisted partitioning. The integration of porin into whole mitochondria occurred with fidelity by the criterion of its resistance to trypsin. Moreover, integration was not inhibited in the presence of the protonophore carbonyl cyanide m-chlorophenyl-hydrazone whereas translocation into the mitochondrial matrix of the in vitro-synthesized gamma subunit of F1-ATPase was inhibited.

  18. Induction of immune responses by two recombinant proteins of brucella abortus, outer membrane proteins 2b porin and Cu/Zn superoxide dismutase, in mouse model.

    PubMed

    Sung, Kyung Yong; Jung, Myunghwan; Shin, Min-Kyoung; Park, Hyun-Eui; Lee, Jin Ju; Kim, Suk; Yoo, Han Sang

    2014-06-28

    The diagnosis of Brucella abortus is mainly based on serological methods using antibody against LPS, which has diagnostic problems. Therefore, to solve this problem, we evaluated two proteins of B. abortus, Cu/Zn superoxide dismutase (SodC) and outer membrane proteins 2b porin (Omp2b). The genes were cloned and expressed in a pMAL system, and the recombinant proteins, rOmp2b and rSodC, were purified as fusion forms with maltosebinding protein. The identity of the proteins was confirmed by SDS-PAGE and Western blot analysis with sera of mice infected with B. abortus. Production of cytokines and nitric oxide (NO) was investigated in RAW 264.7 cells and mouse splenocytes after stimulation with the proteins. Moreover, cellular and humoral immune responses were investigated in BALB/c mice after immunization with the proteins. TNF-α, IL-6, and NO were significantly inducible in RAW 264.7 cells. Splenocytes of naive mice produced IFN-γ and IL-4 significantly by stimulation. Moreover, number of IgG, IFN-γ, and IL-4 producing cells were increased in immunized mice with the two proteins. Production of IgG and IgM with rOmp2b was higher than those with rSodC in immunized mice. These results suggest that the two recombinant proteins of B. abortus may be potential LPS-free proteins for diagnosis.

  19. Immunological characterization of two major secreted forms of recombinant hepatitis B virus e antigens.

    PubMed

    Hwang, G Y; Wang, J C; Wu, C C

    1999-02-01

    Plasmids containing PCR-amplified hepatitis B virus e antigen (HBeAg) genes (HBeAg-MV and HBeAg-SV) were constructed and expressed in E. coli strain DH5alpha. The induced intracellular glutathione S-transferase (GST) fusion proteins of HBeAg-MV and HBeAg-SV were recovered and purified from bacterial lysates by affinity chromatography with glutathione-sepharose beads. The HBeAg-MV protein contained an additional 19 amino acids at its amino terminus. These two proteins were specifically cleaved from GST by the protease factor Xa and recognized by a monoclonal antibody against HBeAg. HBeAg-MV and HBeAg-SV were found to be the two major components of the post-modified HBcAg during viral infection. The antigenic specificities of the fusion and purified HBeAgs (factor Xa-digested) were confirmed by the Abbott HBe enzyme immunoassay (EIA) detection system. Sera from patients with confirmed hepatocellular carcinoma (HCC) specifically reacted only with HBeAg moiety of fusion proteins. HCC sera bound more strongly to the HBeAg-SV protein than to the HBeAg-MV one. This indicates that HBeAg-SV is either more antigenic than -MV or is the major target protein for the elicitation of antibody production after HBV infection. Thus, the two recombinant HBeAgs expressed and obtained in this study are appropriate immunological agents for the diagnostic detection of hepatitis B virus infection in humans.

  20. Protective immunization of horses with a recombinant canarypox virus vectored vaccine co-expressing genes encoding the outer capsid proteins of African horse sickness virus.

    PubMed

    Guthrie, Alan J; Quan, Melvyn; Lourens, Carina W; Audonnet, Jean-Christophe; Minke, Jules M; Yao, Jiansheng; He, Ling; Nordgren, Robert; Gardner, Ian A; Maclachlan, N James

    2009-07-16

    We describe the development and preliminary characterization of a recombinant canarypox virus vectored (ALVAC) vaccine for protective immunization of equids against African horse sickness virus (AHSV) infection. Horses (n=8) immunized with either of two concentrations of recombinant canarypox virus vector (ALVAC-AHSV) co-expressing synthetic genes encoding the outer capsid proteins (VP2 and VP5) of AHSV serotype 4 (AHSV-4) developed variable titres (<10-80) of virus-specific neutralizing antibodies and were completely resistant to challenge infection with a virulent strain of AHSV-4. In contrast, a horse immunized with a commercial recombinant canarypox virus vectored vaccine expressing the haemagglutinin genes of two equine influenza H3N8 viruses was seronegative to AHSV and following infection with virulent AHSV-4 developed pyrexia, thrombocytopenia and marked oedema of the supraorbital fossae typical of the "dikkop" or cardiac form of African horse sickness. AHSV was detected by virus isolation and quantitative reverse transcriptase polymerase chain reaction in the blood of the control horse from 8 days onwards after challenge infection whereas AHSV was not detected at any time in the blood of the ALVAC-AHSV vaccinated horses. The control horse seroconverted to AHSV by 2 weeks after challenge infection as determined by both virus neutralization and ELISA assays, whereas six of eight of the ALVAC-AHSV vaccinated horses did not seroconvert by either assay following challenge infection with virulent AHSV-4. These data confirm that the ALVAC-AHSV vaccine will be useful for the protective immunization of equids against African horse sickness, and avoids many of the problems inherent to live-attenuated AHSV vaccines.

  1. Genome Diversity, Recombination, and Virulence across the Major Lineages of Paracoccidioides

    PubMed Central

    Muñoz, José F.; Desjardins, Christopher A.; Gallo, Juan E.; Sykes, Sean; Sakthikumar, Sharadha; Misas, Elizabeth; Whiston, Emily A.; Bagagli, Eduardo; Soares, Celia M. A.; Teixeira, Marcus de M.; Taylor, John W.; Clay, Oliver K.; McEwen, Juan G.

    2016-01-01

    development of new diagnostics and treatments. In this article, we compared genomes of 31 diverse isolates representing the major lineages of Paracoccidioides spp. and completed the first annotated genome sequences for the PS3 and PS4 lineages. We analyzed the population structure and characterized the genetic diversity among the lineages of Paracoccidioides, including a deep split of S1 into two lineages (S1a and S1b), and differentiated S1b, associated with most clinical cases, as the more highly recombining and diverse lineage. In addition, we found patterns of positive selection in surface proteins and secreted enzymes among the lineages, suggesting diversifying mechanisms of pathogenicity and adaptation across this species complex. These genetic differences suggest associations with the geographic range, pathogenicity, and ecological niches of Paracoccidioides lineages. PMID:27704050

  2. 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

  3. Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

    PubMed

    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 Protochlamydiaouter 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.

  4. Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

    PubMed

    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 Protochlamydiaouter 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

  5. 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

  6. 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

  7. 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.

  8. 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

  9. Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D', I', and L2'.

    PubMed Central

    Dean, D; Patton, M; Stephens, R S

    1991-01-01

    The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp. PMID:1706325

  10. Direct sequence evaluation of the major outer membrane protein gene variant regions of Chlamydia trachomatis subtypes D', I', and L2'.

    PubMed

    Dean, D; Patton, M; Stephens, R S

    1991-04-01

    The nucleotide sequences of variable segments (VS) 1, 2, and 4 for the major outer membrane protein gene (omp1) of Chlamydia trachomatis were determined for serologically defined subtypes D', I', and L2'. Asymmetric DNA amplification was used to produce single-stranded DNA for direct sequencing. Amino acid substitutions were detected in VS1, VS2, and VS4 for I', in VS2 for L2', and in VS4 for D'. DNA sequencing of omp1 variant regions may be an important method for evaluating the molecular epidemiology of Chlamydia spp.

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

    PubMed Central

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

    2016-01-01

    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

  12. An indirect ELISA for detection of Theileria sergenti antibodies in water buffalo using a recombinant major piroplasm surface protein.

    PubMed

    Wang, L X; Zhao, J H; He, L; Liu, Q; Zhou, D N; Zhou, Y Q; Zhao, J L

    2010-06-24

    In this study we investigated the prevalence and enzootic potential of Theileria spp. in water buffalo in the Hubei province in China. An indirect ELISA based on a recombinant major piroplasma surface protein was developed. The complete ORF of the 33-kDa major piroplasma surface protein (p33) was obtained from Theileria sergenti genomic DNA by PCR, cloned into the pET-28(a) vector and expressed in E. coli as a His-fusion protein. Then the recombinant p33 (rp33) was purified and used as the antigen to develop an iELISA. Specificity test showed that there was no cross-reaction with Babesia orientalis, Schistosoma japonicum, Anaplasma marginale and Toxoplasma gondii. 178 water buffaloes raised in different locations in Hubei province in China were detected by this iELISA, all samples were also examined by PCR and microscopy at the same time. The iELISA result showed a higher positive rate (27.5%) than PCR (22.5%) and microscopy (12.9%). This result indicated that the iELISA is a suitable method for the diagnosis of T. sergenti infection and could be used in serological surveys to map out the prevalence of the disease.

  13. Identification of the gene encoding BmpB, a 30 kDa outer envelope lipoprotein of Brachyspira (Serpulina) hyodysenteriae, and immunogenicity of recombinant BmpB in mice and pigs.

    PubMed

    Lee, B J; La, T; Mikosza, A S; Hampson, D J

    2000-10-01

    A gene encoding a 30kDa outer envelope protein of the intestinal spirochaete Brachyspira (Serpulina) hyodysenteriae, was cloned and expressed in Escherichia coli strain XLOLR. Five phagemids containing DNA inserts encoding the protein were established and one clone (pSHA) was sequenced. An 816bp hypothetical open reading frame (ORF) was identified, with a potential ribosome binding site (AGGAG), and putative -10 (TATAAT) and -35 (TTGAAA) promoter regions upstream from the ATG start of the ORF. A 12bp inverted repeat sequence, possibly serving as a transcription terminator, was identified downstream from the TAA stop codon. Analysis of the amino acid sequence identified a 19 residue hydrophobic signal peptide, incorporating a potential signal peptidase cleavage site and membrane lipoprotein lipid attachment site. Further analysis of the amino acid usage of this lipoprotein, designated BmpB, showed its possible outer membrane localisation. Comparison of the gene encoding the lipoprotein, bmpB, with GenBank nucleotide sequences showed that it has homology with the gene (plp3) encoding Plp3, an outer membrane lipoprotein of Pasteurella haemolytica (54% identity in 735bp). Comparison of the deduced amino acid sequence with the SWISS-PROT amino acid database revealed greatest homology with the outer membrane lipoproteins (Plp1, 2, 3) of P. haemolytica (34% identity in 242 aa, 37% identity in 250 aa, and 39% identity in 272 aa, respectively), and lipoproteins (rcsF and lipoprotein-28) of E. coli (40% identity in 267 aa and 36% identity in 263 aa, respectively). Three of the recombinant E. coli clones (pSHA, pSHD, and pSHE) were formalinised and used to immunise mice. A bacterin preparation of one recombinant E. coli clone (pSHA) was used to immunise pigs. Sera from these mice and pigs recognised the 30kDa lipoprotein in outer membrane preparations of B. hyodysenteriae, indicating the immunogenicity of recombinant BmpB. Sera from pigs naturally infected with B

  14. Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation.

    PubMed

    Burdman, S; Dulguerova, G; Okon, Y; Jurkevitch, E

    2001-04-01

    The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

  15. Regional pipeline environmental assessment record: an aggregate analysis of major areas of pipeline concern and impact on the Outer Continental Shelf (Gulf of Mexico)

    SciTech Connect

    Not Available

    1981-03-01

    Oil and gas pipeline activities on the Gulf of Mexico Outer Continental Shelf (OCS) affect a wide range of living and nonliving environmental variables (i.e., water, biologically sensitive areas, and food-web relations). These industrial activities have been increasing steadily over the past 20 years. Much of the information included herein pertains to a description of the existing environment for offshore Louisiana and Texas and an analysis of potential pipeline proposals and alternatives. The document is not as complete as an environmental impact statement (EIS) but it is sufficient in scope to consider the analysis of impacts of the majority of pipeline activities occurring in the northern and western Gulf of Mexico OCS. This report will be useful in evaluating pipeline impacts on the regional environment in the future. Rather than treating each pipeline application extensively, the information included herein will be referenced, thereby reducing the paperwork involved in environmental analysis.

  16. Major surgery in severe haemophilia A with inhibitors using a recombinant factor VIIa and activated prothrombin complex concentrate hybrid regimen.

    PubMed

    van Veen, J J; Maclean, R M; Hampton, K K; Hamer, A; Makris, M

    2014-07-01

    Major surgery in persons with haemophilia A and inhibitors is increasingly being performed. Both recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC) are used to cover surgery but it remains unclear what the optimal dosing schedules are. We describe the use of a hybrid regimen in four inhibitor patients undergoing eight major surgical procedures using rFVIIa in the initial 2-6 postoperative days followed by FEIBA for the remaining period. All patients were also treated with tranexamic acid while receiving rFVIIa. We performed six major orthopaedic procedures, one emergency orchidectomy and one open appendectomy. The dosing schedules were at the higher end of those described in the literature but within the recommendations of the summary of product characteristics. Despite this, we encountered non-surgical bleeding in four of eight episodes. Three of these occurred in one individual suggesting a patient factor. The overall outcome was good for all episodes. The hybrid regimen combines flexibility of dose and dosing frequency of rFVIIa in the immediate postoperative setting with the advantage of a reduced dosing frequency with FEIBA in the subsequent days. This study also emphasizes that surgical procedures in this patient group remain a challenge.

  17. 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

  18. 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.

  19. 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.

  20. 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.

  1. Genomic polymorphism, recombination, and linkage disequilibrium in human major histocompatibility complex-encoded antigen-processing genes

    SciTech Connect

    van Endert, P.M.; Lopez, M.T.; Patel, S.D.; McDevitt, H.O. ); Monaco, J.J. )

    1992-12-01

    Recently, two subunits of a large cytosolic protease and two putative peptide transporter proteins were found to be encoded by genes within the class II region of the major histocompatibility complex (MHC). These genes have been suggested to be involved in the processing of antigenic proteins for presentation by MHC class I molecules. Because of the high degree of polymorphism in MHC genes, and previous evidence for both functional and polypeptide sequence polymorphism in the proteins encoded by the antigen-processing genes, we tested DNA from 27 consanguineous human cell lines for genomic polymorphism by restriction fragment length polymorphism (RFLP) analysis. These studies demonstrate a strong linkage disequilibrium between TAP1 and LMP2 RFLPs. Moreover, RFLPs, as well as a polymorphic stop codon in the telomeric TAP2 gene, appear to be in linkage disequilibrium with HLA-DR alleles and RFLPs in the HLA-DO gene. A high rate of recombination, however, seems to occur in the center of the complex, between the TAP1 and TAP2 genes.

  2. 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.

  3. 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.

  4. 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

  5. The toxR Gene of Vibrio (Listonella) anguillarum Controls Expression of the Major Outer Membrane Proteins but Not Virulence in a Natural Host Model

    PubMed Central

    Okuda, Jun; Nakai, Toshihiro; Chang, Park Se; Oh, Takanori; Nishino, Takeshi; Koitabashi, Tsutomu; Nishibuchi, Mitsuaki

    2001-01-01

    To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen. The toxR gene of V. angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed. The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae. Distribution of the Va-toxR gene sequence in V. anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods. An isogenic toxR mutant of V. anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method. The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods. A 35-kDa major OMP was not produced by the toxR mutant. However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant. For the toxR mutant, the MICs of two β-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain. Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae. The results indicate that the toxR gene is not involved in virulence expression in V. anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae. These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a

  6. 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

  7. Solution Structure, Copper Binding and Backbone Dynamics of Recombinant Ber e 1–The Major Allergen from Brazil Nut

    PubMed Central

    Rundqvist, Louise; Tengel, Tobias; Zdunek, Janusz; Björn, Erik; Schleucher, Jürgen; Alcocer, Marcos J. C.; Larsson, Göran

    2012-01-01

    Background The 2S albumin Ber e 1 is the major allergen in Brazil nuts. Previous findings indicated that the protein alone does not cause an allergenic response in mice, but the addition of components from a Brazil nut lipid fraction were required. Structural details of Ber e 1 may contribute to the understanding of the allergenic properties of the protein and its potential interaction partners. Methodology/Principal Findings The solution structure of recombinant Ber e 1 was solved using NMR spectroscopy and measurements of the protein back bone dynamics at a residue-specific level were extracted using 15N-spin relaxation. A hydrophobic cavity was identified in the structure of Ber e 1. Using the paramagnetic relaxation enhancement property of Cu2+ in conjunction with NMR, it was shown that Ber e 1 is able to specifically interact with the divalent copper ion and the binding site was modeled into the structure. The IgE binding region as well as the copper binding site show increased dynamics on both fast ps-ns timescale as well as slower µs-ms timescale. Conclusions/Significance The overall fold of Ber e 1 is similar to other 2S albumins, but the hydrophobic cavity resembles that of a homologous non-specific lipid transfer protein. Ber e 1 is the first 2S albumin shown to interact with Cu2+ ions. This Cu2+ binding has minimal effect on the electrostatic potential on the surface of the protein, but the charge distribution within the hydrophobic cavity is significantly altered. As the hydrophobic cavity is likely to be involved in a putative lipid interaction the Cu2+ can in turn affect the interaction that is essential to provoke an allergenic response. PMID:23056307

  8. 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

  9. Characterization of the humoral response induced by a synthetic peptide of the major outer membrane protein of Chlamydia trachomatis serovar B.

    PubMed Central

    Villeneuve, A; Brossay, L; Paradis, G; Hébert, J

    1994-01-01

    The major outer membrane protein of Chlamydia trachomatis has been extensively studied and is still considered one of the most promising candidates for development of a synthetic vaccine. Neutralizing epitopes in variable domains I, II, and IV have already been reported. In variable domain I, residues 69 to 78 have been identified as a neutralizing epitope for some of the C- and C-related complex serovars (A, C, I, J, L3, and K). It is not known whether epitopes located at the same position in B-complex serovars are neutralizing. To clarify this point, rabbit polyclonal antibodies directed against the peptide 69TTTGNAVAPS78 from the B serovar were produced. Rabbit antisera were further rendered peptide specific by purification on a peptide-bovine serum albumin-Sepharose affinity column. Peptide-specific rabbit immunoglobulin reacted with five of the B-complex serovars (B, Ba, E, L1, and L2) by immunoblot and by direct-binding enzyme-linked immunosorbent assay. Furthermore, this peptide-specific rabbit immunoglobulin neutralized the chlamydial infectivity of both serovars B and E for HaK cells in a complement-independent in vitro assay. The importance of these results stems from the fact that peptide 69TTTGNAVAPS78 was able to induce an antibody response directed against B- and B-related complex serovars, including serovar E, which is responsible for a high proportion of genital infections. This peptide could therefore be considered for the construction of a multivalent synthetic vaccine. Images PMID:8039926

  10. Using Major Outer Membrane Protein Typing as an Epidemiological Tool To Investigate Outbreaks Caused by Milk-Borne Campylobacter jejuni Isolates in California

    PubMed Central

    Mandrell, Robert E.; Yuan, Jean; Bates, Anna; Manalac, Rosa; Mohle-Boetani, Janet; Kimura, Akiko; Lidgard, Janice; Miller, William G.

    2013-01-01

    We describe using major outer membrane protein (MOMP) typing as a screen to compare the Campylobacter jejuni porA gene sequences of clinical outbreak strains from human stool with the porA sequences of dairy farm strains isolated during two milk-borne campylobacteriosis outbreak investigations in California. The genetic relatedness of clinical and environmental strains with identical or closely related porA sequences was confirmed by multilocus sequence typing and pulsed-field gel electrophoresis analysis. The first outbreak involved 1,644 C. jejuni infections at 11 state correctional facilities and was associated with consumption of pasteurized milk supplied by an on-site dairy (dairy A) at a prison in the central valley. The second outbreak involved eight confirmed and three suspect C. jejuni cases linked to consumption of commercial raw milk and raw chocolate colostrum at another central valley dairy (dairy B). Both dairies bottled fluid milk on the farm and distributed the finished product to off-site locations. Altogether, C. jejuni was isolated from 7 of 15 (46.7%) bovine fecal, 12 of 20 (60%) flush alley water, and 1 of 20 (5%) lagoon samples collected on dairy A. At dairy B, C. jejuni was cultured from 9 of 26 (34.6%) bovine fecal samples. Environmental strains indistinguishable from the clinical outbreak strains were found in five flush alley water samples (dairy A) and four bovine fecal samples (dairy B). The findings demonstrate that MOMP typing is a useful tool to triage environmental isolates prior to conducting more labor-intensive molecular typing methods. PMID:23115263

  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. Homeologous Recombination Plays a Major Role in Chromosome Rearrangements That Occur During Meiosis of Brassica napus Haploids

    PubMed Central

    Nicolas, Stéphane D.; Mignon, Guillaume Le; Eber, Frédérique; Coriton, Olivier; Monod, Hervé; Clouet, Vanessa; Huteau, Virginie; Lostanlen, Antoine; Delourme, Régine; Chalhoub, Boulos; Ryder, Carol D.; Chèvre, Anne Marie; Jenczewski, Eric

    2007-01-01

    Chromosomal rearrangements can be triggered by recombination between distinct but related regions. Brassica napus (AACC; 2n = 38) is a recent allopolyploid species whose progenitor genomes are widely replicated. In this article, we analyze the extent to which chromosomal rearrangements originate from homeologous recombination during meiosis of haploid B. napus (n = 19) by genotyping progenies of haploid × euploid B. napus with molecular markers. Our study focuses on three pairs of homeologous regions selected for their differing levels of divergence (N1/N11, N3/N13, and N9/N18). We show that a high number of chromosomal rearrangements occur during meiosis of B. napus haploid and are transmitted by first division restitution (FDR)-like unreduced gametes to their progeny; half of the progeny of Darmor-bzh haploids display duplications and/or losses in the chromosomal regions being studied. We demonstrate that half of these rearrangements are due to recombination between regions of primary homeology, which represents a 10- to 100-fold increase compared to the frequency of homeologous recombination measured in euploid lines. Some of the other rearrangements certainly result from recombination between paralogous regions because we observed an average of one to two autosyndetic A–A and/or C–C bivalents at metaphase I of the B. napus haploid. These results are discussed in the context of genome evolution of B. napus. PMID:17151256

  14. Characterization of the murine antibody response to peptides representing the variable domains of the major outer membrane protein of Chlamydia pneumoniae.

    PubMed Central

    Peterson, E M; Cheng, X; Qu, Z; de La Maza, L M

    1996-01-01

    In an attempt to gain more knowledge about the immunogenicity of the variable domains (VDs) of the major outer membrane protein (MOMP) of Chlamydia pneumoniae, peptides representing these areas were used to immunize BALB/c and C57BL/6 mice. Antisera to the peptides and to peptides conjugated to keyhole limpet hemocyanin (KLH) were characterized by their ability to recognize the immunizing peptide and elementary bodies (EBs) of C. pneumoniae by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot). In addition, antiserum was analyzed for its molecular specificity by a pepscan as well as its in vitro neutralizing ability. In general, results obtained with antisera to the peptides paralleled the results obtained with the antisera to the KLH-conjugated peptides except that the titers or strength of reaction in the assays was less. Antisera to the VDs in both strains of mice gave ELISA titers to the homologous VD peptide ranging from 1,000 to >64,000. The strength of reactivity with the reduced MOMP as judged by Western blot, in most cases, paralleled the ELISA titer to the peptide. However, only antisera raised in both strains of mice to the VD1 and VD4 peptides reacted strongly with the EBs, suggesting surface exposure of these VDs. In addition, antisera to VD3 from C57BL/6 mice gave strong reactivity to EBs. By pepscan analysis antisera from both strains of mice reacted with several VD1 and VD3 octameric peptides, with weaker reactivity being seen with the octameric peptides in the other two VDs. This was in contrast to antisera raised to EBs of C. pneumoniae TW-183, which identified two immunogenic regions, one in VD1 and the other mapped to VD4. While antisera raised to EBs strongly neutralized the infectivity of C. pneumoniae, none of the peptide antisera was able to neutralize. In addition, peptides to the VDs were not able to block the neutralizing ability of the antisera to EBs of C. pneumoniae. Therefore, these results suggest that the VDs of

  15. Improved Immunogenicity of a Vaccination Regimen Combining a DNA Vaccine Encoding Brucella melitensis Outer Membrane Protein 31 (Omp31) and Recombinant Omp31 Boosting▿

    PubMed Central

    Cassataro, Juliana; Velikovsky, Carlos A.; Bruno, Laura; Estein, Silvia M.; de la Barrera, Silvia; Bowden, Raúl; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2007-01-01

    In the present study, we report an attempt to improve the immunogenicity of the Omp31 antigen by a DNA prime-protein boost immunization regimen. We immunized BALB/c mice with an Omp31 DNA vaccine (pCIOmp31) followed by boosting with recombinant Omp31 (rOmp31) in incomplete Freund's adjuvant and characterized the resulting immune responses and the protective efficacy against Brucella ovis and B. melitensis infection. Immunoglobulin G1 (IgG1) and IgG2a titers were higher in sera from pCIOmp31/rOmp31-immunized mice than in sera from mice immunized with pCIOmp31 or rOmp31 alone. Splenocytes from pCIOmp31/rOmp31-immunized mice produced significantly higher levels of gamma interferon than did those from mice given rOmp31 alone. In contrast, interleukin 2 (IL-2) production levels were comparable between the two groups of immunized mice. Cells from all immunized mice produced undetectable levels of IL-4. Notably, rOmp31 stimulated IL-10 production in the pCIOmp31/rOmp31-immunized group but not in the pCIOmp31- or rOmp31-immunized group. Although the prime-boost regimen induced specific cytotoxic responses, these responses could not reach the levels achieved by the pCIOmp31 immunization. In conclusion, pCIOmp31 priming followed by rOmp31 boosting led to moderately improved protection against a challenge with B. ovis or B. melitensis. PMID:17428946

  16. Protection of pigs from swine dysentery by vaccination with recombinant BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae.

    PubMed

    La, Tom; Phillips, Nyree D; Reichel, Michael P; Hampson, David J

    2004-08-19

    Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study the efficacy of BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae, was evaluated as an SD vaccine. Non-lipidated BmpB was expressed in Escherichia coli as a histidine-tagged protein (His6-BmpB), or as an 8 kDa carboxy-terminal portion fused to maltose-binding protein (MBP-BmpB-F604). The purified proteins were emulsified with oil-based adjuvants for intramuscular (im) administrations. In experiment 1, 20 weaner pigs were vaccinated im with 1 mg of His6-BmpB. After 3 weeks, 10 received 1 mg of the protein orally (im/oral), and 10 received 1 mg im (im/im). Ten acted as unvaccinated controls. In experiment 2, 12 pigs were vaccinated im with 1 mg of His6-BmpB, and 12 with 1 mg of MBP-BmpB-F604. Three weeks later, each was given 1 mg of the same protein orally. Twelve pigs acted as unvaccinated controls. All pigs were challenged orally with B. hyodysenteriae 2 weeks after their second vaccination. In both experiments, all pigs vaccinated with His6-BmpB developed serum antibodies to BmpB, and oral administration provided boosting of im-induced serum antibody titres. In experiment 1, seven non-vaccinated control pigs developed dysentery and severe colitis. Three pigs vaccinated im/oral developed diarrhoea; two had severe colitis and one had mild lesions. Four pigs vaccinated im/im developed diarrhoea; one had severe colitis and the others had mild lesions. In experiment 2, six control pigs developed SD with severe colitis. Two His6-BmpB vaccinated pigs developed SD with mild colitis. Nine pigs vaccinated with MBP-BmpB-F604 developed SD and severe colitis. Overall, 50-70% of controls and 17-40% of His6-BmpB vaccinated pigs developed disease. Vaccination with MBP-BmpB-F604 did not induce serum titres against BmpB, nor confer protection. The incidence of disease for the three His6-BmpB vaccinated groups was significantly less (P = 0

  17. Bacterial fermentation of recombinant major wasp allergen Antigen 5 using oxygen limiting growth conditions improves yield and quality of inclusion bodies.

    PubMed

    Kischnick, Stefanie; Weber, Bernhard; Verdino, Petra; Keller, Walter; Sanders, Ernst A; Anspach, F Birger; Fiebig, Helmut; Cromwell, Oliver; Suck, Roland

    2006-06-01

    A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.

  18. 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.

  19. [Site-directed mutagensis of the major antigen E2 gene of CSFV, its high level expression in Escherichia coli and the immunonicity of recombinant E2 protein].

    PubMed

    Yu, Xing-Long; Tu, Chang-Chun; Xu, Xing-Ran; Zhang, Mao-Lin; Chen, Yi-Xiang; Liu, Bo-Hua

    2003-07-01

    Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The

  20. 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

  1. 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

  2. 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.

  3. Prdm9, a major determinant of meiotic recombination hotspots, is not functional in dogs and their wild relatives, wolves and coyotes.

    PubMed

    Muñoz-Fuentes, Violeta; Di Rienzo, Anna; Vilà, Carles

    2011-01-01

    Meiotic recombination is a fundamental process needed for the correct segregation of chromosomes during meiosis in sexually reproducing organisms. In humans, 80% of crossovers are estimated to occur at specific areas of the genome called recombination hotspots. Recently, a protein called PRDM9 was identified as a major player in determining the location of genome-wide meiotic recombination hotspots in humans and mice. The origin of this protein seems to be ancient in evolutionary time, as reflected by its fairly conserved structure in lineages that diverged over 700 million years ago. Despite its important role, there are many animal groups in which Prdm9 is absent (e.g. birds, reptiles, amphibians, diptera) and it has been suggested to have disruptive mutations and thus to be a pseudogene in dogs. Because of the dog's history through domestication and artificial selection, we wanted to confirm the presence of a disrupted Prdm9 gene in dogs and determine whether this was exclusive of this species or whether it also occurred in its wild ancestor, the wolf, and in a close relative, the coyote. We sequenced the region in the dog genome that aligned to the last exon of the human Prdm9, containing the entire zinc finger domain, in 4 dogs, 17 wolves and 2 coyotes. Our results show that the three canid species possess mutations that likely make this gene non functional. Because these mutations are shared across the three species, they must have appeared prior to the split of the wolf and the coyote, millions of years ago, and are not related to domestication. In addition, our results suggest that in these three canid species recombination does not occur at hotspots or hotspot location is controlled through a mechanism yet to be determined.

  4. 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.

  5. Influence of the maillard reaction on the allergenicity of rAra h 2, a recombinant major allergen from peanut (Arachis hypogaea), its major epitopes, and peanut agglutinin.

    PubMed

    Gruber, Patrick; Becker, Wolf-Meinhard; Hofmann, Thomas

    2005-03-23

    The influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's natural counterpart. ELISA experiments as well as inhibition assays revealed that thermal treatment of rAra h 2 in the presence of reactive carbohydrates and carbohydrate breakdown products induces a strong increase of the IgE-binding activity, thus collaborating with the data reported for the natural protein isolated from peanuts. To localize the Ara h 2 sequences responsible for the formation of highly IgE-affine glycation sites, model peptides have been synthesized mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes. Immunological evaluation of these peptides heated in the absence or presence of reducing sugars and carbonyls, respectively, revealed that neither the two lysine residues of Ara h 2 nor its N-terminus are involved in the formation of IgE-affine structures by Maillard reaction. Also, the cysteine-containing major epitope 3 (aa 27-36) was found to lose its IgE-binding capacity upon heating. By contrast, the overlapping major epitopes 6 and 7, which do not contain any lysine or arginine moieties, showed a distinct higher level of IgE binding when subjected to Maillard reaction, thus giving the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Analogous experiments were performed with peanut agglutinin, considered in the literature as a minor allergen. ELISA experiments revealed that the majority of tested sera samples from peanut-sensitive patients showed a high level of IgE binding to the lectin even after heat treatment. In contradiction to published data, nonenzymatic browning reactions seem to deteriorate the IgE affinity of the lectin. PMID:15769170

  6. Vaccination with recombinant Parasite Surface Antigen 2 from Leishmania major induces a Th1 type of immune response but does not protect against infection.

    PubMed

    Sjölander, A; Baldwin, T M; Curtis, J M; Bengtsson, K L; Handman, E

    1998-12-01

    Vaccination with the native Parasite Surface Antigen 2 of Leishmania major with Corynebacterium parvum as adjuvant protects mice from leishmaniasis through a Th1 mediated response. Here we show that vaccination with a recombinant form of this protein, purified from Escherichia coli and administered in iscoms or with C. parvum as adjuvant, does not induce protective immunity despite the induction of Th1 responses. The results suggest that protective immunity depends on the ability of the vaccinating antigen to induce Th1-like T cells with ability to be recalled by infection. Therefore, the conformation of antigens may play a more major role for the induction of T cell mediated immunity than originally considered. PMID:9796067

  7. Cadaverine covalently linked to peptidoglycan is required for interaction between the peptidoglycan and the periplasm-exposed S-layer-homologous domain of major outer membrane protein Mep45 in Selenomonas ruminantium.

    PubMed

    Kojima, Seiji; Ko, Kyong-Cheol; Takatsuka, Yumiko; Abe, Naoki; Kaneko, Jun; Itoh, Yoshifumi; Kamio, Yoshiyuki

    2010-11-01

    The peptidoglycan of Selenomonas ruminantium is covalently bound to cadaverine (PG-cadaverine), which likely plays a significant role in maintaining the integrity of the cell surface structure. The outer membrane of this bacterium contains a 45-kDa major protein (Mep45) that is a putative peptidoglycan-associated protein. In this report, we determined the nucleotide sequence of the mep45 gene and investigated the relationship between PG-cadaverine, Mep45, and the cell surface structure. Amino acid sequence analysis showed that Mep45 is comprised of an N-terminal S-layer-homologous (SLH) domain followed by α-helical coiled-coil region and a C-terminal β-strand-rich region. The N-terminal SLH domain was found to be protruding into the periplasmic space and was responsible for binding to peptidoglycan. It was determined that Mep45 binds to the peptidoglycan in a manner dependent on the presence of PG-cadaverine. Electron microscopy revealed that defective PG-cadaverine decreased the structural interactions between peptidoglycan and the outer membrane, consistent with the proposed role for PG-cadaverine. The C-terminal β-strand-rich region of Mep45 was predicted to be a membrane-bound unit of the 14-stranded β-barrel structure. Here we propose that PG-cadaverine possesses functional importance to facilitate the structural linkage between peptidoglycan and the outer membrane via specific interaction with the SLH domain of Mep45.

  8. A Recombinant Antibody with the Antigen-Specific, Major Histocompatibility Complex-Restricted Specificity of T Cells

    NASA Astrophysics Data System (ADS)

    Andersen, Peter S.; Stryhn, Anette; Hansen, Bjarke E.; Fugger, Lars; Engberg, Jan; Buus, Soren

    1996-03-01

    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.

  9. One-step purification and porin transport activity of the major outer membrane proteins P2 from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis.

    PubMed

    Kattner, Christof; Pfennig, Sabrina; Massari, Paola; Tanabe, Mikio

    2015-03-01

    Bacterial porins are major outer membrane proteins that function as essential solute transporters between the bacteria and the extracellular environment. Structural features of porins are also recognized by eukaryotic cell receptors involved in innate and adaptive immunity. To better investigate the function of porins, proper refolding is necessary following purification from inclusion bodies [1, 2]. Using a single-step size exclusion chromatographic method, we have purified three major porins from pathogenic bacteria, the OmpP2 (P2) from Haemophilus influenzae, FomA from Fusobacterium nucleatum and PorB from Neisseria meningitidis, at high yield and report their unique solute transport activity with size exclusion limit. Furthermore, we have optimized their purification method and achieved improvement of their thermostability for facilitating functional and structural analyses.

  10. Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species.

    PubMed

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1993-01-01

    DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies.

  11. Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species.

    PubMed Central

    Kaltenboeck, B; Kousoulas, K G; Storz, J

    1993-01-01

    DNA sequences coding for 81% of the ompA gene from 24 chlamydial strains, representing all chlamydial species, were determined from DNA amplified by polymerase chain reactions. Chlamydial strains of serovars and strains with similar chromosomal restriction fragment length polymorphism had identical ompA DNA sequences. The ompA sequences were segregated into 23 different ompA alleles and aligned with each other, and phylogenetic relationships among them were inferred by neighbor-joining and maximum parsimony analyses. The neighbor-joining method produced a single phylogram which was rooted at the branch between two major clusters. One cluster included all Chlamydia trachomatis ompA alleles (trachoma group). The second cluster was composed of three major groups of ompA alleles: psittacosis group (alleles MN, 6BC, A22/M, B577, LW508, FEPN, and GPIC), pneumonia group (Chlamydia pneumoniae AR388 with the allele KOALA), and polyarthritis group (ruminant and porcine chlamydial alleles LW613, 66P130, L71, and 1710S with propensity for polyarthritis). These groups were distinguished through specific DNA sequence signatures. Maximum parsimony analysis yielded two equally most parsimonious phylograms with topologies similar to the ompA tree of neighbor joining. Two phylograms constructed from chlamydial genomic DNA distances had topologies identical to that of the ompA phylogram with respect to branching of the chlamydial species. Human serovars of C. trachomatis with essentially identical genomes represented a single taxonomic unit, while they were divergent in the ompA tree. Consistent with the ompA phylogeny, the porcine isolate S45, previously considered to be Chlamydia psittaci, was identified as C. trachomatis through biochemical characteristics. These data demonstrate that chlamydial ompA allelic relationships, except for human serovars of C. trachomatis, are cognate with chromosomal phylogenies. Images PMID:8419295

  12. Vaccination with the Recombinant Brucella Outer Membrane Protein 31 or a Derived 27-Amino-Acid Synthetic Peptide Elicits a CD4+ T Helper 1 Response That Protects against Brucella melitensis Infection

    PubMed Central

    Cassataro, Juliana; Estein, Silvia M.; Pasquevich, Karina A.; Velikovsky, Carlos A.; de la Barrera, Silvia; Bowden, Raúl; Fossati, Carlos A.; Giambartolomei, Guillermo H.

    2005-01-01

    The immunogenicity and protective efficacy of the recombinant 31-kDa outer membrane protein from Brucella melitensis (rOmp31), administered with incomplete Freund's adjuvant, were evaluated in mice. Immunization of BALB/c mice with rOmp31 conferred protection against B. ovis and B. melitensis infection. rOmp31 induced a vigorous immunoglobulin G (IgG) response, with higher IgG1 than IgG2 titers. In addition, spleen cells from rOmp31-immunized mice produced interleukin 2 (IL-2) and gamma interferon, but not IL-10 or IL-4, after in vitro stimulation with rOmp31, suggesting the induction of a T helper 1 (Th1) response. Splenocytes from rOmp31-vaccinated animals also induced a specific cytotoxic-T-lymphocyte activity, which led to the in vitro lysis of Brucella-infected macrophages. In vitro T-cell subset depletion indicated that rOmp31 immunization elicited specific CD4+ T cells that secrete IL-2 and gamma interferon, while CD8+ T cells induced cytotoxic-T-lymphocyte activity. In vivo depletion of T-cell subsets showed that the rOmp31-elicited protection against B. melitensis infection is mediated by CD4+ T cells while the contribution of CD8+ T cells may be limited. We then evaluated the immunogenicity and protective efficacy of a known exposed region from Omp31 on the Brucella membrane, a peptide that contains amino acids 48 to 74 of Omp31. Immunization with the synthetic peptide in adjuvant did not elicit a specific humoral response but elicited a Th1 response mediated by CD4+ T cells. The peptide in adjuvant induced levels of protection similar to those induced by rOmp31 against B. melitensis but less protection than was induced by rOmp31 against B. ovis. Our results indicate that rOmp31 could be a useful candidate for the development of subunit vaccines against B. melitensis and B. ovis. PMID:16299302

  13. Protective immunity of a Pichia pastoris expressed recombinant iridovirus major capsid protein in the Chinese giant salamander, Andrias davidianus.

    PubMed

    Zhou, Yong; Fan, Yuding; LaPatra, Scott E; Ma, Jie; Xu, Jin; Meng, Yan; Jiang, Nan; Zeng, Lingbing

    2015-10-13

    The major capsid protein (MCP) is the main immunogenic protein of iridoviruses, that has been widely used as an immunogen in vaccination trials. In this study, the codon-optimized giant salamander iridovirus (GSIV) MCP gene (O-MCP) was synthesized and cloned into a pPICZα B vector for secretory expression in the methylotrophic yeast Pichia pastoris after methanol induction. The expression of the O-MCP protein was detected by the Bradford protein assay, SDS-PAGE, Western blotting and electron microscopy. The Bradford protein assay indicated that the concentration of the O-MCP expressed was about 40 μg/ml in culture supernatants. SDS-PAGE analysis revealed that the O-MCP had a molecular weight of about 66 kDa and reacted with a His-specific MAb that was confirmed by Western blotting. Electron microscopy observations revealed that the purified O-MCP could self-assemble into virus-like particles. Healthy giant salamanders were vaccinated by intramuscular injection with the O-MCP antigen at a dose of 20 μg/individual. The numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly at day 3 and reached a peak at day 5 post-immunization. Meanwhile, the differential leukocyte counts of monocytes and neutrophils increased significantly at day 5 post-immunization compared to that of the control group. The percentage of lymphocytes was 71.33 ± 3.57% at day 21 post-immunization. The neutralization assay showed that the serum neutralizing antibody titer reached 321 at day 21 post-immunization. The GSIV challenge test revealed that the relative percent survival of Chinese giant salamanders vaccinated with O-MCP was 78%. These results indicated that the O-MCP antigen expressed by the Pichia pastoris system elicited significant immune response in the Chinese giant salamander against GSIV and might represent a potential yeast-derived vaccine candidate that could be used for the control of disease caused by the

  14. 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

  15. 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.

  16. 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

  17. 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

  18. Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein.

    PubMed

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

    2010-11-15

    To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of 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. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.

  19. Immune response and protection assay of recombinant major surface glycoprotein of Leishmania (rgp63) reconstituted with liposomes in BALB/c mice.

    PubMed

    Jaafari, Mahmoud R; Ghafarian, Attieh; Farrokh-Gisour, Ahsan; Samiei, Afshin; Kheiri, Masoumeh Tavassoti; Mahboudi, Fereidoun; Barkhordari, Farzaneh; Khamesipour, Ali; McMaster, W Robert

    2006-07-17

    In this study the ability of recombinant gp63 entrapped in liposomes to induce immune response and protection against L. major infection in susceptible BALB/c mice was studied. Liposomes containing rgp63 (Lip-rgp63) were prepared from egg lecithin and cholesterol using detergent solubilization method. Immunization of BALB/c mice with rgp63 alone conferred a partial protection while entrapment of rgp63 in liposomes significantly increased the rate of protection (P<0.05). The parasite burden of spleen in mice challenged with L. major was significantly (p<0.001) lower in group of mice immunized with rgp63 alone or Lip-rgp63, however, the least parasite burden was seen in Lip-rgp63 group. Both rgp63 alone and Lip-rgp63 elicited significant delayed-type hypersensitivity (DTH) response compared to controls (p<0.01), however, the DTH response of PBS-rgp63 was less than the Lip-rgp63. Titration of anti-Leishmania IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type of immune response only in mice immunized with Lip-rgp63. The results indicate that liposomes might be used as a suitable immunoadjuvant for development of Leishmania vaccine. PMID:16740346

  20. Protective efficacy of major outer membrane protein-specific immunoglobulin A (IgA) and IgG monoclonal antibodies in a murine model of Chlamydia trachomatis genital tract infection.

    PubMed

    Cotter, T W; Meng, Q; Shen, Z L; Zhang, Y X; Su, H; Caldwell, H D

    1995-12-01

    The protective efficacy of immunoglobulin A (IgA) and IgG monoclonal antibodies (MAbs) specific for the major outer membrane protein of Chlamydia trachomatis MoPn was evaluated in a murine genital tract infection model. MAbs were delivered into serum and vaginal secretions of naive mice by using the backpack hybridoma tumor system, and protective efficacy was assessed over the first 8 days following challenge by quantitative determination of chlamydial recovery from cervicovaginal swabs, histopathological evaluation of genital tract tissue, and immunohistochemical detection of chlamydial inclusions. IgA and IgG significantly reduced the incidence of infection following vaginal challenge with 5 50% infectious doses, but such protection was overwhelmed by 10- and 100-fold higher challenge doses. Both MAbs also consistently reduced vaginal shedding from infected animals with all three challenge doses compared with the negative control MAb, although the magnitude of this effect was marginal. Blinded pathological evaluation of genital tract tissues at 8 days postinfection showed a significant reduction in the severity of the inflammatory infiltrate in oviduct tissue of infected IgA- and IgG-treated animals. Immunohistochemical detection of chlamydial inclusions revealed a marked reduction in the chlamydial burden of the oviduct epithelium; this finding is consistent with the reduced pathological changes observed in this tissue. These studies indicate that the presence of IgA or IgG MAbs specific to major outer membrane proteins has a marginal effect in preventing chlamydial colonization and shedding from the genital tract but has a more pronounced effect on ascending chlamydial infection and accompanying upper genital tract pathology.

  1. 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.

  2. 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

  3. 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.

  4. 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

  5. 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

  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. Disentangling the effects of recombination, selection, and demography on the genetic variation at a major histocompatibility complex class II gene in the alpine chamois.

    PubMed

    Mona, S; Crestanello, B; Bankhead-Dronnet, S; Pecchioli, E; Ingrosso, S; D'Amelio, S; Rossi, L; Meneguz, P G; Bertorelle, G

    2008-09-01

    The major histocompatibility complex (MHC) harbours some of the most polymorphic loci in vertebrate genomes. MHC genes are thought to be subject to some form of balancing selection, most likely pathogen-mediated selection. Hence, MHC genes are excellent candidates for exploring adaptive processes. In this study, we investigated the genetic variation at exon 2 of the DRB class II MHC locus in 191 alpine chamois (Rupicapra rupicapra) from 10 populations in the eastern Alps of Italy. In particular, we were interested in distinguishing and estimating the relative impact of selective and demographic factors, while taking into account the confounding effect of recombination. The extremely high d(n)/d(s) ratio and the presence of trans-species polymorphisms suggest that a strong long-term balancing selection effect has been operating at this locus throughout the evolutionary history of this species. We analysed patterns of genetic variation within and between populations, and the mitochondrial D-loop polymorphism patterns were analysed to provide a baseline indicator of the effects of demographic processes. These analyses showed that (i) the chamois experienced a demographic decline in the last 5000-30 000 years, most likely related to the postglacial elevation in temperature; (ii) this demographic process can explain the results of neutrality tests applied to MHC variation within populations, but cannot justify the much weaker divergence between populations implied by MHC as opposed to mitochondrial DNA; (iii) similar sets of divergent alleles are probably maintained with similar frequencies by balancing selection in different populations, and this mechanism is also operating in small isolated populations, which are strongly affected by drift.

  8. 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.

  9. A vaccine formulated with the major outer membrane protein can protect C3H/HeN, a highly susceptible strain of mice, from a Chlamydia muridarum genital challenge.

    PubMed

    Pal, Sukumar; Tatarenkova, Olga V; de la Maza, Luis M

    2015-11-01

    C3H/HeN female mice were vaccinated with native Chlamydia muridarum major outer membrane protein (MOMP), using Montanide+CpG or Alum+CpG as adjuvants. Negative control groups were immunized with ovalbumin (OVA) and the same adjuvants. As positive control, mice were inoculated intranasally with live Chlamydia. Mice were challenged in the ovarian bursa with 10(5) C. muridarum inclusion forming units. Six weeks after the genital challenge the animals were caged with male mice and monitored for pregnancy. Mice vaccinated with MOMP+Montanide+CpG developed high levels of C. muridarum-specific antibodies, with a high IgG2a/IgG1 ratio and neutralizing titres. Animals immunized using Alum+CpG had low antibody levels. Cellular immune responses were significantly higher in mice vaccinated with MOMP and Montanide+CpG, but not with Alum+CpG, when compared with negative controls. Following the genital challenge, only 20% (4/20) of mice vaccinated with MOMP+CpG+Montanide had positive vaginal cultures whereas 100% (9/9) of mice immunized with MOMP+CpG+Alum had positive cultures. Of the positive control animals inoculated with live Chlamydia only 15% (3/20) had positive vaginal cultures. In contrast, 100% (20/20) of mice immunized with OVA+CpG+Montanide, or minimal essential medium, had positive cultures. Following mating, 80% (16/20) of mice vaccinated with MOMP+CpG+Montanide, and 85% (17/20) of animals inoculated intranasally with live C. muridarum carried embryos in both uterine horns. No protection against infertility was observed in mice immunized with MOMP and CpG+Alum or OVA. In conclusion, this is the first time that a subunit vaccine has been shown to elicit a protective immune response in the highly susceptible C3H/HeN strain of mice against an upper genital challenge.

  10. A vaccine formulated with the major outer membrane protein can protect C3H/HeN, a highly susceptible strain of mice, from a Chlamydia muridarum genital challenge

    PubMed Central

    Pal, Sukumar; Tatarenkova, Olga V; de la Maza, Luis M

    2015-01-01

    C3H/HeN female mice were vaccinated with native Chlamydia muridarum major outer membrane protein (MOMP), using Montanide+CpG or Alum+CpG as adjuvants. Negative control groups were immunized with ovalbumin (OVA) and the same adjuvants. As positive control, mice were inoculated intranasally with live Chlamydia. Mice were challenged in the ovarian bursa with 105 C. muridarum inclusion forming units. Six weeks after the genital challenge the animals were caged with male mice and monitored for pregnancy. Mice vaccinated with MOMP+Montanide+CpG developed high levels of C. muridarum-specific antibodies, with a high IgG2a/IgG1 ratio and neutralizing titres. Animals immunized using Alum+CpG had low antibody levels. Cellular immune responses were significantly higher in mice vaccinated with MOMP and Montanide+CpG, but not with Alum+CpG, when compared with negative controls. Following the genital challenge, only 20% (4/20) of mice vaccinated with MOMP+CpG+Montanide had positive vaginal cultures whereas 100% (9/9) of mice immunized with MOMP+CpG+Alum had positive cultures. Of the positive control animals inoculated with live Chlamydia only 15% (3/20) had positive vaginal cultures. In contrast, 100% (20/20) of mice immunized with OVA+CpG+Montanide, or minimal essential medium, had positive cultures. Following mating, 80% (16/20) of mice vaccinated with MOMP+CpG+Montanide, and 85% (17/20) of animals inoculated intranasally with live C. muridarum carried embryos in both uterine horns. No protection against infertility was observed in mice immunized with MOMP and CpG+Alum or OVA. In conclusion, this is the first time that a subunit vaccine has been shown to elicit a protective immune response in the highly susceptible C3H/HeN strain of mice against an upper genital challenge. PMID:26423798

  11. A major recombination hotspot in the XqYq pseudoautosomal region gives new insight into processing of human gene conversion events.

    PubMed

    Sarbajna, Shriparna; Denniff, Matthew; Jeffreys, Alec J; Neumann, Rita; Soler Artigas, María; Veselis, Amelia; May, Celia A

    2012-05-01

    Recombination plays a fundamental role in meiosis. Non-exchange gene conversion (non-crossover, NCO) may facilitate homologue pairing, while reciprocal crossover (CO) physically connects homologues so they orientate appropriately on the meiotic spindle. In males, X-Y homologous pairing and exchange occurs within the two pseudoautosomal regions (PARs) together comprising <5% of the human sex chromosomes. Successful meiosis depends on an obligatory CO within PAR1, while the nature and role of exchange within PAR2 is unclear. Here, we describe the identification and characterization of a typical ~1 kb wide recombination hotspot within PAR2. We find that both COs and NCOs are strongly modulated in trans by the presumed chromatin remodelling protein PRDM9, and in cis by a single nucleotide polymorphism (SNP) located at the hotspot centre that appears to influence recombination initiation and which causes biased gene conversion in SNP heterozygotes. This, the largest survey to date of human NCOs reveals for the first time substantial inter-individual variation in the NCO:CO ratio. Although the extent of biased transmission at the central marker in COs is similar across men, it is highly variable among NCO recombinants. This suggests that cis-effects are mediated not only through recombination initiation frequencies varying between haplotypes but also through subsequent processing, with the potential to significantly intensify meiotic drive of hotspot-suppressing alleles. The NCO:CO ratio and extent of transmission distortion among NCOs appear to be inter-related, suggesting the existence of two NCO pathways in humans. PMID:22291443

  12. 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.

  13. Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

    PubMed

    Vieths, S; Barber, D; Chapman, M; Costanzo, A; Daas, A; Fiebig, H; Hanschmann, K M; Hrabina, M; Kaul, S; Ledesma, A; Moingeon, P; Reese, G; Schörner, C; van Ree, R; Weber, B; Buchheit, K H

    2012-04-01

    The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the

  14. The leptospiral outer membrane.

    PubMed

    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 to 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.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 outer membrane proteins (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.

  15. 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.

  16. 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…

  17. Outer planet satellites

    NASA Astrophysics Data System (ADS)

    Schenk, Paul M.

    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.

  18. 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.

  19. Evaluation of immune responses and analysis of the effect of vaccination of the Leishmania major recombinant ribosomal proteins L3 or L5 in two different murine models of cutaneous leishmaniasis.

    PubMed

    Ramírez, Laura; Santos, Diego M; Souza, Ana P; Coelho, Eduardo A F; Barral, Aldina; Alonso, Carlos; Escutia, Marta R; Bonay, Pedro; de Oliveira, Camila I; Soto, Manuel

    2013-02-18

    Four new antigenic proteins located in Leishmania ribosomes have been characterized: S4, S6, L3 and L5. Recombinant versions of the four ribosomal proteins from Leishmania major were recognized by sera from human and canine patients suffering different clinical forms of leishmaniasis. The prophylactic properties of these proteins were first studied in the experimental model of cutaneous leishmaniasis caused by L. major inoculation into BALB/c mice. The administration of two of them, LmL3 or LmL5 combined with CpG-oligodeoxynucleotides (CpG-ODN) was able to protect BALB/c mice against L. major infection. Vaccinated mice showed smaller lesions and parasite burden compared to mice inoculated with vaccine diluent or vaccine adjuvant. Protection was correlated with an antigen-specific increased production of IFN-γ paralleled by a decrease of the antigen-specific IL-10 mediated response in protected mice relative to non-protected controls. Further, it was demonstrated that BALB/c mice vaccinated with recombinant LmL3 or LmL5 plus CpG-ODN were also protected against the development of cutaneous lesions following inoculation of L. braziliensis. Together, data presented here indicate that LmL3 or LmL5 ribosomal proteins combined with Th1 inducing adjuvants, may be relevant components of a vaccine against cutaneous leishmaniasis caused by distinct species.

  20. Inhibition of homologous recombination repair with Pentoxifylline targets G2 cells generated by radiotherapy and induces major enhancements of the toxicity of cisplatin and melphalan given after irradiation

    PubMed Central

    Bohm, Lothar

    2006-01-01

    The presentation reviews the modus operandi of the dose modifying drug Pentoxifylline and the dose enhancement factors which can be achieved in different cell types. Preclinical and clinical data show that Pentoxifylline improves the oxygenation of hypoxic tumours and enhances tumour control by irradiation. In vitro experiments demonstrate that Pentoxifylline also operates when oxygen is not limiting and produces dose modifying factors in the region of 1.2 – 2.0. This oxygen independent effect is poorly understood. In p53 mutant cells irradiation induces a G2 block which is abrogated by Pentoxifylline. The enhancement of cell kill observed when Pentoxifylline and irradiation are given together could arise from rapid entry of damaged tumour cells into mitosis and propagation of DNA lesions as the result of curtailment of repair time. Recovery ratios and repair experiments using CFGE after high dose irradiation demonstrate that Pentoxifylline inhibits repair directly and that curtailment of repair time is not the explanation. Use of the repair defective xrs1 and the parental repair competent CHO-K1 cell line shows that Pentoxifylline inhibits homologous recombination repair which operates predominantly in the G2 phase of the cell cycle. When irradiated cells residing in G2 phase are exposed to very low doses of cisplatin at a toxic dose of 5 %. (TC: 0.05) massive toxicity enhancements up to a factor of 80 are observed in melanoma, squamous carcinoma and prostate tumour cell lines. Enhancements of radiotoxicity seen when Pentoxifylline and radiation are applied together are small and do not exceed a factor of 2.0. The capacity of Pentoxifyline to inhibit homologous recombination repair has not as yet been clinically utilized. A suitable application could be in the treatment of cervical carcinoma where irradiation and cisplatin are standard modality. In vitro data also strongly suggest that regimes where irradiation is used in combination with alkylating drugs may

  1. 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

  2. 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

  3. 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.

  4. Channel forming outer membrane porin protein in halophile: expressed as a soluble form in Escherichia coli.

    PubMed

    Tokunaga, Hiroko; Furukawa, Masafumi; Arakawa, Tsutomu; Tokunaga, Masao

    2013-03-01

    We have previously found that the N-terminal sequence of the outer membrane protein from moderate halophile is similar to the sequence of the well-known pore forming porin proteins from other Gram-negative bacteria. This highly expressed outer membrane protein was purified from Halomonas sp. 40 and reconstituted into liposome. It showed a permeability activity in the liposome swelling assay. Based on the N-terminal and internal amino acid sequences of this major outer membrane, we have cloned here the porin gene, hopP (halophilic outer membrane protein), from Halomonas sp. 40. The hopP gene encodes the porin precursor comprising 366 amino acid residues that include a 21 amino acid signal peptide. Mature porin (345 amino acids, 37,611 Da) is a highly acidic protein, just as is so for many halophilic proteins and was soluble when expressed in Escherichia coli with N-terminal His-tag. Purified recombinant His-porin was soluble even after heat-treatment at 95 °C for 5 min in the absence of salt. Circular dichroism analysis of His-porin showed conversion into a β-sheet rich structure by the addition of NaCl at 0.9-2.7 M.

  5. 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.

  6. 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

  7. Refolding and functional assembly of the Vibrio cholerae porin OmpU recombinantly expressed in the cytoplasm of Escherichia coli.

    PubMed

    Khan, Junaid; Gupta, Shelly; Chattopadhyay, Kausik; Mukhopadhaya, Arunika

    2012-10-01

    OmpU is one of the major outer membrane porins of Vibrio cholerae. OmpU has been biochemically characterized previously for its 'porin'-property. However, previous studies have used the OmpU protein extracted from the bacterial outer membrane envelope fractions. Such method of isolation imposes limitations on the availability of the protein reagent, and also enhances the possibility of the OmpU preparation being contaminated with lipid molecules of bacterial outer membrane origin, especially lipopolysaccharides (LPS). Here we report a strategy of purifying the V. cholerae OmpU protein recombinantly overexpressed in heterologous protein expression system in Escherichia coli, without its being incorporated into the bacterial membrane fraction. In our strategy, the majority of the protein was expressed as insoluble inclusion body in the E. coli cytoplasm, the protein was dissolved by denaturation in 8M urea, refolded, and purified to homogeneity in presence of detergent. Our strategy allowed isolation of the recombinant OmpU protein with significantly enhanced yield as compared to that of the wild type protein extracted from the V. cholerae membrane fraction. The recombinant V. cholerae OmpU protein generated in our study displayed functional channel-forming property in the synthetic liposome membrane, thus confirming its 'porin'-property. To the best of our knowledge, this is the first report showing an efficient refolding and functional assembly of the V. cholerae OmpU porin recombinantly expressed as inclusion body in the cytoplasm of a heterologous host E. coli.

  8. Outer membrane protein profiles of Haemophilus pleuropneumoniae.

    PubMed Central

    Rapp, V J; Munson, R S; Ross, R F

    1986-01-01

    Outer membrane protein profiles of Haemophilus pleuropneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells were disrupted by sonication, and outer membrane-enriched fractions were prepared by differential centrifugation and selective solubilization of the inner membrane with sodium N-lauroyl sarcosinate. Colony type, growth medium, time of harvest, and in vitro or in vivo passage had no appreciable effect on the protein profiles of the strains examined. Seven patterns were distinguished among the reference strains of the nine capsular serotypes. These patterns were based on the mobility of the major outer membrane proteins migrating in the 39,000- to 44,000-molecular-weight region of the gel, a 16K to 16.5K protein, and a heat-modifiable 29K protein. Strains of serotypes 1 and 9 had identical outer membrane protein profiles, as did strains of serotypes 2 and 6. The reference strains of the remaining five serotypes each had a distinct pattern. The outer membrane protein profiles of 95 field isolates belonging to serotypes 1, 5, 7, and 9 from swine in the midwestern United States were determined and compared with the reference patterns. The results indicate that the population of H. pleuropneumoniae is clonal, with three predominant clones distinguished by both serotype and outer membrane protein profile responsible for the majority of H. pleuropneumoniae disease occurring in swine in the United States. Images PMID:3699889

  9. The Outer Ejecta

    NASA Astrophysics Data System (ADS)

    Weis, Kerstin

    η Carinae is surrounded by a complex circumstellar nebula ejected during more than one eruption, the great eruption in the 1840s and the second or lesser eruption in the 1890s. Beyond the well-defined edges of its famous bipolar nebula are additional nebulous features and ejecta referred to as the outer ejecta. The outer ejecta includes a variety of structures of very different sizes and morphologies distributed in a region 0.67 pc in diameter with a mass of > 2-4 M⊙. Some individual features in the outer ejecta are moving extremely fast, up to 3,200 km/s, with most of the expansion velocities between 400-900 km/s. As a consequence of these high velocities, 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 follow a Hubble-flow and appear to originate at the central star. The properties of the nebulae associated with other LBVs also are described and compared with η Car. HR Car and AG Car show similar bipolar morphologies but are much older; HR Car's nebula may be η Car's older twin. The larger, extended nebulae detected around the giant eruption LBV P Cygni, and the extended nebulosity associated with AG Car and HR Car could be either from previous eruptions or facsimiles to η Car's outer ejecta.

  10. 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

  11. 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.

  12. 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)

  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. 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

  15. 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

  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. 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

  18. 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.

  19. [Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein].

    PubMed

    Makino, Masahiko; Mukai, Tetsu

    2012-09-01

    To activate naïve T cells convincingly using Mycobacterium bovis BCG (BCG), rBCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of 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. BCG-D70M was more potent in activation of both CD4+ and CD8+ subsets of naïve T cells than rBCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DC) to induce cytokine production and phenotypic changes, and activated CD4+ T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pre-treatment of DC with chloroquine inhibited both surface expression of MMP-II on DC and the activation of T cells by BCG-D70M-infected APCs. The naïve CD8+ T cell activation was inhibited by treatment of DC with brefeldin A and lactacystin so that the T cells was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naïve CD8+ T cells, effector T cells producing perforin and memory T cells having migration markers, were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70, and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II and urease depletion may provide useful tool for inducing better activation of naïve T cells.

  20. 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.

  1. 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.

  2. 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

  3. Spontaneous Regeneration of Human Photoreceptor Outer Segments.

    PubMed

    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. 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

  5. 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)

  6. Salmonella typhimurium delta aroA delta aroD mutants expressing a foreign recombinant protein induce specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes in mice.

    PubMed Central

    Turner, S J; Carbone, F R; Strugnell, R A

    1993-01-01

    Recombinant Salmonella typhimurium aroA aroD mutants which expressed ovalbumin were constructed. The two expression constructs used were based on either pUC18 or pBR322. The pBR322-based construct was more stable in vitro and in vivo than the pUC-based construct. Salmonellae containing the stable pBR322-based plasmid induced major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL), in contrast to salmonellae containing the pUC18-based expression construct. The priming of MHC class I-restricted CTL was increased by multiple immunizations. The study described in this report suggest that S. typhimurium delta aro mutants have the capacity to induce MHC class I-restricted CTL against carried antigens and that MHC class I-restricted CTL responses require stable in vivo expression of the target antigen. Further, the results indicate that the Salmonella typhi delta aro mutants currently undergoing evaluation in studies with humans may be good carriers of viral antigens with CTL determinants. Images PMID:8225611

  7. Bimolecular recombination in organic photovoltaics.

    PubMed

    Lakhwani, Girish; Rao, Akshay; Friend, Richard H

    2014-01-01

    The recombination of electrons and holes is a major loss mechanism in photovoltaic devices that controls their performance. We review scientific literature on bimolecular recombination (BR) in bulk heterojunction organic photovoltaic devices to bring forward existing ideas on the origin and nature of BR and highlight both experimental and theoretical work done to quantify its extent. For these systems, Langevin theory fails to explain BR, and recombination dynamics turns out to be dependent on mobility, temperature, electric field, charge carrier concentration, and trapped charges. Relationships among the photocurrent, open-circuit voltage, fill factor, and morphology are discussed. Finally, we highlight the recent emergence of a molecular-level picture of recombination, taking into account the spin and delocalization of charges. Together with the macroscopic picture of recombination, these new insights allow for a comprehensive understanding of BR and provide design principles for future materials and devices.

  8. 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.

  9. Determination by enzyme-linked immunosorbent assay of immunoglobulin G (IgG), IgM, and IgA to Brucella melitensis major outer membrane proteins and whole-cell heat-killed antigens in sera of patients with brucellosis.

    PubMed Central

    Araj, G F; Kaufmann, A F

    1989-01-01

    An enzyme-linked immunosorbent assay was used to compare Brucella melitensis major outer membrane proteins (MOMP) and whole-cell heat-killed antigens (HK) in measuring antibrucella immunoglobulin G (IgG), IgM, and IgA in sera of brucellosis patients and controls. Antibodies to MOMP were generally similar to those against HK, and the correlation coefficients between the two antigens and IgG, IgM, and IgA in patients varied between 0.73 and 0.94. Both antigens are comparably suitable in detecting antibrucella immunoglobulin isotypes for the serologic diagnosis of patients with brucellosis, with high (greater than or equal to 95%) sensitivity and specificity. PMID:2768476

  10. 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

  11. 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.

  12. Nondisjunction of chromosome 15: origin and recombination.

    PubMed Central

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

    1993-01-01

    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 are utilized. Standard methods of centromere mapping are employed to determine the level 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, most paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome. PMID:8352279

  13. 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.

  14. 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

  15. Controlled release from recombinant polymers.

    PubMed

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

    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.

  16. 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

  17. Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India.

    PubMed

    Khushiramani, Rekha; Tuteja, Urmil; Shukla, Jyoti; Batra, Harsh Vardhan

    2004-05-01

    The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins

  18. 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.

  19. 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.

  20. 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

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

    PubMed

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

    1994-12-01

    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.

  2. Magnetosphere of the outer planets

    NASA Technical Reports Server (NTRS)

    Kennel, C. F.

    1972-01-01

    Scaling laws for possible outer planet magnetospheres are derived. These suggest that convection and its associated auroral effects will play a relatively smaller role than at earth, and that there is a possibility that they could have significant radiation belts of energetic trapped particles.

  3. The View from "Outer Britain"

    ERIC Educational Resources Information Center

    Rees, Gareth

    2007-01-01

    In the UK, there remains an economy of "outer Britain" which has a higher representation of low-pay, low-skill jobs, and correspondingly, lower levels of economic growth than other parts of the country. The author discusses how the distinctive ideological complexions of Scottish and Welsh politics open the possibility of different approaches to…

  4. 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

  5. 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.

  6. Progenitors of Recombining Supernova Remnants

    NASA Astrophysics Data System (ADS)

    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.

  7. 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.

  8. 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.

  9. 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...

  10. 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.

  11. 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…

  12. Chi Enhances Heteroduplex DNA Levels during Recombination

    PubMed Central

    Holbeck, S. L.; Smith, G. R.

    1992-01-01

    The major pathway of homologous recombination in Escherichia coli, the RecBCD pathway, is stimulated by Chi sites. To determine whether Chi enhances an early or late step in recombination, we measured formation of heteroduplex DNA (hDNA) in extracts of lambda-infected E. coli. Chi elevated hDNA levels in these extracts, supporting a role for Chi early (before hDNA formation) in recombination. RecA protein and RecBCD enzyme were both necessary for detection of hDNA, indicating that they, too, act early. Analysis of a panel of recBCD mutants indicated that Chi-nicking activity was needed for Chi's stimulation of hDNA formation. These results support a previously proposed model of recombination. Further results suggested that RecBCD enzyme has an additional role late in recombination. PMID:1459441

  13. The progressive outer retinal necrosis syndrome.

    PubMed

    Holland, G N

    1994-01-01

    The progressive outer retinal necrosis (PORN) syndrome is a recently described clinical variant of necrotizing herpetic retinopathy in patients with the acquired immunodeficiency syndrome (AIDS). It is caused by varicellazoster virus infection of the retina. Its course and clinical features distinguish it from the acute retinal necrosis syndrome and CMV retinopathy. Early disease is characterized by multifocal deep retinal opacification. Lesions rapidly coalesce and progress to total retinal necrosis over a short period of time. Despite aggressive therapy with intravenous antivirial drugs, prognosis is poor; disease progression and/or recurrence is common, and the majority of patients develop no light perception vision. Total retinal detachments are common. Prophylaxis against retinal detachment using laser retinopexy has not been useful in most cases. PORN syndrome is an uncommon, but devastating complication of AIDS.

  14. The progressive outer retinal necrosis syndrome.

    PubMed

    Holland, G N

    1994-01-01

    The progressive outer retinal necrosis (PORN) syndrome is a recently described clinical variant of necrotizing herpetic retinopathy in patients with the acquired immunodeficiency syndrome (AIDS). It is caused by varicellazoster virus infection of the retina. Its course and clinical features distinguish it from the acute retinal necrosis syndrome and CMV retinopathy. Early disease is characterized by multifocal deep retinal opacification. Lesions rapidly coalesce and progress to total retinal necrosis over a short period of time. Despite aggressive therapy with intravenous antivirial drugs, prognosis is poor; disease progression and/or recurrence is common, and the majority of patients develop no light perception vision. Total retinal detachments are common. Prophylaxis against retinal detachment using laser retinopexy has not been useful in most cases. PORN syndrome is an uncommon, but devastating complication of AIDS. PMID:7852023

  15. Protein-Protein Interactions between Intermediate Chains and the Docking Complex of Chlamydomonas Flagellar Outer Arm Dynein

    PubMed Central

    Ide, Takahiro; Owa, Mikito; King, Stephen M.; Kamiya, Ritsu; Wakabayashi, Ken-ichi

    2013-01-01

    Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association. PMID:23747306

  16. CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

    PubMed

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

    2013-11-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

  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. 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.

  19. 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.

  20. Genetic recombination and molecular evolution.

    PubMed

    Charlesworth, B; Betancourt, A J; Kaiser, V B; Gordo, I

    2009-01-01

    Reduced rates of genetic recombination are often associated with reduced genetic variability and levels of adaptation. Several different evolutionary processes, collectively known as Hill-Robertson (HR) effects, have been proposed as causes of these correlates of recombination. Here, we use DNA sequence polymorphism and divergence data from the noncrossing over dot chromosome of Drosophila to discriminate between two of the major forms of HR effects: selective sweeps and background selection. This chromosome shows reduced levels of silent variability and reduced effectiveness of selection. We show that neither model fits the data on variability. We propose that, in large genomic regions with restricted recombination, HR effects among nonsynonymous mutations undermine the effective strength of selection, so that their background selection effects are weakened. This modified model fits the data on variability and also explains why variability in very large nonrecombining genomes is not completely wiped out. We also show that HR effects of this type can produce an individual selection advantage to recombination, as well as greatly reduce the mean fitness of nonrecombining genomes and genomic regions.

  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. Proteases of Treponema denticola outer sheath and extracellular vesicles.

    PubMed Central

    Rosen, G; Naor, R; Rahamim, E; Yishai, R; Sela, M N

    1995-01-01

    Electron microscopical observations of the oral periodontopathogen Treponema denticola show the presence of extracellular vesicles bound to the bacterial surface or free in the surrounding medium. Extracellular vesicles from T. denticola ATCC 35404, 50 to 100 nm in diameter, were isolated and further characterized. Protein and proteolytic patterns of the vesicles were found to be very similar to those of isolated T. denticola outer sheaths. They were enriched with the major outer sheath polypeptides (molecular sizes, 113 to 234 kDa) and with outer sheath proteases of 91, 153, 173, and 228 kDa. These findings indicate that treponemal outer sheath vesicles contain the necessary adhesins and proteolytic arsenal for adherence to and damage of eucaryotic cells and mammalian matrix proteins. The major outer sheath- and vesicle-associated protease of T. denticola ATCC 35404 was purified and characterized. The purified enzyme had a molecular size of 91 kDa, and it dissociated into three polypeptides of 72, 38, and 35 kDa upon heating in the presence of sodium dodecyl sulfate with or without a reducing agent. The activity of the enzyme could be inhibited by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, and phenylboronic acid. The value of the second-order rate constant of the protease inactivation by phenylmethylsulfonyl fluoride was 0.48 x 10(4) M(-1) min-1. Inhibition of the enzyme by phenylboronic acid was rapid (< 1 min) and pH dependent. These data strongly suggest that this major surface proteolytic activity belongs to a family of serine proteases. PMID:7558307

  3. Aft outer rim seal arrangement

    SciTech Connect

    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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    PubMed

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  12. Recombinant baculovirus isolation.

    PubMed

    King, Linda A; Hitchman, Richard; Possee, Robert D

    2007-01-01

    Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac or BaculoDirect, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus. Additionally, many systems require a plaque-assay to separate parental and recombinant virus prior to amplification and use of the recombinant virus. This chapter provides an overview of the historical development of increasingly more efficient systems for the isolation of recombinant baculoviruses (Chapter 3 provides a full account of the different systems and transfer vectors available). The practical details cover: transfection of insect cells with either virus DNA or virus DNA and plasmid transfer vector; a reliable plaque-assay method that can be used to separate recombinant virus from parental (nonrecombinant) virus where this is necessary; methods for the small-scale amplification of recombinant virus; and subsequent titration by plaque-assay. Methods unique to the Bac-to-Bac system are also covered and include the transformation of bacterial cells and isolation of bacmid DNA ready for transfection of insect cells.

  13. 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.

  14. 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... (SOBDs); Correction. SUMMARY: BOEM (formerly the Bureau of Ocean Energy Management, Regulation...

  15. 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

  16. 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.

  17. Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

    PubMed

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

    2014-01-01

    For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

  18. 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.

  19. 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.

  20. Major Advisor.

    ERIC Educational Resources Information Center

    Chatwin, Marshall

    This paper describes a computer program, Major Advisor (MA), which helps students identify college majors. Used in conjunction with career counseling and advising, MA provides information to students who are developing their educational plans. The program matches students' personal preferences and the requirements/characteristics of 130 common…

  1. Recombination and Replication

    PubMed Central

    Syeda, Aisha H.; Hawkins, Michelle; McGlynn, Peter

    2014-01-01

    The links between recombination and replication have been appreciated for decades and it is now generally accepted that these two fundamental aspects of DNA metabolism are inseparable: Homologous recombination is essential for completion of DNA replication and vice versa. This review focuses on the roles that recombination enzymes play in underpinning genome duplication, aiding replication fork movement in the face of the many replisome barriers that challenge genome stability. These links have many conserved features across all domains of life, reflecting the conserved nature of the substrate for these reactions, DNA. PMID:25341919

  2. The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition.

    PubMed

    Ishida, Hiroaki; Garcia-Herrero, Alicia; Vogel, Hans J

    2014-12-01

    Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1-171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180-325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA(180-325)). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA(180-325) with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA(1-325)) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the

  3. [Expression of rice dwarf virus outer coat protein gene(S8) in insect cells].

    PubMed

    Li, S; Liu, H; Chen, Z; Li, Y

    2001-04-01

    Outer coat protein gene(S8) of RDV was cloned into the transfer vector pVL 1393 to construct a recombinant vector pVL1393-S8. The recombinant vector pVL1393-S8 and the linear baculovirus RP23. LacZ were cotransfected into sf9 cells to produce the recombinant virus RP23-S8. RP23-S8 infected sf9 cells were collected and analysed by SDS-PAGE and Western-blot. The results showed that the S8 gene of RDV was expressed in sf9 cells and the expression level of sf9 cells was higher between 72-96 h after infected.

  4. Carbon stars in the outer spheroid of NGC 6822

    NASA Astrophysics Data System (ADS)

    Demers, S.; Battinelli, P.; Artigau, E.

    2006-09-01

    Context: .From a 2°× 2° of NGC 6822 survey we have previously established that this Local Group dwarf irregular galaxy possesses a huge spheroid having more than one degree in length. This spheroid is in rotation but its rotation curve is known only within ~ 15' from the center. It is therefore critical to identify bright stars belonging to the spheroid to characterize, as far as possible, its outer kinematics. Aims: .We use the new wide field near infrared imager CPAPIR, operated by the SMARTS consortium, to acquire J, Ks images of two 34.8'× 34.8' areas in the outer spheroid to search for C stars. Methods: .The colour diagram of the fields allows the identification of 192 C stars candidates but a study of the FWHM of the images permits the rejection of numerous non-stellar objects with colours similar to C stars. Results: . We are left with 75 new C stars, their mean Ks magnitude and mean colour are similar to the bulk of known NGC 6822 C stars. Conclusions: .This outer spheroid survey confirms that the intermediate-age AGB stars are a major contributor to the stellar populations of the spheroid. The discovery of some 50 C stars well beyond the limit of the previously known rotation curve calls for a promising spectroscopic follow-up to a major axis distance of 40'.

  5. 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)

  6. Activated recombinant adenovirus proteinases

    SciTech Connect

    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.

  7. 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.

  8. Evidence of structural genomic region recombination in Hepatitis C virus

    PubMed Central

    Cristina, Juan; Colina, Rodney

    2006-01-01

    Background/Aim Hepatitis C virus (HCV) has been the subject of intense research and clinical investigation as its major role in human disease has emerged. Although homologous recombination has been demonstrated in many members of the family Flaviviridae, to which HCV belongs, there have been few studies reporting recombination on natural populations of HCV. Recombination break-points have been identified in non structural proteins of the HCV genome. Given the implications that recombination has for RNA virus evolution, it is clearly important to determine the extent to which recombination plays a role in HCV evolution. In order to gain insight into these matters, we have performed a phylogenetic analysis of 89 full-length HCV strains from all types and sub-types, isolated all over the world, in order to detect possible recombination events. Method Putative recombinant sequences were identified with the use of SimPlot program. Recombination events were confirmed by bootscaning, using putative recombinant sequence as a query. Results Two crossing over events were identified in the E1/E2 structural region of an intra-typic (1a/1c) recombinant strain. Conclusion Only one of 89 full-length strains studied resulted to be a recombinant HCV strain, revealing that homologous recombination does not play an extensive roll in HCV evolution. Nevertheless, this mechanism can not be denied as a source for generating genetic diversity in natural populations of HCV, since a new intra-typic recombinant strain was found. Moreover, the recombination break-points were found in the structural region of the HCV genome. PMID:16813646

  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. Formaldehyde in the Far Outer Galaxy

    NASA Astrophysics Data System (ADS)

    Lugo, S. K.; Magnani, L.; Brand, J.; Wouterloot, J. G. A.

    2006-06-01

    We present results from an initial survey of the 212-111 transition of formaldehyde (H2CO) in the Far Outer Galaxy (galactocentric distances, Rg > 16 kpc). Formaldehyde is a key prebiotic molecule; determining the outermost extent of its distribution can be used to set a limit to the Galaxy's "Habitable Zone", the region where conditions for the formation of life are most favorable. We surveyed 67 clouds in the outer Galaxy ranging 12 - 23 kpc in distance from the Galactic Center. Formaldehyde emission at 140.8 GHz was detected from 44 of 67 lines of sight, including 7 clouds at Rg > 20 kpc. Formaldehyde is readily detectable even in the Far Outer Galaxy beyond the edge of the stellar disk. The widespread distribution of H2CO in the Far Outer Galaxy is a positive first step in determining how favorable are conditions in this large region towards the formation of life.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii.

    PubMed

    Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye Yeon

    2011-12-01

    Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2(1), with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å(3) Da(-1).

  19. 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

  20. 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

  1. Planet Nine From Outer Space

    NASA Astrophysics Data System (ADS)

    Batygin, Konstantin; Brown, Michael E.

    2016-10-01

    All known Kuiper belt objects with orbital periods longer than 4,000 years have orbits that are clustered in physical space. Statistically, the chances of such alignment being coincidental are smaller than a hundredth of a percent. In this talk, we show that the observed clustering of Kuiper belt orbits can be explained by a distant, eccentric, Neptune-like planet, whose orbit lies in approximately the same plane as those of the distant Kuiper belt objects, but whose perihelion is 180° away from the perihelia of the minor bodies. In addition to accounting for the observed grouping of orbital trajectories, the existence of such a planet naturally explains the presence of high-perihelion Sedna-like objects, as well as the known collection of high semi-major axis objects with inclinations between 60° and 150°.

  2. 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

  3. 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

  4. 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.

  5. 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

  6. 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

  7. Propagation of Interplanetary Disturbances in the Outer Heliosphere

    NASA Technical Reports Server (NTRS)

    Wang, Chi

    2005-01-01

    Contents include the following: 1. We have developed a one-dimensional, spherically symmetric, multi-fluid MHD model that includes solar wind protons and electrons, pickup ions, and interstellar neutral hydrogen. This model advances the existing solar wind models for the outer heliosphere in two important ways: one is that it distinguishes solar wind protons from pickup ions, and the other is that it allows for energy transfer from pickup ions to the solar wind protons. Model results compare favorably with the Voyager 2 observations. 2. 2. Solar wind slowdown and interstellar neutral density. The solar wind in the outer heliosphere is fundamentally different from that in the inner heliosphere since the effects of interstellar neutrals become significant. 3. ICME propagation from the inner to outer heliosphere. Large coronal mass ejections (CMEs) have major effects on the structure of the solar wind and the heliosphere. The plasma and magnetic field can be compressed ahead of interplanetary CMEs. 4. During the current solar cycle (Cycle 23), several major CMEs associated with solar flares produced large transient shocks which were observed by widely-separated spacecraft such as Wind at Earth and Voyager 2 beyond 60 AU. Using data from these spacecraft, we use the multi-fluid model to investigate shock propagation and interaction in the heliosphere. Specifically, we studied the Bastille Day 2000, April 2001 and Halloween 2003 events. 5. Statistical properties of the solar wind in the outer heliosphere. In a collaboration with L.F. Burlaga of GSFC, it is shown that the basic statistical properties of the solar wind in the outer heliosphere can be well produced by our model. We studied the large-scale heliospheric magnetic field strength fluctuations as a function of distance from the Sun during the declining phase of a solar cycle, using our numerical model with observations made at 1 AU during 1995 as input. 6. Radial heliospheric magnetic field events. The

  8. Mitogenic effects of purified outer membrane proteins from Pseudomonas aeruginosa.

    PubMed Central

    Chen, Y H; Hancock, R E; Mishell, R I

    1980-01-01

    Three major outer membrane proteins from Pseudomonas aeruginosa PAO1 were purified and tested for their ability to stimulate resting murine lymphocytes to proliferate. It was demonstrated that picomole amounts of all three proteins were mitogenic for both intact and T-lymphocyte-depleted populations of spleen cells from C3H/HeJ mice. In contrast, they had no activity against either mature or immature thymocytes. Since the strain of mice used is unable to respond to lipopolysaccharide, we condlude that the three proteins are B-cell mitogens. Images Fig. 2 PMID:6769818

  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. DETECTING THE RAPIDLY EXPANDING OUTER SHELL OF THE CRAB NEBULA: WHERE TO LOOK

    SciTech Connect

    Wang Xiang; Ferland, G. J.; Baldwin, J. A.; Loh, E. D.; Richardson, C. T.

    2013-09-10

    We present a range of steady-state photoionization simulations, corresponding to different assumed shell geometries and compositions, of the unseen postulated rapidly expanding outer shell to the Crab Nebula. The properties of the shell are constrained by the mass that must lie within it, and by limits to the intensities of hydrogen recombination lines. In all cases the photoionization models predict very strong emissions from high ionization lines that will not be emitted by the Crab's filaments, alleviating problems with detecting these lines in the presence of light scattered from brighter parts of the Crab. The near-NIR [Ne VI] {lambda}7.652 {mu}m line is a particularly good case; it should be dramatically brighter than the optical lines commonly used in searches. The C IV {lambda}1549 doublet is predicted to be the strongest absorption line from the shell, which is in agreement with Hubble Space Telescope observations. We show that the cooling timescale for the outer shell is much longer than the age of the Crab, due to the low density. This means that the temperature of the shell will actually ''remember'' its initial conditions. However, the recombination time is much shorter than the age of the Crab, so the predicted level of ionization should approximate the real ionization. In any case, it is clear that IR observations present the best opportunity to detect the outer shell and so guide future models that will constrain early events in the original explosion.

  11. Deciphering the roles of BamB and its interaction with BamA in outer membrane biogenesis, T3SS expression and virulence in Salmonella.

    PubMed

    Namdari, Fatémeh; Hurtado-Escobar, Genaro Alejandro; Abed, Nadia; Trotereau, Jérôme; Fardini, Yann; Giraud, Etienne; Velge, Philippe; Virlogeux-Payant, Isabelle

    2012-01-01

    The folding and insertion of β-barrel proteins in the outer membrane of Gram-negative bacteria is mediated by the BAM complex, which is composed of the outer membrane protein BamA and four lipoproteins BamB to BamE. In Escherichia coli and/or Salmonella, the BamB lipoprotein is involved in (i) β-barrel protein assembly in the outer membrane, (ii) outer membrane permeability to antibiotics, (iii) the control of the expression of T3SS which are major virulence factors and (iv) the virulence of Salmonella. In E. coli, this protein has been shown to interact directly with BamA. In this study, we investigated the structure-function relationship of BamB in order to assess whether the roles of BamB in these phenotypes were inter-related and whether they require the interaction of BamB with BamA. For this purpose, recombinant plasmids harbouring point mutations in bamB were introduced in a ΔSalmonella bamB mutant. We demonstrated that the residues L173, L175 and R176 are crucial for all the roles of BamB and for the interaction of BamB with BamA. Moreover, the results obtained with a D229A BamB variant, which is unable to immunoprecipitate BamA, suggest that the interaction of BamB with BamA is not absolutely necessary for BamB function in outer-membrane protein assembly, T3SS expression and virulence. Finally, we showed that the virulence defect of the ΔbamB mutant is not related to its increased susceptibility to antimicrobials, as the D227A BamB variant fully restored the virulence of the mutant while having a similar antibiotic susceptibility to the ΔbamB strain. Overall, this study demonstrates that the different roles of BamB are not all inter-related and that L173, L175 and R176 amino-acids are privileged sites for the design of BamB inhibitors that could be used as alternative therapeutics to antibiotics, at least against Salmonella.

  12. Deciphering the Roles of BamB and Its Interaction with BamA in Outer Membrane Biogenesis, T3SS Expression and Virulence in Salmonella

    PubMed Central

    Namdari, Fatémeh; Hurtado-Escobar, Genaro Alejandro; Abed, Nadia; Trotereau, Jérôme; Fardini, Yann; Giraud, Etienne; Velge, Philippe; Virlogeux-Payant, Isabelle

    2012-01-01

    The folding and insertion of β-barrel proteins in the outer membrane of Gram-negative bacteria is mediated by the BAM complex, which is composed of the outer membrane protein BamA and four lipoproteins BamB to BamE. In Escherichia coli and/or Salmonella, the BamB lipoprotein is involved in (i) β-barrel protein assembly in the outer membrane, (ii) outer membrane permeability to antibiotics, (iii) the control of the expression of T3SS which are major virulence factors and (iv) the virulence of Salmonella. In E. coli, this protein has been shown to interact directly with BamA. In this study, we investigated the structure-function relationship of BamB in order to assess whether the roles of BamB in these phenotypes were inter-related and whether they require the interaction of BamB with BamA. For this purpose, recombinant plasmids harbouring point mutations in bamB were introduced in a ΔSalmonella bamB mutant. We demonstrated that the residues L173, L175 and R176 are crucial for all the roles of BamB and for the interaction of BamB with BamA. Moreover, the results obtained with a D229A BamB variant, which is unable to immunoprecipitate BamA, suggest that the interaction of BamB with BamA is not absolutely necessary for BamB function in outer-membrane protein assembly, T3SS expression and virulence. Finally, we showed that the virulence defect of the ΔbamB mutant is not related to its increased susceptibility to antimicrobials, as the D227A BamB variant fully restored the virulence of the mutant while having a similar antibiotic susceptibility to the ΔbamB strain. Overall, this study demonstrates that the different roles of BamB are not all inter-related and that L173, L175 and R176 amino-acids are privileged sites for the design of BamB inhibitors that could be used as alternative therapeutics to antibiotics, at least against Salmonella. PMID:23144780

  13. Deciphering the Function of the Outer Membrane Protein OprD Homologue of Acinetobacter baumannii

    PubMed Central

    Catel-Ferreira, Manuella; Nehmé, Rony; Molle, Virginie; Aranda, Jesús; Bouffartigues, Emeline; Chevalier, Sylvie; Bou, Germán; Jouenne, Thierry

    2012-01-01

    The increasing number of carbapenem-resistant Acinetobacter baumannii isolates is a major cause for concern which restricts therapeutic options to treat severe infections caused by this emerging pathogen. To identify the molecular mechanisms involved in carbapenem resistance, we studied the contribution of an outer membrane protein homologue of the Pseudomonas aeruginosa OprD porin. Suspected to be the preferred pathway of carbapenems in A. baumannii, the oprD homologue gene was inactivated in strain ATCC 17978. Comparison of wild-type and mutant strains did not confirm the expected increased resistance to any antibiotic tested. OprD homologue sequence analysis revealed that this protein actually belongs to an OprD subgroup but is closer to the P. aeruginosa OprQ protein, with which it could share some functions, e.g., allowing bacterial survival under low-iron or -magnesium growth conditions or under poor oxygenation. We thus overexpressed and purified a recombinant OprD homologue protein to further examine its functional properties. As a specific channel, this porin presented rather low single-channel conductance, i.e., 28 pS in 1 M KCl, and was partially closed by micro- and millimolar concentrations of Fe3+ and Mg2+, respectively, but not by imipenem and meropenem or basic amino acids. The A. baumannii OprD homologue is likely not involved in the carbapenem resistance mechanism, but as an OprQ-like protein, it could contribute to the adaptation of this bacterium to magnesium- and/or iron-depleted environments. PMID:22564848

  14. Comparison of Genetic Variability at Multiple Loci across the Genomes of the Major Subtypes of Kaposi’s Sarcoma-Associated Herpesvirus Reveals Evidence for Recombination and for Two Distinct Types of Open Reading Frame K15 Alleles at the Right-Hand End

    PubMed Central

    Poole, Lynn J.; Zong, Jian-Chao; Ciufo, Dolores M.; Alcendor, Donald J.; Cannon, Jennifer S.; Ambinder, Richard; Orenstein, Jan M.; Reitz, Marvin S.; Hayward, Gary S.

    1999-01-01

    Kaposi’s sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV8) DNA is found consistently in nearly all classical, endemic, transplant, and AIDS-associated KS lesions, as well as in several AIDS-associated lymphomas. We have previously sequenced the genes for the highly variable open reading frame K1 (ORF-K1) protein from more than 60 different HHV8 samples and demonstrated that they display up to 30% amino acid variability and cluster into four very distinct evolutionary subgroups (the A, B, C, and D subtypes) that correlate with the major migrationary diasporas of modern humans. Here we have extended this type of analysis to six other loci across the HHV8 genome to further evaluate overall genotype patterns and the potential for chimeric genomes. Comparison of the relatively conserved ORF26, T0.7/K12, and ORF75 gene regions at map positions 0.35, 0.85, and 0.96 revealed typical ORF-K1-linked subtype patterns, except that between 20 and 30% of the genomes analyzed proved to be either intertypic or intratypic mosaics. In addition, a 2,500-bp region found at the extreme right-hand side of the unique segment in 45 HHV8 genomes proved to be highly diverged from the 3,500-bp sequence found at this position in the other 18 HHV8 genomes examined. Furthermore, these previously uncharacterized “orphan” region sequences proved to encompass multiexon latent-state mRNAs encoding two highly diverged alleles of the novel ORF-K15 protein. The predominant (P) and minor (M) forms of HHV8 ORF-K15 are structurally related integral membrane proteins that have only 33% overall amino acid identity to one another but retain conserved likely tyrosine kinase signaling motifs and may be distant evolutionary relatives of the LMP2 latency protein of Epstein-Barr virus. The M allele of ORF-K15 is also physically linked to a distinctive M subtype of the adjacent ORF75 gene locus, and in some cases, this linkage extends as far back as the T0.7 locus also. Overall, the results

  15. 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

  16. Recombinant DNA for Teachers.

    ERIC Educational Resources Information Center

    Duvall, James G., III

    1992-01-01

    A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

  17. Recombineering Pseudomonas syringae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we report the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecE...

  18. Recombinant renewable polyclonal antibodies.

    PubMed

    Ferrara, Fortunato; D'Angelo, Sara; Gaiotto, Tiziano; Naranjo, Leslie; Tian, Hongzhao; Gräslund, Susanne; Dobrovetsky, Elena; Hraber, Peter; Lund-Johansen, Fridtjof; Saragozza, Silvia; Sblattero, Daniele; Kiss, Csaba; Bradbury, Andrew R M

    2015-01-01

    Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.

  19. Baseline Microstructural Characterization of Outer 3013 Containers

    SciTech Connect

    Zapp, Phillip E.; Dunn, Kerry A

    2005-07-31

    Three DOE Standard 3013 outer storage containers were examined to characterize the microstructure of the type 316L stainless steel material of construction. Two of the containers were closure-welded yielding production-quality outer 3013 containers; the third examined container was not closed. Optical metallography and Knoop microhardness measurements were performed to establish a baseline characterization that will support future destructive examinations of 3013 outer containers in the storage inventory. Metallography revealed the microstructural features typical of this austenitic stainless steel as it is formed and welded. The grains were equiaxed with evident annealing twins. Flow lines were prominent in the forming directions of the cylindrical body and flat lids and bottom caps. No adverse indications were seen. Microhardness values, although widely varying, were consistent with annealed austenitic stainless steel. The data gathered as part of this characterization will be used as a baseline for the destructive examination of 3013 containers removed from the storage inventory.

  20. MHD processes in the outer heliosphere

    NASA Technical Reports Server (NTRS)

    Burlaga, L. F.

    1984-01-01

    The magnetic field measurements from Voyager and the magnetohydrodynamic (MHD) processes in the outer heliosphere are reviewed. A bibliography of the experimental and theoretical work concerning magnetic fields and plasmas observed in the outer heliosphere is given. Emphasis in this review is on basic concepts and dynamical processes involving the magnetic field. The theory that serves to explain and unify the interplanetary magnetic field and plasma observations is magnetohydrodynamics. Basic physical processes and observations that relate directly to solutions of the MHD equations are emphasized, but obtaining solutions of this complex system of equations involves various assumptions and approximations. The spatial and temporal complexity of the outer heliosphere and some approaches for dealing with this complexity are discussed.

  1. Geometry of Comet Halley's outer plasma environment

    NASA Astrophysics Data System (ADS)

    Perez de Tejada, H.

    A study of Comet Halley's outer plasma environment is presented. It is argued that the intermediate transition detected nearly halfway between the comet's bow shock and the cometopause represents the outer boundary of a thick friction layer that develops along the flanks of the cometopause. In the subsolar region the cometopause and the intermediate transition are expected to reach nearly the same (about 350,000 km) distance from the nucleus. The plasma changes seen in the outer ionosheath (between the bow shock and the intermediate transition) are further interpreted as due to an intrinsic axial asymmetry of the cometopause which may have a highly oblate cross-sectional shape with respect to the solar wind direction.

  2. Isolation and characterization of outer membrane permeability mutants in Escherichia coli K-12.

    PubMed Central

    Benson, S A; Decloux, A

    1985-01-01

    Escherichia coli normally requires the lamB gene for the uptake of maltodextrins. We have identified and characterized three independent mutations that allow E. coli to grow on maltodextrin in the absence of a functional lamB gene by allowing maltodextrins with a molecular weight greater than 1,000 to cross the outer membrane barrier. Two of the mutations map to the structural gene for the outer membrane porin OmpF, and the remaining mutation maps to the structural gene for the second major outer membrane porin, OmpC. These mutations increase the permeability of the outer membrane to small hydrophilic substances, antibiotics, and detergents. These mutations alter the electrophoretic mobility of the respective porin proteins. Images PMID:2981807

  3. Edwardsiella tarda Outer Membrane Protein C: An Immunogenic Protein Induces Highly Protective Effects in Flounder (Paralichthys olivaceus) against Edwardsiellosis.

    PubMed

    Liu, Fuguo; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

    2016-01-01

    Outer membrane protein C of Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder (Paralichthys olivaceus) anti-recombinant OmpC (rOmpC), and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1) the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2) rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4-5 weeks after immunization; and (3) the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection. PMID:27420049

  4. Edwardsiella tarda Outer Membrane Protein C: An Immunogenic Protein Induces Highly Protective Effects in Flounder (Paralichthys olivaceus) against Edwardsiellosis

    PubMed Central

    Liu, Fuguo; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

    2016-01-01

    Outer membrane protein C of Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder (Paralichthys olivaceus) anti-recombinant OmpC (rOmpC), and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1) the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2) rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4–5 weeks after immunization; and (3) the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection. PMID:27420049

  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. Ptolemy's treatment of the outer planets

    NASA Astrophysics Data System (ADS)

    Duke, Dennis

    2005-01-01

    The purpose of this paper is to investigate whether for the outer planets Ptolemy followed his otherwise consistent custom of describing a scenario that did not happen as he says, or whether, at least for the outer planets, he left us a more accurate rendition of events. The detailed reconstructions of Ptolemy's calculations that follow show that, at least in the Almagest, Ptolemy is a writer with consistent habits when it comes to observations. We begin by reviewing, with minimal editorial comment, Ptolemy's calculations for each planet.

  7. Outer planet probe cost estimates: First impressions

    NASA Technical Reports Server (NTRS)

    Niehoff, J.

    1974-01-01

    An examination was made of early estimates of outer planetary atmospheric probe cost by comparing the estimates with past planetary projects. Of particular interest is identification of project elements which are likely cost drivers for future probe missions. Data are divided into two parts: first, the description of a cost model developed by SAI for the Planetary Programs Office of NASA, and second, use of this model and its data base to evaluate estimates of probe costs. Several observations are offered in conclusion regarding the credibility of current estimates and specific areas of the outer planet probe concept most vulnerable to cost escalation.

  8. Cochlear amplification, outer hair cells and prestin

    PubMed Central

    Dallos, Peter

    2008-01-01

    Mechanical amplification of acoustic signals is apparently a common feature of vertebrate auditory organs. In non-mammalian vertebrates amplification is produced by stereociliary processes, related to the mechanotransducer channel complex and probably to the phenomenon of fast adaptation. The extended frequency range of the mammalian cochlea has likely co-evolved with a novel hair cell type, the outer hair cell and its constituent membrane protein, prestin. Cylindrical outer hair cells are motile and their somatic length changes are voltage driven and powered by prestin. One of the central outstanding problems in mammalian cochlear neurobiology is the relation between the two amplification processes. PMID:18809494

  9. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    PubMed

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.

  10. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    PubMed

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

  11. Prevention of the Outer Space Weaponization

    NASA Astrophysics Data System (ADS)

    Zhukov, Gennady P.

    2002-01-01

    9 states. The satellites of various functions (early warning, communication, data acquisition, reconnaissance and navigation) were actively used and continue to be used with the purposes of raising efficiency of ground armed forces, especially in fight against international terrorism. At the same time such satellites are not a weapon in the sense of that word since they do not create the threats of armed attack in outer space or from outer space. Moreover, they promote maintaining of stability in the international relations. For this reason the reconnaissance and data acquisition satellites used for the verification of observance by States of the arms limitation agreements are under international protection as national technical means of the control. Similar protection is enjoyed by the early warning satellites. With the help of space communication facilities the more reliable operative connection of the statesmen is organized in the strained situations. By this way the probability of making of the incorrect retaliatory decisions in critical political situations is reduced. At the same time it's necessary to take into consideration that the activities of such satellite systems are tightly connected with ground armed forces of the states. the earth, what from the point of view of international law may be qualified as establishing a partial demilitarization regime in outer space. After the prohibition of anti-satellite weapons (ASAT) and anti-satellite (ASAT) weapons it will be possible to speak about establishing of an international legal regime of complete demilitarization in outer space eliminating any kinds of weapon from outer space. in a peaceful time. weaponization.The main task of this paper is to analyze and to discuss the present binding regime of the outer space deweaponization and particular measures on consolidation and strengthening of this regime. agreements of the Russian Federation and the USA into multilateral Treaties. Such "immunity" would cover

  12. Syrtis Major

    NASA Technical Reports Server (NTRS)

    2002-01-01

    (Released 6 June 2002) The Science This image, located near the equator and 288W (72E), is near the southern edge of a low, broad volcanic feature called Syrtis Major. A close look at this image reveals a wrinkly texture that indicates a very rough surface that is associated with the lava flows that cover this region. On a larger scale, there are numerous bright streaks that trail topographic features such as craters. These bright streaks are in the wind shadows of the craters where dust that settles onto the surface is not as easily scoured away. It is important to note that these streaks are only bright in a relative sense to the surrounding image. Syrtis Major is one of the darkest regions on Mars and it is as dark as fresh basalt flows or dunes are on Earth. The Story Cool! It almost looks as if nature has 'painted' comets on the surface of Mars, using craters as comet cores and dust as streaky tails. Of course, that's just an illusion. As in many areas of Mars, the wind is behind the creation of such fantastic landforms. The natural phenomenon seen here gives this particular surface of Mars a very dynamic, fast-moving, almost luminous 'cosmic personality.' The bright, powdery-looking streaks of dust are in the 'wind shadows' of craters, where dust that settles onto the surface is not as easily scoured away. That's because the wind moves across the land in a particular direction, and a raised surface like the rim of a crater 'protects' dust from being completely blown away on the other side. The raised landforms basically act as a buffer. From the streaks seen above, you can tell the wind was blowing in a northeast to southwest direction. Why are the streaks so bright? Because they contrast with the really dark underlying terrain in this volcanic area of Mars. Syrtis Major is one of the darkest regions on Mars because it is made of basalt. Basalt is typically dark gray or black, and forms when a certain type of molten lava cools. The meaning of the word basalt

  13. Lysyl-derived aldehydes in outer membrane proteins of Escherichia coli.

    PubMed Central

    Diedrich, D L; Schnaitman, C A

    1978-01-01

    The major outer membrane proteins from Escherichia coli K-12 are modified to contain alpha-aminoadipic acid delta-semialdehyde (allysine). The allysine was found to be derived from lysine and it was identified by derivatizing it to chloronorleucine by reduction, alpha-aminoadipic acid by oxidation, and to alpha,epsilon-diaminopimelic acid by reacting it with CN- and NH3. The alpha-aminoadipic acid was identified by mass spectrometry. Two major outer membrane proteins were found to possess allysine, a modified lysine characteristically found to connective tissue. PMID:358196

  14. Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli.

    PubMed

    Abdeljabbar, Diya M; Song, Hank J; Link, A James

    2012-01-01

    Cellulose degradation is essential for the future production of many advanced biofuels. Cellulases from the filamentous fungus Trichoderma reesei are among the most efficient enzymes for the hydrolysis of cellulosic materials. One of the cellulases from T. reesei, cellobiohydrolase II (CBH2), was studied because of its industrial relevance and proven enzymatic activity. Using both crude and rigorous membrane fractionation methods we show that full length T. reesei CBH2 is exclusively localized to the outer membrane when expressed recombinantly in Escherichia coli. Even fusing signal sequence-free maltose-binding protein to the N-terminus of CBH2, which has been shown to increase solubility of other proteins, did not prevent the outer membrane localization of CBH2. These results highlight the difficulties in producing fungal cellulases in bacterial hosts and provide a stepping stone for future cellulase engineering efforts.

  15. Ground Based Studies of the Outer Planets

    NASA Technical Reports Server (NTRS)

    Trafton, Laurence M.

    2005-01-01

    This report covers progress to date under this grant on our continuing program to conduct ground based studies of the outer solar system planets and satellites, with emphasis on spectroscopy and atmospheric phenomena. The research continues under our new PAST grant, NNG04G131G beginning 5/1/2004. The original period of performance of the subject grant was 3/1/2001 to 2/28/2004, but was extended one year at no cost. Although there is some overlap in the scientific projects conducted during the extended year with those of the new grant, this report is confined to the portion of the work funded under NAG5-10435. The primary goals for this grant period were a comparative study of outer planet thermospheres/ionospheres near solar maximum, extended to the mid-IR, and the investigation of molecular dimers in outer solar system atmospheres. This project supports NASA's planned space missions, Jupiter Polar Orbiter, outer Planet Microprobes, and the recent Cassini flyby of Jupiter. It also supports the OSS strategic plan themes, The Exploration of the Solar System and The Sun-Earth Connection/ Understanding comparative planetary space environments.

  16. BIOREACTOR DESIGN - OUTER LOOP LANDFILL, LOUISVILLE, KY

    EPA Science Inventory

    Bioreactor field demonstration projects are underway at the Outer Loop Landfill in Louisville, KY, USA. The research effort is a cooperative research effort between US EPA and Waste Management Inc. Two primary kinds of municipal waste bioreactors are under study at this site. ...

  17. THE ORBITS OF THE OUTER URANIAN SATELLITES

    SciTech Connect

    Brozovic, M.; Jacobson, R. A.

    2009-04-15

    We report on the numerically integrated orbits for the nine outer Uranian satellites. The orbits are calculated based on fits to the astrometric observations for the period from 1984 to 2006. The results include the state vectors, post-fit residuals, and mean orbital elements. We also assess the accuracy of the orbital fits and discuss the need for future measurements.

  18. The magnetospheres of the outer planets

    SciTech Connect

    Mcnutt, R.L., Jr. )

    1991-01-01

    Research on the magnetospheres of all of the outer planets including Jupiter, Uranus, Neptune, and Pluto is reviewed for the 1987-1990 time period. Particular attention is given to magnetospheric structure, plasma transport, Jovian aurora, Io and the plasma torus, Titan and its magnetospheric interactions, rings and dusty plasmas, magnetospheric convection, and satellite interactions.

  19. Assembly Platform For Use In Outer Space

    NASA Technical Reports Server (NTRS)

    Rao, Niranjan S.; Buddington, Patricia A.

    1995-01-01

    Report describes conceptual platform or framework for use in assembling other structures and spacecraft in outer space. Consists of three fixed structural beams comprising central beam and two cross beams. Robotic manipulators spaced apart on platform to provide telerobotic operation of platform by either space-station or ground crews. Platform and attached vehicles function synergistically to achieve maximum performance for intended purposes.

  20. The processing of materials in outer space

    NASA Technical Reports Server (NTRS)

    Gelles, S. H.; Colling, E. W.

    1977-01-01

    Zero-gravity environment may lead to fabrication of new and improved materials. According to comprehensive study of application of this promising technology to superconducting and electrical contact materials, outer space processing could improve microstructure and homogeneity of many single and multicomponent systems formed from solidification of fluid phases. New structures that are impossible to form terrestrially may also be accessible in space environment.

  1. Site directed recombination

    DOEpatents

    Jurka, Jerzy W.

    1997-01-01

    Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

  2. The recombination epoch revisited

    NASA Technical Reports Server (NTRS)

    Krolik, Julian H.

    1989-01-01

    Previous studies of cosmological recombination have shown that this process produces as a by-product a highly superthermal population of Ly-alpha photons which retard completion of recombination. Cosmological redshifting was thought to determine the frequency distribution of the photons, while two-photon decay of hydrogen's 2s state was thought to control their numbers. It is shown here that frequency diffusion due to photon scattering dominate the cosmological redshift in the frequency range near line center which fixes the ratio of ground state to excited state population, while incoherent scattering into the far-red damping wing effectively destroys Ly-alpha photons as a rate which is competitive with two-photon decay. The former effect tends to hold back recombination, while the latter tends to accelerate it; the net results depends on cosmological parameters, particularly the combination Omega(b) h/sq rt (2q0), where Omega(b) is the fraction of the critical density provided by baryons.

  3. Crystallization of proteins from crude bovine rod outer segments.

    PubMed

    Baker, Bo Y; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L; Palczewski, Krzysztof

    2015-01-01

    Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques.

  4. Crystallization of Proteins from Crude Bovine Rod Outer Segments☆

    PubMed Central

    Baker, Bo Y.; Gulati, Sahil; Shi, Wuxian; Wang, Benlian; Stewart, Phoebe L.; Palczewski, Krzysztof

    2015-01-01

    Obtaining protein crystals suitable for X-ray diffraction studies comprises the greatest challenge in the determination of protein crystal structures, especially for membrane proteins and protein complexes. Although high purity has been broadly accepted as one of the most significant requirements for protein crystallization, a recent study of the Escherichia coli proteome showed that many proteins have an inherent propensity to crystallize and do not require a highly homogeneous sample (Totir et al., 2012). As exemplified by RPE65 (Kiser, Golczak, Lodowski, Chance, & Palczewski, 2009), there also are cases of mammalian proteins crystallized from less purified samples. To test whether this phenomenon can be applied more broadly to the study of proteins from higher organisms, we investigated the protein crystallization profile of bovine rod outer segment (ROS) crude extracts. Interestingly, multiple protein crystals readily formed from such extracts, some of them diffracting to high resolution that allowed structural determination. A total of seven proteins were crystallized, one of which was a membrane protein. Successful crystallization of proteins from heterogeneous ROS extracts demonstrates that many mammalian proteins also have an intrinsic propensity to crystallize from complex biological mixtures. By providing an alternative approach to heterologous expression to achieve crystallization, this strategy could be useful for proteins and complexes that are difficult to purify or obtain by recombinant techniques. PMID:25950977

  5. Two protein lysine methyltransferases methylate outer membrane protein B from Rickettsia.

    PubMed

    Abeykoon, Amila H; Chao, Chien-Chung; Wang, Guanghui; Gucek, Marjan; Yang, David C H; Ching, Wei-Mei

    2012-12-01

    Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Its outer membrane protein B (OmpB) is an immunodominant antigen and plays roles as protective envelope and as adhesins. The observation of the correlation between methylation of lysine residues in rickettsial OmpB and bacterial virulence has suggested the importance of an enzymatic system for the methylation of OmpB. However, no rickettsial lysine methyltransferase has been characterized. Bioinformatic analysis of genomic DNA sequences of Rickettsia identified putative lysine methyltransferases. The genes of the potential methyltransferases were synthesized, cloned, and expressed in Escherichia coli, and expressed proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The methyltransferase activities of the purified proteins were analyzed by methyl incorporation of radioactively labeled S-adenosylmethionine into recombinant fragments of OmpB. Two putative recombinant methyltransferases (rRP789 and rRP027-028) methylated recombinant OmpB fragments. The specific activity of rRP789 is 10- to 30-fold higher than that of rRP027-028. Western blot analysis using specific antibodies against trimethyl lysine showed that both rRP789 and rRP027-028 catalyzed trimethylation of recombinant OmpB fragments. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis showed that rRP789 catalyzed mono-, di-, and trimethylation of lysine, while rRP027-028 catalyzed exclusively trimethylation. To our knowledge, rRP789 and rRP027-028 are the first biochemically characterized lysine methyltransferases of outer membrane proteins from Gram-negative bacteria. The production and characterization of rickettsial lysine methyltransferases provide new tools to investigate the mechanism of methylation of OmpB, effects of methylation on the structure and function of OmpB, and development of methylated OmpB-based diagnostic assays and vaccine candidates.

  6. HERSCHEL OBSERVATIONS OF THE T CHA TRANSITION DISK: CONSTRAINING THE OUTER DISK PROPERTIES

    SciTech Connect

    Cieza, Lucas A.; Olofsson, Johan; Henning, Thomas; Harvey, Paul M.; Evans II, Neal J.; Pinte, Christophe; Augereau, Jean-Charles; Menard, Francois; Najita, Joan

    2011-11-10

    T Cha is a nearby (d {approx} 100 pc) transition disk known to have an optically thin gap separating optically thick inner and outer disk components. Huelamo et al. recently reported the presence of a low-mass object candidate within the gap of the T Cha disk, giving credence to the suspected planetary origin of this gap. Here we present the Herschel photometry (70, 160, 250, 350, and 500 {mu}m) of T Cha from the 'Dust, Ice, and Gas in Time' Key Program, which bridges the wavelength range between existing Spitzer and millimeter data and provide important constraints on the outer disk properties of this extraordinary system. We model the entire optical to millimeter wavelength spectral energy distribution (SED) of T Cha (19 data points between 0.36 and 3300 {mu}m without any major gaps in wavelength coverage). T Cha shows a steep spectral slope in the far-IR, which we find clearly favors models with outer disks containing little or no dust beyond {approx}40 AU. The full SED can be modeled equally well with either an outer disk that is very compact (only a few AU wide) or a much larger one that has a very steep surface density profile. That is, T Cha's outer disk seems to be either very small or very tenuous. Both scenarios suggest a highly unusual outer disk and have important but different implications for the nature of T Cha. Spatially resolved images are needed to distinguish between the two scenarios.

  7. Performance of the LHCb Outer Tracker

    NASA Astrophysics Data System (ADS)

    2014-01-01

    The LHCb Outer Tracker is a gaseous detector covering an area of 5 × 6 m2 with 12 double layers of straw tubes. The detector with its services are described together with the commissioning and calibration procedures. Based on data of the first LHC running period from 2010 to 2012, the performance of the readout electronics and the single hit resolution and efficiency are presented. The efficiency to detect a hit in the central half of the straw is estimated to be 99.2%, and the position resolution is determined to be approximately 200 μm. The Outer Tracker received a dose in the hottest region corresponding to 0.12 C/cm, and no signs of gain deterioration or other ageing effects are observed.

  8. Irradiation chemistry in the outer solar system

    NASA Astrophysics Data System (ADS)

    Brown, Michael E.

    2014-11-01

    The dark, reddish tinged surfaces of icy bodies in the outer solar are usually attributed to the long term irradiation of simple hydrocarbons such as methane leading to the loss of hydrogen and the production of long carbon chains. While methane is stable and detected on the most massive bodies in the Kuiper belt, evidence of active irradiation chemistry is scant except for the presence of ethane on methane-rich Makemake and possible detections of ethane on more methane-poor Pluto and Quaoar. We have obtained deep high signal-to-noise spectra of Makemake from 1.5 to 2.5 microns in an attempt to trace the radiation chemistry in the outer solar system beyond the initial ethane formation. We present the first astrophysical detections of solid ethylene, acetylene, and possibly propane -- all expected products of the continued irradiation of methane, and use these species to map the chemical pathway from methane to long-chain hydrocarbons.

  9. Cryovolcanism in the outer solar system

    USGS Publications Warehouse

    Geissler, Paul E.

    2015-01-01

    Cryovolcanism is defined as the extrusion of liquids and vapors of materials that would be frozen solid at the planetary surface temperatures of the icy bodies of the outer solar system. Active cryovolcanism is now known to occur on Saturn's moon Enceladus and on Neptune's moon Triton and is suspected on Jupiter's moon Europa, while evidence for past cryovolcanic activity is widespread throughout the outer solar system. This chapter examines the mechanisms and manifestations of cryovolcanism, beginning with a review of the materials that make up these unusual ‘‘magmas’’ and the means by which they might erupt and concluding with a volcanologist's tour of the farthest reaches of the solar system.

  10. Outer crust of nonaccreting cold neutron stars

    NASA Astrophysics Data System (ADS)

    Rüster, Stefan B.; Hempel, Matthias; Schaffner-Bielich, Jürgen

    2006-03-01

    The properties of the outer crust of nonaccreting cold neutron stars are studied by using modern nuclear data and theoretical mass tables, updating in particular the classic work of Baym, Pethick, and Sutherland. Experimental data from the atomic mass table from Audi, Wapstra, and Thibault of 2003 are used and a thorough comparison of many modern theoretical nuclear models, both relativistic and nonrelativistic, is performed for the first time. In addition, the influences of pairing and deformation are investigated. State-of-the-art theoretical nuclear mass tables are compared to check their differences concerning the neutron drip line, magic neutron numbers, the equation of state, and the sequence of neutron-rich nuclei up to the drip line in the outer crust of nonaccreting cold neutron stars.

  11. Outer crust of nonaccreting cold neutron stars

    SciTech Connect

    Ruester, Stefan B.; Hempel, Matthias; Schaffner-Bielich, Juergen

    2006-03-15

    The properties of the outer crust of nonaccreting cold neutron stars are studied by using modern nuclear data and theoretical mass tables, updating in particular the classic work of Baym, Pethick, and Sutherland. Experimental data from the atomic mass table from Audi, Wapstra, and Thibault of 2003 are used and a thorough comparison of many modern theoretical nuclear models, both relativistic and nonrelativistic, is performed for the first time. In addition, the influences of pairing and deformation are investigated. State-of-the-art theoretical nuclear mass tables are compared to check their differences concerning the neutron drip line, magic neutron numbers, the equation of state, and the sequence of neutron-rich nuclei up to the drip line in the outer crust of nonaccreting cold neutron stars.

  12. The DarkSide-50 outer detectors

    NASA Astrophysics Data System (ADS)

    Westerdale, S.; Agnes, P.; Agostino, L.; Albuquerque, I. F. M.; Alexander, T.; Alton, A. K.; Arisaka, K.; Back, H. O.; Baldin, B.; Biery, K.; Bonfini, G.; Bossa, M.; Bottino, B.; Brigatti, A.; Brodsky, J.; Budano, F.; Bussino, S.; Cadeddu, M.; Cadonati, L.; Cadoni, M.; Calaprice, F.; Canci, N.; Candela, A.; Cao, H.; Cariello, M.; Carlini, M.; Catalanotti, S.; Cavalcante, P.; Chepurnov, A.; Cocco, A. G.; Covone, G.; D’Angelo, D.; D’Incecco, M.; Davini, S.; De Cecco, S.; De Deo, M.; De Vincenzi, M.; Derbin, A.; Devoto, A.; Di Eusanio, F.; Di Pietro, G.; Edkins, E.; Empl, A.; Fan, A.; Fiorillo, G.; Fomenko, K.; Foster, G.; Franco, D.; Gabriele, F.; Galbiati, C.; Giganti, C.; Goretti, A. M.; Granato, F.; Grandi, L.; Gromov, M.; Guan, M.; Guardincerri, Y.; Hackett, B. R.; Herner, K. R.; Hungerford, E. V.; Aldo, Ianni; Andrea, Ianni; James, I.; Jollet, C.; Keeter, K.; Kendziora, C. L.; Kobychev, V.; Koh, G.; Korablev, D.; Korga, G.; Kubankin, A.; Li, X.; Lissia, M.; Lombardi, P.; Luitz, S.; Ma, Y.; Machulin, I. N.; Mandarano, A.; Mari, S. M.; Maricic, J.; Marini, L.; Martoff, C. J.; Meregaglia, A.; Meyers, P. D.; Miletic, T.; Milincic, R.; Montanari, D.; Monte, A.; Montuschi, M.; Monzani, M. E.; Mosteiro, P.; Mount, B. J.; Muratova, V. N.; Musico, P.; Napolitano, J.; Orsini, M.; Ortica, F.; Pagani, L.; Pallavicini, M.; Pantic, E.; Parmeggiano, S.; Pelczar, K.; Pelliccia, N.; Perasso, S.; Pocar, A.; Pordes, S.; Pugachev, D. A.; Qian, H.; Randle, K.; Ranucci, G.; Razeto, A.; Reinhold, B.; Renshaw, A. L.; Romani, A.; Rossi, B.; Rossi, N.; Rountree, S. D.; Sablone, D.; Saggese, P.; Saldanha, R.; Sands, W.; Sangiorgio, S.; Savarese, C.; Segreto, E.; Semenov, D. A.; Shields, E.; Singh, P. N.; DSkorokhvatov, M.; Smirnov, O.; Sotnikov, A.; Stanford, C.; Suvorov, Y.; Tartaglia, R.; Tatarowicz, J.; Testera, G.; Tonazzo, A.; Trinchese, P.; Unzhakov, E. V.; Vishneva, A.; Vogelaar, B.; Wada, M.; Walker, S.; Wang, H.; Wang, Y.; Watson, A. W.; Wilhelmi, J.; Wojcik, M. M.; Xiang, X.; Xu, J.; Yang, C.; Yoo, J.; Zavatarelli, S.; Zec, A.; Zhong, W.; Zhu, C.; Zuzel, G.; The DarkSide Collaboration

    2016-05-01

    DarkSide-50 is a dark matter detection experiment searching for Weakly Interacting Massive Particles (WIMPs), in Gran Sasso National Laboratory. For experiments like DarkSide-50, neutrons are one of the primary backgrounds that can mimic WIMP signals. The experiment consists of three nested detectors: a liquid argon time projection chamber surrounded by two outer detectors. The outermost detector is a 10 m by 11 m cylindrical water Cherenkov detector with 80 PMTs, designed to provide shielding and muon vetoing. Inside the water Cherenkov detector is the 4 m diameter spherical boron-loaded liquid scintillator veto, with a cocktail of pseudocumene, trimethyl borate, and PPO wavelength shifter, designed to provide shielding, neutron vetoing, and in situ measurements of the TPC backgrounds. We present design and performance details of the DarkSide-50 outer detectors.

  13. Boundary layers of the earth's outer magnetosphere

    NASA Technical Reports Server (NTRS)

    Eastman, T. E.; Frank, L. A.

    1984-01-01

    The magnetospheric boundary layer and the plasma-sheet boundary layer are the primary boundary layers of the earth's outer magnetosphere. Recent satellite observations indicate that they provide for more than 50 percent of the plasma and energy transport in the outer magnetosphere although they constitute less than 5 percent by volume. Relative to the energy density in the source regions, plasma in the magnetospheric boundary layer is predominantly deenergized whereas plasma in the plasma-sheet boundary layer has been accelerated. The reconnection hypothesis continues to provide a useful framework for comparing data sampled in the highly dynamic magnetospheric environment. Observations of 'flux transfer events' and other detailed features near the boundaries have been recently interpreted in terms of nonsteady-state reconnection. Alternative hypotheses are also being investigated. More work needs to be done, both in theory and observation, to determine whether reconnection actually occurs in the magnetosphere and, if so, whether it is important for overall magnetospheric dynamics.

  14. Evolution of recombination in eutherian mammals: insights into mechanisms that affect recombination rates and crossover interference.

    PubMed

    Segura, Joana; Ferretti, Luca; Ramos-Onsins, Sebastián; Capilla, Laia; Farré, Marta; Reis, Fernanda; Oliver-Bonet, Maria; Fernández-Bellón, Hugo; Garcia, Francisca; Garcia-Caldés, Montserrat; Robinson, Terence J; Ruiz-Herrera, Aurora

    2013-11-22

    Recombination allows faithful chromosomal segregation during meiosis and contributes to the production of new heritable allelic variants that are essential for the maintenance of genetic diversity. Therefore, an appreciation of how this variation is created and maintained is of critical importance to our understanding of biodiversity and evolutionary change. Here, we analysed the recombination features from species representing the major eutherian taxonomic groups Afrotheria, Rodentia, Primates and Carnivora to better understand the dynamics of mammalian recombination. Our results suggest a phylogenetic component in recombination rates (RRs), which appears to be directional, strongly punctuated and subject to selection. Species that diversified earlier in the evolutionary tree have lower RRs than those from more derived phylogenetic branches. Furthermore, chromosome-specific recombination maps in distantly related taxa show that crossover interference is especially weak in the species with highest RRs detected thus far, the tiger. This is the first example of a mammalian species exhibiting such low levels of crossover interference, highlighting the uniqueness of this species and its relevance for the study of the mechanisms controlling crossover formation, distribution and resolution.

  15. Photopolarimetry team outer planets mission definition phase

    NASA Technical Reports Server (NTRS)

    1972-01-01

    The work is reported of the Photopolarimetry Team in identifying scientific objectives for photometer/polarimeter experiments for outer planet flyby missions. A discussion of the scientific objectives which can be attained with a photometer/polarimeter experiment, and summaries of the special studies which were performed for the Photopolarimetry Team are presented along with a description of the photometer/polarimeter design which was developed for the Meteoroid Detection Team.

  16. A model environment for outer zone electrons

    NASA Technical Reports Server (NTRS)

    Singley, G. W.; Vette, J. I.

    1972-01-01

    A brief morphology of outer zone electrons is given to illustrate the nature of the phenomena that we are attempting to model. This is followed by a discussion of the data processing that was done with the various data received from the experimenters before incorporating it into the data base from which this model was ultimately derived. The details of the derivation are given, and several comparisons of the final model with the various experimental measurements are presented.

  17. Stratigraphic evolution of Blake Outer Ridge

    SciTech Connect

    Markl, R.G.; Bryan, G.M.

    1983-04-01

    Multichannel seismic data from a reconnaissance survey of the Blake Outer Ridge reveal the seismic stratigraphy down to oceanic basement, as well as intracrustal and mantle reflections. The depositional history of the outer ridge can now be subdivided into four principal phases, based on seismic stratigraphic style: (1) Jurassic-Early Cretaceous sedimentation which filled in basement irregularities and leveled the sea floor by horizon ..beta.. (Barremian) time, (2) Early Cretaceous-Late Cretaceous deposition of a seaward-thinning wedge typical of Atlantic-type margins; its deeply eroded surface is probably equivalent to the Late Cretaceous-Miocene hiatus reflected by horizon A'', (3) earlies Miocene current-controlled deposits preferentially deposited along the axis of the incipient Blake Outer Ridge; these prograding strata, which thin and dip downridge and laterally away from the ridge axis, are attributed to the interaction of the Florida Current and Western Boundary Undercurrent, and (4) continuing early Miocene preferential deposition on the ridge axis and sculpting by the Western Boundary Undercurrent; this phase is characterized by strata thinning and dipping upridge and toward the ridge axis. The reversal of dip is explained to a first approximation by a steady-state contour-current model in which the current position is progressively shifted by the deposition. Five angular unconformities and associated bathymetric terraces west of the ridge crest are mapped using all Lamont-Doherty seismic data and interpreted as having formed penecontemporaneously during phase four. The areal extent of the gas hydrate (clathrate) horizon is also delineated; it is continuous across the northern Blake Outer Ridge, and the multichannel seismic data presented here show it to extend onto the shallow Blake Plateau as well.

  18. Planetary magnetism in the outer solar system.

    NASA Technical Reports Server (NTRS)

    Sonett, C. P.

    1973-01-01

    A brief review of the salient considerations which apply to the existence of magnetic fields in connection with planetary and subplanetary objects in the outer solar system is given. Consideration is given to internal dynamo fields, fields which might originate from interaction with the solar wind or magnetospheres (externally driven dynamos) and lastly fossil magnetic fields such as have been discovered on the moon. Where possible, connection is made between magnetism, means of detection, and internal body properties.

  19. Evidence for an oxygen-depleted liquid outer core of the Earth.

    PubMed

    Huang, Haijun; Fei, Yingwei; Cai, Lingcang; Jing, Fuqian; Hu, Xiaojun; Xie, Hongsen; Zhang, Lianmeng; Gong, Zizheng

    2011-11-24

    On the basis of geophysical observations, cosmochemical constraints, and high-pressure experimental data, the Earth's liquid outer core consists of mainly liquid iron alloyed with about ten per cent (by weight) of light elements. Although the concentrations of the light elements are small, they nevertheless affect the Earth's core: its rate of cooling, the growth of the inner core, the dynamics of core convection, and the evolution of the geodynamo. Several light elements-including sulphur, oxygen, silicon, carbon and hydrogen-have been suggested, but the precise identity of the light elements in the Earth's core is still unclear. Oxygen has been proposed as a major light element in the core on the basis of cosmochemical arguments and chemical reactions during accretion. Its presence in the core has direct implications for Earth accretion conditions of oxidation state, pressure and temperature. Here we report new shockwave data in the Fe-S-O system that are directly applicable to the outer core. The data include both density and sound velocity measurements, which we compare with the observed density and velocity profiles of the liquid outer core. The results show that we can rule out oxygen as a major light element in the liquid outer core because adding oxygen into liquid iron would not reproduce simultaneously the observed density and sound velocity profiles of the outer core. An oxygen-depleted core would imply a more reduced environment during early Earth accretion.

  20. Dielectronic recombination theory

    SciTech Connect

    LaGattuta, K.J.

    1991-12-31

    A theory now in wide use for the calculation of dielectronic recombination cross sections ({sigma}{sup DR}) and rate coefficients ({alpha}{sup DR}) was one introduced originally by Feshbach for nuclear physics applications, and then later adapted for atomic scattering problems by Hahn. In the following, we briefly review this theory in a very general form, which allows one to account for the effects of overlapping and interacting resonances, as well as continuum-continuum coupling. An extension of our notation will then also allow for the inclusion of the effects of direct radiative recombination, along with a treatment of the interference between radiative and dielectronic recombination. Other approaches to the calculation of {sigma}{sup DR} have been described by Fano and by Seaton. We will not consider those theories here. Calculations of {alpha}{sup DR} have progressed considerably over the last 25 years, since the early work of Burgess. Advances in the reliability of theoretical predictions have also been promoted recently b a variety of direct laboratory measurements of {sigma}{sup DR}. While the measurements of {sigma}{sup DR} for {delta}n {ne} 0 excitations have tended to agree very well with calculations, the case of {delta}n = 0 has been much problematic. However, by invoking a mechanism originally proposed by Jacobs, which takes into account the effect of stray electric fields on high Rydberg states (HRS) participating in the DR process, new calculations have improved the agreement between theory and experiment for these cases. Nevertheless, certain discrepancies still remain.

  1. Recombinant Collagenlike Proteins

    NASA Technical Reports Server (NTRS)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  2. CLOSURE WELD DEVELOPMENT FOR 3013 OUTER CONTAINERS

    SciTech Connect

    Daugherty, W.; Howard, S.; Peterson, K.; Stokes, M.

    2009-11-10

    Excess plutonium materials in the DOE complex are packaged and stored in accordance with DOE-STD-3013. This standard specifies requirements for the stabilization of such materials and subsequent packaging in dual nested seal-welded containers. Austenitic stainless steels have been selected for container fabrication. The inner 3013 container provides contamination control while the outer 3013 container is the primary containment vessel and is the focus of this paper. Each packaging site chose a process for seal welding the outer 3013 containers in accordance with its needs and expertise. The two processes chosen for weld closure were laser beam welding (LBW) and gas tungsten arc welding (GTAW). Following development efforts, each system was qualified in accordance with DOE-STD-3013 prior to production use. The 3013 outer container closure weld joint was designed to accommodate the characteristics of a laser weld. This aspect of the joint design necessitated some innovative process and equipment considerations in the application of the GTAW process. Details of the weld requirements and the development processes are presented and several potential enhancements for the GTAW system are described.

  3. Outer membrane proteins of pathogenic spirochetes

    PubMed Central

    Cullen, Paul A.; Haake, David A.; Adler, Ben

    2009-01-01

    Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

  4. Innovation by homologous recombination.

    PubMed

    Trudeau, Devin L; Smith, Matthew A; Arnold, Frances H

    2013-12-01

    Swapping fragments among protein homologs can produce chimeric proteins with a wide range of properties, including properties not exhibited by the parents. Computational methods that use information from structures and sequence alignments have been used to design highly functional chimeras and chimera libraries. Recombination has generated proteins with diverse thermostability and mechanical stability, enzyme substrate specificity, and optogenetic properties. Linear regression, Gaussian processes, and support vector machine learning have been used to model sequence-function relationships and predict useful chimeras. These approaches enable engineering of protein chimeras with desired functions, as well as elucidation of the structural basis for these functions.

  5. Expression of Recombinant Antibodies

    PubMed Central

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transgenic plants and animals. Currently, almost all therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to altered, non-human glycosylation patterns. However, recent developments of glycosylation-engineered yeast, insect cell lines, and transgenic plants are promising to obtain antibodies with “human-like” post-translational modifications. Furthermore, smaller antibody fragments including bispecific antibodies without any glycosylation are successfully produced in bacteria and have advanced to clinical testing. The first therapeutic antibody products from a non-mammalian source can be expected in coming next years. In this review, we focus on current antibody production systems including their usability for different applications. PMID:23908655

  6. Recombinant electric storage battery

    SciTech Connect

    Flicker, R.P.; Fenstermacher, S.

    1989-10-10

    This patent describes a recombinant storage battery. It comprises: a plurality of positive plates containing about 2 to 4 percent of antimony based upon the total weight of the alloy and positive active material, and essentially antimony free negative plates in a closed case; a fibrous sheet plate separator between adjacent ones of the plates, and a body of an electrolyte to which the sheet separators are inert absorbed by each of the separators and maintained in contact with each of the adjacent ones of the plates. Each of the separator sheets comprising first fibers which impart to the sheet a given absorbency greater than 90 percent relative to the electrolyte and second fibers which impart to the sheet a different absorbency less than 80 percent relative to the electrolyte. The first and second fibers being present in such proportions that each of the sheet separators has an absorbency with respect to the electrolyte of from 75 to 95 percent and the second fibers being present in such proportions that the battery has a recombination rate adequate to compensate for gassing.

  7. 75 FR 61512 - Outer Continental Shelf Official Protraction Diagrams

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-10-05

    ... Outer Continental Shelf Official Protraction Diagrams (OPDs) located within Atlantic Ocean areas, with... Bureau of Ocean Energy Management, Regulation and Enforcement Outer Continental Shelf Official Protraction Diagrams AGENCY: Bureau of Ocean Energy Management, Regulation and Enforcement, Interior....

  8. Involvement of a 43-kilodalton outer membrane protein in beta-lactam resistance of Shigella dysenteriae.

    PubMed Central

    Kar, A K; Ghosh, A S; Chauhan, K; Ahamed, J; Basu, J; Chakrabarti, P; Kundu, M

    1997-01-01

    A beta-lactam-sensitive strain (C152) of Shigella dysenteriae showed two major outer membrane proteins (OMPs) with M(r)s of 43,000 and 38,000, while the clinical isolate M2 lacked the 43,000-Mr OMP, which acted as a channel for beta-lactam antibiotics. Permeability of beta-lactams across the outer membrane (OM) of M2 was lower than that across the OM of C152. Mutants deficient in the 43-kDa OMP could be selected in vitro from strain C152 in the presence of cefoxitin. All beta-lactam-resistant strains were sensitive to imipenem. PMID:9333070

  9. Functional Activity of Antibodies against the Recombinant OpaJ Protein from Neisseria meningitidis

    PubMed Central

    de Jonge, M. I.; Vidarsson, G.; van Dijken, H. H.; Hoogerhout, P.; van Alphen, L.; Dankert, J.; van der Ley, P.

    2003-01-01

    The opacity proteins belong to the major outer membrane proteins of the pathogenic Neisseria and are involved in adhesion and invasion. We studied the functional activity of antibodies raised against the OpaJ protein from strain H44/76. Recombinant OpaJ protein was obtained from Escherichia coli in two different ways: cytoplasmic expression in the form of inclusion bodies followed by purification and refolding and cell surface expression followed by isolation of outer membrane complexes (OMCs). Immunization with purified protein and Quillaja saponin A (QuilA) induced high levels of Opa-specific antibodies, whereas the E. coli OMC preparations generally induced lower levels of antibodies. Two chimeric Opa proteins, hybrids between OpaB and OpaJ, were generated to demonstrate that the hypervariable region 2 is immunodominant. Denatured OpaJ with QuilA induced high levels of immunoglobulin G2a (IgG2a) in addition to IgG1, whereas refolded OpaJ with QuilA induced IgG1 exclusively. These sera did not induce significant complement-mediated killing. However, all sera blocked the interaction of OpaJ-expressing bacteria to CEACAM1-transfected cells. In addition, cross-reactive blocking of OpaB-expressing bacteria to both CEACAM1- and CEA-transfected cells was found for all sera. Sera raised against purified OpaJ and against OpaJ-containing meningococcal OMCs also blocked the nonopsonic interaction of Opa-expressing meningococci with human polymorphonuclear leukocytes. PMID:12704102

  10. Formaldehyde in the far outer galaxy: constraining the outer boundary of the galactic habitable zone.

    PubMed

    Blair, Samantha K; Magnani, Loris; Brand, Jan; Wouterloot, Jan G A

    2008-02-01

    We present results from an initial survey of the 2(12)-1(11) transition of formaldehyde (H2CO) at 140.8 GHz in giant molecular clouds in the far outer Galaxy (RG >or= 16 kpc). Formaldehyde is a key prebiotic molecule that likely plays an important role in the development of amino acids. Determining the outermost extent of the H2CO distribution can constrain the outer limit of the Galactic Habitable Zone, the region where conditions for the formation of life are thought to be most favorable. We surveyed 69 molecular clouds in the outer Galaxy, ranging from 12 to 23.5 kpc in galactocentric radius. Formaldehyde emission at 140.8 GHz was detected in 65% of the clouds. The H2CO spectral line was detected in 26 of the clouds with RG > 16 kpc (detection rate of 59%), including 6 clouds with RG > 20 kpc (detection rate of 55%). Formaldehyde is readily found in the far outer Galaxy-even beyond the edge of the old stellar disk. Determining the relatively widespread distribution of H2CO in the far outer Galaxy is a first step in establishing how favorable an environment this vast region of the Galaxy may be toward the formation of life.

  11. Comparison of marine productivity among Outer Continental Shelf planning areas

    SciTech Connect

    Darnell, R.M.

    1991-04-01

    Continental Shelf Associates was contracted to update and expand an earlier work on a comparison of primary productivity among Outer Continental Shelf (OCS) planning areas. The update consists of four general objectives. This report addresses the fourth objective, the potential to use measures of marine secondary productivity in determining relative rankings of the OCS planning area. Ideally, comparisons of secondary productivity among diverse geographic areas would take an ecosystem perspective. However, there is not enough ecosystem-level understanding to allow a comparison on that basis. The report focuses on individual species and group of species. Zooplankton and benthic communities represent major portions of the marine ecosystems and would be the preferred subjects for comparative studies. At this time comparisons of secondary productivity among planning areas would be difficult with existing databases. It is unlikely this situation will improve in the near future.

  12. The outer mucus layer hosts a distinct intestinal microbial niche

    PubMed Central

    Li, Hai; Limenitakis, Julien P.; Fuhrer, Tobias; Geuking, Markus B.; Lawson, Melissa A.; Wyss, Madeleine; Brugiroux, Sandrine; Keller, Irene; Macpherson, Jamie A.; Rupp, Sandra; Stolp, Bettina; Stein, Jens V.; Stecher, Bärbel; Sauer, Uwe; McCoy, Kathy D.; Macpherson, Andrew J.

    2015-01-01

    The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host. PMID:26392213

  13. Progressive outer retinal necrosis in a patient with nephrotic syndrome.

    PubMed

    Shinoda, K; Inoue, M; Ishida, S; Kawashima, S; Wakabayashi, T; Suzuki, S; Katsura, H

    2001-01-01

    Progressive outer retinal necrosis syndrome (PORN) is a variant of necrotizing herpetic retinopathy and the majority of the described cases were related to acquired immunodeficiency syndrome. We present a patient who is HIV negative with nephrotic syndrome and prednisolone use for 4 months who showed clinical features of PORN. Low CD4 counts and lymphocytopenia suggested immunosuppression. In the left eye, tractional retinal detachment at the posterior pole followed by incomplete posterior vitreous detachment developed. In addition to intravenous administration of acyclovir, vitreous surgeries including stripping of the posterior hyaloid and silicone-oil tamponade were successfully performed to repair the retinal detachment in the left eye and to prevent it in the right eye.

  14. 46 CFR 154.170 - Outer hull steel plating.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Outer hull steel plating. 154.170 Section 154.170... Structure § 154.170 Outer hull steel plating. (a) Except as required in paragraph (b) of this section, the outer hull steel plating, including the shell and deck plating must meet the material standards of...

  15. 46 CFR 154.170 - Outer hull steel plating.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Outer hull steel plating. 154.170 Section 154.170... Structure § 154.170 Outer hull steel plating. (a) Except as required in paragraph (b) of this section, the outer hull steel plating, including the shell and deck plating must meet the material standards of...

  16. 46 CFR 154.170 - Outer hull steel plating.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Outer hull steel plating. 154.170 Section 154.170... Structure § 154.170 Outer hull steel plating. (a) Except as required in paragraph (b) of this section, the outer hull steel plating, including the shell and deck plating must meet the material standards of...

  17. 46 CFR 154.170 - Outer hull steel plating.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 5 2010-10-01 2010-10-01 false Outer hull steel plating. 154.170 Section 154.170... Structure § 154.170 Outer hull steel plating. (a) Except as required in paragraph (b) of this section, the outer hull steel plating, including the shell and deck plating must meet the material standards of...

  18. 46 CFR 154.170 - Outer hull steel plating.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Outer hull steel plating. 154.170 Section 154.170... Structure § 154.170 Outer hull steel plating. (a) Except as required in paragraph (b) of this section, the outer hull steel plating, including the shell and deck plating must meet the material standards of...

  19. 33 CFR 117.323 - Outer Clam Bay

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Outer Clam Bay 117.323 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.323 Outer Clam Bay The drawspan of the Outer Clam Bay Boardwalk Drawbridge shall open on signal if at least 30 minutes advance notice is given....

  20. 33 CFR 117.323 - Outer Clam Bay.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Outer Clam Bay. 117.323 Section... DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.323 Outer Clam Bay. The drawspan of the Outer Clam Bay Boardwalk Drawbridge shall open on signal if at least 30 minutes advance notice is given....

  1. Unraveling recombination rate evolution using ancestral recombination maps

    PubMed Central

    Munch, Kasper; Schierup, Mikkel H; Mailund, Thomas

    2014-01-01

    Recombination maps of ancestral species can be constructed from comparative analyses of genomes from closely related species, exemplified by a recently published map of the human-chimpanzee ancestor. Such maps resolve differences in recombination rate between species into changes along individual branches in the speciation tree, and allow identification of associated changes in the genomic sequences. We describe how coalescent hidden Markov models are able to call individual recombination events in ancestral species through inference of incomplete lineage sorting along a genomic alignment. In the great apes, speciation events are sufficiently close in time that a map can be inferred for the ancestral species at each internal branch - allowing evolution of recombination rate to be tracked over evolutionary time scales from speciation event to speciation event. We see this approach as a way of characterizing the evolution of recombination rate and the genomic properties that influence it. PMID:25043668

  2. Rv1698 of Mycobacterium tuberculosis Represents a New Class of Channel-forming Outer Membrane Proteins*S⃞

    PubMed Central

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

    2008-01-01

    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

  3. The outer haloes of massive, elliptical galaxies

    NASA Astrophysics Data System (ADS)

    Das, Payel; Gerhard, Ortwin; de Lorenzi, Flavio; McNeil, Emily; Churazov, Eugene; Coccato, Lodovico

    2010-11-01

    The outer haloes of massive elliptical galaxies are dark-matter dominated regions where stellar orbits have longer dynamical timescales than the central regions and therefore better preserve their formation history. Dynamical models out to large radii suffer from a degeneracy between mass and orbital structure, as the outer kinematics are unable to resolve higher moments of the line-of-sight velocity distribution. We mitigate this degeneracy for a sample of quiescent, massive, nearby ellipticals by determining their mass distributions independently using a non-parametric method on X-ray observations of the surrounding hot interstellar medium. We then create dynamical models using photometric and kinematic constraints consisting of integral-eld, long-slit and planetary nebulae (PNe) data extending to ~50 kpc. The rst two galaxies of our sample, NGC 5846 and NGC 1399, were found to have very shallow pro jected light distributions with a power law index of ~1.5 and a dark matter content of 70-80% at 50 kpc. Spherical Jeans models of the data show that, in the outer haloes of both galaxies, the pro jected velocity dispersions are almost inde- pendent of the anisotropy and that the PNe prefer the lower end of the range of mass distributions consistent with the X-ray data. Using the N-body code NMAGIC, we cre- ated axisymmetric models of NGC 5846 using the individual PNe radial velocities in a likelihood method and found them to be more constraining than the binned velocity dispersions. Characterising the orbital structure in terms of spherically averaged proles of the velocity dispersions we nd σψ > σr > σθ.

  4. The Young Outer Disk of M83

    NASA Astrophysics Data System (ADS)

    Davidge, T. J.

    2010-08-01

    Deep near-infrared images recorded with NICI on Gemini South are used to investigate the evolved stellar content in the outer southeast quadrant of the spiral galaxy M83. A diffuse population of asymptotic giant branch (AGB) stars is detected, indicating that there are stars outside of the previously identified young and intermediate age star clusters in the outer disk. The brightest AGB stars have M K >= -8, and the AGB luminosity function (LF) is well matched by model LFs that assume ages <=1 Gyr. The specific star formation rate (SFR) during the past few Gyr estimated from AGB star counts is consistent with that computed from mid-infrared observations of star clusters at similar radii, and it is concluded that the disruption timescale for star clusters in the outer disk is Lt1 Gyr. The LF and specific frequency of AGB stars vary with galactocentric radius, in a manner that is indicative of lower luminosity-weighted ages at larger radii. Modest numbers of red supergiants are also found, indicating that there has been star formation during the past 100 Myr, while the ratio of C stars to M giants is consistent with that expected for a solar metallicity system that has experienced a constant SFR for the past few Gyr. The results drawn from the properties of resolved AGB stars are broadly consistent with those deduced from integrated light observations in the UV. Based on observations obtained at the Gemini Observatory, which is operated by the Association of Universities for Research in Astronomy, Inc., under a co-operative agreement with the NSF on behalf of the Gemini partnership: the National Science Foundation (United States), the Science and Technology Facilities Council (United Kingdom), the National Research Council of Canada (Canada), CONICYT (Chile), the Australian Research Council (Australia), the Ministerio da Ciencia e Technologia (Brazil), and the Ministerio de Ciencia, Tecnologia e Innovacion Productiva (Argentina).

  5. Extra-mitochondrial aerobic metabolism in retinal rod outer segments: new perspectives in retinopathies.

    PubMed

    Panfoli, I; Calzia, D; Ravera, S; Morelli, A M; Traverso, C E

    2012-04-01

    Vertebrate retinal rods are photoreceptors for dim-light vision. They display extreme sensitivity to light thanks to a specialized subcellular organelle, the rod outer segment. This is filled with a stack of membranous disks, expressing the proteins involved in visual transduction, a very energy demanding process. Our previous proteomic and biochemical studies have shed new light on the chemical energy processes that supply ATP to the outer segment, suggesting the presence of an extra-mitochondrial aerobic metabolism in rod outer segment, devoid of mitochondria, which would account for a quantitatively adequate ATP supply for phototransduction. Here the functional presence of an oxidative phosphorylation in the rod outer limb is examined for its relationship to many physiological and pathological data on the rod outer segment. We hypothesize that the rod outer limb is at risk of oxidative stress, in any case of impairment in the respiratory chain functioning, or of blood supply. In fact, the electron transfer chain is a major source of reactive O(2) species, known to produce severe alteration to the membrane lipids, especially those of the outer segment that are rich in polyunsaturated fatty acids. We propose that the disk membrane may become the target of reactive oxygen species that may be released by the electron transport chain under pathologic conditions. For example, during aging reactive oxygen species production increases, while cellular antioxidant capacity decreases. Also the apoptosis of the rod observed after exposure to bright or continuous illumination can be explained considering that an overfunctioning of phototransduction may damage the disk membrane to a point at which cytochrome c escapes from the intradiskal space, where it is presently supposed to be, activating a putative caspase 9 and the apoptosome. A pathogenic mechanism for many inherited and acquired retinal degenerations, representing a major problem in clinical ophthalmology, is

  6. Outer atmospheres of giant and supergiant stars

    NASA Technical Reports Server (NTRS)

    Brown, A.

    1984-01-01

    The properties of the chromospheres, transition regions and coronas of cool evolved stars are reviewed based primarily on recent ultraviolet and X-ray studies. Determinations of mass loss rates using new observational techniques in the ultraviolet and radio spectral regions are discussed and observations indicating general atmospheric motions are considered. The techniques available for the quantitative modeling of these atmospheres are outlined and recent results discussed. Finally, the current rudimentary understanding of the evolution of these outer atmospheres and its causes are considered.

  7. Dark matter in the outer solar system

    NASA Technical Reports Server (NTRS)

    Owen, T.; Cruikshank, D.; De Bergh, C.; Geballe, T.

    1994-01-01

    There are now a large number of small bodies in the outer solar system that are known to be covered with dark material. Attempts to identify that material have been thwarted by the absence of discrete absorption features in the reflection spectra of these planetesimals. An absorption at 2.2 micrometers that appeared to be present in several objects has not been confirmed by new observations. Three absorptions in the spectrum of the unusually red planetesimal 5145 Pholus are well-established, but their identity remains a mystery.

  8. Dishwasher For Earth Or Outer Space

    NASA Technical Reports Server (NTRS)

    Tromble, Jon D.

    1991-01-01

    Dishwashing machine cleans eating utensils in either Earth gravity or zero gravity of outer space. Cycle consists of three phases: filling, washing, and draining. Rotation of tub creates artificial gravity aiding recirculation of water during washing phase in absence of true gravity. Centrifugal air/water separator helps system function in zero gravity. Self-cleaning filter contains interdigitating blades catching solid debris when water flows between them. Later, blades moved back and forth in scissor-like manner to dislodge debris, removed by backflow of water.

  9. Eccentric features in Saturn's outer C ring

    SciTech Connect

    Porco, C.C.; Nicholson, P.D.

    1987-11-01

    The present search for possible eccentric and inclined features in the outer C ring of Saturn measured all sharp-edged feature radii in Voyager C ring data. The Maxwell ringlet and two other narrow ringlets, 1.470R(s) and 1.495R(s) are found to be eccentric; the latter is best fitted by a model describing a freely precessing Keplerian ellipse, while the former is not conclusively fitted by either a resonant forcing or a free precession model. These two eccentric ringlets are compared with the Titan and Maxwell ringlets. 51 references.

  10. Primordial magnetogenesis before recombination

    NASA Astrophysics Data System (ADS)

    Fabre, Ophélia; Shankaranarayanan, S.

    2016-04-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order 10-49 G on a coherence scale of 10 kpc. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications. Our seed magnetic fields are generated on small scales whereas the main mechanisms studied in the literature are on scale bigger than 1 Mpc. However, compared to more exotic theories generating seed magnetic fields on similar scales, the strength of our fields are generally smaller.

  11. Recombinant Human Erythropoietin

    PubMed Central

    Bartels, Claudia; Späte, Kira; Krampe, Henning

    2008-01-01

    Treatment of multiple sclerosis (MS) is still unsatisfactory and essentially non-existing for the progressive course of the disease. Recombinant human erythropoietin (EPO) may be a promising neuroprotective/neuroregenerative treatment of MS. In the nervous system, EPO acts anti-apoptotic, antioxidative, anti-inflammatory, neurotrophic and plasticity-modulating. Beneficial effects have been shown in animal models of various neurological and psychiatric diseases, including different models of experimental autoimmune encephalomyelitis. EPO is also effective in human brain disease, as shown in double-blind placebo-controlled clinical studies on ischemic stroke and chronic schizophrenia. An exploratory study on chronic progressive MS yielded lasting improvement in motor and cognitive performance upon high-dose long-term EPO treatment. PMID:21180577

  12. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  13. Identification of recombination in the NS1 and VPs genes of parvovirus B19.

    PubMed

    Shen, Hongxing; Zhang, Wen; Wang, Hua; Shao, Shihe

    2016-08-01

    Human parvovirus B19 (B19V), a member of the genus Erythrovirus of the family Parvoviridae, is a pathogenic virus distributed worldwide in the human population. In this study, we performed phylogenetic and recombination analysis of B19V based on the available nonstructural gene (NS1) and capsid proteins (VPs) genes in GenBank. Results indicated that recombination occurred between genotypes 3 and 1, leading to the recombinant cluster genotype 2. Other three inter-genotype recombination events were also discovered. Moreover, our results showed that among the four recombinant events in the present study, all of the major parents belonged to genotype 1, the minor parents were from genotypes 3 or 2, and all of the recombinants belonged to genotype 2. These recombinant events were confirmed by SimPlot Program and phylogenetic analysis. J. Med. Virol. 88:1457-1461, 2016. © 2016 Wiley Periodicals, Inc.

  14. Human norovirus genogroup II recombinants in Thailand, 2009-2014.

    PubMed

    Phumpholsup, Tikumporn; Chieochansin, Thaweesak; Vongpunsawad, Sompong; Vuthitanachot, Viboonsuk; Payungporn, Sunchai; Poovorawan, Yong

    2015-10-01

    Norovirus (NoV) is a major cause of nonbacterial acute gastroenteritis worldwide. New strains emerge partly due to viral recombination. In Thailand, there is a lack of data on NoV recombinants among clinical isolates. We screened stool samples from pediatric diarrheal patients for norovirus by RT-PCR and found GII.4 to be the most prevalent genotype. Phylogenetic and SimPlot analyses detected seven intra-genogroup recombinant strains: three GII.21/GII.3, two GII.12/GII.3, and two GII.12/GII.1 recombinants. Maximum chi-square analysis indicated that all had similar breakpoints near the ORF1/ORF2 junction (p < 0.001), either slightly upstream within the C-terminus of RdRp or downstream within the N-terminal domain of VP1.

  15. Iron ionization and recombination rates and ionization equilibrium

    NASA Technical Reports Server (NTRS)

    Arnaud, M.; Raymond, J.

    1992-01-01

    In the past few years important progress has been made on the knowledge of ionization and recombination rates of iron, an astrophysically abundant heavy element and a major impurity in laboratory fusion devices. We make a critical review of the existing data on ionization and dielectronic recombination and present new computations of radiative recombination rate coefficients of Fe(+14) through Fe(+25) using the photoionization cross sections of Clark et al. (1986). We provide analytical fits to the recommended data (direct ionization and excitation-autoionization cross sections; radiative and dielectronic recombination rate coefficients). Finally we determine the iron ionic fractions at ionization equilibrium and compare them with previous computations as well as with observational data.

  16. Atmospheric entry probes for outer planet exploration. Outer planet entry probe technical summary

    NASA Technical Reports Server (NTRS)

    1974-01-01

    The use of unmanned space probes for investigating the conditions existing on and around the outer planets of the solar system is discussed. The subjects included in the report are: (1) the design of a common entry probe for outer planet missions, (2) the significant trades related to the development of a common probe design, (3) the impact of bus selection on probe design, (4) the impact of probe requirements on bus modifications, and (5) the key technology elements recommended for advanced development. Drawings and illustrations of typical probes are included to show the components and systems used in the space probes.

  17. Radioiodination of an outer membrane protein in intact Rickettsia prowazekii

    SciTech Connect

    Smith, D.K.; Winkler, H.H.

    1980-08-01

    Intact Rickettsia prowazekii was radiolabeled with the glucose oxidase-lactoperoxidase method of iodination. Separation of the rickettsial extract into cytoplasmic, outer and inner membrane fractions demonstrated that the outer membrane was preferentially labeled. Analysis of the polypeptides of these fractions on high-resolution slab polyacrylamide gels showed that most of the /sup 125/I was in polypeptide T49, an outer membrane constituent. Additional outer membrane polypeptides were iodinated in broken envelope preparations, demonstrating that T49 is uniquely accessible to the external environment and the asymmetric polypeptide organization of the outer membrane.

  18. A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

    PubMed

    Gornicka, Agnieszka; Bragoszewski, Piotr; Chroscicki, Piotr; Wenz, Lena-Sophie; Schulz, Christian; Rehling, Peter; Chacinska, Agnieszka

    2014-12-15

    Mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria with the help of protein translocases. For the majority of precursor proteins, the role of the translocase of the outer membrane (TOM) and mechanisms of their transport across the outer mitochondrial membrane are well recognized. However, little is known about the mode of membrane translocation for proteins that are targeted to the intermembrane space via the redox-driven mitochondrial intermembrane space import and assembly (MIA) pathway. On the basis of the results obtained from an in organello competition import assay, we hypothesized that MIA-dependent precursor proteins use an alternative pathway to cross the outer mitochondrial membrane. Here we demonstrate that this alternative pathway involves the protein channel formed by Tom40. We sought a translocation intermediate by expressing tagged versions of MIA-dependent proteins in vivo. We identified a transient interaction between our model substrates and Tom40. Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22. Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

  19. Outer-rise stress regime post 2004 mega-thrust rupture: Constraints from source process of selected Sumatra - Andaman outer-rise earthquakes.

    NASA Astrophysics Data System (ADS)

    Earnest, A.; K, S.

    2014-12-01

    The outer-rise region of a subducting margin will be in a compressional state prior to a megathrust event and in a tensional state afterwards due to the large co-seismic displacements. In coupled subduction zones, tensional earthquakes are occasionally observed post a megathrust rupture, as tensional stress from slab pull is temporarily transmitted to the outer-rise. In this study we analyze some of the significant outer-rise events along the Sumatra-Andaman margin post 26 December 2004 Sumatra-Andaman earthquake, like those occurred on January 2005 (Mw=6.5), July 2005 (Mw=7.1), August 2009 (Mw=7.5 ), March 2010 (Mw=6.6) and June 2010 (Mw=7.5). We will be discussing the source parameters,kinematic rupture process, directivity and the average stress drop of these events which are derived using seismic body wave inversion. We adopted teleseismic body wave inversion methodology of Kikuchi and Kanamori (1982, 1986 and 1991). We will be discussing the stress drop patterns and a possible asperity model for this margin and compare with other major outer-rise events post Tohoku-Oki (2011) and Maule (2010) events. The stress regimes inferred from the P & T axis orientations and its temporal evolution from inter-seismic to post-seismic phase also will be discussed.

  20. SDC conceptual design: Scintillating fiber outer tracker

    SciTech Connect

    Adams, D.; Baumbaugh, A.; Bird, F.; SDC Collaboration

    1992-01-22

    The authors propose an all-scintillating fiber detector for the purpose of outer tracking for the SDC. The objectives of this tracking system are to: (1) provide a first level trigger for {vert_bar}{eta}{vert_bar} < 2.3 with sharp p{sub T} threshold with the ability to resolve individual beam crossings; (2) provide pattern recognition capability and momentum resolution which complements and extends the capabilities of the inner silicon tracking system; (3) provide three dimensional linkage with outer detection systems including the shower maximum detector, muon detectors, and calorimetry; (4) provide robust tracking and track-triggering at the highest luminosities expected at the SSC. The many attractive features of a fiber tracker include good position resolution, low occupancy, low mass in the active volume, and excellent resistance to radiation damage. An additional important feature, especially at the SSC, is the intrinsically prompt response time of a scintillating fiber. This property is exploited in the construction of a level 1 trigger sensitive to individual beam crossings.

  1. Stellar evolution in the outer Galaxy

    NASA Astrophysics Data System (ADS)

    Szczerba, Ryszard; Siódmiak, Natasza; Leśniewska, Aleksandra; Karska, Agata; Sewiło, Marta

    2016-07-01

    We investigate the distribution of different classes of spectroscopically identified sources and theoretical models in the color-color diagrams (CCDs) combining the near-infrared (NIR) and mid-infrared (MIR) data to develop a method to classify Outer Galaxy sources detected with the Spitzer Space Telescope (hereafter Spitzer) SMOG survey in the IRAC 3.68.0 µm and MIPS 24 µm bands. We supplement the Spitzer data with the data from other satellite and ground-based surveys. The main goal of our study is to discover and characterize the population of intermediate- and low-mass young stellar objects (YSOs) in the Outer Galaxy and use it to study star formation in a significantly different environment than the Galaxy inside the solar circle. Since the YSOs can be confused with evolved stars in the MIR, these classes of objects need to be carefully separated. Here we present the initial results of our analysis using the Ks-[8.0] vs. Ks-[24] CCD as an example. The evolved stars separated from YSOs in the YSO selection process will be investigated in detail in the follow-up study.

  2. Fabrication of Spherical Reflectors in Outer Space

    NASA Technical Reports Server (NTRS)

    Wang, Yu; Dooley, Jennifer; Dragovan, Mark; Serivens, Wally

    2005-01-01

    A process is proposed for fabrication of lightweight spherical reflectors in outer space for telescopes, radio antennas, and light collectors that would be operated there. The process would obviate the relatively massive substrates and frames needed to support such reflectors in normal Earth gravitation. According to the proposal, fabrication of a reflector would begin with blowing of a bubble to the specified reflector radius. Taking advantage of the outer-space vacuum as a suitable environment for evaporative deposition of metal, a metal-evaporation source would be turned on and moved around the bubble to deposit a reflective metal film over the specified reflector area to a thickness of several microns. Then the source would be moved and aimed to deposit more metal around the edge of the reflector area, increasing the thickness there to approximately equal to 100 micron to form a frame. Then the bubble would be deflated and peeled off the metal, leaving a thin-film spherical mirror having an integral frame. The mirror would then be mounted for use. The feasibility of this technology has been proved by fabricating a prototype at JPL. As shown in the figure, a 2-in. (.5-cm) diameter hemispherical prototype reflector was made from a polymer bubble coated with silver, forming a very smooth surface.

  3. Residual Stress Testing of Outer 3013 Containers

    SciTech Connect

    Dunn, K.

    2004-02-12

    A Gas Tungsten Arc Welded (GTAW) outer 3013 container and a laser welded outer 3013 container have been tested for residual stresses according to the American Society for Testing Materials (ASTM) Standard G-36-94 [1]. This ASTM standard describes a procedure for conducting stress-corrosion cracking tests in boiling magnesium chloride (MgCl2) solution. Container sections in both the as-fabricated condition as well as the closure welded condition were evaluated. Significantly large residual stresses were observed in the bottom half of the as-fabricated container, a result of the base to can fabrication weld because through wall cracks were observed perpendicular to the weld. This observation indicates that regardless of the closure weld technique, sufficient residual stresses exist in the as-fabricated container to provide the stress necessary for stress corrosion cracking of the container, at the base fabrication weld. Additionally, sufficiently high residual stresses were observed in both the lid and the body of the GTAW as well as the laser closure welded containers. The stresses are oriented perpendicular to the closure weld in both the container lid and the container body. Although the boiling MgCl2 test is not a quantitative test, a comparison of the test results from the closure welds shows that there are noticeably more through wall cracks in the laser closure welded container than in the GTAW closure welded container.

  4. Retroviral recombination during reverse transcription.

    PubMed

    Goodrich, D W; Duesberg, P H

    1990-03-01

    After mixed infection, up to half of related retroviruses are recombinants. During infection, retroviral RNA genomes are first converted to complementary DNA (cDNA) and then to double-stranded DNA. Thus recombination could occur during reverse transcription, by RNA template switching, or after reverse transcription, by breakage and reunion of DNA. It has not been possible to distinguish between these two potential mechanisms of recombination because both single-stranded cDNA and double-stranded proviral DNA exist in infected cells during the eclipse period. Therefore we have analyzed for recombinant molecules among cDNA products transcribed in vitro from RNA of disrupted virions. Since recombinants from allelic parents can only be distinguished from parental genomes by point mutations, we have examined the cDNAs from virions with distinct genetic structures for recombinant-specific size and sequence markers. The parents share a common internal allele that allows homology-directed recombination, but each contains specific flanking sequences. One parent is a synthetically altered Harvey murine sarcoma virus RNA that lacks a retroviral 3' terminus but carries a Moloney murine retrovirus-derived envelope gene (env) fragment 3' of its transforming ras gene. The other parent is intact Moloney virus. Using a Harvey-specific 5' primer and a Moloney-specific 3' primer, we have found recombinant cDNAs with the polymerase chain reaction, proving directly that retroviruses can recombine during reverse transcription unassisted by cellular enzymes, probably by template switching during cDNA synthesis. The recombinants that were obtained in vitro were identical with those obtained in parallel experiments in vivo.

  5. “Genome-wide recombination and chromosome segregation in human oocytes and embryos reveal selection for maternal recombination rates”

    PubMed Central

    Natesan, Senthilkumar A.; Joshi, Hrishikesh A.; Cimadomo, Danilo; Griffin, Darren K.; Sage, Karen; Summers, Michael C.; Thornhill, Alan R.; Housworth, Elizabeth; Herbert, Alex D.; Rienzi, Laura; Ubaldi, Filippo M.; Handyside, Alan H.; Hoffmann, Eva R.

    2015-01-01

    Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here, we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping > 4 million informative single-nucleotide polymorphisms (SNPs) from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a novel reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germline by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings reveal that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II. PMID:25985139

  6. Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

    PubMed

    Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

    2012-03-01

    Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

  7. Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses

    PubMed Central

    Saleem, Huma; Nahid, Nazia; Shakir, Sara; Ijaz, Sehrish; Murtaza, Ghulam; Khan, Asif Ali; Mubin, Muhammad; Nawaz-ul-Rehman, Muhammad Shah

    2016-01-01

    The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent. PMID:26963635

  8. Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells.

    PubMed

    Fan, Guo-cheng; Gao, Fang-luan; Wei, Tai-yun; Huang, Mei-ying; Xie, Li-yan; Wu, Zu-jian; Lin, Qi-ying; Xie, Lian-hui

    2010-12-01

    To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.

  9. Green biofactories: recombinant protein production in plants.

    PubMed

    Ahmad, Adil; Pereira, Eridan O; Conley, Andrew J; Richman, Alex S; Menassa, Rima

    2010-11-01

    Until recently, low accumulation levels have been the major bottleneck for plant-made recombinant protein production. However, several breakthroughs have been described in the past few years allowing for very high accumulation levels, mainly through chloroplast transformation and transient expression, coupled with subcellular targeting and protein fusions. Another important factor influencing our ability to use plants for the production of recombinant proteins is the availability of quick and simple purification strategies. Recent developments using oleosin, zein, ELP and hydrophobin fusion tags have shown promise as efficient and cost-effective methods for non-chromatographic separation. Furthermore, plant glycosylation is a major barrier to the parenteral administration of plant-made biopharmaceuticals because of potential immunogenicity concerns. A major effort has been invested in humanizing plant glycosylation, and several groups have been able to reduce or eliminate immunogenic glycans while introducing mammalian-specific glycans. Finally, biosafety issues and public perception are essential for the acceptance of plants as bioreactors for the production of proteins. Over recent years, it has become clear that food and feed plants carry an inherent risk of contaminating our food supply, and thus much effort has focused on the use of non-food plants. Presently, Nicotiana benthamiana has emerged as the preferred host for transient expression, while tobacco is most frequently used for chloroplast transformation. In this review, we focus on the main issues hindering the economical production of recombinant proteins in plants, describing the current efforts for addressing these limitations, and we include an extensive list of recent patents generated with the intention of solving these limitations. PMID:21171961

  10. Can outer-to-outer diameter be used alone in diagnosing appendicitis on 128-slice MDCT?

    PubMed Central

    Yaqoob, Jamal; Idris, Muhammad; Alam, Muhammad Shahbaz; Kashif, Nazia

    2014-01-01

    AIM: To assess the frequency of visualization, position and diameter of normal appendix on 128-slice multidetector computed tomography (MDCT) in adult population. METHODS: Retrospective cross sectional study conducted at Radiology Department, Dallah Hospital, Riyadh, Saudi Arabia from March 2013 to October 2013. Non-enhanced computed tomography scans of abdomen and pelvis of 98 patients presenting with hematuria (not associated with abdominal pain, fever or colonic disease) were reviewed by two radiologists, blinded to patient history. The study group included 55 females and 43 males with overall mean age of 54.7 years (range 21 to 94 years). The coronal reformatted images were reviewed in addition to the axial images. The frequency of visualization of appendix was recorded with assessment of position, diameter and luminal contents. RESULTS: The appendix was recorded as definitely visualized in 99% of patients and mean outer-to-outer diameter of the appendix was 5.6 ± 1.3 mm (range 3.0-11.0 mm). CONCLUSION: MDCT with its multiplanar reformation display is extremely useful for visualization of normal appendix. The normal appendix is very variable in its position and diameter. In the absence of other signs, the diagnosis of acute appendix should not be made solely on outer-to-outer appendiceal diameter. PMID:25550996

  11. Antibiotic-mediated recombination: ciprofloxacin stimulates SOS-independent recombination of divergent sequences in Escherichia coli.

    PubMed

    López, Elena; Elez, Marina; Matic, Ivan; Blázquez, Jesús

    2007-04-01

    The widespread use and abuse of antibiotics as therapeutic agents has produced a major challenge for bacteria, leading to the selection and spread of antibiotic resistant variants. However, antibiotics do not seem to be mere selectors of these variants. Here we show that the fluoroquinolone antibiotic ciprofloxacin, an inhibitor of type II DNA topoisomerases, stimulates intrachromosomal recombination of DNA sequences. The stimulation of recombination between divergent sequences occurs via either the RecBCD or RecFOR pathways and is, surprisingly, independent of SOS induction. Additionally, this stimulation also occurs in a hyperrecombinogenic mismatch repair mutS mutant. It is worth noting that ciprofloxacin also stimulates the conjugational recombination of an antibiotic resistance gene. Finally, we demonstrate that Escherichia coli is able to recover from treatments with recombination-stimulating concentrations of the antibiotic. Thus, fluoroquinolones can increase genetic variation by the stimulation of the recombinogenic capability of treated bacteria (via an SOS-independent mechanism) and consequently may favour the acquisition, evolution and spread of antibiotic resistance determinants. PMID:17376074

  12. The Impact of Recombination Hotspots on Genome Evolution of a Fungal Plant Pathogen.

    PubMed

    Croll, Daniel; Lendenmann, Mark H; Stewart, Ethan; McDonald, Bruce A

    2015-11-01

    Recombination has an impact on genome evolution by maintaining chromosomal integrity, affecting the efficacy of selection, and increasing genetic variability in populations. Recombination rates are a key determinant of the coevolutionary dynamics between hosts and their pathogens. Historic recombination events created devastating new pathogens, but the impact of ongoing recombination in sexual pathogens is poorly understood. Many fungal pathogens of plants undergo regular sexual cycles, and sex is considered to be a major factor contributing to virulence. We generated a recombination map at kilobase-scale resolution for the haploid plant pathogenic fungus Zymoseptoria tritici. To account for intraspecific variation in recombination rates, we constructed genetic maps from two independent crosses. We localized a total of 10,287 crossover events in 441 progeny and found that recombination rates were highly heterogeneous within and among chromosomes. Recombination rates on large chromosomes were inversely correlated with chromosome length. Short accessory chromosomes often lacked evidence for crossovers between parental chromosomes. Recombination was concentrated in narrow hotspots that were preferentially located close to telomeres. Hotspots were only partially conserved between the two crosses, suggesting that hotspots are short-lived and may vary according to genomic background. Genes located in hotspot regions were enriched in genes encoding secreted proteins. Population resequencing showed that chromosomal regions with high recombination rates were strongly correlated with regions of low linkage disequilibrium. Hence, genes in pathogen recombination hotspots are likely to evolve faster in natural populations and may represent a greater threat to the host.

  13. THE OUTER DISKS OF DWARF IRREGULAR GALAXIES

    SciTech Connect

    Hunter, Deidre A.; Massey, Philip; Wilsey, Nick; Riabokin, Malanka; Elmegreen, Bruce G.; Oh, Se-Heon; Anderson, Ed; Nordgren, Tyler E. E-mail: phil.massey@lowell.edu E-mail: riabokin@msu.edu E-mail: seheon-oh@ast.uct.ac.za E-mail: tyler_nordgren@redlands.edu

    2011-10-15

    In order to explore the properties of extreme outer stellar disks, we obtained ultra-deep V and GALEX ultraviolet (UV) images of four dwarf irregular galaxies and one blue compact dwarf galaxy, and ultra-deep B images of three of these. Our V-band surface photometry extends to 29.5 mag arcsec{sup -2}. We convert the FUV and V-band photometry, along with H{alpha} photometry obtained in a larger survey, into radial star formation rate profiles that are sensitive to timescales from 10 Myr to the lifetime of the galaxy. We also obtained H I-line emission data and compare the stellar distributions, surface brightness profiles, and star formation rate profiles to H I-line emission maps, gas surface density profiles, and gas kinematics. Our data lead us to two general observations. First, the exponential disks in these irregular galaxies are extraordinarily regular. We observe that the stellar disks continue to decline exponentially as far as our measurements extend. In spite of lumpiness in the distribution of young stars and H I distributions and kinematics that have significant unordered motions, sporadic processes that have built the disks-star formation, radial movement of stars, and perhaps even perturbations from the outside-have, nevertheless, conspired to produce standard disk profiles. Second, there is a remarkable continuity of star formation throughout these disks over time. In four out of five of our galaxies the star formation rate in the outer disk measured from the FUV tracks that determined from the V-band, to within factors of five, requiring star formation at a fairly steady rate over the galaxy's lifetime. Yet, the H I surface density profiles generally decline with radius more shallowly than the stellar light, and the gas is marginally gravitationally stable against collapse into clouds. Outer stellar disks are challenging our concepts of star formation and disk growth and provide a critical environment in which to understand processes that mold

  14. Delayed recombination and standard rulers

    SciTech Connect

    De Bernardis, Francesco; Melchiorri, Alessandro; Bean, Rachel; Galli, Silvia; Silk, Joseph I.; Verde, Licia

    2009-02-15

    Measurements of baryonic acoustic oscillations (BAOs) in galaxy surveys have been recognized as a powerful tool for constraining dark energy. However, this method relies on the knowledge of the size of the acoustic horizon at recombination derived from cosmic microwave background (CMB) anisotropy measurements. This estimate is typically derived assuming a standard recombination scheme; additional radiation sources can delay recombination altering the cosmic ionization history and the cosmological inferences drawn from CMB and BAO data. In this paper we quantify the effect of delayed recombination on the determination of dark energy parameters from future BAO surveys such as the Baryon Oscillation Spectroscopic Survey and the Wide-Field Multi-Object Spectrograph. We find the impact to be small but still not negligible. In particular, if recombination is nonstandard (to a level still allowed by CMB data), but this is ignored, future surveys may incorrectly suggest the presence of a redshift-dependent dark energy component. On the other hand, in the case of delayed recombination, adding to the analysis one extra parameter describing deviations from standard recombination does not significantly degrade the error bars on dark energy parameters and yields unbiased estimates. This is due to the CMB-BAO complementarity.

  15. Analysis of interchromosomal mitotic recombination.

    PubMed

    McGill, C B; Shafer, B K; Higgins, D R; Strathern, J N

    1990-07-01

    A novel synthetic locus is described that provides a simple assay system for characterizing mitotic recombinants. The locus consists of the TRP1 and HIS3 genes inserted into chromosome III of S. cerevisiae between the CRY1 and MAT loci. Defined trp1 and his3 alleles have been generated that allow the selection of interchromosomal recombinants in this interval. Trp+ or His+ recombinants can be divided into several classes based on coupling of the other alleles in the interval. The tight linkage of the CRY1 and MAT loci, combined with the drug resistance and cell type phenotypes that they respectively control, facilitates the classification of the recombinants without resorting to tetrad dissection. We present the distribution of spontaneous recombinants among the classes defined by this analysis. The data suggest that the recombination intermediate can have regions of symmetric strand exchange and that co-conversion tracts can extend over 1-3 kb. Continuous conversion tracts are favored over discontinuous tracts. The distribution among the classes defined by this analysis is altered in recombinants induced by UV irradiation.

  16. Role of the Outer Membrane and Porins in Susceptibility of β-Lactamase-Producing Enterobacteriaceae to Ceftazidime-Avibactam.

    PubMed

    Pagès, Jean-Marie; Peslier, Sabine; Keating, Thomas A; Lavigne, Jean-Philippe; Nichols, Wright W

    2016-03-01

    This study examined the activity of the novel antimicrobial combination ceftazidime-avibactam against Enterobacteriaceae exhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of avibactam alone, ceftazidime alone, and ceftazidime-avibactam against the characterized clinical isolates of Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine β-naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which avibactam crossed the outer membranes of E. coli and K. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of avibactam across the outer membrane of E. aerogenes, although other diffusion channels for avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-avibactam. Nevertheless, the MICs of ceftazidime-avibactam (with 4 mg/liter avibactam) against the ceftazidime-resistant clinical isolates of the three species of Enterobacteriaceae studied were ≤ 8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.

  17. Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

    PubMed Central

    Smith, D K; Winkler, H H

    1979-01-01

    Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels. Images PMID:106046

  18. Aspergillus: sex and recombination.

    PubMed

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli.

  19. Aspergillus: sex and recombination.

    PubMed

    Varga, János; Szigeti, Gyöngyi; Baranyi, Nikolett; Kocsubé, Sándor; O'Gorman, Céline M; Dyer, Paul S

    2014-12-01

    The genus Aspergillus is one of the most widespread groups of fungi on Earth, comprised of about 300-350 species with very diverse lifestyles. Most species produce asexual propagula (conidia) on conidial heads. Despite their ubiquity, a sexual cycle has not yet been identified for most of the aspergilli. Where sexual reproduction is present, species exhibit either homothallic (self fertile) or heterothallic (obligate outcrossing) breeding systems. A parasexual cycle has also been described in some Aspergillus species. As in other fungi, sexual reproduction is governed by mating-type (MAT) genes, which determine sexual identity and are involved in regulating later stages of sexual development. Previous population genetic studies have indicated that some supposedly asexual aspergilli exhibit evidence of a recombining population structure, suggesting the presence of a cryptic sexual cycle. In addition, genome analyses have revealed networks of genes necessary for sexual reproduction in several Aspergillus species, again consistent with latent sexuality in these fungi. Knowledge of MAT gene presence has then successfully been applied to induce sexual reproduction between MAT1-1 and MAT1-2 isolates of certain supposedly asexual aspergilli. Recent progress in understanding the extent and significance of sexual reproduction is described here, with special emphasis on findings that are relevant to clinically important aspergilli. PMID:25118872

  20. Advantage of recombinant borrelial proteins for serodiagnosis of neuroborreliosis.

    PubMed

    Kaiser, R; Rauer, S

    1999-01-01

    Two enzyme immunoassay (EIA) systems were compared for their ability to detect Borrelia burgdorferi sensu lato specific IgG and IgM antibodies and to differentiate between symptomatic (83 patients with neuroborreliosis) and asymptomatic seropositive subjects (80 healthy controls). Antibody concentrations were determined by EIA; the antigens used were either a sonicate of B. burgdorferi or three recombinant borrelial proteins: the 14-kDa flagellin fragment, the outer surface protein C (22 kDa) and the high molecular mass protein p83 (83 kDa). In the sonicate, EIA, IgG or IgM antibodies to B. burgdorferi, or both, were detected in all patients with neuroborreliosis and in all controls. Pre-absorption of sera with Treponema phagedenis sonicate diminished the sensitivity of detection of borrelial specific IgG (IgG or IgM or both) antibodies in patients with neuroborreliosis from 80 to 57% (100 to 82%) and in the controls from 100 to 32% (100 to 37%). While being specific for B. burgdorferi, the recombinant EIAs proved to be significantly more sensitive than the sonicate EIA: IgG or IgM, or both antibodies against any of the recombinant antigens were detected in 92% of patients with neuroborreliosis and in 24% of controls. The increase in sensitivity in patients with neuroborreliosis was mostly due to the higher detection rate of IgM antibodies in the recombinant EIA (77% versus 48% in the sonicate EIA), while IgG antibodies were demonstrated with similar frequencies in both EIA systems (57% versus 60%). It was concluded that the recombinant EIAs are superior to the sonicate EIA with pre-absorption of cross-reactive antibodies in the confirmation of an acute borrelial infection and in the differentiation between symptomatic and asymptomatic infections.

  1. Recurrent Neural Network for Computing Outer Inverse.

    PubMed

    Živković, Ivan S; Stanimirović, Predrag S; Wei, Yimin

    2016-05-01

    Two linear recurrent neural networks for generating outer inverses with prescribed range and null space are defined. Each of the proposed recurrent neural networks is based on the matrix-valued differential equation, a generalization of dynamic equations proposed earlier for the nonsingular matrix inversion, the Moore-Penrose inversion, as well as the Drazin inversion, under the condition of zero initial state. The application of the first approach is conditioned by the properties of the spectrum of a certain matrix; the second approach eliminates this drawback, though at the cost of increasing the number of matrix operations. The cases corresponding to the most common generalized inverses are defined. The conditions that ensure stability of the proposed neural network are presented. Illustrative examples present the results of numerical simulations.

  2. Organic Matter in the Outer Solar System

    NASA Technical Reports Server (NTRS)

    Cruiskshank, Dale P.; DeVincenzi, Donald L. (Technical Monitor)

    2000-01-01

    Many solid bodies in the outer Solar System are covered with ices of various compositions, including water, carbon dioxide, methane, nitrogen, and other molecules that are solid at the low temperatures that prevail there. These ices have all been detected by remote sensing observations made with telescopes on Earth, or more recently, spacecraft in orbit (notably Galileo at Jupiter). The data also reveal other solid materials that could be minerals or complex carbon-bearing organic molecules. A study in progress using large ground-based telescopes to acquire infrared spectroscopic data, and laboratory results on the optical properties of complex organic matter, seeks to identify the non-icy materials on several satellites of Saturn, Uranus, and Neptune. The work on the satellites of Saturn is in part preparatory to the Cassini spacecraft investigation of the Saturn system, which will begin in 2004 and extend for four years.

  3. Outer Hair Cell Electromotility in vivo

    NASA Astrophysics Data System (ADS)

    Ramamoorthy, Sripriya; Nuttall, Alfred L.

    2011-11-01

    The effectiveness of outer hair cell (OHC) electro-motility in vivo has been challenged by the expected low-pass filtering of the transmembrane potential due to the cell's own capacitance. The OHC electromotility is characterized here by an electromechanical ratio defined as the ratio of the OHC contraction to the transmembrane potential. This ratio has been measured in isolated cells to be approximately 26 nm/mV. We estimate the OHC electromechanical ratio in vivo from the recently measured displacements of the reticular lamina and the basilar membrane near the 19 kHz characteristic frequency in the basal region of guinea pig cochlea. Our analysis strongly suggests OHC electromotility process is effective for cochlear amplification in vivo at least around the characteristic frequency of the basal location in spite of the low-pass filtering.

  4. Seismic characteristics of outer-rise earthquakes in the different seismic coupling subduction zones

    NASA Astrophysics Data System (ADS)

    Lee, Hsin-Hua; Lin, Jing-Yi

    2013-04-01

    Characterizing the seismogenic zone of major subduction plate boundaries provides us a possible to reduce large earthquakes hazard. In the past several decades, many scientists have analyzed various geophysical methods and datasets, such as seismic and geodetic ground motion data, historical tsunami deposits, aftershock distributions, and seafloor bathymetry, trying to understand the mechanisms behind great devastating earthquakes, and to estimate the probability of a major earthquake occurrence in the future. In this study, by using the global earthquake catalog (GCMT) from January 1, 1976 to December 31, 2011. We firstly re-examines the outer-rise earthquake model proposed by the Christensen (1988) at the subduction zones suggested to have different coupling levels. The compressive stress cumulated during the subducting processes are often reflected by the occurrence of compressional outer-rise earthquakes. Thus, in the region where the compressional outer-rise earthquakes take place without any corresponding large underthrusting earthquakes, the seismic potential is usually considered to be high. We re-examined the high seismic potential areas determined by this criteria in Christensen (1988) and confirm that the large underthrusting earthquakes did really occur in the 30 years following the appearance of compressional outer-rise events, such as in Tonga region in the vicinity of 20S, a Mw 8.3 large earthquake occurred in 2006. This result represents that the outer-rise earthquake model could be an indicator for the generation of large earthquakes along subduction zones. In addition, to have a more accurate estimation for the seismic potential, we discuss the relationship between the generation of earthquakes and the change of cumulative gravitational potential energy caused by earthquakes (ΔGPE) over time. Our result shows an acceleration of ΔGPE before large earthquakes. Our result also shows that the extensional outer-rise events for strong seismic coupling

  5. Multiwavelength diagnostics for newly discovered PNe in the outer LMC

    NASA Astrophysics Data System (ADS)

    Reid, W.; Parker, Q. A.

    2014-04-01

    We have extended our PNe survey to the outer ~64deg^2 of the LMC using maps from the Magellanic Cloud Emission Line Survey (MCELS) and the UKST H-alpha and red surveys. Although the MCELS survey has poorer ~5 arcsecond resolution than the UKST survey, it has the advantage of additional narrow-band filters at H-alpha, [OIII] and [SII], providing improved diagnostic capabilities. The UKST survey has a similar angular resolution (~2 arcsec from 0.67 arcsec pixels) to our original median stacked map of the central 25deg^2 of the central LMC, but lacks the depth advantages provided by median stacking. Using these data to uncover new emission line candidates we have so far spectroscopically confirmed an extra 63 LMC PNe which we present here. We have also independently recovered and spectroscopically confirmed 107 of the 109 (98%) PNe that were previously known to exist in the outer LMC. The majority of our newly discovered and previously known PNe were confirmed using the AAOmega, multi-object fibre spectroscopy system on the 3.9-m Anglo-Australian Telescope (AAT) and the 6dF multi-object spectrograph on the UK Schmidt Telescope (UKST). These newly identified PNe were cross-checked using extant multi-wavelength imaging surveys in the near and mid-infrared in particular and against the latest emission-line ratio diagnostic plots for improved confidence in PNe identification. These new identifications raise the number of known PNe in the LMC to 715, approaching the number of 900 predicted by stellar evolutionary theory. We present accurate positions, diagnostic plots based on de-reddened emission lines, multi-wavelength SEDs and mid-infrared plots based on SAGE spitzer data.

  6. Nuclear Electric Propulsion for Outer Space Missions

    NASA Technical Reports Server (NTRS)

    Barret, Chris

    2003-01-01

    Today we know of 66 moons in our very own Solar System, and many of these have atmospheres and oceans. In addition, the Hubble (optical) Space Telescope has helped us to discover a total of 100 extra-solar planets, i.e., planets going around other suns, including several solar systems. The Chandra (X-ray) Space Telescope has helped us to discover 33 Black Holes. There are some extremely fascinating things out there in our Universe to explore. In order to travel greater distances into our Universe, and to reach planetary bodies in our Solar System in much less time, new and innovative space propulsion systems must be developed. To this end NASA has created the Prometheus Program. When one considers space missions to the outer edges of our Solar System and far beyond, our Sun cannot be relied on to produce the required spacecraft (s/c) power. Solar energy diminishes as the square of the distance from the Sun. At Mars it is only 43% of that at Earth. At Jupiter, it falls off to only 3.6% of Earth's. By the time we get out to Pluto, solar energy is only .066% what it is on Earth. Therefore, beyond the orbit of Mars, it is not practical to depend on solar power for a s/c. However, the farther out we go the more power we need to heat the s/c and to transmit data back to Earth over the long distances. On Earth, knowledge is power. In the outer Solar System, power is knowledge. It is important that the public be made aware of the tremendous space benefits offered by Nuclear Electric Propulsion (NEP) and the minimal risk it poses to our environment. This paper presents an overview of the reasons for NEP systems, along with their basic components including the reactor, power conversion units (both static and dynamic), electric thrusters, and the launch safety of the NEP system.

  7. Outer Planet Mission Studies Neptune Aerocapture

    NASA Technical Reports Server (NTRS)

    Wercinski, Paul F.; Langhoff, Steven R. (Technical Monitor)

    1997-01-01

    Current and previous studies of orbiter missions to the outer planets have clearly identified high-energy aerocapture as a critical and enabling technology. Aerocapture involves the use of aerodynamic lift to fly a trajectory through a planet's atmosphere to sufficiently decelerate an entry vehicle to capture into planetary orbit. In the past, numerous studies of different configurations of lifting entry vehicles were studied for various planetary orbiter missions which identified aerocapture as a feasible concept yet complex and technically challenging. In order to determine the feasibility of high-speed aerocapture at the outer planets, an accurate trajectory simulation of the flight vehicle is the critical first step in the proposed research. Vehicle response to aerodynamic loading must be predicted accurately in the trajectory simulations. For several Neptune orbiter missions currently under study at the Jet Propulsion Laboratory (JPL), entry velocities relative to the rotating atmosphere ranging from 25 to 30 km/sec, are to be expected. Preliminary trajectory analysis has identified the various flow regimes the entry vehicle is expected to fly in the 8 1% H2 and 19% He atmosphere of Neptune. The size and mass of the vehicle are also determined by the launch vehicle constraints and orbiter spacecraft requirements. For a given baseline arrival conditions of an inertial entry velocity of 28 km/sec and an entry mass of 400 kg, a medium lift (L/D = 1), axisymmetric biconic shaped vehicle was selected in order to satisfy entry corridor width requirements expected for Neptune aerocapture. The analysis summarized in this study indicates that a biconic entry vehicle is a feasible concept for a Neptune aerocapture orbiter mission. The preliminary entry trajectory simulations has demonstrated adequate entry corridor control authority. Furthermore, estimates of the stagnation point heating environment has enabled the preliminary selection of candidate lightweight ceramic

  8. Lateral interactions in the outer retina

    PubMed Central

    Thoreson, Wallace B.; Mangel, Stuart C.

    2012-01-01

    Lateral interactions in the outer retina, particularly negative feedback from horizontal cells to cones and direct feed-forward input from horizontal cells to bipolar cells, play a number of important roles in early visual processing, such as generating center-surround receptive fields that enhance spatial discrimination. These circuits may also contribute to post-receptoral light adaptation and the generation of color opponency. In this review, we examine the contributions of horizontal cell feedback and feed-forward pathways to early visual processing. We begin by reviewing the properties of bipolar cell receptive fields, especially with respect to modulation of the bipolar receptive field surround by the ambient light level and to the contribution of horizontal cells to the surround. We then review evidence for and against three proposed mechanisms for negative feedback from horizontal cells to cones: 1) GABA release by horizontal cells, 2) ephaptic modulation of the cone pedicle membrane potential generated by currents flowing through hemigap junctions in horizontal cell dendrites, and 3) modulation of cone calcium currents (ICa) by changes in synaptic cleft proton levels. We also consider evidence for the presence of direct horizontal cell feed-forward input to bipolar cells and discuss a possible role for GABA at this synapse. We summarize proposed functions of horizontal cell feedback and feed-forward pathways. Finally, we examine the mechanisms and functions of two other forms of lateral interaction in the outer retina: negative feedback from horizontal cells to rods and positive feedback from horizontal cells to cones. PMID:22580106

  9. FBL Outer Can Welder Acquisition Software

    2004-01-16

    The Outer Can Welder Data Acquisition Software (OCWDAS) was originally developed by SRTC for use at Hanford to assist in the storage of their excess plutonium in the DOE standard 3013 containers until it can be properly dispositioned using one of the approved DOE methods. After Hanford OCWDAS software was the starting point for the new version developed for FB-Line. New graphical display formats and features were added to this software to make it moremore » robust and operator friendly. Several hardware changes were also made at this time and the software was modified to accommodate these as well. During the welding process, critical weld parameters such as weld current and voltage, can give valuable information about the weld. In the past, weld data from the TIG welding process, such as the bagless transfer system in FB-Line, has been monitored using strip chart recorders. The data from the weld process, recorded on the strip chart recorder traces, are reviewed to analyze the weld. The OCWDAS improves this technology by digitizing the weld data which allows for automation of the analysis process. The OCWDAS performs the necessary functions to perform the data acquisition functions during the 3013 Outer Can Welding Process. It is important to monitor the critical weld parameters, current and voltage, during a weld as they can be used to set acceptance criteria for weld acceptance. The software monitors and records the weld current, voltage, and RPM data. It also records the absolute position of the weld head during the weld process from a quadrature encoder. Digital handshaking between the AMI Welding unit and the OCWDAS ensure that both systems are operational and ready prior to the weld initiation taking place.« less

  10. Cloning, expression of Vibrio alginolyticus outer membrane protein-OmpU gene and its potential application as vaccine in crimson snapper, Lutjanus erythropterus Bloch.

    PubMed

    Cai, S H; Lu, Y S; Wu, Z H; Jian, J C

    2013-08-01

    The outer membrane proteins of the marine aquatic animal pathogen, Vibrio alginolyticus, play an important role in the virulence of the bacterium and are potential candidates for vaccine development. In this study, the gene encoding an outer membrane protein-OmpU was cloned and expressed in Escherichia coli. Polyclonal antibodies were raised in rabbits against the purified recombinant OmpU, and the reaction of the antibody was confirmed by Western blotting using the isolated OmpU and the recombinant OmpU of V. alginolyticus. To analyze the immunogenicity of the recombinant OmpU, crimson snapper, Lutjanus erythropterus Bloch, were immunized by intraperitoneal injection, and antibody response was assessed by the enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the recombinant OmpU produced an observable antibody response in all sera of the vaccinated fish. The vaccinated fish were challenged by virulent V. alginolyticus and observed to have high resistance to infection. These results indicate that the recombinant OmpU is an effective vaccine candidate against V. alginolyticus in L. erythropterus.

  11. RPA homologs and ssDNA processing during meiotic recombination.

    PubMed

    Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

    2016-06-01

    Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

  12. Genetic diversity and recombination analysis of sweepoviruses from Brazil

    PubMed Central

    2012-01-01

    Background Monopartite begomoviruses (genus Begomovirus, family Geminiviridae) that infect sweet potato (Ipomoea batatas) around the world are known as sweepoviruses. Because sweet potato plants are vegetatively propagated, the accumulation of viruses can become a major constraint for root production. Mixed infections of sweepovirus species and strains can lead to recombination, which may contribute to the generation of new recombinant sweepoviruses. Results This study reports the full genome sequence of 34 sweepoviruses sampled from a sweet potato germplasm bank and commercial fields in Brazil. These sequences were compared with others from public nucleotide sequence databases to provide a comprehensive overview of the genetic diversity and patterns of genetic exchange in sweepoviruses isolated from Brazil, as well as to review the classification and nomenclature of sweepoviruses in accordance with the current guidelines proposed by the Geminiviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV). Co-infections and extensive recombination events were identified in Brazilian sweepoviruses. Analysis of the recombination breakpoints detected within the sweepovirus dataset revealed that most recombination events occurred in the intergenic region (IR) and in the middle of the C1 open reading frame (ORF). Conclusions The genetic diversity of sweepoviruses was considerably greater than previously described in Brazil. Moreover, recombination analysis revealed that a genomic exchange is responsible for the emergence of sweepovirus species and strains and provided valuable new information for understanding the diversity and evolution of sweepoviruses. PMID:23082767

  13. Phospholipid molecular species of frog rod outer segment membranes.

    PubMed

    Wiegand, R D; Anderson, R E

    1983-08-01

    Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were isolated from frog retinal rod outer segment (ROS) membranes and their major molecular species determined. Saturated fatty acids are predominantly located on position-1 and polyunsaturates on position-2, although this assignment is not absolute. One-fifth of the species of PC are of the saturated and monoenoic type, compared to only trace amounts of these species in PE. On the other hand, PE contains at least 50% dipolyunsaturated species, while PC has 6%. The predominant molecular species of frog PC are: 18:0-22:6 omega 3 (39%), 16:0-22:6 omega 3 (17%), 16:0-16:1 (6%), 16:0-18:1 (5%) and 16:0-16:0 (4%). The major molecular species of frog PE are: 22:6 omega 3-22:6 omega 3 (21%), 18:0-22:6 omega 3 (18%), 18:1-22:6 omega 3 (14%), 22:4 omega 6-22:6 omega 3 (10%), 22:5 omega (3 + 6)-22:6 omega 3 (10%), 16:0-22:6 omega 3 (7%) and 20:4 omega 6-22:6 omega 3 (5%). The specific molecular species are discussed in relation to their effect on fluidity and asymmetry of ROS disc membranes.

  14. Three Decades of Recombinant DNA.

    ERIC Educational Resources Information Center

    Palmer, Jackie

    1985-01-01

    Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

  15. Recombinant DNA means and method

    SciTech Connect

    Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

    1987-05-19

    This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

  16. Recombination system for storage batteries

    SciTech Connect

    Bopp, B.; Ledjeff, K.; Winsel, A.

    1983-03-29

    A recombination system for catalytic oxidation of hydrogen in storage battery gases includes a gas supply duct which makes it possible for the combustible gas flowing through it to aspirate from the ambient the necessary combustion air, following the principle of a bunsen burner, and to entrain it to the recombination catalyst. In case of over-supply of gas, an acid separator positioned in the gas supply pipe counteracts the gas aspiration by means of its flow impedance and thereby makes the recombination system safe from overload. It can also be connected following a conventional recombiner, thereby increasing its effectiveness, by receiving the excess hydrogen from same and reacting it with the aid of the air aspiration.

  17. Influenza Vaccine, Inactivated or Recombinant

    MedlinePlus

    ... die from flu, and many more are hospitalized.Flu vaccine can:keep you from getting flu, make flu ... inactivated or recombinant influenza vaccine?A dose of flu vaccine is recommended every flu season. Children 6 months ...

  18. Recombination device for storage batteries

    DOEpatents

    Kraft, Helmut; Ledjeff, Konstantin

    1985-01-01

    A recombination device including a gas-tight enclosure connected to receive he discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  19. Recombination device for storage batteries

    DOEpatents

    Kraft, H.; Ledjeff, K.

    1984-01-01

    A recombination device including a gas-tight enclosure connected to receive the discharge gases from a rechargeable storage battery. Catalytic material for the recombination of hydrogen and oxygen to form water is supported within the enclosure. The enclosure is sealed from the atmosphere by a liquid seal including two vertical chambers interconnected with an inverted U-shaped overflow tube. The first chamber is connected at its upper portion to the enclosure and the second chamber communicates at its upper portion with the atmosphere. If the pressure within the enclosure differs as overpressure or vacuum by more than the liquid level, the liquid is forced into one of the two chambers and the overpressure is vented or the vacuum is relieved. The recombination device also includes means for returning recombined liquid to the battery and for absorbing metal hydrides.

  20. [Enzymatic control of homologous recombination in Escherichia coli cells and hyper-recombination].

    PubMed

    Bakhlanova, I V; Dudkina, A V; Baĭtin, D M

    2013-01-01

    The RecA protein is a major enzyme of homologous recombination in bacterial cell. Forming a right-handed helical filament on ssDNA, it provides a homology search between two DNA molecules and homologous strand exchange. The RecA protein not only defends the cell from exposure to ionizing radiation and UV-irradiation, but also ensures the recombination process in the course of normal cell growth. A number of wild-type or mutant RecA proteins demonstrate increased recombinogenic properties in vitro and in vivo as compared with the wild-type RecA protein from Escherichia coli, which leads to hyper-recombination. The hyper-rec activity of RecA proteins during the recombination process in many depends on the filamentation dynamics on ssDNA and DNA-transferase properties. Changes in filamentation and DNA-transferase abilities of RecA protein may be the result of not only specific amino-acid substitutions, but also the functioning of the cell enzymatic apparatus, including such proteins as RecO, RecR, RecF, RecX, DinI, SSB, PsiB. To date, the function of each of these proteins is identified at the molecular level. However, the role of some of them in the cell metabolism remains to be seen. Increase in recombination in vivo is not always useful for a cell and faces various limitations. Moreover, in the bacterial cell some mechanisms are activated, that cause genomic reorganization, directed to suppress the expression of hyper-active RecA protein. The ways of hyper-active RecA protein regulation are very interesting, and they are studied in different model systems. PMID:23808153

  1. Fermentations with new recombinant organisms

    SciTech Connect

    Bothast, R.J.; Nichols, N.N.; Dien, B.S.

    1999-10-01

    US fuel ethanol production in 1998 exceeded the record production of 1.4 billion gallons set in 1995. Most of this ethanol was produced from over 550 million bushels of corn. Expanding fuel ethanol production will require developing lower-cost feedstocks, and only lignocellulosic feedstocks are available in sufficient quantities to substitute for corn starch. Major technical hurdles to converting lignocellulose to ethanol include the lack of low-cost efficient enzymes for saccharification of biomass to fermentable sugars and the development of microorganisms for the fermentation of these mixed sugars. To date, the most successful research approaches to develop novel biocatalysts that will efficiently ferment mixed sugar syrups include isolation of novel yeasts that ferment xylose, genetic engineering of Escherichia coli and other gram negative bacteria for ethanol production, and genetic engineering of Saccharomyces cerevisiae and Zymomonas mobilis for pentose utilization. The authors have evaluated the fermentation of corn fiber hydrolyzates by the various strains developed. E. coli K011, E. coli SL40, E. coli FBR3, Zymomonas CP4 (pZB5), and Saccharomyces 1400 (pLNH32) fermented corn fiber hydrolyzates to ethanol in the range of 21--34 g/L with yields ranging from 0.41 to 0.50 g of ethanol per gram of sugar consumed. Progress with new recombinant microorganisms has been rapid and will continue with the eventual development of organisms suitable for commercial ethanol production. Each research approach holds considerable promise, with the possibility existing that different industrially hardened strains may find separate applications in the fermentation of specific feedstocks.

  2. Differential Compartmentalization of HIV-Targeting Immune Cells in Inner and Outer Foreskin Tissue

    PubMed Central

    Liu, Lu; Meng, Zhefeng; Li, Liangzhu; Qiu, Chao; Xu, Jianqing; Zhang, Xiaoyan

    2014-01-01

    Ex vivo foreskin models have demonstrated that inner foreskin is more susceptible to HIV-1 infection than outer foreskin. In the present study we characterized the compartition of HIV-1 target cells and quantified these cells in the epidermis and dermis of inner and outer foreskins using immunohistochemistry and flow cytometry. Our data showed that the epidermis of the inner foreskin was more enriched with CD4+ T cells and Langerhans cells (LCs), with the co-expression of CCR5 and α4β7 receptors, than the outer foreskin. Interestingly, the vast majority of CD4+ T cells and LCs expressed CCR5, but not CXCR4, indicating that the inner foreskin might capture and transmit R5-tropic HIV strains more efficiently. In addition, lymphoid aggregates, composed of T cells, macrophages and dendritic cells (DCs) in the dermis, were closer to the epithelial surface in the inner foreskin than in the outer foreskin. As dendritic cells are able to capture and pass HIV particles to susceptible target cells, HIV may be able to more efficiently infect the inner foreskin by hijacking the augmented immune communication pathways in this tissue. After the inoculation of HIV-1 particles in a foreskin explant culture model, the level of p24 antigen in the supernatant from the inner foreskin was slightly higher than that from the outer foreskin, although this difference was not significant. The present study is the first to employ both CCR5 and α4β7 to identify HIV target cells in the foreskin. Our data demonstrated that the inner foreskin was more enriched with HIV target immune cells than the outer foreskin, and this tissue was structured for efficient communication among immune cells that may promote HIV transmission and replication. In addition, our data suggests the R5-tropism of HIV sexual transmission is likely shaped through the inherent receptor composition on HIV target cells in the mucosa. PMID:24454812

  3. Registration of 'RU9101001'/'Katy' recombinant inbred lines of rice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The cross of RU9101001/'Katy' rice (Oryza sativa L.) was used to develop a mapping population consisting of 238 F9 generation recombinant inbred lines of rice (Oryza sativa L.) (GSOR100361 to GSOR100600). This population has been used to map major genes that provide resistance to the rice blast pat...

  4. Comparative and Evolutionary Analysis of the Bacterial Homologous Recombination Systems

    PubMed Central

    Rocha, Eduardo P. C; Cornet, Emmanuel; Michel, Bénédicte

    2005-01-01

    Homologous recombination is a housekeeping process involved in the maintenance of chromosome integrity and generation of genetic variability. Although detailed biochemical studies have described the mechanism of action of its components in model organisms, there is no recent extensive assessment of this knowledge, using comparative genomics and taking advantage of available experimental data on recombination. Using comparative genomics, we assessed the diversity of recombination processes among bacteria, and simulations suggest that we missed very few homologs. The work included the identification of orthologs and the analysis of their evolutionary history and genomic context. Some genes, for proteins such as RecA, the resolvases, and RecR, were found to be nearly ubiquitous, suggesting that the large majority of bacterial genomes are capable of homologous recombination. Yet many genomes show incomplete sets of presynaptic systems, with RecFOR being more frequent than RecBCD/AddAB. There is a significant pattern of co-occurrence between these systems and antirecombinant proteins such as the ones of mismatch repair and SbcB, but no significant association with nonhomologous end joining, which seems rare in bacteria. Surprisingly, a large number of genomes in which homologous recombination has been reported lack many of the enzymes involved in the presynaptic systems. The lack of obvious correlation between the presence of characterized presynaptic genes and experimental data on the frequency of recombination suggests the existence of still-unknown presynaptic mechanisms in bacteria. It also indicates that, at the moment, the assessment of the intrinsic stability or recombination isolation of bacteria in most cases cannot be inferred from the identification of known recombination proteins in the genomes. PMID:16132081

  5. Corrugated outer sheath gas-insulated transmission line

    DOEpatents

    Kemeny, George A.; Cookson, Alan H.

    1981-01-01

    A gas-insulated transmission line includes two transmission line sections each of which are formed of a corrugated outer housing enclosing an inner high-voltage conductor disposed therein, with insulating support means supporting the inner conductor within the outer housing and an insulating gas providing electrical insulation therebetween. The outer housings in each section have smooth end sections at the longitudinal ends thereof which are joined together by joining means which provide for a sealing fixed joint.

  6. Delayed recombination and cosmic parameters

    SciTech Connect

    Galli, Silvia; Melchiorri, Alessandro; Bean, Rachel; Silk, Joseph

    2008-09-15

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, n{sub s}, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z{sub *}=1078{+-}11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1{sigma} to R=1.734{+-}0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: {epsilon}{sub {alpha}}<0.39 and {epsilon}{sub i}<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  7. Ethanol production by recombinant hosts

    DOEpatents

    Ingram, Lonnie O.; Beall, David S.; Burchhardt, Gerhard F. H.; Guimaraes, Walter V.; Ohta, Kazuyoshi; Wood, Brent E.; Shanmugam, Keelnatham T.

    1995-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  8. Ethanol production by recombinant hosts

    DOEpatents

    Fowler, David E.; Horton, Philip G.; Ben-Bassat, Arie

    1996-01-01

    Novel plasmids comprising genes which code for the alcohol dehydrogenase and pyruvate decarboxylase are described. Also described are recombinant hosts which have been transformed with genes coding for alcohol dehydrogenase and pyruvate. By virtue of their transformation with these genes, the recombinant hosts are capable of producing significant amounts of ethanol as a fermentation product. Also disclosed are methods for increasing the growth of recombinant hosts and methods for reducing the accumulation of undesirable metabolic products in the growth medium of these hosts. Also disclosed are recombinant host capable of producing significant amounts of ethanol as a fermentation product of oligosaccharides and plasmids comprising genes encoding polysaccharases, in addition to the genes described above which code for the alcohol dehydrogenase and pyruvate decarboxylase. Further, methods are described for producing ethanol from oligomeric feedstock using the recombinant hosts described above. Also provided is a method for enhancing the production of functional proteins in a recombinant host comprising overexpressing an adhB gene in the host. Further provided are process designs for fermenting oligosaccharide-containing biomass to ethanol.

  9. Delayed recombination and cosmic parameters

    NASA Astrophysics Data System (ADS)

    Galli, Silvia; Bean, Rachel; Melchiorri, Alessandro; Silk, Joseph

    2008-09-01

    Current cosmological constraints from cosmic microwave background anisotropies are typically derived assuming a standard recombination scheme, however additional resonance and ionizing radiation sources can delay recombination, altering the cosmic ionization history and the cosmological inferences drawn from the cosmic microwave background data. We show that for recent observations of the cosmic microwave background anisotropy, from the Wilkinson microwave anisotropy probe satellite mission (WMAP) 5-year survey and from the arcminute cosmology bolometer array receiver experiment, additional resonance radiation is nearly degenerate with variations in the spectral index, ns, and has a marked effect on uncertainties in constraints on the Hubble constant, age of the universe, curvature and the upper bound on the neutrino mass. When a modified recombination scheme is considered, the redshift of recombination is constrained to z*=1078±11, with uncertainties in the measurement weaker by 1 order of magnitude than those obtained under the assumption of standard recombination while constraints on the shift parameter are shifted by 1σ to R=1.734±0.028. From the WMAP5 data we obtain the following constraints on the resonance and ionization sources parameters: γα<0.39 and γi<0.058 at 95% c.l.. Although delayed recombination limits the precision of parameter estimation from the WMAP satellite, we demonstrate that this should not be the case for future, smaller angular scales measurements, such as those by the Planck satellite mission.

  10. A universal BMV-based RNA recombination system--how to search for general rules in RNA recombination.

    PubMed

    Alejska, Magdalena; Figlerowicz, Magdalena; Malinowska, Nelli; Urbanowicz, Anna; Figlerowicz, Marek

    2005-07-07

    At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication.

  11. Phosphatidylserine Converts Immunogenic Recombinant Human Acid Alpha-Glucosidase to a Tolerogenic Form in a Mouse Model of Pompe Disease.

    PubMed

    Schneider, Jennifer L; Balu-Iyer, Sathy V

    2016-10-01

    Development of unwanted immune responses against therapeutic proteins is a major clinical complication. Recently, we have shown that exposure of Factor VIII in the presence of phosphatidylserine (PS) induces antigen-specific hyporesponsiveness to Factor VIII rechallenge, suggesting that PS is not immune suppressive, but rather immune regulatory in that PS converts an immunogen to a tolerogen. Since PS is exposed in the outer leaflet during apoptosis, we hypothesize that PS imparts tolerogenic activity to this natural process. Thus, immunization with PS containing liposomes would mimic this natural process. Here, we investigate the immune regulatory effects of PS in inducing tolerance toward recombinant human acid alpha-glucosidase (rhGAA). rhGAA was found to complex with PS liposomes through hydrophobic interactions, and incubation PS-rhGAA with dendritic cells resulted in the increased secretion of transforming growth factor-β. Immunization with PS-rhGAA or O-phospho-L-serine-rhGAA led to a reduction in anti-rhGAA antibody response which persisted despite rechallenge with free rhGAA. Importantly, the titer levels in a majority of these animals remained unchanged after rechallenge and can be considered nonresponders. These data provide evidence that PS liposomes can be used to induce tolerance toward therapeutic proteins, in general. PMID:27488899

  12. Tectonics of the Outer Planet Satellites

    NASA Technical Reports Server (NTRS)

    McKinnon, W. B.; Collins, G. C.; Moore, J. M.; Nimmo, F.; Pappalardo, R. T.; Prockter, L. M.; Schenk, P. M.

    2010-01-01

    Tectonic features on the satellites of the outer planets range from the familiar, such as clearly recognizable graben on many satellites, to the bizarre, such as the ubiquitous double ridges on Europa, the twisting sets of ridges on Triton, or the isolated giant mountains rising from Io's surface. All of the large and middle-sized outer planet satellites except Io are dominated by water ice near their surfaces. Though ice is a brittle material at the cold temperatures found in the outer solar system, the amount of energy it takes to bring it close to its melting point is lower than for a rocky body. Therefore, some unique features of icy satellite tectonics may be influenced by a near-surface ductile layer beneath the brittle surface material, and several of the icy satellites may possess subsurface oceans. Sources of stress to drive tectonism are commonly dominated by the tides that deform these satellites as they orbit their primary giant planets. On several satellites, the observed tectonic features may be the result of changes in their tidal figures, or motions of their solid surfaces with respect to their tidal figures. Other driving mechanisms for tectonics include volume changes due to ice or water phase changes in the interior, thermoelastic stress, deformation of the surface above rising diapirs of warm ice, and motion of subsurface material toward large impact basins as they fill in and relax. Most satellites exhibit evidence for extensional deformation, and some exhibit strike-slip faulting, whereas contractional tectonism appears to be rare. Io s surface is unique, exhibiting huge isolated mountains that may be blocks of crust tilting and foundering into the rapidly emptying interior as the surface is constantly buried by deposits from hyperactive volcanoes. Of the satellites, diminutive Enceladus is spectacularly active; its south polar terrain is a site of young tectonism, copious heat flow, and tall plumes venting into space. Europa's surface is

  13. Purification and Bicelle Crystallization for Structure Determination of the E. coli Outer Membrane Protein TamA.

    PubMed

    Gruss, Fabian; Hiller, Sebastian; Maier, Timm

    2015-01-01

    TamA is an Omp85 protein involved in autotransporter assembly in the outer membrane of Escherichia coli. It comprises a C-terminal 16-stranded transmembrane β-barrel as well as three periplasmic POTRA domains, and is a challenging target for structure determination. Here, we present a method for crystal structure determination of TamA, including recombinant expression in E. coli, detergent extraction, chromatographic purification, and bicelle crystallization in combination with seeding. As a result, crystals in space group P21212 are obtained, which diffract to 2.3 Å resolution. This protocol also serves as a template for structure determination of other outer membrane proteins, in particular of the Omp85 family.

  14. Sex in a test tube: testing the benefits of in vitro recombination.

    PubMed

    Pesce, Diego; Lehman, Niles; de Visser, J Arjan G M

    2016-10-19

    The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'.

  15. Statistical Analysis on Detecting Recombination Sites in DNA-β Satellites Associated with Old World Geminiviruses

    PubMed Central

    Xu, Kai; Yoshida, Ruriko

    2010-01-01

    Although exchange of genetic information by recombination plays an important role in the evolution of viruses, it is not clear how it generates diversity. Understanding recombination events helps with the study of the evolution of new virus strains or new viruses. Geminiviruses are plant viruses which have ambisense single-stranded circular DNA genomes and are one of the most economically important plant viruses in agricultural production. Small circular single-stranded DNA satellites, termed DNA-β, have recently been found to be associated with some geminivirus infections. In this paper we analyze several DNA-β sequences of geminiviruses for recombination events using phylogenetic and statistical analysis and we find that one strain from ToLCMaB has a recombination pattern and is a recombinant molecule between two strains from two species, PaLCuB-[IN:Chi:05] (major parent) and ToLCB-[IN:CP:04] (minor parent). We propose that this recombination event contributed to the evolution of the strain of ToLCMaB in South India. The Hidden Markov Chain (HMM) method developed by Webb et al. (2009) estimating phylogenetic tree through out the whole alignment provide us a recombination history of these DNA-β strains. It is the first time that this statistic method has been used on DNA-β recombination study and give a clear recombination history of DNA-β recombination. PMID:21423447

  16. Sex in a test tube: testing the benefits of in vitro recombination.

    PubMed

    Pesce, Diego; Lehman, Niles; de Visser, J Arjan G M

    2016-10-19

    The origin and evolution of sex, and the associated role of recombination, present a major problem in biology. Sex typically involves recombination of closely related DNA or RNA sequences, which is fundamentally a random process that creates but also breaks up beneficial allele combinations. Directed evolution experiments, which combine in vitro mutation and recombination protocols with in vitro or in vivo selection, have proved to be an effective approach for improving functionality of nucleic acids and enzymes. As this approach allows extreme control over evolutionary conditions and parameters, it also facilitates the detection of small or position-specific recombination benefits and benefits associated with recombination between highly divergent genotypes. Yet, in vitro approaches have been largely exploratory and motivated by obtaining improved end products rather than testing hypotheses of recombination benefits. Here, we review the various experimental systems and approaches used by in vitro studies of recombination, discuss what they say about the evolutionary role of recombination, and sketch their potential for addressing extant questions about the evolutionary role of sex and recombination, in particular on complex fitness landscapes. We also review recent insights into the role of 'extracellular recombination' during the origin of life.This article is part of the themed issue 'Weird sex: the underappreciated diversity of sexual reproduction'. PMID:27619693

  17. Depositional history of Louisiana-Mississippi outer continental shelf

    USGS Publications Warehouse

    Kindinger, J.L.; Miller, R.J.; Stelting, C.E.

    1982-01-01

    A geological study was undertaken in 1981 in the Louisiana-Mississippi outer continental shelf for the Bureau of Land Management. The study included a high-resolution seismic reflection survey, surficial sediment sampling and surface current drifter sampling. Approximately 7100 sq km of the Louisiana-Mississippi shelf and upper slope were surveyed. The sea floor of the entire area is relatively smooth except for occasional areas of uplift produced by diapiric intrusion along the upper slope. Characteristics of the topography and subsurface shelf sediments are the result of depositional sequences due to delta outbuilding over transgressive sediments with intervening periods of erosion during low sea level stands. Little evidence of structural deformation such as faults, diapirs, and shallow gas is present on the shelf and only a few minor faults and scarps are found on the slope. Minisparker seismic records in combination with air gun (40 and 5 cu in) and 3.5-kHz subbottom profile records reveal that seven major stages of shelf development have occurred since the middle Pleistocene. The shelf development has been controlled by the rise and fall of sea level. These stages are defined by four major unconformities, several depositions of transgressive sediments, sequences of river channeling and progradational delta deposits. Surficial sediment sample and seismic records indicate tat the last major depositional event was the progradation of the St. Bernard Delta lobe. This delta lobe covered the northwestern and central regions. Surficial sediments in most of the study area are the product of the reworking of the San Bernard Delta lobe and previous progradations.

  18. Outer table craniotomy for frontal sinus mucocele.

    PubMed

    Wang, Kuo-Wei; Hsu, Huan-Chen; Lu, Kang; Chen, Han-Jung; Liang, Cheng-Loong

    2004-09-01

    Variants of the external osteoplastic flap procedure or endoscopy have been used to approach the frontal sinus mucocele. The authors introduce a modified external approach for radical resection of the mucocele. Using the bicoronal skin incision, the skin flap exposed the right upper orbital rim. The outer table craniotomy was then performed to expose the frontal sinus cavity while carefully preserving the inner table, with radical removal of the mucocele mucosa. The sinus cavity was irrigated with hyper-oxide solution to ensure adequate destruction of possible residual mucosa. The fascia of the frontalis muscle was split, with one part placed into the mucocele cavity to plug the nasofrontal duct. The advantages of the procedures for mucocele removal include clear visualization of the frontal sinus for radical resection of the mucosa, preservation of the inner table avoiding dura manipulation, prevention of central nervous system infection, possibly lower rates of recurrence, prevention of mucosal ingrowth by plugging of the nasofrontal ducts with fascia, and favorable cosmetic outcome. The disadvantages are more intensive surgery comparable to the endoscopic approaches and execution difficulties when the frontal mucocele is small. Additional clinical studies are needed to evaluate the efficiency and safety of this procedure.

  19. Religion and fertility in the Outer Hebrides.

    PubMed

    Clegg, E J; Cross, J F

    1995-01-01

    Estimates of fertility in Protestant Barra and Catholic Harris, islands in the Outer Hebrides, over the period 1856-1985, show that in both islands fertility declined, although marital fertility was generally greater than in Scotland as a whole, and illegitimate fertility was less. However, in Barra during 1966-75 there were pronounced rises in all the indices; illegitimate fertility showed the smallest rise. The publication of the papal encyclical Humanae Vitae may have played a part in this change in fertility, although other, probably local, factors must have been acting, as the first rise in numbers of births occurred before the publication of the encyclical. Other than this transient rise, the religious difference between Harris and Barra had little effect on changes in fertility over the whole period. Indices of proportions married showed declines until after World War 2, followed by steep increases. The marked decline in fertility in both islands may be due in part to this low nuptiality. In addition, a trend of occupation away from traditional crofting and fishing towards more skilled mercantile and professional employment may have played a role.

  20. Modelling turbulence in the outer heliosphere

    NASA Astrophysics Data System (ADS)

    Macek, Wieslaw

    2016-07-01

    Turbulence is complex behaviour that is ubiquitous both in laboratory and astrophysical magnetized plasmas. Notwithstanding the progress in simulation of turbulence in various continuous media, its mechanism is still not sufficiently clear. Therefore, following the basic idea of Kolmogorov, some phenomenological models of scaling behaviour have been proposed, including fractal and multifractal modelling, that can reveal the intermittent character of turbulence. Based on wealth of data provided by deep spacecraft missions including Voyager 1 and 2, these models show that the turbulence in the entire heliosphere is intermittent and multifractal. Moreover, the degree of multifractality decreases with the heliocentric distance and is modulated by the phases of the solar cycles, also beyond the heliospheric termination shock, i. e. in the heliosheath. However, in the very local interstellar medium beyond the heliopause turbulence becomes rather weak and less intermittent, as shown by recent measurements from Voyager 1. This suggests that the heliosphere is immersed in a relatively quiet environment. Hence these studies of turbulence, especially at the heliospheric boundaries, demonstrate that the outer heliosphere provides an interesting possibility to look into turbulence in various media.

  1. Bacterial Outer Membrane Vesicles and Vaccine Applications

    PubMed Central

    Acevedo, Reinaldo; Fernández, Sonsire; Zayas, Caridad; Acosta, Armando; Sarmiento, Maria Elena; Ferro, Valerie A.; Rosenqvist, Einar; Campa, Concepcion; Cardoso, Daniel; Garcia, Luis; Perez, Jose Luis

    2014-01-01

    Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates. PMID:24715891

  2. Outer membrane vesicles as platform vaccine technology

    PubMed Central

    Stork, Michiel; van der Ley, Peter

    2015-01-01

    Abstract Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications. PMID:26912077

  3. The Outer Heliosphere: The Next Frontier

    NASA Astrophysics Data System (ADS)

    Heber, B.; Zank, G. P.

    Only a few decades after the formulation of the concept of a continuous solar corpus- cular radiation by Ludwig Biermann and a solar wind by Eugene Parker, heliospheric physics has evolved into an important branch of astrophysical research. The solar wind forms a bubble, called the heliosphere, in the local interstellar medium, within which the solar system resides and whose size and properties are determined by the manner in which the solar wind and the partially ionized local interstellar medium are coupled. In the last decade, great progress has been made in our understanding of the physical processes thought to describe the outer heliosphere. Numerous spacecraft missions have increased our knowledge about the large-scale structure, the properties, and the character of the heliosphere tremendously. of these, the ageing spacecraft Voyager 1,2 might encounter the inner border of the heliospheric boundary region in the not too distant future. Now, at the beginning of a new millenium, it seems possible, by newly developed technologies to send an interstellar probe beyond the boundaries of the he- liosphere, in order to explore the local interstellar medium in situ. We shall review our basic understanding of global heliospheric structure, emphasizing the importance of the LISM-solar wind coupling, emphasizing the demands and constraints that this places on a mission to the edge of interstellar space.

  4. The Outer Heliosphere: The Next Frontiers

    NASA Astrophysics Data System (ADS)

    Scherer, K.; Fichtner, Horst; Fahr, Hans Jörg; Marsch, Eckart

    The 11th COSPAR Colloquium "The Outer Heliosphere: The Next Frontiers" was held in Potsdam, Germany, from July 24 to 28, 2000, and is the second dedicated to this subject after the first one held in Warsaw, Poland in 1989. Roughly a century has passed after the first ideas by Oliver Lodge, George Francis Fitzgerald and Kristan Birkeland about particle clouds emanating from the Sun and interacting with the Earth environment. Only a few decades after the formulation of the concepts of a continuous solar corpuscular radiation by Ludwig Bierman and a solar wind by Eugene Parker, heliospheric physics has evolved into an important branch of astrophysical research. Numerous spacecraft missions have increased the knowledge about the heliosphere tremendously. Now, at the beginning of a new millenium it seems possible, by newly developed propulasion technologies to send a spacecraft beyond the boundaries of the heliosphere. Such an Interstellar Proce will start the in-situ exploration of interstellar space and, thus, can be considered as the first true astrophysical spacecraft. The year 2000 appeared to be a highly welcome occassion to review the achievements since the last COSPAR Colloquia 11 years ago, to summarize the present developments and to give new impulse for future activities in heliospheric research.

  5. Recombination at the DNA level. Abstracts

    SciTech Connect

    Not Available

    1984-01-01

    Abstracts of papers in the following areas are presented: (1) chromosome mechanics; (2) yeast systems; (3) mammalian homologous recombination; (4) transposons; (5) Mu; (6) plant transposons/T4 recombination; (7) topoisomerase, resolvase, and gyrase; (8) Escherichia coli general recombination; (9) recA; (10) repair; (11) eucaryotic enzymes; (12) integration and excision of bacteriophage; (13) site-specific recombination; and (14) recombination in vitro. (ACR)

  6. Characteristics of the molecular diversity of the outer membrane protein A gene of Haemophilus parasuis

    PubMed Central

    Tang, Cheng; Zhang, Bin; Yue, Hua; Yang, Falong; Shao, Guoqing; Hai, Quan; Chen, Xiaofei; Guo, Dingqian

    2010-01-01

    The molecular diversity of the gene encoding the outer membrane protein A (OmpA) of Haemophilus parasuis has been unclear. In this study, the structural characteristics, sequence types, and genetic diversity of ompA were investigated in 15 H. parasuis reference strains of different serovars and 20 field isolates. Three nucleotide lengths of the complete open reading frame (ORF) of ompA were found: 1098 base pairs (bp), 1104 bp, and 1110 bp. The OmpA contained 4 hypervariable domains, mainly encoding the 4 putative surface-exposed loops, which makes it a potential molecular marker for genotyping. Western blot analysis showed that the recombinant OmpAs of serovars 4 and 5 could cross-react with antiserum to all 15 serovars. Hence, although ompA of H. parasuis exhibited high variation among serovars, this variation did not seem to affect the strong antigenic characteristics of OmpA. PMID:20885850

  7. Evaluation of the possibility to use thick slabs of reconstructed outer breast tomosynthesis slice images

    NASA Astrophysics Data System (ADS)

    Petersson, Hannie; Dustler, Magnus; Tingberg, Anders; Timberg, Pontus

    2016-03-01

    The large image volumes in breast tomosynthesis (BT) have led to large amounts of data and a heavy workload for breast radiologists. The number of slice images can be decreased by combining adjacent image planes (slabbing) but the decrease in depth resolution can considerably affect the detection of lesions. The aim of this work was to assess if thicker slabbing of the outer slice images (where lesions seldom are present) could be a viable alternative in order to reduce the number of slice images in BT image volumes. The suggested slabbing (an image volume with thick outer slabs and thin slices between) were evaluated in two steps. Firstly, a survey of the depth of 65 cancer lesions within the breast was performed to estimate how many lesions would be affected by outer slabs of different thicknesses. Secondly, a selection of 24 lesions was reconstructed with 2, 6 and 10 mm slab thickness to evaluate how the appearance of lesions located in the thicker slabs would be affected. The results show that few malignant breast lesions are located at a depth less than 10 mm from the surface (especially for breast thicknesses of 50 mm and above). Reconstruction of BT volumes with 6 mm slab thickness yields an image quality that is sufficient for lesion detection for a majority of the investigated cases. Together, this indicates that thicker slabbing of the outer slice images is a promising option in order to reduce the number of slice images in BT image volumes.

  8. Finite element modelling of the canine and feline outer ear canal: benefits for local drug delivery?

    PubMed

    Wefstaedt, Patrick; Behrens, Bernd-Arno; Nolte, Ingo; Bouguecha, Anas

    2011-01-01

    Current therapeutic regimes of outer ear infections in dogs and cats aim at the application of efficient local therapeutics after cleaning of the acoustic meatus. One so far insufficiently answered question is if the local application of these substances results in an individually suitable drug concentration in the external ear canal. Thus, the purpose of the present study was to develop a finite element model to calculate the values of the different areas of the external acoustic meatus in dogs and cats in order to provide a tool for the benefit of an appropriate local drug dosage determination. A 3D finite element model (FEM), based on computer tomographic (CT) data sets of four dogs and two cats, was generated to determine areas and volumes of the outer ear canal. Furthermore, various ear therapeutics and cleansers were tested concerning their optimal distribution on 5 cm2 dog and cat skin. The data shows major variations of the area values of the external auditory canal in case of the different dogs but not in the examined cats. These results suggest that manufacturer's recommendations of the pharmaceuticals might be insufficient in terms of achieving an optimal drug concentration in the outer ear canal especially in larger dogs. In conclusion, the developed finite element model has shown to be suitable to calculate areas of the outer ear canal in cats and dogs and could be of help in context with the definition of optimal drug concentrations for a local drug delivery.

  9. Electron-Beam Recombination Lasers

    NASA Astrophysics Data System (ADS)

    Rhoades, Robert Lewis

    1992-01-01

    The first known instance of electron-beam pumping of the 546.1 nm mercury laser is reported. This has been achieved using high-energy electrons to create intense ionization in a coaxial diode chamber containing a mixture of noble gases with a small amount of mercury vapor. Also reported are the results of a study of the 585.3 nm neon laser in He:Ne:Ar mixtures under similar experimental conditions. Both of these lasers are believed to be predominantly pumped by recombination. For the mercury laser, kinetic processes in the partially ionized plasma following the excitation pulse of high-energy electrons should favor the production of atomic mercury ions and molecular ions containing mercury. Subsequent recombination with electrons heavily favors the production of the 7^3S and 6^3 D states of Hg, of which 7^3S is the upper level of the reported laser. For the neon laser, the dominant recombining ion has been previously shown to be Ne_2^{+}. One of the dominant roles of helium in recombination lasers is inferred from the data for the neon laser at low helium concentrations. Helium appears to be necessary for the rapid relaxation of the electron energy which then increases the reaction rates for all known recombination processes thus increasing the pump rate into the upper state.

  10. 38 CFR 38.629 - Outer Burial Receptacle Allowance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... section provides for payment of a monetary allowance for an outer burial receptacle for any interment in a... casket will not be damaged. (d) Payment of monetary allowance. VA will pay a monetary allowance for each... privately purchased the outer burial receptacle will be paid the monetary allowance. For burials during...

  11. Newborns' Face Recognition: Role of Inner and Outer Facial Features

    ERIC Educational Resources Information Center

    Turati, Chiara; Macchi Cassia, Viola; Simion, Francesca; Leo, Irene

    2006-01-01

    Existing data indicate that newborns are able to recognize individual faces, but little is known about what perceptual cues drive this ability. The current study showed that either the inner or outer features of the face can act as sufficient cues for newborns' face recognition (Experiment 1), but the outer part of the face enjoys an advantage…

  12. 33 CFR 117.323 - Outer Clam Bay

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Outer Clam Bay 117.323 Section 117.323 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.323 Outer Clam Bay The drawspan of...

  13. 33 CFR 117.323 - Outer Clam Bay

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Outer Clam Bay 117.323 Section 117.323 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.323 Outer Clam Bay The drawspan of...

  14. 33 CFR 117.323 - Outer Clam Bay.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Outer Clam Bay. 117.323 Section 117.323 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Florida § 117.323 Outer Clam Bay. The drawspan of...

  15. Long-Lived Glass Mirrors For Outer Space

    NASA Technical Reports Server (NTRS)

    Bouquet, Frank L.; Maag, Carl R.; Heggen, Philip M.

    1988-01-01

    Paper summarizes available knowledge about glass mirrors for use in outer space. Strengths and weaknesses of various types of first and second reflective surfaces identified. Second-surface glass mirrors used in outer space designed to different criteria more stringent for terrestrial mirrors. Protons, electrons, cosmic rays, meteorites, and orbiting space debris affect longevities of components. Contamination also factor in space.

  16. Prestin is the motor protein of cochlear outer hair cells

    NASA Astrophysics Data System (ADS)

    Zheng, Jing; Shen, Weixing; He, David Z. Z.; Long, Kevin B.; Madison, Laird D.; Dallos, Peter

    2000-05-01

    The outer and inner hair cells of the mammalian cochlea perform different functions. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. Here we have identified an abundant complementary DNA from a gene, designated Prestin, which is specifically expressed in outer hair cells. Regions of the encoded protein show moderate sequence similarity to pendrin and related sulphate/anion transport proteins. Voltage-induced shape changes can be elicited in cultured human kidney cells that express prestin. The mechanical response of outer hair cells to voltage change is accompanied by a `gating current', which is manifested as nonlinear capacitance. We also demonstrate this nonlinear capacitance in transfected kidney cells. We conclude that prestin is the motor protein of the cochlear outer hair cell.

  17. Far Outer Galaxy H II Regions

    NASA Technical Reports Server (NTRS)

    Rudolph, A. L.; deGues, E. J.; Brand, J.; Wouterloot, J. G. A.; Gross, Anthony R. (Technical Monitor)

    1994-01-01

    We have made a multifrequency (6, 3.6, and 2 cm), high-resolution (3"-6"), radio continuum survey of IRAS selected sources from the catalogue of Wouterloot & Brand (1989) to search for and study H II regions in the far outer Galaxy. We identified 31 sources in this catalog with well determined galactocentric distances, and with R approx.. greater than 15 kpc and L(sub FIR) approx.greater than 10(exp 4) solar luminosity, indicating the presence of high-mass star-formation. We have observed 11 of these sources with the Very Large Array (VLA). We observed the sources at 6 and 2 cm using "scaled arrays", making possible a direct and reliable comparison of the data at these two wavelengths for the determination of spectral indices. We detected a total of 12 radio sources, of which 10 have spectral indices consistent with optically-thin free-free emission from H II regions. Combined with previous VLA observations by other investigators, we have data on a total of 15 H II regions at galactocentric distances of 15 to 18.2kpc, among the most remote H II regions found in our Galaxy. The sizes of the H II regions range from approx. less than 0.10 to 2.3 pc. Using the measured fluxes and sizes, we determine the electron densities, emission measures, and excitation parameters of the H II regions, as well as the fluxes of Lyman continuum photons needed to keep the nebulae ionized. The sizes and electron densities are consistent with most of the sources detected in this survey being compact or ultracompact H II regions. Seven of the fifteen H II regions have sizes approx. less than 0.20 pc. Assuming simple pressure-driven expansion of the H II regions, these sizes indicate ages approx. less than 5 x 10(exp 4) yr, or only 1% of the lifetime of an O star, which implies an unlikely overabundance of O stars in the outer Galaxy. Thus, the large number of compact H II regions suggests that the time these regions spend in a compact phase must be much longer than their dynamical

  18. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles

    PubMed Central

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  19. Proteomics of Aggregatibacter actinomycetemcomitans Outer Membrane Vesicles.

    PubMed

    Kieselbach, Thomas; Zijnge, Vincent; Granström, Elisabeth; Oscarsson, Jan

    2015-01-01

    Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we not only confirmed the vesicle-associated release of LtxA, and the presence of proteins, which are known to act as immunoreactive antigens in the human host, but we also identified numerous additional putative virulence-related proteins in the A. actinomycetemcomitans OMV proteome. The known and putative functions of these proteins include immune evasion, drug targeting, and iron/nutrient acquisition. In summary, our findings are consistent with an OMV-associated proteome that exhibits several offensive and defensive functions, and they provide a comprehensive basis to further disclose roles of A. actinomycetemcomitans OMVs in periodontal and systemic disease. PMID:26381655

  20. FBL Outer Can Welder Analysis Software

    2003-04-21

    The Outer Can Welder Analysis Software (OCWAS) was originally developed by SRTC for use at Hanford to assist in the storage of their excess plutonium in the DOE standard 3013 containers until it can be properly dispositioned using one of the approved DOE methods. After that development effort, SRS funded SRTC to develop a similar system for FB-Line for use with 3013 canister welding at SRS. More robust analysis routines and techniques were used withmore » this new version as well as enhanced graphical representation of the data. During the welding process, critical weld parameters such as weld current and voltage, can give valuable information about the weld. In the past, weld data from the TIG welding process, has been monitored using strip chart recorders. The data from the weld process, recorded on the strip chart recorder traces, are reviewed to analyze the weld. To improve this process, another software package developed by SRTC, the OCW DAS, digitizes the weld data and stores the data in a file. The OCWAS automates the weld analysis process by analyzing the data obtained during the weld process. The OCWAS reads a data file that was previously collected using the OCW DAS software. The software will read the file and parse the data. The user is first prompted to enter the file name. The file is then opened and the operator name, canister identification, and Date/Time of Acquisition are read from the file and displayed on the screen. The binary weld data is then read from the file into an array until the end of the file is reached. The current, voltage, and position data are displayed on the screen in graphical format on the front panel. The weld power and resistance are calculated and are also displayed in graphical format on the front panel.« less

  1. A Second Outer-Core Region in Klebsiella pneumoniae Lipopolysaccharide†

    PubMed Central

    Regué, Miguel; Izquierdo, Luis; Fresno, Sandra; Piqué, Núria; Corsaro, Maria Michela; Naldi, Teresa; De Castro, Cristina; Waidelich, Dietmar; Merino, Susana; Tomás, Juan M.

    2005-01-01

    Up to now only one major type of core oligosaccharide has been found in the lipopolysaccharide of all Klebsiella pneumoniae strains analyzed. Applying a different screening approach, we identified a novel Klebsiella pneumoniae core (type 2). Both Klebsiella core types share the same inner core and the outer-core-proximal disaccharide, GlcN-(1,4)-GalA, but they differ in the GlcN substituents. In core type 2, the GlcpN residue is substituted at the O-4 position by the disaccharide β-Glcp(1-6)-α-Glcp(1, while in core type 1 the GlcpN residue is substituted at the O-6 position by either the disaccharide α-Hep(1-4)-α-Kdo(2 or a Kdo residue (Kdo is 3-deoxy-d-manno-octulosonic acid). This difference correlates with the presence of a three-gene region in the corresponding core biosynthetic clusters. Engineering of both core types by interchanging this specific region allowed studying the effect on virulence. The replacement of Klebsiella core type 1 in a highly type 2 virulent strain (52145) induces lower virulence than core type 2 in a murine infection model. PMID:15937181

  2. Regulation of peptidoglycan synthesis by outer membrane proteins

    PubMed Central

    Typas, Athanasios; Banzhaf, Manuel; van den Berg van Saparoea, Bart; Verheul, Jolanda; Biboy, Jacob; Nichols, Robert J.; Zietek, Matylda; Beilharz, Katrin; Kannenberg, Kai; von Rechenberg, Moritz; Breukink, Eefjan; den Blaauwen, Tanneke; Gross, Carol A.; Vollmer, Waldemar

    2011-01-01

    Summary Growth of the meshlike peptidoglycan (PG) sacculus located between the bacterial inner and outer membranes (OM) is tightly regulated to ensure cellular integrity, maintain cell shape and orchestrate division. Cytoskeletal elements direct placement and activity of PG synthases from inside the cell but precise spatiotemporal control over this process is poorly understood. We demonstrate that PG synthases are also controlled from outside the sacculus. Two OM lipoproteins, LpoA and LpoB, are essential for the function respectively of PBP1A and PBP1B, the major E. coli bifunctional PG synthases. Each Lpo protein binds specifically to its cognate PBP and stimulates its transpeptidase activity, thereby facilitating attachment of new PG to the sacculus. LpoB shows partial septal localization and our data suggest that the LpoB-PBP1B complex contributes to OM constriction during cell division. LpoA/ LpoB and their PBP docking regions are restricted to γ-proteobacteria, providing models for niche-specific regulation of sacculus growth. PMID:21183073

  3. Recombinative generalization of within-syllable units in prereading children.

    PubMed Central

    Mueller, M M; Olmi, D J; Saunders, K J

    2000-01-01

    This study demonstrates recombinative generalization of within-syllable units in prereading children. Three kindergarten children learned to select printed consonant-vowel-consonant words upon hearing the corresponding spoken words. The words were taught in sets; there were six sets, presented consecutively. Within sets, the four words that were taught had overlapping letters, for example, sat, mat, sop, and sug. Tests for recombinative generalization determined whether the children selected novel words with the same components as the trained words (e.g., mop and mug). Two children demonstrated recombinative generalization after one training set, and the 3rd demonstrated it after two training sets. In contrast, 2 other children, who received tests but no training, showed low accuracy across six sets. The 3 experimental children then demonstrated highly accurate printed-word-to-picture matching, and named the majority of the printed words. These findings are a promising step in the development of a computerized instructional technology for reading. PMID:11214028

  4. Current Drive in Recombining Plasma

    SciTech Connect

    P.F. Schmit and N.J. Fisch

    2012-05-15

    The Langevin equations describing the average collisional dynamics of suprathermal particles in nonstationary plasma remarkably admit an exact analytical solution in the case of recombining plasma. The current density produced by arbitrary particle fluxes is derived including the effect of charge recombination. Since recombination has the effect of lowering the charge density of the plasma, thus reducing the charged particle collisional frequencies, the evolution of the current density can be modified substantially compared to plasma with fixed charge density. The current drive efficiency is derived and optimized for discrete and continuous pulses of current, leading to the discovery of a nonzero "residual" current density that persists indefinitely under certain conditions, a feature not present in stationary plasmas.

  5. DNA recombination: the replication connection.

    PubMed

    Haber, J E

    1999-07-01

    Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself. Homologous recombination plays a critical role in repair of such DSBs. There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication. Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur. There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans.

  6. The Dissociative Recombination of OH(+)

    NASA Technical Reports Server (NTRS)

    Guberman, Steven L.

    1995-01-01

    Theoretical quantum chemical calculations of the cross sections and rates for the dissociative recombination of the upsilon = 0 level of the ground state of OH(+) show that recombination occurs primarily along the 2 (2)Pi diabatic route. The products are 0((1)D) and a hot H atom with 6.1 eV kinetic energy. The coupling to the resonances is very small and the indirect recombination mechanism plays only a minor role. The recommended value for the rate coefficient is (6.3 +/- 0.7) x 10(exp -9)x (T(e)/1300)(exp -0.48) cu.cm/s for 10 less than T(e) less than 1000 K.

  7. Recombinant snake venom prothrombin activators.

    PubMed

    Lövgren, Ann

    2013-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need for additional cofactors, but does not discriminate non-carboxylated prothrombin from biologically active γ-carboxylated prothrombin. Here we report that recombinant trocarin and oscutarin could not efficiently generate thrombin without additional protein co-factors. We confirm that both trocarin and oscutarin are similar to human coagulation Factor X (FX), explaining the need for additional cofactors. Sequencing of a genomic fragment containing 7 out of the 8 exons coding for oscutarin further confirmed the similarity to human FX. PMID:23111318

  8. Proteomic analysis of Lawsonia intracellularis reveals expression of outer membrane proteins during infection.

    PubMed

    Watson, Eleanor; Alberdi, M Pilar; Inglis, Neil F; Lainson, Alex; Porter, Megan E; Manson, Erin; Imrie, Lisa; Mclean, Kevin; Smith, David G E

    2014-12-01

    Lawsonia intracellularis is the aetiological agent of the commercially significant porcine disease, proliferative enteropathy. Current understanding of host-pathogen interaction is limited due to the fastidious microaerophilic obligate intracellular nature of the bacterium. In the present study, expression of bacterial proteins during infection was investigated using a mass spectrometry approach. LC-ESI-MS/MS analysis of two isolates of L. intracellularis from heavily-infected epithelial cell cultures and database mining using fully annotated L. intracellularis genome sequences identified 19 proteins. According to the Clusters of Orthologous Groups (COG) functional classification, proteins were identified with roles in cell metabolism, protein synthesis and oxidative stress protection; seven proteins with putative or unknown function were also identified. Detailed bioinformatic analyses of five uncharacterised proteins, which were expressed by both isolates, identified domains and motifs common to other outer membrane-associated proteins with important roles in pathogenesis including adherence and invasion. Analysis of recombinant proteins on Western blots using immune sera from L. intracellularis-infected pigs identified two proteins, LI0841 and LI0902 as antigenic. The detection of five outer membrane proteins expressed during infection, including two antigenic proteins, demonstrates the potential of this approach to interrogate L. intracellularis host-pathogen interactions and identify novel targets which may be exploited in disease control.

  9. Leptospiral outer membrane protein LipL32 induces inflammation and kidney injury in zebrafish larvae

    PubMed Central

    Chang, Ming-Yang; Cheng, Yi-Chuan; Hsu, Shen-Hsing; Ma, Tsu-Lin; Chou, Li-Fang; Hsu, Hsiang-Hao; Tian, Ya-Chung; Chen, Yung-Chang; Sun, Yuh-Ju; Hung, Cheng-Chieh; Pan, Rong-Long; Yang, Chih-Wei

    2016-01-01

    Leptospirosis is an often overlooked cause of acute kidney injury that can lead to multiple organ failure and even death. The principle protein that conserved in many pathogenic leptospires is the outer membrane protein LipL32. However, the role of LipL32 in the pathogenesis of renal injury in leptospirosis is not entirely clear. Here we studied the effects of LipL32 on the developing kidney in zebrafish larvae. Incubation of zebrafish larvae with Leptospira santarosai serovar Shermani induced acute tubular injury predominantly in the proximal pronephric ducts. Furthermore, microinjection of lipl32 mRNA or recombinant LipL32 protein into zebrafish larvae increased macrophage accumulation and disrupted the basolateral location of NA-K-ATPase in pronephric ducts. These changes led to substantial impairment of the pronephric kidney structure. We further demonstrated that morpholino knockdown of tlr2, but not tlr4, reduced the LipL32-induced leukocyte infiltration and kidney injury. These data demonstrate that LipL32 contributes to the renal pathology in leptospirosis and gives some clues to the potential virulence of LipL32. Our results support the use of zebrafish as a model organism for studying the disease mechanism of leptospiral infection. This model might permit the future exploration of the virulence and molecular pathways of different leptospiral outer membrane proteins. PMID:27278903

  10. Leptospiral outer membrane protein LipL32 induces inflammation and kidney injury in zebrafish larvae.

    PubMed

    Chang, Ming-Yang; Cheng, Yi-Chuan; Hsu, Shen-Hsing; Ma, Tsu-Lin; Chou, Li-Fang; Hsu, Hsiang-Hao; Tian, Ya-Chung; Chen, Yung-Chang; Sun, Yuh-Ju; Hung, Cheng-Chieh; Pan, Rong-Long; Yang, Chih-Wei

    2016-01-01

    Leptospirosis is an often overlooked cause of acute kidney injury that can lead to multiple organ failure and even death. The principle protein that conserved in many pathogenic leptospires is the outer membrane protein LipL32. However, the role of LipL32 in the pathogenesis of renal injury in leptospirosis is not entirely clear. Here we studied the effects of LipL32 on the developing kidney in zebrafish larvae. Incubation of zebrafish larvae with Leptospira santarosai serovar Shermani induced acute tubular injury predominantly in the proximal pronephric ducts. Furthermore, microinjection of lipl32 mRNA or recombinant LipL32 protein into zebrafish larvae increased macrophage accumulation and disrupted the basolateral location of NA-K-ATPase in pronephric ducts. These changes led to substantial impairment of the pronephric kidney structure. We further demonstrated that morpholino knockdown of tlr2, but not tlr4, reduced the LipL32-induced leukocyte infiltration and kidney injury. These data demonstrate that LipL32 contributes to the renal pathology in leptospirosis and gives some clues to the potential virulence of LipL32. Our results support the use of zebrafish as a model organism for studying the disease mechanism of leptospiral infection. This model might permit the future exploration of the virulence and molecular pathways of different leptospiral outer membrane proteins. PMID:27278903

  11. Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa.

    PubMed Central

    Ankenbauer, R G

    1992-01-01

    Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa. Images PMID:1320609

  12. Selenium incorporation using recombinant techniques

    SciTech Connect

    Walden, Helen

    2010-04-01

    An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein. Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in prokaryal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.

  13. Epistasis and the selective advantage of sex and recombination

    NASA Astrophysics Data System (ADS)

    de Oliveira, Viviane M.; da Silva, Juliana K.; Campos, Paulo R. A.

    2008-09-01

    The understanding of the central mechanisms favoring sex and recombination in real populations is one of the fundamental issues in evolutionary biology. Based on a previous stochastic formulation for the study of sex, here we aim to investigate the conditions under which epistasis favors the fixation of the sexual mode of reproduction in a given population. In addition, we try to identify the evolutionary forces which contribute to this process. One considers a finite population model which assumes the existence of a recombination modifier allele that can activate the recombination mechanism. We have found that sex is very little favored in a scenario of antagonistic epistasis, and this advantage only occurs in a narrow range of values of the selection coefficient sd . On the other hand, synergistic epistasis favors recombination in a very broad domain. However, the major mechanism contributing to the spreading of the modifier allele depends on the range of values of sd . At large sd , background selection favors recombination since it increases the efficacy of selection, while at low sd Muller’s ratchet is the leading mechanism.

  14. The recombination landscape in Arabidopsis thaliana F2 populations.

    PubMed

    Salomé, P A; Bomblies, K; Fitz, J; Laitinen, R A E; Warthmann, N; Yant, L; Weigel, D

    2012-04-01

    Recombination during meiosis shapes the complement of alleles segregating in the progeny of hybrids, and has important consequences for phenotypic variation. We examined allele frequencies, as well as crossover (XO) locations and frequencies in over 7000 plants from 17 F(2) populations derived from crosses between 18 Arabidopsis thaliana accessions. We observed segregation distortion between parental alleles in over half of our populations. The potential causes of distortion include variation in seed dormancy and lethal epistatic interactions. Such a high occurrence of distortion was only detected here because of the large sample size of each population and the number of populations characterized. Most plants carry only one or two XOs per chromosome pair, and therefore inherit very large, non-recombined genomic fragments from each parent. Recombination frequencies vary between populations but consistently increase adjacent to the centromeres. Importantly, recombination rates do not correlate with whole-genome sequence differences between parental accessions, suggesting that sequence diversity within A. thaliana does not normally reach levels that are high enough to exert a major influence on the formation of XOs. A global knowledge of the patterns of recombination in F(2) populations is crucial to better understand the segregation of phenotypic traits in hybrids, in the laboratory or in the wild. PMID:22072068

  15. Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins.

    PubMed

    Freshour, Judy; Yokoyama, Ruth; Mitchell, David R

    2007-02-23

    We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

  16. Classifications and comparisons of multilocus recombination distributions

    PubMed Central

    Karlin, Samuel; Liberman, Uri

    1978-01-01

    Various classifications and representations of multilocus recombination structures are delineated based on generalized notions of linkage values and recombination rates. An important class of recombination distributions (called the count-location chiasma process) is parameterized by a distribution of the number of crossover events and, for each such crossover count, by a conditional distribution of crossover locations. A number of properties of this recombination structure are developed. A multilocus definition of a “natural” recombination range is set forth. Orderings among recombination distributions in the multilocus setting are also discussed. Comparisons are made in terms of complete linkage, free assortment and noninterference schemes serving as standards. PMID:16592601

  17. Regulation of DNA Pairing in Homologous Recombination

    PubMed Central

    Daley, James M.; Gaines, William A.; Kwon, YoungHo; Sung, Patrick

    2014-01-01

    Homologous recombination (HR) is a major mechanism for eliminating DNA double-strand breaks from chromosomes. In this process, the break termini are resected nucleolytically to form 3′ ssDNA (single-strand DNA) overhangs. A recombinase (i.e., a protein that catalyzes homologous DNA pairing and strand exchange) assembles onto the ssDNA and promotes pairing with a homologous duplex. DNA synthesis then initiates from the 3′ end of the invading strand, and the extended DNA joint is resolved via one of several pathways to restore the integrity of the injured chromosome. It is crucial that HR be carefully orchestrated because spurious events can create cytotoxic intermediates or cause genomic rearrangements and loss of gene heterozygosity, which can lead to cell death or contribute to the development of cancer. In this review, we will discuss how DNA motor proteins regulate HR via a dynamic balance of the recombination-promoting and -attenuating activities that they possess. PMID:25190078

  18. It's Major! College Major Selection & Success

    ERIC Educational Resources Information Center

    Byers, Jenny; Mattern, Krista D.; Shaw, Emily J.; Springall, Robert

    2011-01-01

    Presented at the College Board National Forum, October 26, 2011. Choosing a college major is challenging enough, without stopping to consider the impact it has on a student's college experience and career choice. To provide support during this major decision, participants in this session will develop strategies to facilitate students in making an…

  19. The development of the mammalian outer and middle ear.

    PubMed

    Anthwal, Neal; Thompson, Hannah

    2016-02-01

    The mammalian ear is a complex structure divided into three main parts: the outer; middle; and inner ear. These parts are formed from all three germ layers and neural crest cells, which have to integrate successfully in order to form a fully functioning organ of hearing. Any defect in development of the outer and middle ear leads to conductive hearing loss, while defects in the inner ear can lead to sensorineural hearing loss. This review focuses on the development of the parts of the ear involved with sound transduction into the inner ear, and the parts largely ignored in the world of hearing research: the outer and middle ear. The published data on the embryonic origin, signalling, genetic control, development and timing of the mammalian middle and outer ear are reviewed here along with new data showing the Eustachian tube cartilage is of dual embryonic origin. The embryonic origin of some of these structures has only recently been uncovered (Science, 339, 2013, 1453; Development, 140, 2013, 4386), while the molecular mechanisms controlling the growth, structure and integration of many outer and middle ear components are hardly known. The genetic analysis of outer and middle ear development is rather limited, with a small number of genes often affecting either more than one part of the ear or having only very small effects on development. This review therefore highlights the necessity for further research into the development of outer and middle ear structures, which will be important for the understanding and treatment of conductive hearing loss.

  20. Patterns of recombination on human chromosome 22

    SciTech Connect

    Schlumpf, K.S.; Kim, D.; Haines, J.L.

    1994-09-01

    Virtually all genetic linkage maps generated to date are gross averages across individuals, ages, and (often) sexes. In addition, although some level of positive interference has been assumed, until recently little evidence to support this in humans has been available. The major stumbling block has been the quality of the data available, since even a few genotypic errors can have drastic effects on both the map length and the number of apparent recombinants. In addition, variation in recombination by factors other than sex have pretty much been ignored. To explore recombination in more detail, we have generated a microsatellite marker map of human chromosome 22. This map includes 32 markers genotyped through 46 sibships of the Venezuelan Reference Pedigree (VRP). Extensive error checking and regenotyping was performed to remove as many genotypic errors as possible, but no genotypes were removed simply because they created unlikely events. The following 1000:1 odds map has been obtained: cen--F8VWFP1--11--S264--3-S311--4--S257--2--TOP1P2--3--S156--1--CRYB2--1--S258--2--S310--6--S193--1--S275--3--S268--1--S280--4--S304--3--S283--2--LiR1--3--IL2RB--3--S299--1--S302--1--S537--2--S270--4--PDGF--8--S274--qter. The female map (91 cM) is twice as long as the male map (46 cM) and the log-likelihood difference in the maps (22.3) is highly significant (P=0.001, df=22) and appears constant across the chromosome. Analysis of recombination with age showed no particular trends for either males or females when chromosomes were grouped into three categories (20, 20-30, 30+) by parental age at birth of child. Positive interference was found in maternally derived chromosomes ({chi}{sup 2}=30.5 (4), p<0.005), but not in paternally derived chromosomes ({chi}{sup 2}=6.24 (3), P=0.10). This contrasts to data from chromosomes 9 and 21 where positive interference was found for both sexes. More detailed analyses are in progress.

  1. Ad 2.0: a novel recombineering platform for high-throughput generation of tailored adenoviruses

    PubMed Central

    Mück-Häusl, Martin; Solanki, Manish; Zhang, Wenli; Ruzsics, Zsolt; Ehrhardt, Anja

    2015-01-01

    Recombinant adenoviruses containing a double-stranded DNA genome of 26–45 kb were broadly explored in basic virology, for vaccination purposes, for treatment of tumors based on oncolytic virotherapy, or simply as a tool for efficient gene transfer. However, the majority of recombinant adenoviral vectors (AdVs) is based on a small fraction of adenovirus types and their genetic modification. Recombineering techniques provide powerful tools for arbitrary engineering of recombinant DNA. Here, we adopted a seamless recombineering technology for high-throughput and arbitrary genetic engineering of recombinant adenoviral DNA molecules. Our cloning platform which also includes a novel recombination pipeline is based on bacterial artificial chromosomes (BACs). It enables generation of novel recombinant adenoviruses from different sources and switching between commonly used early generation AdVs and the last generation high-capacity AdVs lacking all viral coding sequences making them attractive candidates for clinical use. In combination with a novel recombination pipeline allowing cloning of AdVs containing large and complex transgenes and the possibility to generate arbitrary chimeric capsid-modified adenoviruses, these techniques allow generation of tailored AdVs with distinct features. Our technologies will pave the way toward broader applications of AdVs in molecular medicine including gene therapy and vaccination studies. PMID:25609697

  2. Unraveling a Region-Specific Hyper-Recombination Phenomenon: Genetic Control and Modalities of Terminal Recombination in Escherichia Coli

    PubMed Central

    Corre, J.; Cornet, F.; Patte, J.; Louarn, J. M.

    1997-01-01

    The propensity of the terminus of the Escherichia coli chromosome for recombination has been further explored, using a test based on the selectable loss of a λ prophage inserted between repeated sequences from Tn10. Terminal recombination appears region-specific and unrelated to replication termination in a strain harboring a major chromosomal rearrangement. It requires RecBC(D) activity and must therefore occur between sister chromosomes, to conserve genomic integrity in spite of DNA degradation by RecBCD. Terminal recombination is maximal in the dif region and its intensity on either side of this recombination site depends on the orientation of the repeated sequences, probably because of the single χ site present in each repeat. Additional observations support the model that the crossover is initiated by single-strand invasion between sister chromosomes followed by RecBCD action as a consequence of DNA breakage due to the initial invasion event. Crossover location within repeats inserted at dif position supports the possibility that sister chromosomes are tightly paired in the centre of the terminal recombination zone. These data reinforce the model that postreplicative reconstruction of nucleoid organization creates a localized synapsis between the termini of sister chromosomes. PMID:9383046

  3. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane. PMID:26621472

  4. Self-sterilization of bodies during outer planet entry

    NASA Technical Reports Server (NTRS)

    Hoffman, A. R.; Jaworski, W.; Taylor, D. M.

    1974-01-01

    A body encountering the atmosphere of an outer planet is subjected to heat loads which could result in high temperature conditions that render terrestrial organisms on or within the body nonviable. To determine whether an irregularly shaped entering body, consisting of several different materials, would be sterilized during inadvertent entry at high velocity, the thermal response of a typical outer planet spacecraft instrument was studied. The results indicate that the Teflon insulated cable and electronic circuit boards may not experience sterilizing temperatures during a Jupiter, Saturn, or Titan entry. Another conclusion of the study is that small plastic particles entering Saturn from outer space have wider survival corridors than do those at Jupiter.

  5. Radioisotope Reduction Using Solar Power for Outer Planetary Missions

    NASA Technical Reports Server (NTRS)

    Fincannon, James

    2008-01-01

    Radioisotope power systems have historically been (and still are) the power system of choice from a mass and size perspective for outer planetary missions. High demand for and limited availability of radioisotope fuel has made it necessary to investigate alternatives to this option. Low mass, high efficiency solar power systems have the potential for use at low outer planetary temperatures and illumination levels. This paper documents the impacts of using solar power systems instead of radioisotope power for all or part of the power needs of outer planetary spacecraft and illustrates the potential fuel savings of such an approach.

  6. Structural Aspects of Bacterial Outer Membrane Protein Assembly.

    PubMed

    Calmettes, Charles; Judd, Andrew; Moraes, Trevor F

    2015-01-01

    The outer membrane of Gram-negative bacteria is predominantly populated by β-Barrel proteins and lipid anchored proteins that serve a variety of biological functions. The proper folding and assembly of these proteins is essential for bacterial viability and often plays a critical role in virulence and pathogenesis. The β-barrel assembly machinery (Bam) complex is responsible for the proper assembly of β-barrels into the outer membrane of Gram-negative bacteria, whereas the localization of lipoproteins (Lol) system is required for proper targeting of lipoproteins to the outer membrane.

  7. Recombinant expression and epitope mapping of grass pollen allergens.

    PubMed

    Suphioglu, C; Smith, P M; Ong, E K; Knox, R B; Singh, M B

    1996-01-01

    We have studied the expression of recombinant forms of Group 1 allergens from rye-grass and Bermuda grass pollens. Recombinant Lol p 1 expressed in bacteria bound serum IgE from allergic patients. Based on analysis of fragments of the Lol p 1 cDNA clone, the major IgE-reactive epitope has been mapped to the C-terminus. However, although SDS-denatured natural Cyn d 1 (from Bermuda grass) bound IgE, the full or partial recombinant proteins expressed in bacteria did not bind IgE. We have since expressed Cyn d 1 in the yeast Pichia pastoris and restored IgE binding. cDNA clones encoding two isoforms of Lol p 5, Lol p 5A and Lol p 5B, have been expressed in bacteria and resulting polypeptides show IgE-binding. Random fragments of these clones have been generated and when expressed as partial recombinant proteins in bacteria, allowed us to identify the major IgE-binding epitopes. The allergenic epitopes were localised towards the C-terminal half of the molecule. Although both isoforms shared similar IgE-reactive epitopes, Lol p 5B did not recognise the Lol p 5A-specific monoclonal antibody A7. At sequence level, there appear to be several amino acid differences between the antigenic epitopes of these two isoallergens. These results aid in the design of diagnostics and in grass pollen immunotherapy.

  8. The role of outer membrane in Serratia marcescens intrinsic resistance to antibiotics.

    PubMed

    Sánchez, L; Ruiz, N; Leranoz, S; Viñas, M; Puig, M

    1997-09-01

    Three different porins from Serratia marcescens were described. They were named Omp1, Omp2 and Omp3 and their molecular weights were 42, 40 and 39 kDa respectively. Omp2 and Omp3 showed osmoregulation and thermoregulation in a similar way to OmpC and OmpF of Escherichia coli. Permeability coefficients of the outer membrane of this species were calculated following the Zimmermann and Rosselet method. P values were similar to those obtained in Escherichia coli, which suggests that the chromosomal beta-lactamase would play a major role in the resistance of Serratia marcescens to beta-lactam antibiotics. Both MIC values and permeabilities were modified by salycilates and acetylsalycilate. Synergism between the outer membrane and the beta-lactamase was also evaluated. When bacteria grew in the presence of a beta-lactam in the medium, the beta-lactamase accounted for most of the resistance.

  9. Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria.

    PubMed

    Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

    2014-01-01

    Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors.

  10. [Analysis of the 4th generation outer space bred Angelica dahurica by FTIR spectroscopy].

    PubMed

    Zhu, Yan-ying; Wu, Peng-le; Liu, Mei-yi; Wang, Zhi-zhou; Guo, Xi-hua; Guan, Ying

    2012-03-01

    The major components of the 4th generation outer space bred angelica and the ground group were determined and analyzed by Fourier transform infrared spectroscopy (FTIR) and second derivative spectrum, considering the large mutation of the plants with space mutagenesis. The results show that the content of the coumarin (1741 cm(-1)), which is the main active components of the space angelica dahurica increased, and the content of the protein (1 459, 1 419 cm(-1)) and the fat (930 cm(-1)) increased slightly, whereas the content of the starch and the dietary fiber reduced drastically. There are obvious differences between the peak values of the second derivative spectra of the plants, revealing that the outer space angelica dahurica contained amine component at 1 279 cm(-1). Space mutation breeding is favor of breeding angelica with better idiosyncrasy.

  11. Epitaxial thin film growth in outer space

    NASA Technical Reports Server (NTRS)

    Ignatiev, Alex; Chu, C. W.

    1988-01-01

    A new concept for materials processing in space exploits the ultravacuum component of space for thin-film epitaxial growth. The unique LEO space environment is expected to yield 10-ftorr or better pressures, semiinfinite pumping speeds, and large ultravacuum volume (about 100 cu m) without walls. These space ultravacuum properties promise major improvement in the quality, unique nature, and throughput of epitaxially grown materials, including semiconductors, magnetic materials, and thin-film high-temperature superconductors.

  12. Evolution and recombination of bovine DNA repeats.

    PubMed

    Jobse, C; Buntjer, J B; Haagsma, N; Breukelman, H J; Beintema, J J; Lenstra, J A

    1995-09-01

    The history of the abundant repeat elements in the bovine genome has been studied by comparative hybridization and PCR. The Bov-A and Bov-B SINE elements both emerged just after the divergence of the Camelidae and the true ruminants. A 31-bp subrepeat motif in satellites of the Bovidae species cattle, sheep, and goat is also present in Cervidae (deer) and apparently predates the Bovidae. However, the other components of the bovine satellites were amplified after the divergence of the cattle and the Caprinae (sheep and goat). A 23-bp motif, which as subrepeat of two major satellites occupies 5% of the cattle genome, emerged only after the split of the water buffalo and other cattle species. During the evolution of the Bovidae the satellite repeat units were shaped by recombination events involving subrepeats, other satellite components, and SINE elements. Differences in restriction sites of homologous satellites indicate a continuing rapid horizontal spread of new sequence variants.

  13. SNAREs Interact with Retinal Degeneration Slow and Rod Outer Segment Membrane Protein-1 during Conventional and Unconventional Outer Segment Targeting

    PubMed Central

    Zulliger, Rahel; Conley, Shannon M.; Mwoyosvi, Maggie L.; Stuck, Michael W.; Azadi, Seifollah; Naash, Muna I.

    2015-01-01

    Mutations in the photoreceptor protein peripherin-2 (also known as RDS) cause severe retinal degeneration. RDS and its homolog ROM-1 (rod outer segment protein 1) are synthesized in the inner segment and then trafficked into the outer segment where they function in tetramers and covalently linked larger complexes. Our goal is to identify binding partners of RDS and ROM-1 that may be involved in their biosynthetic pathway or in their function in the photoreceptor outer segment (OS). Here we utilize several methods including mass spectrometry after affinity purification, in vitro co-expression followed by pull-down, in vivo pull-down from mouse retinas, and proximity ligation assay to identify and confirm the SNARE proteins Syntaxin 3B and SNAP-25 as novel binding partners of RDS and ROM-1. We show that both covalently linked and non-covalently linked RDS complexes interact with Syntaxin 3B. RDS in the mouse is trafficked from the inner segment to the outer segment by both conventional (i.e., Golgi dependent) and unconventional secretory pathways, and RDS from both pathways interacts with Syntaxin3B. Syntaxin 3B and SNAP-25 are enriched in the inner segment (compared to the outer segment) suggesting that the interaction with RDS/ROM-1 occurs in the inner segment. Syntaxin 3B and SNAP-25 are involved in mediating fusion of vesicles carrying other outer segment proteins during outer segment targeting, so could be involved in the trafficking of RDS/ROM-1. PMID:26406599

  14. Recombinant DNA: History of the Controversy.

    ERIC Educational Resources Information Center

    Vigue, Charles L.; Stanziale, William G.

    1979-01-01

    The hazards associated with recombinant DNA research are presented along with some social implications and the development of recombinant DNA research guidelines by the National Institutes of Health. (SA)

  15. Meiotic Recombination: The Essence of Heredity.

    PubMed

    Hunter, Neil

    2015-10-28

    The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.

  16. Mineral resource management of the Outer Continental Shelf : leasing procedures, evaluation of resources, and supervision of production operations on leased lands of the Outer Continental Shelf

    USGS Publications Warehouse

    Adams, Maurice V.; John, C.B.; Kelly, R.F.; LaPointe, A.E.; Meurer, R.W.

    1975-01-01

    An important function of the Geological Survey is the evaluation and management of the mineral resources of the Outer Continental Shelf, particularly with respect to oil and gas, salt, and sulfur. Production of oil and gas from the Outer Continental Shelf of the United States has increased substantially over the past 20 years and represents an increasing percentage of total United States production. As discovery of major onshore production of oil and gas has become more difficult, the search has moved into the surrounding waters where submerged sedimentary formations are conducive to the accumulation of oil and gas. Increased energy demands of recent years have accelerated the pace of offshore operations with a corresponding improvement in technology as exploration and development have proceeded farther from shore and into deeper water. While improved technology and enforcement of more stringent regulations have made offshore operations safer, it is unrealistic to believe that completely accident-free operations can ever be achieved. Only slightly more than six percent of the world's continental terrace is adjacent to the United States, but less than one percent has been explored for oil and gas. Since the lead time for the development of offshore oil and gas resources can be as much as a decade, they do not provide an immediate energy supply but should be viewed in the light of a near-term source with a potential of becoming a medium-range source of supply pending the development of alternative energy sources. Revenues from the Outer Continental Shelf are deposited to the general fund of the United States Treasury. A major portion of these funds is allocated to the Land and Water Conservation Fund, the largest Federal grant-in-aid program of assistance to States, counties, and cities for the acquisition and development of public parks, open space, and recreation lands and water.

  17. Molecular parasitism in the Escherichia coli-Bdellovibrio bacteriovorus system: translocation of the matrix protein from the host to the parasite outer membrane.

    PubMed Central

    Guerrini, F; Romano, V; Valenzi, M; Di Giulio, M; Mupo, M R; Sacco, M

    1982-01-01

    During the intracellular maturation in Escherichia coli of the parasite Bdellovibrio bacteriovorus the outer membrane, major protein I of E. coli (i.e., the matrix protein) becomes associated with the outer membrane of the emerging parasite cells. The binding properties of this protein with the outer membrane of the host and of the parasite are identical. An analogous phenomenon also occurs during Bdellovibrio parasitism on Klebsiella pneumoniae and on Salmonella typhimurium. Possible roles for this scavenging action of Bdellovibrio, and similar phenomena in other parasitic systems, are discussed. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:6765198

  18. Outer Continental Shelf Oil and Gas Leasing/Production Program

    SciTech Connect

    Not Available

    1988-01-01

    This annual report on the Outer Continental Shelf (OCS) Oil and Gas Leasing and Production program summarizes receipts and expenditures, and includes information on OCS safety violations as reported by the US Coast Guard. 3 figs., 12 tabs.

  19. Viewing the outer heliosphere in energetic neutral atoms

    NASA Technical Reports Server (NTRS)

    Hsieh, K. C.; Gruntman, M. A.

    1993-01-01

    The use of energetic neutral atoms (ENAs) to study the outer heliosphere is discussed. The detection of ENAs under stringent observational conditions is addressed. Imaging ENA instruments are examined.

  20. Decreasing Outer Hair Cell Membrane Cholesterol Increases Cochlear Electromechanics

    NASA Astrophysics Data System (ADS)

    Brownell, William E.; Jacob, Stefan; Hakizimana, Pierre; Ulfendahl, Mats; Fridberger, Anders

    2011-11-01

    The effect of decreasing membrane cholesterol on the mechanical response of the cochlea to acoustic and/or electrical stimulation was monitored using laser interferometry. In contrast to pharmacological interventions that typically decrease cochlear electromechanics, reducing membrane cholesterol increased the response. The electromechanical response in untreated preparations was asymmetric with greater displacements in response to positive currents and cholesterol depletion increased the asymmetry. The results confirm that outer hair cell electromotility is enhanced by low membrane cholesterol. The asymmetry of the response indicates the outer hair cell resting membrane potential is hyperpolarized relative to the voltage of maximum gain for the outer hair cell voltage-displacement function. The magnitude of the response increase suggests a non-uniform distribution of cholesterol along the lateral wall of normal adult outer hair cells.

  1. 6. OUTER BLAST DOOR, WEST REAR. Edwards Air Force ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    6. OUTER BLAST DOOR, WEST REAR. - Edwards Air Force Base, South Base Sled Track, Firing & Control Blockhouse for 10,000-foot Track, South of Sled Track at midpoint of 20,000-foot track, Lancaster, Los Angeles County, CA

  2. Nuclear power sources in outer space. [spacecraft propulsion legal aspects

    NASA Technical Reports Server (NTRS)

    Hosenball, S. N.

    1978-01-01

    Legal problems associated with nuclear power sources in space are discussed with particular reference to the Cosmos 954 incident. Deliberations of the Legal and Scientific and Technical Subcommittees on the Peaceful Uses of Outer Space on this subject are discussed.

  3. Radioisotope Electric Propulsion for Fast Outer Planetary Orbiters

    NASA Technical Reports Server (NTRS)

    Oleson, Steven; Benson, Scott; Gefert, Leon; Patterson, Michael; Schreiber, Jeffrey

    2002-01-01

    Recent interest in outer planetary targets by the Office of Space Science has spurred the search for technology options to enable relatively quick missions to outer planetary targets. Several options are being explored including solar electric propelled stages combined with aerocapture at the target and nuclear electric propulsion. Another option uses radioisotope powered electric thrusters to reach the outer planets. Past work looked at using this technology to provide faster flybys. A better use for this technology is for outer planet orbiters. Combined with medium class launch vehicles and a new direct trajectory these small, sub-kilowatt ion thrusters and Stirling radioisotope generators were found to allow missions as fast as 5 to 12 years for objects from Saturn to Pluto, respectively. Key to the development is light spacecraft and science payload technologies.

  4. Electron beam seals outer surfaces of porous bodies

    NASA Technical Reports Server (NTRS)

    Herz, W. H.; Kurtz, A. D.; Kurtz, R. A.

    1966-01-01

    Porous tungsten plugs provide even airflow for frictionless bearings used in air bearing supported gyros. The plugs have their outer cylindrical surface sealed by an electron beam process to ensure unidirectional airflow through their exit ends.

  5. Comparative Proteome Analysis of Spontaneous Outer Membrane Vesicles and Purified Outer Membranes of Neisseria meningitidis

    PubMed Central

    Otto, Andreas; Becher, Dörte; Vogel, Ulrich

    2013-01-01

    Outer membrane vesicles (OMVs) of Gram-negative bacteria receive increasing attention because of various biological functions and their use as vaccines. However, the mechanisms of OMV release and selective sorting of proteins into OMVs remain unclear. Comprehensive quantitative proteome comparisons between spontaneous OMVs (SOMVs) and the outer membrane (OM) have not been conducted so far. Here, we established a protocol for metabolic labeling of neisserial proteins with 15N. SOMV and OM proteins labeled with 15N were used as an internal standard for proteomic comparison of the SOMVs and OMs of two different strains. This labeling approach, coupled with high-sensitivity mass spectrometry, allowed us to comprehensively unravel the proteome of the SOMVs and OMs. We quantified the relative distribution of 155 proteins between SOMVs and the OM. Complement regulatory proteins, autotransporters, proteins involved in iron and zinc acquisition, and a two-partner secretion system were enriched in SOMVs. The highly abundant porins PorA and PorB and proteins connecting the OM with peptidoglycan or the inner membrane, such as RmpM, MtrE, and PilQ, were depleted in SOMVs. Furthermore, the three lytic transglycosylases MltA, MltB, and Slt were less abundant in SOMVs. In conclusion, SOMVs are likely to be released from surface areas with a low local abundance of membrane-anchoring proteins and lytic transglycosylases. The enrichment of complement regulatory proteins, autotransporters, and trace metal binding and transport proteins needs to be explored in the context of the pathogenesis of meningococcal disease. PMID:23893116

  6. TRACING THE OUTER HALO IN A GIANT ELLIPTICAL TO 25 R {sub eff}

    SciTech Connect

    Rejkuba, M.; Harris, W. E.; Greggio, L.; Harris, G. L. H.; Jerjen, H.; Gonzalez, O. A.

    2014-08-10

    We have used the Advanced Camera for Surveys and Wide Field Camera 3 cameras on board the Hubble Space Telescope to resolve stars in the halo of the nearest giant elliptical (gE) galaxy NGC 5128 out to a projected distance of 140 kpc (25 effective radii, R {sub eff}) along the major axis and 90 kpc (16 R {sub eff}) along the minor axis. This data set provides an unprecedented radial coverage of the stellar halo properties in any gE galaxy. Color-magnitude diagrams clearly reveal the presence of the red giant branch stars belonging to the halo of NGC 5128, even in our most distant fields. The star counts demonstrate increasing flattening of the outer halo, which is elongated along the major axis of the galaxy. The V – I colors of the red giants enable us to measure the metallicity distribution in each field and so map the gradient out to ∼16 R {sub eff} from the galaxy center along the major axis. A median metallicity is obtained even for the outermost fields along both axes. We observe a smooth transition from a metal-rich ([M/H] ∼0.0) inner galaxy to lower metallicity in the outer halo, with the metallicity gradient slope along the major axis of Δ[M/H]/ΔR ≅ –0.0054 ± 0.0006 dex kpc{sup –1}. In the outer halo, beyond ∼10 R {sub eff}, the number density profile follows a power law, but also significant field-to-field metallicity and star count variations are detected. The metal-rich component dominates in all observed fields, and the median metallicity is [M/H] >–1 dex in all fields.

  7. The Velocity Structure of SN 1987A's Outer Circumstellar Envelope

    NASA Astrophysics Data System (ADS)

    Crotts, A. P. S.; Heathcote, S. R.

    1997-12-01

    We present high-resolution optical spectroscopy, (obtained with the CTIO 4-meter/echelle spectrograph over many epochs between 1989 and 1997) of the circumstellar nebula of SN 1987A, including the outer rings (within 3 arcsec of the SN), the inner (equatorial) ring, and fainter features at larger radii never studied before spectroscopically. We report velocity displacements for portions of the outer rings, up to 26 km s(-1) with respect the SN centroid velocity, with blueshifted components in the location of the southern outer ring and the redshifted portions of the northern outer ring. The largest shifts are near the SN, as predicted by a model in which the outer rings are the crowns of an expanding, bipolar nebula with the inner ring at its waist. We also confirm that the inner ring shows a velocity full-width of about 13 km s(-1) , which, along with the geometry of the rings and our outer ring velocity measurements, allows us to estimate a characteristic timescale of about 20,000 y for each of the three rings, implying that all are coeval. This contrasts with measurements by others of compositional ratios in the inner versus outer rings indicating that they were, perhaps, ejected at different times from the progenitor's star's outer envelope. Additionally, we measure the velocity of low surface brightness features at larger radii indicating that circumstellar material even farther from the SN was ejected up to 400,000 y before the explosion. Finally, we note the presence of transient emission features within the circumstellar nebula and describe their behaviour, and consider what implications our observations may have for the coming transformation of this nebula into Supernova Remnant 1987A.

  8. Science payload. [of outer planet atmospheric entry probe

    NASA Technical Reports Server (NTRS)

    Myers, H.

    1974-01-01

    The representative science payload of an outer planet atmospheric entry probe is described. The instrumental details are based on experiments that have been successfully flown in the atmospheres of Earth and Venus. The incorporation of these instruments into an outer planet probe requires a strong interaction between instrument designer and probe designer. The installation of the instruments into a 250 lb entry probe is illustrated.

  9. Star Formation in the Outer Disk of Spiral Galaxies

    NASA Astrophysics Data System (ADS)

    Barnes, Kate L.; van Zee, Liese; Côté, Stéphanie; Schade, David

    2012-09-01

    We combine new deep and wide field of view Hα imaging of a sample of eight nearby (d ≈ 17 Mpc) spiral galaxies with new and archival H I and CO imaging to study the star formation and the star formation regulation in the outer disk. We find that, in agreement with previous studies, star formation in the outer disk has low covering fractions, and star formation is typically organized into spiral arms. The star formation in the outer disk is at extremely low levels, with typical star formation rate surface densities of ~10-5 to 10-6 M ⊙ yr-1 kpc-2. We find that the ratio of the radial extent of detected H II regions to the radius of the H I disk is typically gsim85%. This implies that in order to further our understanding of the implications of extended star formation, we must further our understanding of the formation of extended H I disks. We measure the gravitational stability of the gas disk, and find that the outer gaseous disk is typically a factor of ~2 times more stable than the inner star-forming disk. We measure the surface density of outer disk H I arms, and find that the disk is closer to gravitational instability along these arms. Therefore, it seems that spiral arms are a necessary, but not sufficient, requirement for star formation in the outer disk. We use an estimation of the flaring of the outer gas disk to illustrate the effect of flaring on the Schmidt power-law index; we find that including flaring increases the agreement between the power-law indices of the inner and outer disks.

  10. Observation of enhanced electron-ion recombination rates at very low energies

    SciTech Connect

    Gao, H.; DeWitt, D.R.; Schuch, R.; Zong, W.; Asp, S.; Pajek, M. |

    1995-12-01

    A strong enhancement of electron-ion recombination over expected rates at sub-eV relative energies is revealed for highly charged ions. Measured Ne{sup 10+} and Ar{sup 13+} recombination rates were found to be enhanced at low ({lt}1 meV) energies by factors of 4 and 10 over theoretical predictions of radiative recombination rates, respectively. It is shown that the majority of the enhancement in Ar{sup 13+} is caused by very low lying {Delta}{ital n}=0 dielectronic recombination resonances; nevertheless, the nonresonant contributions to the measured recombination rates for both systems were found to be significantly higher than predicted by presently available models. {copyright} {ital 1995 The American Physical Society.}

  11. MosaicSolver: a tool for determining recombinants of viral genomes from pileup data

    PubMed Central

    Wood, Graham R.; Ryabov, Eugene V.; Fannon, Jessica M.; Moore, Jonathan D.; Evans, David J.; Burroughs, Nigel

    2014-01-01

    Viral recombination is a key evolutionary mechanism, aiding escape from host immunity, contributing to changes in tropism and possibly assisting transmission across species barriers. The ability to determine whether recombination has occurred and to locate associated specific recombination junctions is thus of major importance in understanding emerging diseases and pathogenesis. This paper describes a method for determining recombinant mosaics (and their proportions) originating from two parent genomes, using high-throughput sequence data. The method involves setting the problem geometrically and the use of appropriately constrained quadratic programming. Recombinants of the honeybee deformed wing virus and the Varroa destructor virus-1 are inferred to illustrate the method from both siRNAs and reads sampling the viral genome population (cDNA library); our results are confirmed experimentally. Matlab software (MosaicSolver) is available. PMID:25120266

  12. Theoretical prediction of the impact of Auger recombination on charge collection from an ion track

    NASA Technical Reports Server (NTRS)

    Edmonds, Larry D.

    1991-01-01

    A recombination mechanism that significantly reduces charge collection from very dense ion tracks in silicon devices was postulated by Zoutendyk et al. The theoretical analysis presented here concludes that Auger recombination is such a mechanism and is of marginal importance for higher density tracks produced by 270-MeV krypton, but of major importance for higher density tracks. The analysis shows that recombination loss is profoundly affected by track diffusion. As the track diffuses, the density and recombination rate decrease so fast that the linear density (number of electron-hole pairs per unit length) approaches a non-zero limiting value as t yields infinity. Furthermore, the linear density is very nearly equal to this limiting value in a few picoseconds or less. When Auger recombination accompanies charge transport processes that have much longer time scales, it can be simulated by assigning a reduced linear energy transfer to the ion.

  13. jpHMM: improving the reliability of recombination prediction in HIV-1.

    PubMed

    Schultz, Anne-Kathrin; Zhang, Ming; Bulla, Ingo; Leitner, Thomas; Korber, Bette; Morgenstern, Burkhard; Stanke, Mario

    2009-07-01

    Previously, we developed jumping profile hidden Markov model (jpHMM), a new method to detect recombinations in HIV-1 genomes. The jpHMM predicts recombination breakpoints in a query sequence and assigns to each position of the sequence one of the major HIV-1 subtypes. Since incorrect subtype assignment or recombination prediction may lead to wrong conclusions in epidemiological or vaccine research, information about the reliability of the predicted parental subtypes and breakpoint positions is valuable. For this reason, we extended the output of jpHMM to include such information in terms of 'uncertainty' regions in the recombination prediction and an interval estimate of the breakpoint. Both types of information are computed based on the posterior probabilities of the subtypes at each query sequence position. Our results show that this extension strongly improves the reliability of the jpHMM recombination prediction. The jpHMM is available online at http://jphmm.gobics.de/.

  14. Proteome analysis of mitochondrial outer membrane from Neurospora crassa

    SciTech Connect

    Schmitt, Simone; Prokisch, Holger; Schlunk, Tilman; Camp, David G.; Ahting, Uwe; Waizenegger, Thomas; Scharfe, Curt M.; Meitinger, Thomas; Imhof, Axel; Neupert, Walter; Oefner, Peter J.; Rapaport, Doron

    2006-01-01

    The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of liquid chromatography tandem mass spectrometry of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS peptide fingerprinting. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (Porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

  15. A model of the dynamical structure of Earth's outer core

    NASA Astrophysics Data System (ADS)

    Loper, David E.

    2000-01-01

    The dynamical state of the outer core is quantified, assuming that the convective pattern consists of buoyant parcels which rise to the top, forcing a broad descending flow in the bulk of the outer core. This convective circulation is assumed sufficiently rapid that the outer core is close to a well-mixed adiabatic state. Small compositional deviations from this state, resulting from the secular evolution of composition as the solid inner core grows, make the descending portion of the outer core stably stratified. Thermal deviations result from the mismatch between effective volumetric heating and the divergence of conducted heat. Effective volumetric heating is the sum of secular cooling, compressional heating and Ohmic heating. Divergence of heat is negative and larger in magnitude than effective volumetric heating throughout the core, resulting in a stabilizing thermal gradient in the descending fluid which reinforces the compositional gradient. The compositional and thermal contributions to the stable stratification are of comparable magnitudes. The strength of stratification depends on the strength of the convective circulation, which is unquantified. This stratification, coupled with Coriolis and Lorentz forces, has the potential to inhibit turbulence in the descending portions of the outer core. The thermal and compositional perturbations arising from processes at the inner-core boundary and core-mantle boundary are quantified and the dynamic behavior of the rising parcels is discussed and quantified. Depending on the sign and magnitude of exchanges of heat and composition, convective motions may be inhibited or suppressed near the top of the outer core.

  16. Biochemical and immunological characterization of recombinant allergen Lol p 1.

    PubMed

    Tamborini, E; Faccini, S; Lidholm, J; Svensson, M; Brandazza, A; Longhi, R; Groenlund, H; Sidoli, A; Arosio, P

    1997-11-01

    Pollen from perennial rye grass (Lolium perenne), a major cause of type-I allergy worldwide, contains a complex mixture of allergenic proteins among which Lol p 1 is one of the most important. We describe the expression, purification and characterization of a recombinant Lol p 1 overproduced in Escherichia coli. The recombinant allergen, expressed in high yields and purified in milligram amounts, bound to specific IgE antibodies from human sera, induced histamine release from sensitized human basophils, and elicited rabbit antisera that recognize specifically recombinant Lol p 1 and natural Lol p 1 of pollen extract. Recombinant Lol p 1 was used to develop ImmunoCAP assays for analysis of 150 sera that were Radioallergosorbent test positive to L. perenne pollen. In 130 of them (87%) the assay detected a significant level of IgE antibodies to Lol p 1, reaching on average 37% of the level obtained with a test for IgE to the whole grass pollen extract. To map epitopes on Lol p 1, we produced three deletion mutants [des-(116-240)-Lol p 1, des-(1-88)-Lol p 1 and des-(133-189)-Lol p 1], which were efficiently expressed in bacteria. These all showed a strong reactivity with the specific rabbit IgG antibodies, but lacked most or all the allergenic properties of recombinant Lol p 1. A study of the antigenic structure of Lol p 1 was performed using the three deletion mutants and a set of 17-18-residue overlapping synthetic peptides covering the whole allergen sequence. The results indicate that human IgE and rabbit IgG antibodies bind to distinct regions of Lol p 1, and that at least some important IgE epitopes are mainly conformational. The findings suggest that recombinant allergens constitute useful reagents for further development of serological diagnosis of allergy, and that it should be possible to produce immunogenic fragments of allergenic proteins without allergenic properties.

  17. Recombinant production of mecasermin in E. coli expression system

    PubMed Central

    Jafari, S.; Babaeipour, V.; Seyedi, H.A. Eslampanah; Rahaie, M.; Mofid, M.R.; Haddad, L.; Namvaran, M.M.; Fallah, J.

    2014-01-01

    Human Insulin-like growth factor 1 (hIGF-1) consists of 70 amino acids in a single chain with three intermolecular disulfide bridges possessing valuable therapeutic effects. To date, numerous variants of specifically engineered hIGF-1 have been produced so as to improve hIGF-1 biological activity, stability and stronger binding to IGF-1 receptor. Mecasermin is one of the modified variants with one amino acid substitution near the N-terminal (T4I) approved for the treatment of growth failure diabetes, wound healing, amyotrophic lateral sclerosis and severe primary IGF-1 deficiency. No scientific report for recombinant production of mecasermin in Escherichia coli (E. coli) expression system has been sofar reported. In the present study, we therefore investigated the overexpression of mecasermin in two different E. coli strains in order to obtain higher yield of recombinant protein. To achieve this goal, mecasermin DNA encoding sequence was designed based on polypeptide sequence, optimized according to E. coli codon preference, and cloned in pET15b. Recombinant vector, pET15-mecasermin, transferred into two E. coli strains rigami B (DE3) and BL21 (DE3) and induced for expression in a small scale. Results revealed the E. coli Origami B (DE3) expression system was a preferable host for mecasermin production due to its high expression level being around twice as much as BL21 (DE3). Large scale mecasermin production was performed in batch culture and produced recombinant protein specifically confirmed by western blotting and mass spectroscopy. Since major part of recombinant mecasermin was expressed as inclusion body, isolation and refolding was accomplished through developed purification procedure, and finally recombinant protein was successfully purified by gel filtration chromatography. PMID:26339260

  18. Functional insights into recombinant TROSPA protein from Ixodes ricinus.

    PubMed

    Figlerowicz, Marek; Urbanowicz, Anna; Lewandowski, Dominik; Jodynis-Liebert, Jadwiga; Sadowski, Czeslaw

    2013-01-01

    Lyme disease (also called borreliosis) is a prevalent chronic disease transmitted by ticks and caused by Borrelia burgdorferi s. l. spirochete. At least one tick protein, namely TROSPA from I. scapularis, commonly occurring in the USA, was shown to be required for colonization of the vector by bacteria. Located in the tick gut, TROSPA interacts with the spirochete outer surface protein A (OspA) and initiates the tick colonization. Ixodes ricinus is a primary vector involved in B. burgdorferi s. l. transmission in most European countries. In this study, we characterized the capacities of recombinant TROSPA protein from I. ricinus to interact with OspA from different Borrelia species and to induce an immune response in animals. We also showed that the N-terminal part of TROSPA (a putative transmembrane domain) is not involved in the interaction with OspA and that reduction of the total negative charge on the TROSPA protein impaired TROSPA-OspA binding. In general, the data presented in this paper indicate that recombinant TROSPA protein retains the capacity to form a complex with OspA and induces a significant level of IgG in orally immunized rats. Thus, I. ricinus TROSPA may be considered a good candidate component for an animal vaccine against Borrelia. PMID:24204685

  19. Recombinant protein polymers in biomaterials.

    PubMed

    Kim, Wookhyun

    2013-01-01

    Naturally occurring protein-based materials have been found that function as critical components in biomechanical response, fibers and adhesives. A relatively small but growing number of recombinant protein-based materials that mimic the desired features of their natural sources, such as collagens, elastins and silks, are considered as an alternative to conventional synthetic polymers. Advances in genetic engineering have facilitated the synthesis of repetitive protein polymers with precise control of molecular weights which are designed by using synthetic genes encoding tandem repeats of oligopeptide originating from a modular domain of natural proteins. Many repeat sequences as protein polymer building blocks adopt a well-defined secondary structure and undergo self-assembly to result in physically cross-linked networks or with chemical cross-linking so that further form three-dimensional architectures similar to natural counterparts. In this review, recombinant protein polymers currently developed will be presented that have emerged as promising class of next generation biomaterials. PMID:23276922

  20. Recombinant bacteriophage lysins as antibacterials

    PubMed Central

    Fenton, Mark; Ross, Paul; McAuliffe, Olivia; O'Mahony, Jim

    2010-01-01

    With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential. PMID:21327123