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Sample records for altered recombination specificity

  1. Recombinant feline leukemia virus (FeLV) variants establish a limited infection with altered cell tropism in specific-pathogen-free cats in the absence of FeLV subgroup A helper virus.

    Science.gov (United States)

    Bechtel, M K; Hayes, K A; Mathes, L E; Pandey, R; Stromberg, P C; Roy-Burman, P

    1999-03-01

    Feline leukemia virus subgroup B (FeLV-B) is commonly associated with feline lymphosarcoma and arises through recombination between endogenous retroviral elements inherited in the cat genome and corresponding regions of the envelope (env) gene from FeLV subgroup A (FeLV-A). In vivo infectivity for FeLV-B is thought to be inefficient in the absence of FeLV-A. Proposed FeLV-A helper functions include enhanced replication efficiency, immune evasion, and replication rescue for defective FeLV-B virions. In vitro analysis of the recombinant FeLV-B-like viruses (rFeLVs) employed in this study confirmed these viruses were replication competent prior to their use in an in vivo study without FeLV-A helper virus. Eight specific-pathogen-free kittens were inoculated with the rFeLVs alone. Subsequent hematology and histology results were within normal limits, however, in the absence of detectable viremia, virus expression, or significant seroconversion, rFeLV proviral DNA was detected in bone marrow tissue of 4/4 (100%) cats at 45 weeks postinoculation (pi), indicating these rFeLVs established a limited but persistent infection in the absence of FeLV-A. Altered cell tropism was also noted. Focal infection was seen in T-cell areas of the splenic follicles in 3/4 (75%) rFeLV-infected cats analyzed, while an FeLV-A-infected cat showed focal infection in B-cell areas of the splenic follicles. Nucleotide sequence analysis of the surface glycoprotein portion of the rFeLV env gene amplified from bone marrow tissue collected at 45 weeks pi showed no sequence alterations from the original rFeLV inocula.

  2. Recombinant protein hydrazides: application to site-specific protein PEGylation.

    Science.gov (United States)

    Thom, Jennifer; Anderson, David; McGregor, Joanne; Cotton, Graham

    2011-06-15

    Here, we describe a novel method for the site-specific C-terminal PEGylation of recombinant proteins. This general approach exploits chemical cleavage of precursor intein-fusion proteins with hydrazine to directly produce recombinant protein hydrazides. This unique functionality within the protein sequence then facilitates site-specific C-terminal modification by hydrazone-forming ligation reactions. This approach was used to generate folded, site-specifically C-terminal PEGylated IFNalpha2b and IFNbeta1b, which retained excellent antiviral activity, demonstrating the utility of this technology in the PEGylation of therapeutic proteins. As this methodology is straightforward to perform, is compatible with disulfide bonds, and is exclusively selective for the protein C-terminus, it shows great potential as general technology for the site-specific engineering and labeling of recombinant proteins.

  3. Temporally-controlled site-specific recombination in zebrafish.

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    Stefan Hans

    Full Text Available Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM or its active metabolite, 4-hydroxy-tamoxifen (4-OHT. Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

  4. [Progress on XerCD/dif site-specific recombination].

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    Tian, De-Qiao; Wang, Yu-Min; Zheng, Tao

    2012-08-01

    In Escherichia coli, 10% to 15% of growing bacteria produce chromosome dimers during DNA replication. These dimers are resolved by XerC and XerD, two chromosome recombinases that target the dif sequence in the replication terminus of chromosome. Phage CTXΦ integrates into vibrio cholerae chromosome in a site-specific manner. However, CTXΦ genome does not encode any recombinase, while recombinase XerC and XerD, which is coded by vibrio cholerae chromosome are required for the integration of CTXΦ into the vibrio cholerae chromosome. The CTXΦ integration site overlaps with the dif site. The wide distribution of XerCD recombinase and dif site among bacteria genome suggests that it may be universal in resolve of chromosome dimers and phage integration. In this article, we reviewed the research progresses on chromosome dimer resolve and phage integration through XerCD/dif site-specific recombination.

  5. Inhibitor specificity of recombinant and endogenous caspase-9.

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    Ryan, Ciara A; Stennicke, Henning R; Nava, Victor E; Burch, Jennifer B; Hardwick, J Marie; Salvesen, Guy S

    2002-01-01

    Apoptosis triggered through the intrinsic pathway by radiation and anti-neoplastic drugs is initiated by the activation of caspase-9. To elucidate control mechanisms in this pathway we used a range of synthetic and natural reagents. The inhibitory potency of acetyl-Asp-Glu-Val-Asp-aldehyde ('Ac-DEVD-CHO'), benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone ('Z-VAD-FMK') and the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis protein ('XIAP') against recombinant caspase-9 were predictive of the efficacy of these compounds in a cell-free system. However, the viral proteins CrmA and p35, although potent inhibitors of recombinant caspase-9, had almost no ability to block caspase-9 in this system. These findings were also mirrored in cell expression studies. We hypothesize that the viral inhibitors CrmA and p35 are excluded from reacting productively with the natural form of active caspase-9 in vivo, making the potency of inhibitors highly context-dependent. This is supported by survival data from a mouse model of apoptosis driven by Sindbis virus expressing either p35 or a catalytic mutant of caspase-9. These results consolidate previous findings that CrmA is a potent inhibitor of caspase-9 in vitro, yet fails to block caspase-9-mediated cell death. PMID:12067274

  6. Genomic sequence of a mutant strain of Caenorhabditis elegans with an altered recombination pattern

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    Marra Marco

    2010-02-01

    Full Text Available Abstract Background The original sequencing and annotation of the Caenorhabditis elegans genome along with recent advances in sequencing technology provide an exceptional opportunity for the genomic analysis of wild-type and mutant strains. Using the Illumina Genome Analyzer, we sequenced the entire genome of Rec-1, a strain that alters the distribution of meiotic crossovers without changing the overall frequency. Rec-1 was derived from ethylmethane sulfonate (EMS-treated strains, one of which had a high level of transposable element mobility. Sequencing of this strain provides an opportunity to examine the consequences on the genome of altering the distribution of meiotic recombination events. Results Using Illumina sequencing and MAQ software, 83% of the base pair sequence reads were aligned to the reference genome available at Wormbase, providing a 21-fold coverage of the genome. Using the software programs MAQ and Slider, we observed 1124 base pair differences between Rec-1 and the reference genome in Wormbase (WS190, and 441 between the mutagenized Rec-1 (BC313 and the wild-type N2 strain (VC2010. The most frequent base-substitution was G:C to A:T, 141 for the entire genome most of which were on chromosomes I or X, 55 and 31 respectively. With this data removed, no obvious pattern in the distribution of the base differences along the chromosomes was apparent. No major chromosomal rearrangements were observed, but additional insertions of transposable elements were detected. There are 11 extra copies of Tc1, and 8 of Tc2 in the Rec-1 genome, most likely the remains of past high-hopper activity in a progenitor strain. Conclusion Our analysis of high-throughput sequencing was able to detect regions of direct repeat sequences, deletions, insertions of transposable elements, and base pair differences. A subset of sequence alterations affecting coding regions were confirmed by an independent approach using oligo array comparative genome

  7. Oral Salmonella: malaria circumsporozoite recombinants induce specific CD8+ cytotoxic T cells

    OpenAIRE

    1990-01-01

    Oral immunization with an attenuated Salmonella typhimurium recombinant containing the full-length Plasmodium berghei circumsporozoite (CS) gene induces protective immunity against P. berghei sporozoite challenge in the absence of antibody. We found that this immunity was mediated through the induction of specific CD8+ T cells since in vivo elimination of CD8+ cells abrogated protection. In vitro studies revealed that this Salmonella-P. berghei CS recombinant induced class I- restricted CD8+ ...

  8. Altered biochemical specificity of G-quadruplexes with mutated tetrads

    Czech Academy of Sciences Publication Activity Database

    Švehlová, Kateřina; Lawrence, M. S.; Bednárová, Lucie; Curtis, Edward A.

    2016-01-01

    Roč. 44, č. 22 (2016), s. 10789-10803 ISSN 0305-1048 Institutional support: RVO:61388963 Keywords : G-quadruplex * G motif GTP aptamer * peroxidase deoxyribozyme Subject RIV: CE - Biochemistry Impact factor: 10.162, year: 2016 https://academic.oup.com/nar/article/44/22/10789/2333933/Altered-biochemical-specificity-of-G-quadruplexes

  9. FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori.

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    Ding-Pei Long

    Full Text Available A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.

  10. FLP Recombinase-Mediated Site-Specific Recombination in Silkworm, Bombyx mori

    Science.gov (United States)

    Long, Ding-Pei; Zhao, Ai-Chun; Chen, Xue-Jiao; Zhang, Yang; Lu, Wei-Jian; Guo, Qing; Handler, Alfred M.; Xiang, Zhong-Huai

    2012-01-01

    A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species. PMID:22768245

  11. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

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    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  12. phiC31 integrase-mediated site-specific recombination in barley.

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    Eszter Kapusi

    Full Text Available The Streptomyces phage phiC31 integrase was tested for its feasibility in excising transgenes from the barley genome through site-specific recombination. We produced transgenic barley plants expressing an active phiC31 integrase and crossed them with transgenic barley plants carrying a target locus for recombination. The target sequence involves a reporter gene encoding green fluorescent protein (GFP, which is flanked by the attB and attP recognition sites for the phiC31 integrase. This sequence disruptively separates a gusA coding sequence from an upstream rice actin promoter. We succeeded in producing site-specific recombination events in the hybrid progeny of 11 independent barley plants carrying the above target sequence after crossing with plants carrying a phiC31 expression cassette. Some of the hybrids displayed fully executed recombination. Excision of the GFP gene fostered activation of the gusA gene, as visualized in tissue of hybrid plants by histochemical staining. The recombinant loci were detected in progeny of selfed F(1, even in individuals lacking the phiC31 transgene, which provides evidence of stability and generative transmission of the recombination events. In several plants that displayed incomplete recombination, extrachromosomal excision circles were identified. Besides the technical advance achieved in this study, the generated phiC31 integrase-expressing barley plants provide foundational stock material for use in future approaches to barley genetic improvement, such as the production of marker-free transgenic plants or switching transgene activity.

  13. Vika/vox, a novel efficient and specific Cre/loxP-like site-specific recombination system

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    Karimova, Madina; Abi-Ghanem, Josephine; Berger, Nicolas; Surendranath, Vineeth; Pisabarro, Maria Teresa; Buchholz, Frank

    2013-01-01

    Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo. However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/vox. Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/vox is a novel powerful and safe instrument in the ‘genetic toolbox’ that can be used alone or in combination with other SSRs in heterologous hosts. PMID:23143104

  14. Immunoglobulin A-specific serodiagnosis of acute human cytomegalovirus infection by using recombinant viral antigens

    NARCIS (Netherlands)

    Vornhagen, R; Hinderer, W; Sonneborn, HH; Bein, G; Matter, L; The, TH; Jahn, G; Plachter, B

    The immunoglobulin A-specific reactivities of recombinant viral proteins from nine different reading frames of human cytomegalovirus were evaluated in enzyme-linked immunosorbent assay experiments. Antigen fragments of reading frames pUL32, pUL44, and pUL57 were identified as preferable antigens for

  15. A dominant, recombination-defective allele of Dmc1 causing male-specific sterility

    NARCIS (Netherlands)

    Bannister, Laura A.; Pezza, Roberto J.; Donaldson, Janet R.; de Rooij, Dirk G.; Schimenti, Kerry J.; Camerini-Otero, R. Daniel; Schimenti, John C.

    2007-01-01

    DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant

  16. Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

    Science.gov (United States)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, Pimmune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  17. Pressure for Pattern-Specific Intertypic Recombination between Sabin Polioviruses: Evolutionary Implications

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    Ekaterina Korotkova

    2017-11-01

    Full Text Available Complete genomic sequences of a non-redundant set of 70 recombinants between three serotypes of attenuated Sabin polioviruses as well as location (based on partial sequencing of crossover sites of 28 additional recombinants were determined and compared with the previously published data. It is demonstrated that the genomes of Sabin viruses contain distinct strain-specific segments that are eliminated by recombination. The presumed low fitness of these segments could be linked to mutations acquired upon derivation of the vaccine strains and/or may have been present in wild-type parents of Sabin viruses. These “weak” segments contribute to the propensity of these viruses to recombine with each other and with other enteroviruses as well as determine the choice of crossover sites. The knowledge of location of such segments opens additional possibilities for the design of more genetically stable and/or more attenuated variants, i.e., candidates for new oral polio vaccines. The results also suggest that the genome of wild polioviruses, and, by generalization, of other RNA viruses, may harbor hidden low-fitness segments that can be readily eliminated only by recombination.

  18. Phenotype- and genotype-specific structural alterations in spasmodic dysphonia.

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    Bianchi, Serena; Battistella, Giovanni; Huddleston, Hailey; Scharf, Rebecca; Fleysher, Lazar; Rumbach, Anna F; Frucht, Steven J; Blitzer, Andrew; Ozelius, Laurie J; Simonyan, Kristina

    2017-04-01

    Spasmodic dysphonia is a focal dystonia characterized by involuntary spasms in the laryngeal muscles that occur selectively during speaking. Although hereditary trends have been reported in up to 16% of patients, the causative etiology of spasmodic dysphonia is unclear, and the influences of various phenotypes and genotypes on disorder pathophysiology are poorly understood. In this study, we examined structural alterations in cortical gray matter and white matter integrity in relationship to different phenotypes and putative genotypes of spasmodic dysphonia to elucidate the structural component of its complex pathophysiology. Eighty-nine patients with spasmodic dysphonia underwent high-resolution magnetic resonance imaging and diffusion-weighted imaging to examine cortical thickness and white matter fractional anisotropy in adductor versus abductor forms (distinct phenotypes) and in sporadic versus familial cases (distinct genotypes). Phenotype-specific abnormalities were localized in the left sensorimotor cortex and angular gyrus and the white matter bundle of the right superior corona radiata. Genotype-specific alterations were found in the left superior temporal gyrus, supplementary motor area, and the arcuate portion of the left superior longitudinal fasciculus. Our findings suggest that phenotypic differences in spasmodic dysphonia arise at the level of the primary and associative areas of motor control, whereas genotype-related pathophysiological mechanisms may be associated with dysfunction of regions regulating phonological and sensory processing. Identification of structural alterations specific to disorder phenotype and putative genotype provides an important step toward future delineation of imaging markers and potential targets for novel therapeutic interventions for spasmodic dysphonia. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

  19. Screening strategy to generate cell specific recombination: a case report with the RIP-Cre mice.

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    Spinelli, Valeria; Martin, Céline; Dorchies, Emilie; Vallez, Emmanuelle; Dehondt, Hélène; Trabelsi, Mohamed-Sami; Tailleux, Anne; Caron, Sandrine; Staels, Bart

    2015-10-01

    Conditional gene knockout technology is a powerful tool to study the function of a gene in a specific tissue, organ or cell lineage. The most commonly used procedure applies the Cre-LoxP strategy, where the choice of the Cre driver promoter is critical to determine the efficiency and specificity of the system. However, a considered choice of an appropriate promoter does not always protect against the risk of unwanted recombination and the consequent deletion of the gene in other tissues than the desired one(s), due to phenomena of non-specific activation of the Cre transgene. Furthermore, the causes of these phenomena are not completely understood and this can potentially affect every strain of Cre-mice. In our study on the deletion of a same gene in two different tissues, we show that the incidence rate of non-specific recombination in unwanted tissues depends on the Cre driver strain, ranging from 100%, rendering it useless (aP2-Cre strain), to ~5%, which is still compatible with their use (RIP-Cre strain). The use of a simple PCR strategy conceived to detect this occurrence is indispensable when producing a tissue-specific knockout mouse. Therefore, when choosing the Cre-driver promoter, researchers not only have to be careful about its tissue-specificity and timing of activation, but should also include a systematical screening in order to exclude mice in which atypical recombination has occurred and to limit the unnecessary use of laboratory animals in uninterpretable experiments.

  20. Histology-Specific MicroRNA Alterations in Melanoma

    Science.gov (United States)

    Poliseno, Laura; Haimovic, Adele; Segura, Miguel F.; Hanniford, Douglas; Christos, Paul J.; Darvishian, Farbod; Wang, Jinhua; Shapiro, Richard L.; Pavlick, Anna C.; Berman, Russell S.; Hernando, Eva; Zavadil, Jiri; Osman, Iman

    2013-01-01

    We examined the microRNA signature that distinguishes the most common melanoma histological subtypes, superficial spreading melanoma (SSM) and nodular melanoma (NM). We also investigated the mechanisms underlying the differential expression of histology-specific microRNAs. MicroRNA array performed on a training cohort of 82 primary melanoma tumors (26 SSM, 56 NM), and nine congenital nevi (CN) revealed 134 microRNAs differentially expressed between SSM and NM (Pmelanoma cases (38 SSM, 59 NM). Our data support a molecular classification in which SSM and NM are two molecularly distinct phenotypes. Therapeutic strategies that take into account subtype-specific alterations might improve the outcome of melanoma patients. PMID:22551973

  1. Recombinant breast carcinoma-associated mucins expressed in a baculovirus system contain a tumor specific epitope.

    Science.gov (United States)

    Hu, P; Wright, S E

    1998-10-01

    Mucins are highly immunogenic glycoproteins that are abundantly expressed by breast and other adenocarcinomas. In order to progress in the understanding of the structure immunity relationship of the breast tumor associated mucin and normal tissue mucin, two forms of breast carcinoma associated mucin, muc7-BV and pem-BV, were expressed in a baculovirus expression system. The muc7-BV was constructed by inserting the seven tandem repeats of mucin core cDNA fragment into transfer vector pAc360, forming a fusion protein containing 14 amino acids of the baculovirus polyhedrin N-terminus. The pem-BV was constructed by cloning full-length mucin cDNA into the transfer vector pVL1392. The recombinant mucins were purified using immunoaffinity chromatography. The purified muc7-BV and pem-BV had molecular weights of 28 and 59 kd, respectively. No carbohydrate was detected on these recombinant mucins and is speculated to explain why both forms of recombinant mucin showed strong affinity to tumor-specific monoclonal antibody SM3. These recombinant mucins may have the potential value to develop vaccines against breast and other adenocarcinomas and to induce cytotoxic T-lymphocyte lines for immunotherapy of the same.

  2. Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

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    Lee, Hong Jo; Lee, Hyung Chul; Kim, Young Min; Hwang, Young Sun; Park, Young Hyun; Park, Tae Sub; Han, Jae Yong

    2016-02-01

    Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence. © FASEB.

  3. Female site-specific transposase-induced recombination: a high-efficiency method for fine mapping mutations on the X chromosome in Drosophila.

    OpenAIRE

    Marcus, Jeffrey M

    2003-01-01

    P-element transposons in the Drosophila germline mobilize only in the presence of the appropriate transposase enzyme. Sometimes, instead of mobilizing completely, P elements will undergo site-specific recombination with the homologous chromosome. Site-specific recombination is the basis for male recombination mapping, since the male germline does not normally undergo recombination. Site-specific recombination also takes place in females, but this has been difficult to study because of the obs...

  4. In vivo recombination efficiency of two site-specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes).

    Science.gov (United States)

    Kishimoto, Kenta; Nakayama, Manabu; Kinoshita, Masato

    2016-08-01

    The present study delineates the in vivo efficiency of two site-specific recombination systems, VCre/VloxP and SCre/SloxP, in medaka (Oryzias latipes). VCre, SCre, and Cre RNA was microinjected into fertilized medaka eggs belonging to three transgenic lines harboring VloxP, SloxP, and loxP cassette. VCre induced site-specific recombination specifically at VloxP sequence and SCre at SloxP sequence without any cross-reactivity. These findings provide two novel alternative recombination systems in vivo in addition to the existing Cre/loxP and Flp/FRT systems, thus enabling sophisticated gene expression in model organisms. © 2016 Japanese Society of Developmental Biologists.

  5. Engineered Cpf1 variants with altered PAM specificities.

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    Gao, Linyi; Cox, David B T; Yan, Winston X; Manteiga, John C; Schneider, Martin W; Yamano, Takashi; Nishimasu, Hiroshi; Nureki, Osamu; Crosetto, Nicola; Zhang, Feng

    2017-08-01

    The RNA-guided endonuclease Cpf1 is a promising tool for genome editing in eukaryotic cells. However, the utility of the commonly used Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) and Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1) is limited by their requirement of a TTTV protospacer adjacent motif (PAM) in the DNA substrate. To address this limitation, we performed a structure-guided mutagenesis screen to increase the targeting range of Cpf1. We engineered two AsCpf1 variants carrying the mutations S542R/K607R and S542R/K548V/N552R, which recognize TYCV and TATV PAMs, respectively, with enhanced activities in vitro and in human cells. Genome-wide assessment of off-target activity using BLISS indicated that these variants retain high DNA-targeting specificity, which we further improved by introducing an additional non-PAM-interacting mutation. Introducing the identified PAM-interacting mutations at their corresponding positions in LbCpf1 similarly altered its PAM specificity. Together, these variants increase the targeting range of Cpf1 by approximately threefold in human coding sequences to one cleavage site per ∼11 bp.

  6. The Xer/dif site-specific recombination system of Campylobacter jejuni.

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    Leroux, Maxime; Rezoug, Zoulikha; Szatmari, George

    2013-10-01

    Chromosome dimers, which form during the bacterial life cycle, represent a problem that must be solved by the bacterial cell machinery so that chromosome segregation can occur effectively. The Xer/dif site-specific recombination system, utilized by most bacteria, resolves chromosome dimers into monomers using two tyrosine recombinases, XerC and XerD, to perform the recombination reaction at the dif site which consists of 28-30 bp. However, single Xer recombinase systems have been recently discovered in several bacterial species. In Streptococci and Lactococci a single recombinase, XerS, is capable of completing the monomerisation reaction by acting at an atypical dif site called dif SL (31 bp). It was recently shown that a subgroup of ε-proteobacteria including Campylobacter spp. and Helicobacter spp. had a phylogenetically distinct Xer/dif recombination system with only one recombinase (XerH) and an atypical dif motif (difH). In order to biochemically characterize this system in greater detail, Campylobacter jejuni XerH was purified and its DNA-binding activity was characterized. The protein showed specific binding to the complete difH site and to both halves separately. It was also shown to form covalent complexes with difH suicide substrates. In addition, XerH was able to catalyse recombination between two difH sites located on a plasmid in Escherichia coli in vivo. This indicates that this XerH protein performs a similar function as the related XerS protein, but shows significantly different binding characteristics.

  7. Enrichment of intersubtype HIV-1 recombinants in a dual infection system using HIV-1 strain-specific siRNAs

    Science.gov (United States)

    2011-01-01

    Background Intersubtype HIV-1 recombinants in the form of unique or stable circulating recombinants forms (CRFs) are responsible for over 20% of infections in the worldwide epidemic. Mechanisms controlling the generation, selection, and transmission of these intersubtype HIV-1 recombinants still require further investigation. All intersubtype HIV-1 recombinants are generated and evolve from initial dual infections, but are difficult to identify in the human population. In vitro studies provide the most practical system to study mechanisms, but the recombination rates are usually very low in dual infections with primary HIV-1 isolates. This study describes the use of HIV-1 isolate-specific siRNAs to enrich intersubtype HIV-1 recombinants and inhibit the parental HIV-1 isolates from a dual infection. Results Following a dual infection with subtype A and D primary HIV-1 isolates and two rounds of siRNA treatment, nearly 100% of replicative virus was resistant to a siRNA specific for an upstream target sequence in the subtype A envelope (env) gene as well as a siRNA specific for a downstream target sequence in the subtype D env gene. Only 20% (10/50) of the replicating virus had nucleotide substitutions in the siRNA-target sequence whereas the remaining 78% (39/50) harbored a recombination breakpoint that removed both siRNA target sequences, and rendered the intersubtype D/A recombinant virus resistant to the dual siRNA treatment. Since siRNAs target the newly transcribed HIV-1 mRNA, the siRNAs only enrich intersubtype env recombinants and do not influence the recombination process during reverse transcription. Using this system, a strong bias is selected for recombination breakpoints in the C2 region, whereas other HIV-1 env regions, most notably the hypervariable regions, were nearly devoid of intersubtype recombination breakpoints. Sequence conservation plays an important role in selecting for recombination breakpoints, but the lack of breakpoints in many conserved

  8. A diagnostic test for scabies: IgE specificity for a recombinant allergen of Sarcoptes scabiei.

    Science.gov (United States)

    Jayaraj, Rama; Hales, Belinda; Viberg, Linda; Pizzuto, Susan; Holt, Deborah; Rolland, Jennifer M; O'Hehir, Robyn E; Currie, Bart J; Walton, Shelley F

    2011-12-01

    Scabies infestations are difficult to diagnose clinically and current serologic tests have less than 50% accuracy. To develop more reliable diagnosis of scabies, specific IgE antibodies to a major scabies antigen recombinant Sar s 14.3 (rSar s 14.3) were measured in 140 plasma samples from scabies-infested and control subject groups using dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA). Levels of rSar s 14.3-specific IgE were quantified, and cross-reactivity with its house dust mite homologue, Der p 14, was assessed. The rSar s 14.3 DELFIA showed excellent diagnostic capability, with 100% sensitivity and 93.75% specificity for distinguishing subjects with current scabies infestation from control, uninfested subjects. Recombinant Der p 14 preparation was ineffective at inhibiting IgE binding to rSar s 14.3. This study shows that quantification of levels of IgE antibody to rSar s 14.3 is a highly sensitive method for diagnosis of scabies infestation in clinical practice. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Altering coenzyme specificity of Pichia stipitis xylose reductase by the semi-rational approach CASTing

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    Zhang Jingqing

    2007-11-01

    Full Text Available Abstract Background The NAD(PH-dependent Pichia stipitis xylose reductase (PsXR is one of the key enzymes for xylose fermentation, and has been cloned into the commonly used ethanol-producing yeast Saccharomyces cerevisiae. In order to eliminate the redox imbalance resulting from the preference of this enzyme toward NADPH, efforts have been made to alter the coenzyme specificity of PsXR by site-directed mutagenesis, with limited success. Given the industrial importance of PsXR, it is of interest to investigate further ways to create mutants of PsXR that prefers NADH rather than NADPH, by the alternative directed evolution approach. Results Based on a homology model of PsXR, six residues were predicted to interact with the adenine ribose of NAD(PH in PsXR and altered using a semi-rational mutagenesis approach (CASTing. Three rounds of saturation mutagenesis were carried to randomize these residues, and a microplate-based assay was applied in the screening. A best mutant 2-2C12, which carried four mutations K270S, N272P, S271G and R276F, was obtained. The mutant showed a preference toward NADH over NADPH by a factor of about 13-fold, or an improvement of about 42-fold, as measured by the ratio of the specificity constant kcat/Kmcoenzyme. Compared with the wild-type, the kcatNADH for the best mutant was only slightly lower, while the kcatNADPH decreased by a factor of about 10. Furthermore, the specific activity of 2-2C12 in the presence of NADH was 20.6 U·mg-1, which is highest among PsXR mutants reported. Conclusion A seemingly simplistic and yet very effective mutagenesis approach, CASTing, was applied successfully to alter the NAD(PH preference for Pichia stipitis xylose reductase, an important enzyme for xylose-fermenting yeast. The observed change in the NAD(PH preference for this enzyme seems to have resulted from the altered active site that is more unfavorable for NADPH than NADH in terms of both Km and kcat. There are potentials for

  10. Altering the ribosomal subunit ratio in yeast maximizes recombinant protein yield

    Directory of Open Access Journals (Sweden)

    Poyner David R

    2009-01-01

    Full Text Available Abstract Background The production of high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences that has yet to be addressed in a truly rational manner. Typically eukaryotic protein production experiments have relied on varying expression construct cassettes such as promoters and tags, or culture process parameters such as pH, temperature and aeration to enhance yields. These approaches require repeated rounds of trial-and-error optimization and cannot provide a mechanistic insight into the biology of recombinant protein production. We published an early transcriptome analysis that identified genes implicated in successful membrane protein production experiments in yeast. While there has been a subsequent explosion in such analyses in a range of production organisms, no one has yet exploited the genes identified. The aim of this study was to use the results of our previous comparative transcriptome analysis to engineer improved yeast strains and thereby gain an understanding of the mechanisms involved in high-yielding protein production hosts. Results We show that tuning BMS1 transcript levels in a doxycycline-dependent manner resulted in optimized yields of functional membrane and soluble protein targets. Online flow microcalorimetry demonstrated that there had been a substantial metabolic change to cells cultured under high-yielding conditions, and in particular that high yielding cells were more metabolically efficient. Polysome profiling showed that the key molecular event contributing to this metabolically efficient, high-yielding phenotype is a perturbation of the ratio of 60S to 40S ribosomal subunits from approximately 1:1 to 2:1, and correspondingly of 25S:18S ratios from 2:1 to 3:1. This result is consistent with the role of the gene product of BMS1 in ribosome biogenesis. Conclusion This work demonstrates the power of a rational approach to recombinant protein production by using the results of

  11. Recombinant human parvovirus B19 vectors: erythroid cell-specific delivery and expression of transduced genes.

    Science.gov (United States)

    Ponnazhagan, S; Weigel, K A; Raikwar, S P; Mukherjee, P; Yoder, M C; Srivastava, A

    1998-06-01

    A novel packaging strategy combining the salient features of two human parvoviruses, namely the pathogenic parvovirus B19 and the nonpathogenic adeno-associated virus type 2 (AAV), was developed to achieve erythroid cell-specific delivery as well as expression of the transduced gene. The development of such a chimeric vector system was accomplished by packaging heterologous DNA sequences cloned within the inverted terminal repeats of AAV and subsequently packaging the DNA inside the capsid structure of B19 virus. Recombinant B19 virus particles were assembled, as evidenced by electron microscopy as well as DNA slot blot analyses. The hybrid vector failed to transduce nonerythroid human cells, such as 293 cells, as expected. However, MB-02 cells, a human megakaryocytic leukemia cell line which can be infected by B19 virus following erythroid differentiation with erythropoietin (N. C. Munshi, S. Z. Zhou, M. J. Woody, D. A. Morgan, and A. Srivastava, J. Virol. 67:562-566, 1993) but lacks the putative receptor for AAV (S. Ponnazhagan, X.-S. Wang, M. J. Woody, F. Luo, L. Y. Kang, M. L. Nallari, N. C. Munshi, S. Z. Zhou, and A. Srivastava, J. Gen. Virol. 77:1111-1122, 1996), were readily transduced by this vector. The hybrid vector was also found to specifically target the erythroid population in primary human bone marrow cells as well as more immature hematopoietic progenitor cells following erythroid differentiation, as evidenced by selective expression of the transduced gene in these target cells. Preincubation with anticapsid antibodies against B19 virus, but not anticapsid antibodies against AAV, inhibited transduction of primary human erythroid cells. The efficiency of transduction of primary human erythroid cells by the recombinant B19 virus vector was significantly higher than that by the recombinant AAV vector. Further development of the AAV-B19 virus hybrid vector system should prove beneficial in gene therapy protocols aimed at the correction of inherited and

  12. Generation and characterization of a novel recombinant scFv antibody specific for Campylobacter jejuni.

    Science.gov (United States)

    Nzuma, Ruramayi M; Liu, Fuquan; Grant, Irene R

    2018-04-07

    Campylobacter jejuni is a leading cause of foodborne illness worldwide, mainly due to consumption and handling of contaminated raw chicken. Rapid detection methods for C. jejuni are vital for monitoring contamination levels in chicken products and reducing human Campylobacteriosis cases. The 'gold standard' culture-based method of Campylobacter detection takes 3-5 days and is too slow to permit effective intervention. Immuno-based methods are faster, but usually necessitate use of animals or hybridoma technology to produce antibodies; making them difficult and expensive to produce. Here, we report the generation and characterization of recombinant single-chain variable fragment (scFv) antibodies specific for C. jejuni cells, and evaluation of one scFv antibody for an immunomagnetic separation-quantitative PCR (IMS-qPCR) method to rapidly, sensitively, and specifically detect low numbers of C. jejuni. An scFv antibody phage-display library was constructed using spleen mRNA derived from a rabbit immunized with gamma-irradiated C. jejuni cells. This library was screened by surface biopanning against C. jejuni whole cells. Enriched clones were analyzed by enzyme-linked immunosorbent assay (ELISA). Two scFv antibodies that strongly and specifically recognized C. jejuni cell were expressed in Escherichia coli. Western blot analysis showed that one antibody, scFv80, was expressed as a soluble protein and retained its specific and strong binding to C. jejuni cells. This recombinant monoclonal scFv antibody was purified and used to covalently coat paramagnetic beads to be used for IMS-qPCR. The IMS-qPCR method was able to specifically and sensitively detect C. jejuni in mixed cultures within 3 h.

  13. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  14. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    Science.gov (United States)

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  15. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2014-01-01

    Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  16. Mutations within ICP4 acquired during in vitro attenuation do not alter virulence of recombinant Marek's disease viruses in vivo

    Directory of Open Access Journals (Sweden)

    Evin Hildebrandt

    2015-12-01

    Full Text Available Marek's disease (MD is a T-cell lymphoma of chickens caused by the oncogenic Marek's disease virus (MDV. MD is primarily controlled by live-attenuated vaccines generated by repeated in vitro serial passage. Previous efforts to characterize attenuated MDVs identified numerous mutations, particularly a convergence of high-frequency mutations around amino acids 60–63 within ICP4 (RS1, therefore, ICP4 was considered a candidate gene deserving further characterization. Recombinant MDVs were generated containing a single Q63H mutation or double Q63H + S1630P mutations. Despite the repetitive nature of mutations within ICP4, neither recombinant virus decreased virulence, although one mutant reduced in vivo replication and failed to transmit horizontally. Our results indicate that these mutations are insufficient to reduce disease incidence in infected birds, and suggest that variants in ICP4 do not directly alter virulence, but rather may enhance MDV replication rates in vitro, offering an explanation for the widespread occurrence of ICP4 mutations in a variety of attenuated herpesviruses.

  17. Induction of influenza-specific mucosal immunity by an attenuated recombinant Sendai virus.

    Directory of Open Access Journals (Sweden)

    Thuc-vy L Le

    2011-04-01

    Full Text Available Many pathogens initiate infection at the mucosal surfaces; therefore, induction of mucosal immune responses is a first level of defense against infection and is the most powerful means of protection. Although intramuscular injection is widely used for vaccination and is effective at inducing circulating antibodies, it is less effective at inducing mucosal antibodies.Here we report a novel recombinant, attenuated Sendai virus vector (GP42-H1 in which the hemagglutinin (HA gene of influenza A virus was introduced into the Sendai virus genome as an additional gene. Infection of CV-1 cells by GP42-H1 resulted in cell surface expression of the HA protein. Intranasal immunization of mice with 1,000 plaque forming units (pfu of GP42-H1 induced HA-specific IgG and IgA antibodies in the blood, bronchoalveolar lavage fluid, fecal pellet extracts and saliva. The HA-specific antibody titer induced by GP42-H1 closely resembles the titer induced by sublethal infection by live influenza virus; however, in contrast to infection by influenza virus, immunization with GP42-H1 did not result in disease symptoms or the loss of body weight. In mice that were immunized with GP42-H1 and then challenged with 5LD(50 (1250 pfu of influenza virus, no significant weight loss was observed and other visual signs of morbidity were not detected.These results demonstrate that the GP42-H1 Sendai virus recombinant is able to confer full protection from lethal infection by influenza virus, supporting the conclusion that it is a safe and effective mucosal vaccine vector.

  18. The rad52-Y66A allele alters the choice of donor template during spontaneous chromosomal recombination

    DEFF Research Database (Denmark)

    de Mayolo, A.A.; Sunjevaric, I.; Reid, R.

    2010-01-01

    Spontaneous mitotic recombination is a potential source of genetic changes Such as loss of heterozygosity and chromosome translocations, which may lead to genetic disease. In this study we have used a rad52 hyper-recombination mutant, rad52-Y66A, to investigate the process of spontaneous...... heteroallelic recombination in the yeast Soccharomyces cerevisiae. We find that spontaneous recombination has different genetic requirements, depending on whether the recombination event occurs between chromosomes or between chromosome and plasmid sequences. The hyper-recombination phenotype of the rad52-Y66A...... that spontaneous DNA lesions that require recombinational repair occur at the same frequency in wild-type and rad52-Y66A cells, but that the recombination process is slow in rad52-Y66A cells. Taken together, we propose that the slow recombinational DNA repair in the rad52-Y66A mutant leads to a by...

  19. Generation of Recombinant Schmallenberg Virus Nucleocapsid Protein in Yeast and Development of Virus-Specific Monoclonal Antibodies

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    Justas Lazutka

    2014-01-01

    Full Text Available Schmallenberg virus (SBV, discovered in continental Europe in late 2011, causes mild clinical signs in adult ruminants, including diarrhoea and reduced milk yield. However, fetal infection can lead to severe malformation in newborn offspring. To develop improved reagents for SBV serology, a high-level yeast expression system was employed to produce recombinant SBV nucleocapsid (N protein. Recombinant SBV N protein was investigated as an antigen in SBV-specific IgG enzyme immunoassay and used for generation of monoclonal antibodies (MAbs. Yeast-expressed SBV N protein was reactive with anti-SBV IgG-positive cow serum specimens collected from different farms of Lithuania. After immunization of mice with recombinant SBV N protein, four MAbs were generated. The MAbs raised against recombinant SBV N protein reacted with native viral nucleocapsids in SBV-infected BHK cells by immunofluorescence assay. The reactivity of recombinant N protein with SBV-positive cow serum specimens and the ability of the MAbs to recognize virus-infected cells confirm the antigenic similarity between yeast-expressed SBV N protein and native viral nucleocapsids. Our study demonstrates that yeast expression system is suitable for high-level production of recombinant SBV N protein and provides the first evidence on the presence of SBV-specific antibodies in cow serum specimens collected in Lithuania.

  20. A rapid method for recombination and site-specific mutagenesis by placing homologous ends on DNA using polymerase chain reaction.

    Science.gov (United States)

    Jones, D H; Howard, B H

    1991-01-01

    We have developed a novel polymerase chain reaction (PCR) method that permits the rapid generation of site-specific mutants and recombinant DNA constructs with a minimum number of steps and primers. DNA segments are modified by using amplifying primers that add homologous ends to the polymerase chain reaction product(s). These homologous ends undergo recombination in vivo following transformation of recA-E. coli strains used routinely in cloning. In vivo circularization of PCR products containing plasmid sequences with a selective marker permits the rapid cloning of the desired mutant or recombinant. In the mutagenesis protocol, 7 of the 12 clones contained the product of interest, and 6 of these clones had no detected error (50% of the clones without detected errors). In each of several recombination protocols, at least 50% of the clones tested contained the insert of interest without detected errors.

  1. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Science.gov (United States)

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  2. Deciphering bartonella diversity, recombination, and host specificity in a rodent community.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Buffet

    Full Text Available Host-specificity is an intrinsic feature of many bacterial pathogens, resulting from a long history of co-adaptation between bacteria and their hosts. Alpha-proteobacteria belonging to the genus Bartonella infect the erythrocytes of a wide range of mammal orders, including rodents. In this study, we performed genetic analysis of Bartonella colonizing a rodent community dominated by bank voles (Myodes glareolus and wood mice (Apodemus sylvaticus in a French suburban forest to evaluate their diversity, their capacity to recombine and their level of host specificity. Following the analysis of 550 rodents, we detected 63 distinct genotypes related to B. taylorii, B. grahamii, B. doshiae and a new B. rochalimae-like species. Investigating the most highly represented species, we showed that B. taylorii strain diversity was markedly higher than that of B. grahamii, suggesting a possible severe bottleneck for the latter species. The majority of recovered genotypes presented a strong association with either bank voles or wood mice, with the exception of three B. taylorii genotypes which had a broader host range. Despite the physical barriers created by host specificity, we observed lateral gene transfer between Bartonella genotypes associated with wood mice and Bartonella adapted to bank voles, suggesting that those genotypes might co-habit during their life cycle.

  3. Evaluation of Cocktails with Recombinant Proteins of Mycobacterium bovis for a Specific Diagnosis of Bovine Tuberculosis

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    María Laura Mon

    2014-01-01

    Full Text Available The Delayed type hypersensitivity skin test (DTH and interferon-gamma assay are used for the diagnosis of bovine tuberculosis (TBB. The specificity of these diagnoses, however, is compromised because both are based on the response against purified protein derivative of Mycobacterium bovis (PPD-B. In this study, we assessed the potential of two cocktails containing M. bovis recombinant proteins: cocktail 1 (C1: ESAT-6, CFP-10 and MPB83 and cocktail 2 (C2: ESAT-6, CFP-10, MPB83, HspX, TB10.3, and MPB70. C1, C2, and PPD-B showed similar response by DTH in M. bovis-sensitized guinea pigs. Importantly, C1 induced a lower response than PPD-B in M. avium-sensitized guinea pigs. In cattle, C1 displayed better performance than PPD-B and C2; indeed, C1 showed the least detection of animals either vaccinated or Map-infected. To optimize the composition of the cocktails, we obtained protein fractions from PPD-B and tested their immunogenicity in experimentally M. bovis-infected cattle. In one highly reactive fraction, seven proteins were identified. The inclusion of FixB in C1 enhanced the recognition of M. bovis-infected cattle without compromising specificity. Our data provide a promising basis for the future development of a cocktail for TBB detection without interference by the presence of sensitized or infected animals with other mycobacteria.

  4. Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions.

    Science.gov (United States)

    Schrader, Carol E; Vardo, Joycelyn; Linehan, Erin; Twarog, Michael Z; Niedernhofer, Laura J; Hoeijmakers, Jan H J; Stavnezer, Janet

    2004-08-02

    The structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a mismatch repair (MMR) protein involved in class switch recombination (CSR). Therefore, ERCC1-XPF has abilities that might be useful for antibody CSR. We tested whether ERCC1 is involved in CSR and found that Ercc1(-)(/)(-) splenic B cells show moderately reduced CSR in vitro, demonstrating that ERCC1-XPF participates in, but is not required for, CSR. To investigate the role of ERCC1 in CSR, the nucleotide sequences of switch (S) regions were determined. The mutation frequency in germline Smicro segments and recombined Smicro-Sgamma3 segments cloned from Ercc1(-)(/)(-) splenic B cells induced to switch in culture was identical to that of wild-type (WT) littermates. However, Ercc1(-)(/)(-) cells show increased targeting of the mutations to G:C bp in RGYW/WRCY hotspots and mutations occur at sites more distant from the S-S junctions compared with WT mice. The results indicate that ERCC1 is not epistatic with MMR and suggest that ERCC1 might be involved in processing or repair of DNA lesions in S regions during CSR.

  5. Screening of recombinant glycosyltransferases reveals the broad acceptor specificity of stevia UGT-76G1.

    Science.gov (United States)

    Dewitte, Griet; Walmagh, Maarten; Diricks, Margo; Lepak, Alexander; Gutmann, Alexander; Nidetzky, Bernd; Desmet, Tom

    2016-09-10

    UDP-glycosyltransferases (UGTs) are a promising class of biocatalysts that offer a sustainable alternative for chemical glycosylation of natural products. In this study, we aimed to characterize plant-derived UGTs from the GT-1 family with an emphasis on their acceptor promiscuity and their potential application in glycosylation processes. Recombinant expression in E. coli provided sufficient amounts of enzyme for the in-depth characterization of the salicylic acid UGT from Capsella rubella (UGT-SACr) and the stevia UGT from Stevia rebaudiana (UGT-76G1Sr). The latter was found to have a remarkably broad specificity with activities on a wide diversity of structures, from aliphatic and branched alcohols, over small phenolics to larger flavonoids, terpenoids and even higher glycoside compounds. As an example for its industrial potential, the glycosylation of curcumin was thoroughly evaluated. Under optimized conditions, 96% of curcumin was converted within 24h into the corresponding curcumin β-glycosides. In addition, the reaction was performed in a coupled system with sucrose synthase from Glycine max, to enable the cost-efficient (re)generation of UDP-Glc from sucrose as abundant and renewable resource. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Polyol specificity of recombinant Arabidopsis thaliana sorbitol dehydrogenase studied by enzyme kinetics and in silico modeling

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    María Francisca eAguayo

    2015-02-01

    Full Text Available Polyols are enzymatically-produced plant compounds which can act as compatible solutes during periods of abiotic stress. NAD+-dependent SORBITOL DEHYDROGENASE (SDH, E.C. 1.1.1.14 from Arabidopsis thaliana L. (AtSDH is capable of oxidizing several polyols including sorbitol, ribitol and xylitol. In the present study, enzymatic assays using recombinant AtSDH demonstrated a higher specificity constant for xylitol compared to sorbitol and ribitol, all of which are C2 (S and C4 (R polyols. Enzyme activity was reduced by preincubation with ethylenediaminetetraacetic acid (EDTA, indicating a requirement for zinc ions. In humans, it has been proposed that sorbitol becomes part of a pentahedric coordination sphere of the catalytic zinc during the reaction mechanism. In order to determine the validity of this pentahedric coordination model in a plant SDH, homology modeling and Molecular Dynamics simulations of AtSDH ternary complexes with the three polyols were performed using crystal structures of human and Bemisia argentifolii (Genn. (Hemiptera: Aleyrodidae SDHs as scaffolds. The results indicate that the differences in interaction with structural water molecules correlate very well with the observed enzymatic parameters, validate the proposed pentahedric coordination of the catalytic zinc ion in a plant SDH, and provide an explanation for why AtSDH shows a preference for polyols with a chirality of C2 (S and C4 (R.

  7. Improved adhesive properties of recombinant bifidobacteria expressing the Bifidobacterium bifidum-specific lipoprotein BopA

    Directory of Open Access Journals (Sweden)

    Gleinser Marita

    2012-06-01

    Full Text Available Abstract Background Bifidobacteria belong to one of the predominant bacterial groups in the intestinal microbiota of infants and adults. Several beneficial effects on the health status of their human hosts have been demonstrated making bifidobacteria interesting candidates for probiotic applications. Adhesion of probiotics to the intestinal epithelium is discussed as a prerequisite for colonisation of and persistence in the gastrointestinal tract. Results In the present study, 15 different strains of bifidobacteria were tested for adhesion. B. bifidum was identified as the species showing highest adhesion to all tested intestinal epithelial cell (IEC lines. Adhesion of B. bifidum S17 to IECs was strongly reduced after treatment of bacteria with pronase. These results strongly indicate that a proteinaceous cell surface component mediates adhesion of B. bifidum S17 to IECs. In silico analysis of the currently accessible Bifidobacterium genomes identified bopA encoding a lipoprotein as a B. bifidum-specific gene previously shown to function as an adhesin of B. bifidum MIMBb75. The in silico results were confirmed by Southern Blot analysis. Furthermore, Northern Blot analysis demonstrated that bopA is expressed in all B. bifidum strains tested under conditions used to cultivate bacteria for adhesion assays. The BopA gene was successfully expressed in E. coli and purified by Ni-NTA affinity chromatography as a C-terminal His6-fusion. Purified BopA had an inhibitory effect on adhesion of B. bifidum S17 to IECs. Moreover, bopA was successfully expressed in B. bifidum S17 and B. longum/infantis E18. Strains overexpressing bopA showed enhanced adhesion to IECs, clearly demonstrating a role of BopA in adhesion of B. bifidum strains. Conclusions BopA was identified as a B. bifidum-specific protein involved in adhesion to IECs. Bifidobacterium strains expressing bopA show enhanced adhesion. Our results represent the first report on recombinant

  8. Cloning-free genome engineering in Sinorhizobium meliloti advances applications of Cre/loxP site-specific recombination.

    Science.gov (United States)

    Döhlemann, Johannes; Brennecke, Meike; Becker, Anke

    2016-09-10

    The soil-dwelling α-proteobacterium Sinorhizobium meliloti serves as model for studies of symbiotic nitrogen fixation, a highly important process in sustainable agriculture. Here, we report advancements of the genetic toolbox accelerating genome editing in S. meliloti. The hsdMSR operon encodes a type-I restriction-modification (R-M) system. Transformation of S. meliloti is counteracted by the restriction endonuclease HsdR degrading DNA which lacks the appropriate methylation pattern. We provide a stable S. meliloti hsdR deletion mutant showing enhanced transformation with Escherichia coli-derived plasmid DNA and demonstrate that using an E. coli plasmid donor, expressing S. meliloti methyl transferase genes, is an alternative strategy of increasing the transformation efficiency of S. meliloti. Furthermore, we devise a novel cloning-free genome editing (CFGE) method for S. meliloti, Agrobacterium tumefaciens and Xanthomonas campestris, and demonstrate the applicability of this method for intricate applications of the Cre/lox recombination system in S. meliloti. An enhanced Cre/lox system, allowing for serial deletions of large genomic regions, was established. An assay of lox spacer mutants identified a set of lox sites mediating specific recombination. The availability of several non-promiscuous Cre recognition sites enables simultaneous specific Cre/lox recombination events. CFGE combined with Cre/lox recombination is put forward as powerful approach for targeted genome editing, involving serial steps of manipulation to expedite the genetic accessibility of S. meliloti as chassis. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Molecular and macromolecular alterations of recombinant adenoviral vectors do not resolve changes in hepatic drug metabolism during infection

    Directory of Open Access Journals (Sweden)

    Croyle Maria A

    2008-09-01

    Full Text Available Abstract In this report we test the hypothesis that long-term virus-induced alterations in CYP occur from changes initiated by the virus that may not be related to the immune response. Enzyme activity, protein expression and mRNA of CYP3A2, a correlate of human CYP3A4, and CYP2C11, responsive to inflammatory mediators, were assessed 0.25, 1, 4, and 14 days after administration of several different recombinant adenoviruses at a dose of 5.7 × 1012 virus particles (vp/kg to male Sprague Dawley rats. Wild type adenovirus, containing all viral genes, suppressed CYP3A2 and 2C11 activity by 37% and 39%, respectively within six hours. Levels fell to 67% (CYP3A2 and 79% (CYP2C11 of control by 14 days (p ≤ 0.01. Helper-dependent adenovirus, with all viral genes removed, suppressed CYP3A2 (43% and CYP2C11 (55% within six hours. CYP3A2 remained significantly suppressed (47%, 14 days, p ≤ 0.01 while CYP2C11 returned to baseline at this time. CYP3A2 and 2C11 were reduced by 45 and 42% respectively 6 hours after treatment with PEGylated adenovirus, which has a low immunological profile (p ≤ 0.05. CYP3A2 remained suppressed (34%, p ≤ 0.05 for 14 days while CYP2C11 recovered. Inactivated virus suppressed CYP3A2 activity by 25–50% for 14 days (p ≤ 0.05. CYP2C11 was affected similar manner but recovered by day 14. Microarray and in vitro studies suggest that changes in cellular signaling pathways initiated early in virus infection contribute to changes in CYP.

  10. A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity.

    Science.gov (United States)

    Nautiyal, Kalpana; Kuroda, Yutaka

    2018-02-12

    Tobacco Etch Virus (TEV) protease is used in the purification of recombinant proteins, but its usage is often hampered by solubility issues. Here, we report a short, 12-residue solubility enhancing peptide (SEP) tag attached at the C-terminus of TEV (TEV-C9R). We assessed the effects of the C9R tag on the biophysical and biochemical characteristics of TEV. The yield of HPLC purified TEV-C9R expressed in E. coli grown in 200 mL LB or TB media was between 10 and 13 mg, which was up to 6.5 times higher than the yield of the untagged TEV (untagged-TEV). TEV-C9R was active over a pH range of 5-8, which was wider than that of the commonly used thrombin, and it remained active upon incubation at 60 °C much longer than the untagged-TEV, which aggregated at this temperature. Static and dynamic light scattering demonstrated the higher solubility of purified TEV-C9R. Furthermore, the thermal unfolding of TEV-C9R, as assessed by circular dichroism at pH 4.7, was almost perfectly reversible, in contrast to that of untagged-TEV, which aggregated at high temperature. These results demonstrate the improved biophysical and biochemical characteristics of TEV-C9R originating from higher solubility and provide another example of how SEP tags can enhance enzyme solubility without altering its activity. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Isolation of Specific Clones from Nonarrayed BAC Libraries through Homologous Recombination

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    Mikhail Nefedov

    2011-01-01

    Full Text Available We have developed a new approach to screen bacterial artificial chromosome (BAC libraries by recombination selection. To test this method, we constructed an orangutan BAC library using an E. coli strain (DY380 with temperature inducible homologous recombination (HR capability. We amplified one library segment, induced HR at 42∘C to make it recombination proficient, and prepared electrocompetent cells for transformation with a kanamycin cassette to target sequences in the orangutan genome through terminal recombineering homologies. Kanamycin-resistant colonies were tested for the presence of BACs containing the targeted genes by the use of a PCR-assay to confirm the presence of the kanamycin insertion. The results indicate that this is an effective approach for screening clones. The advantage of recombination screening is that it avoids the high costs associated with the preparation, screening, and archival storage of arrayed BAC libraries. In addition, the screening can be conceivably combined with genetic engineering to create knockout and reporter constructs for functional studies.

  12. Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L.

    Science.gov (United States)

    Sansri, Veerawat; Meemon, Krai; Changklungmoa, Narin; Kueakhai, Pornanan; Chantree, Pathanin; Chaichanasak, Pannigan; Lorsuwannarat, Natcha; Itagaki, Tadashi; Sobhon, Prasert

    2015-03-24

    Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its

  13. Isotope-specific and amino acid-specific heavy atom substitutions alter barrier crossing in human purine nucleoside phosphorylase.

    Science.gov (United States)

    Suarez, Javier; Schramm, Vern L

    2015-09-08

    Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

  14. Passive protection of piglets by recombinant baculovirus induced transmissible gastroenteritis virus specific antibodies.

    OpenAIRE

    Tuboly, T; Nagy, E; Derbyshire, J B

    1995-01-01

    Sera of pigs immunized with parts of the transmissible gastroenteritis virus (TGEV) spike (S) protein expressed by recombinant baculoviruses were tested, together with a TGEV hyperimmune antiserum, for their abilities to protect three-day-old piglets against TGEV infection. The piglets were infected with virulent TGEV and the sera were given orally 3 h before infection, together with the virus, and every 6 h postinfection during the 30 h of the experiment. Virus shedding was monitored by TGEV...

  15. Specific Roles of XRCC4 Paralogs PAXX and XLF during V(DJ Recombination

    Directory of Open Access Journals (Sweden)

    Chloé Lescale

    2016-09-01

    Full Text Available Paralog of XRCC4 and XLF (PAXX is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ, a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(DJ recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(DJ recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(DJ recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.

  16. XerCD-Mediated Site-Specific Recombination Leads to Loss of the 57-Kilobase Gonococcal Genetic Island▿

    OpenAIRE

    Domínguez, Nadia M.; Hackett, Kathleen T.; Dillard, Joseph P.

    2010-01-01

    Most strains of Neisseria gonorrhoeae carry the 57-kb gonococcal genetic island (GGI), as do a few strains of Neisseria meningitidis. The GGI is inserted into the chromosome at the dif site (difA) and is flanked by a partial repeat of the dif site (difB). Since dif is a sequence recognized by the site-specific recombinases XerC and XerD and the GGI shows evidence of horizontal acquisition, we hypothesized that the GGI may be acquired or lost by XerCD-mediated site-specific recombination. We s...

  17. Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo

    DEFF Research Database (Denmark)

    Mikkelsen, J G; Lund, Anders Henrik; Duch, M

    2000-01-01

    synthesis in newly infected cells. We have previously shown that template shifts within the 5' leader of murine leukemia viruses occur preferentially within the kissing stem-loop motif, a cis element crucial for in vitro RNA dimer formation. By use of a forced recombination approach based on single......-cycle transfer of Akv murine leukemia virus-based vectors harboring defective primer binding site sequences, we now report that modifications of the kissing-loop structure, ranging from a deletion of the entire sequence to introduction of a single point mutation in the loop motif, significantly disturb site...... specificity of recombination within the highly structured 5' leader region. In addition, we find that an intact kissing-loop sequence favors optimal RNA encapsidation and vector transduction. Our data are consistent with the kissing-loop dimerization model and suggest that a direct intermolecular RNA...

  18. Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

    Science.gov (United States)

    Yong-Su Jin; Thomas W. Jeffries

    2003-01-01

    We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes(XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into...

  19. An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

    Science.gov (United States)

    Ieyasu, Aki; Ishida, Reiko; Kimura, Takaharu; Morita, Maiko; Wilkinson, Adam C; Sudo, Kazuhiro; Nishimura, Toshinobu; Ohehara, Jun; Tajima, Yoko; Lai, Chen-Yi; Otsu, Makoto; Nakamura, Yukio; Ema, Hideo; Nakauchi, Hiromitsu; Yamazaki, Satoshi

    2017-03-14

    Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors

    Directory of Open Access Journals (Sweden)

    Aki Ieyasu

    2017-03-01

    Full Text Available Hematopoietic stem cells (HSCs are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.

  1. Purification of polyclonal IgG specific for Camelid’s antibodies and their recombinant nanobodies

    Directory of Open Access Journals (Sweden)

    Haddad Muhammad

    2016-01-01

    Full Text Available Camelid’ s heavy-chain antibody (HCAb consists of only two heavy chains and lacks the two light chains together with the CH1 domain usually found in conventional immunoglobulins. A recombinant single antigen-binding entity, named VHH (or Nanobody® was generated by reengineering the variable domains from HCAb. This study focuses on the detection of camelid´s immunoglobulins as well as their derivative nanobodies using a universal anti-camel antibody produced in rabbit (rIgG. Starting from a crude rabbit serum, a standard stock of rIgG (1 mg/ml was prepared after purification by affinity chromatography using protein-A column. As expected, rIgG was able to detect camel antibodies in ELISA and immunoblotting, and its reactivity was equal against all different camel IgG subclasses, which were purified from serum by differential affinity chromatography on protein-G and -A. Interestingly, rIgG also recognized nanobodies since they were originally part of camel HCAbs, providing an alternative method to detect the corpus of these recombinant proteins rather than targeting their artificial tags. These data suggest that the anti-camel rIgG described here could be efficiently applied at different stages of nanobody technology, including the quantitation of the issued nanobodies and their detection when bound to target antigens.

  2. Exposure to Hycanthone alters chromatin structure around specific gene functions and specific repeats in Schistosoma mansoni

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    David eRoquis

    2014-07-01

    Full Text Available Schistosoma mansoni is a parasitic plathyhelminth responsible for intestinal schistosomiasis (or bilharziasis, a disease affecting 67 million people worldwide and causing an important economic burden. The schistosomicides hycanthone, and its later proxy oxamniquine, were widely used for treatments in endemic areas during the 20th century. Recently, the mechanism of action, as well as the genetic origin of a stably and Mendelian inherited resistance for both drugs was elucidated in two strains. However, several observations suggested early on that alternative mechanisms might exist, by which resistance could be induced for these two drugs in sensitive lines of schistosomes. This induced resistance appeared rapidly, within the first generation, but was metastable (not stably inherited. Epigenetic inheritance could explain such a phenomenon and we therefore re-analyzed the historical data with our current knowledge of epigenetics. In addition, we performed new experiments such as ChIP-seq on hycanthone treated worms. We found distinct chromatin structure changes between sensitive worms and induced resistant worms from the same strain. No specific pathway was discovered, but genes in which chromatin structure modification were observed are mostly associated with transport and catabolism, which makes sense in the context of the elimination of the drug. Specific differences were observed in the repetitive compartment of the genome. We finally describe what types of experiments are needed to understand the complexity of heritability that can be based on genetic and/or epigenetic mechanisms for drug resistance in schistosomes.

  3. Induction of antigen-specific Th1-type immune responses by gamma-irradiated recombinant Brucella abortus RB51.

    Science.gov (United States)

    Sanakkayala, Neelima; Sokolovska, Anna; Gulani, Jatinder; Hogenesch, Harm; Sriranganathan, Nammalwar; Boyle, Stephen M; Schurig, Gerhardt G; Vemulapalli, Ramesh

    2005-12-01

    Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli beta-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize beta-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a beta-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.

  4. The dock-and-lock method combines recombinant engineering with site-specific covalent conjugation to generate multifunctional structures.

    Science.gov (United States)

    Rossi, Edmund A; Goldenberg, David M; Chang, Chien-Hsing

    2012-03-21

    Advances in recombinant protein technology have facilitated the production of increasingly complex fusion proteins with multivalent, multifunctional designs for use in various in vitro and in vivo applications. In addition, traditional chemical conjugation remains a primary choice for linking proteins with polyethylene glycol (PEG), biotin, fluorescent markers, drugs, and others. More recently, site-specific conjugation of two or more interactive modules has emerged as a valid approach to expand the existing repertoires produced by either recombinant engineering or chemical conjugation alone, thus advancing the range of potential applications. Five such methods, each involving a specific binding event, are highlighted in this review, with a particular focus on the Dock-and-Lock (DNL) method, which exploits the natural interaction between the dimerization and docking domain (DDD) of cAMP-dependent protein kinase (PKA) and the anchoring domain (AD) of A-kinase anchoring proteins (AKAP). The various enablements of DNL to date include trivalent, tetravalent, pentavalent, and hexavalent antibodies of monospecificity or bispecificity; immnocytokines comprising multiple copies of interferon-alpha (IFNα); and site-specific PEGylation. These achievements attest to the power of the DNL platform technology to develop novel therapeutic and diagnostic agents from both proteins and nonproteins for unmet medical needs.

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

    Science.gov (United States)

    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.

  6. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

    Directory of Open Access Journals (Sweden)

    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  7. Controlled Cre/loxP site-specific recombination in the developing brain in medaka fish, Oryzias latipes.

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    Teruhiro Okuyama

    Full Text Available BACKGROUND: Genetic mosaic techniques have been used to visualize and/or genetically modify a neuronal subpopulation within complex neural circuits in various animals. Neural populations available for mosaic analysis, however, are limited in the vertebrate brain. METHODOLOGY/PRINCIPAL FINDINGS: To establish methodology to genetically manipulate neural circuits in medaka, we first created two transgenic (Tg medaka lines, Tg (HSP:Cre and Tg (HuC:loxP-DsRed-loxP-GFP. We confirmed medaka HuC promoter-derived expression of the reporter gene in juvenile medaka whole brain, and in neuronal precursor cells in the adult brain. We then demonstrated that stochastic recombination can be induced by micro-injection of Cre mRNA into Tg (HuC:loxP-DsRed-loxP-GFP embryos at the 1-cell stage, which allowed us to visualize some subpopulations of GFP-positive cells in compartmentalized regions of the telencephalon in the adult medaka brain. This finding suggested that the distribution of clonally-related cells derived from single or a few progenitor cells was restricted to a compartmentalized region. Heat treatment of Tg(HSP:Cre x HuC:loxP-DsRed-loxP-GFP embryos (0-1 day post fertilization [dpf] in a thermalcycler (39°C led to Cre/loxP recombination in the whole brain. The recombination efficiency was notably low when using 2-3 dpf embyos compared with 0-1 dpf embryos, indicating the possibility of stage-dependent sensitivity of heat-inducible recombination. Finally, using an infrared laser-evoked gene operator (IR-LEGO system, heat shock induced in a micro area in the developing brains led to visualization of clonally-related cells in both juvenile and adult medaka fish. CONCLUSIONS/SIGNIFICANCE: We established a noninvasive method to control Cre/loxP site-specific recombination in the developing nervous system in medaka fish. This method will broaden the neural population available for mosaic analyses and allow for lineage tracing of the vertebrate nervous system

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

    DEFF Research Database (Denmark)

    Andersen, P S; Stryhn, A; Hansen, B E

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might ...

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

    DEFF Research Database (Denmark)

    Andersen, P S; Stryhn, A; Hansen, B E

    1996-01-01

    Specific recognition of peptide/major histocompatibility complex (MHC) molecule complexes by the T-cell receptor is a key reaction in the specific immune response. Antibodies against peptide/MHC complexes would therefore be valuable tools in studying MHC function and T-cell recognition and might...... peptide/MHC complexes....

  10. Identification of a putative Crf splice variant and generation of recombinant antibodies for the specific detection of Aspergillus fumigatus.

    Directory of Open Access Journals (Sweden)

    Mark Schütte

    Full Text Available BACKGROUND: Aspergillus fumigatus is a common airborne fungal pathogen for humans. It frequently causes an invasive aspergillosis (IA in immunocompromised patients with poor prognosis. Potent antifungal drugs are very expensive and cause serious adverse effects. Their correct application requires an early and specific diagnosis of IA, which is still not properly achievable. This work aims to a specific detection of A. fumigatus by immunofluorescence and the generation of recombinant antibodies for the detection of A. fumigatus by ELISA. RESULTS: The A. fumigatus antigen Crf2 was isolated from a human patient with proven IA. It is a novel variant of a group of surface proteins (Crf1, Asp f9, Asp f16 which belong to the glycosylhydrolase family. Single chain fragment variables (scFvs were obtained by phage display from a human naive antibody gene library and an immune antibody gene library generated from a macaque immunized with recombinant Crf2. Two different selection strategies were performed and shown to influence the selection of scFvs recognizing the Crf2 antigen in its native conformation. Using these antibodies, Crf2 was localized in growing hyphae of A. fumigatus but not in spores. In addition, the antibodies allowed differentiation between A. fumigatus and related Aspergillus species or Candida albicans by immunofluorescence microscopy. The scFv antibody clones were further characterized for their affinity, the nature of their epitope, their serum stability and their detection limit of Crf2 in human serum. CONCLUSION: Crf2 and the corresponding recombinant antibodies offer a novel approach for the early diagnostics of IA caused by A. fumigatus.

  11. Immunization against recombinant GnRH-I alters ultrastructure of gonadotropin cell in an experimental boar model.

    Science.gov (United States)

    Fang, Fugui; Su, Shiping; Liu, Ya; Zhang, Yunhai; Pu, Yong; Zhao, Xijie; Li, Yunsheng; Cao, Hongguo; Wang, Juhua; Zhou, Jie; Zhang, Xiaorong

    2013-07-15

    Gonadotropin cell is the main responsible for the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and immunocastration reduces the concentrations of serum FSH and LH. A few studies have reported the histological structure of gonadotropin cells obtained from immunocastration animals at the light microscopy level. However, the ultrastructure of gonadotropin cells remains largely unexplored. The aim of this study was to evaluate and to compare ultrastructure of gonadotropin cell in gonadally intact boars and immunologically castrated male animals. In this study, serum and adenohypophysis tissue were collected from nine gonadally intact boars and nine male pigs treated with recombinant gonadotropin releasing hormone I (GnRH-I). Anti-GnRH-I antibodies in serum and the ultrastructure of gonadotropin cell in adenohypophysis were determined by enzymelinked immunosorbent assay and electron microscopy, respectively. The results demonstrated that active immunization against recombinant GnRH-I increased serum GnRH-I antibody levels (Pimmunized animals. We conclude that immunization against recombinant GnRH-I induces severe atrophy of granules in gonadotropin cell of boars, possibly reflecting GnRH-I regulation of gonadotropin cell.

  12. Sonographic alteration of lenticular nucleus in focal task-specific dystonia of musicians.

    Science.gov (United States)

    Walter, Uwe; Buttkus, Franziska; Benecke, Reiner; Grossmann, Annette; Dressler, Dirk; Altenmüller, Eckart

    2012-01-01

    In distinct movement disorders, transcranial sonography detects alterations of deep brain structures with higher sensitivity than other neuroimaging methods. Lenticular nucleus hyperechogenicity on transcranial sonography, thought to be caused by increased local copper content, has been reported as a characteristic finding in primary spontaneous dystonia. Here, we wanted to find out whether deep brain structures are altered in task-specific dystonia. The frequency of sonographic brainstem and basal ganglia changes was studied in an investigator-blinded setting in 15 musicians with focal task-specific hand dystonia, 15 musicians without dystonia, and 15 age- and sex-matched nonmusicians without dystonia. Lenticular nucleus hyperechogenicity was found in 12 musicians with task-specific dystonia, but only in 3 nondystonic musicians (Fisher's exact test, p = 0.001) and 2 nonmusicians (p musicians correlated with age, but not with duration of music practice or duration of dystonia. In 2 of 3 affected musicians with normal echogenic lenticular nucleus, substantia nigra hyperechogenicity was found. Our findings support the idea of a pathogenetic link between primary spontaneous and task-specific dystonia. Sonographic basal ganglia alteration might indicate a risk factor that in combination with extensive fine motor training promotes the manifestation of task-specific dystonia. Copyright © 2011 S. Karger AG, Basel.

  13. A novel recombinant antibody specific to full-length stromal derived factor-1 for potential application in biomarker studies.

    Directory of Open Access Journals (Sweden)

    Daniel I Bromage

    Full Text Available Stromal derived factor-1α (SDF-1α/CXCL12 is a chemokine that is up-regulated in diseases characterised by tissue hypoxia, including myocardial infarction, ischaemic cardiomyopathy and remote ischaemic conditioning (RIC, a technique of cyclical, non-injurious ischaemia applied remote from the heart that protects the heat from lethal ischaemia-reperfusion injury. Accordingly, there is considerable interest in SDF-1α as a potential biomarker of such conditions. However, SDF-1α is rapidly degraded and inactivated by dipeptidyl peptidase 4 and other peptidases, and the kinetics of intact SDF-1α remain unknown.To facilitate investigation of full-length SDF-1α we established an ELISA using a novel recombinant human antibody we developed called HCI.SDF1. HCI.SDF1 is specific to the N-terminal sequence of all isoforms of SDF-1 and has a comparable KD to commercially available antibodies. Together with a detection antibody specific to the α-isoform, HCI.SDF1 was used to specifically quantify full-length SDF-1α in blood for the first time. Using RIC applied to the hind limb of Sprague-Dawley rats or the arms of healthy human volunteers, we demonstrate an increase in SDF-1α using a commercially available antibody, as previously reported, but an unexpected decrease in full-length SDF-1α after RIC in both species.We report for the first time the development of a novel recombinant antibody specific to full-length SDF-1. Applied to RIC, we demonstrate a significant decrease in SDF-1α that is at odds with the literature and suggests a need to investigate the kinetics of full-length SDF-1α in conditions characterised by tissue hypoxia.

  14. Evaluation of Serum Specific Antibody against Recombinant ESAT-6 Antigen in Patients with Tuberculosis and Comparing to Normal Controls

    Directory of Open Access Journals (Sweden)

    Homeira Izadi

    2017-02-01

    Full Text Available Background & Objective: Tuberculosis (TB is a zoonotic disease which is caused by Mycobacterium tuberculosis. Because of common structural and secretory antigens between pathogen and nonpathogenic mycobacterium, the specific diagnosis of TB is difficult. Therefore, it is very important to find a new method with high specificity and sensitivity for accurate and rapid diagnosis of tuberculosis. In this study, the serodiagnostic potential of Mycobacterium tuberculosis recombinant ESAT-6 in TB infected patients was evaluated by Enzyme Linked Immunosorbent Assay (ELISA. Materials & Methods: 55 TB patients with active disease and 28 healthy controls have been collected and evaluated in different dilutions in ELISA methods for the presence of specific anti-ESAT-6 antibody. The specificity and the sensitivity of this method was compared with the culture test. Results: TB patients have high levels of specific antibody against ESAT-6 antigens. The specificity and the sensitivity of this method was calculated as 80.90% and 85.45%, respectively. Conclusion: These findings provide useful information on the importance of ESAT-6 protein and suggested this serologic test as a good alternative method for rapid and prefect diagnosis of tuberculosis.

  15. Recombinant adenovirus expressing ICP47 gene suppresses the ability of dendritic cells by restricting specific T cell responses.

    Science.gov (United States)

    Wang, Peng; Kan, Quancheng; Yu, Zujiang; Li, Ling; Zhang, Zhenxiang; Pan, Xue; Feng, Ting

    2013-04-01

    Adenoviral vectors have been demonstrated to be one of the most effective vehicles to deliver foreign DNA into dendritic cells (DCs). However, the response of host immune systems against foreign gene products is a major obstacle to successful gene therapy. Infected cell protein 47 (ICP47) inhibits MHC Ⅰ antigen presentation pathway by binding to host transporter associated with antigen presentation (TAP), and thereby attenuates of specific cytotoxic T lymphocytes (CTLs) responses and evades the host immune clearance. This subject was designed to construct a recombinant adenovirus expressing His-tag-ICP47 fusion protein to investigate further the role of ICP47 in the elimination of transgene expression. Consequently, a recombinant adenovirus expressing the His-tag-ICP47 fusion protein was successfully constructed and it had the abilities of attenuating the stimulatory capacity of DCs by reducing the proliferation of lymphocytes and cytokine production of perforin compared with those of the r-track group and the control group. Our observations provide the first evidence of the regulation mechanism of ICP47 on DC-based immunotherapy for long-term persistence. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Immunization with recombinantly expressed glycan antigens from Schistosoma mansoni induces glycan-specific antibodies against the parasite.

    Science.gov (United States)

    Prasanphanich, Nina Salinger; Luyai, Anthony E; Song, Xuezheng; Heimburg-Molinaro, Jamie; Mandalasi, Msano; Mickum, Megan; Smith, David F; Nyame, A Kwame; Cummings, Richard D

    2014-07-01

    Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Peptide specific expansion of CD8(+) T cells by recombinant plate bound MHC/peptide complexes

    DEFF Research Database (Denmark)

    Schmidt, Esben G W; Buus, Soren; Thorn, Mette

    2009-01-01

    Development of methods for efficient in vitro stimulation and expansion of peptide specific CD8(+) T cells is compelling not only with respect to adoptive T cell therapy but also regarding analysis of T cell responses and search for new immunogenic peptides. In the present study, a new approach...... cells (NA-PBMCs) with PB-MHC/CMVp resulted in significant expansion of CMVp specific CD8(+) T cells, which was comparable to that achieved by CMVp pulsed mature dendritic cells (DCs). By repeated exposure of NA-PBMCs to PB-MHC/CMVp more than 60% CMVp specific CD8(+) T cells, representing a 240-fold...... expansion, were reached after only two stimulations. Although stimulation with PB-MHC/CMVp clearly demonstrated efficient peptide specific expansion of CD8(+) T cells, there was a tendency to proliferative exhaustion of the cells after 3-4 stimulations. Thus, it will be of interest to examine the effect...

  18. Recombinant jacalin-like plant lectins are produced at high levels in Nicotiana benthamiana and retain agglutination activity and sugar specificity.

    Science.gov (United States)

    Fernandez-del-Carmen, Asun; Juárez, Paloma; Presa, Silvia; Granell, Antonio; Orzáez, Diego

    2013-02-20

    The plant kingdom is an underexplored source of valuable proteins which, like plant lectins, display unique interacting specificities. Furthermore, plant protein diversity remains under-exploited due to the low availability and heterogeneity of native sources. All these hurdles could be overcome with recombinant production. A narrow phylogenetic gap between the native source and the recombinant platform is likely to facilitate proper protein processing and stability; therefore, the plant cell chassis should be specially suited for the recombinant production of many plant native proteins. This is illustrated herein with the recombinant production of two representatives of the plant jacalin-related lectin (JRLs) protein family in Nicotiana benthamiana using state-of-the-art magnICON technology. Mannose-specific Banlec JRL was produced at very high levels in leaves, reaching 1.0mg of purified protein per gram of fresh weight and showing strong agglutination activity. Galactose-specific jacalin JRL, with its complicated processing requirements, was also successfully produced in N. benthamiana at levels of 0.25 mg of purified protein per gram of fresh weight. Recombinant Jacalin (rJacalin) proved efficient in the purification of human IgA1, and was able to discriminate between plant-made and native IgA1 due to their differential glycosylation status. Together, these results show that the plant cell factory should be considered a primary option in the recombinant production of valuable plant proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Maternal Western diet age-specifically alters female offspring voluntary physical activity and dopamine- and leptin-related gene expression.

    Science.gov (United States)

    Ruegsegger, Gregory N; Grigsby, Kolter B; Kelty, Taylor J; Zidon, Terese M; Childs, Thomas E; Vieira-Potter, Victoria J; Klinkebiel, David L; Matheny, Michael; Scarpace, Phillip J; Booth, Frank W

    2017-12-01

    Prenatal overnutrition affects development into adulthood and influences risk of obesity. We assessed the transgenerational effect of maternal Western diet (WD) consumption on offspring physical activity. Voluntary wheel running was increased in juvenile (4-7 wk of age), but decreased in adult (16-19 wk of age), F 1 female WD offspring In contrast, no wheel-running differences in F 1 male offspring were observed. Increased wheel running in juvenile female WD offspring was associated with up-regulated dopamine receptor (DRD)-1 and -2 in the nucleus accumbens (NAc) and with down-regulated Lepr in the ventral tegmental area (VTA). Conversely, decreased wheel running by adult female WD offspring was associated with down-regulated DRD1 in the NAc and with up-regulated Lepr in the VTA. Body fat, leptin, and insulin were increased in male, but not in female, F 1 WD offspring. Recombinant virus (rAAV) leptin antagonism in the VTA decreased wheel running in standard diet but not in WD F 1 female offspring. Analysis of F 2 offspring found no differences in wheel running or adiposity in male or female offspring, suggesting that changes in the F 1 generation were related to in utero somatic reprogramming. Our findings indicate prenatal WD exposure leads to age-specific changes in voluntary physical activity in female offspring that are differentially influenced by VTA leptin antagonism.-Ruegsegger, G. N., Grigsby, K. B., Kelty, T. J., Zidon, T. M., Childs, T. E., Vieira-Potter, V. J., Klinkebiel, D. L., Matheny, M., Scarpace, P. J., Booth, F. W. Maternal Western diet age-specifically alters female offspring voluntary physical activity and dopamine- and leptin-related gene expression. © FASEB.

  20. Development of pronuclear injection-based targeted transgenesis in mice through Cre-loxP site-specific recombination.

    Science.gov (United States)

    Ohtsuka, Masato

    2014-01-01

    Microinjection of DNA into the pronuclei of zygotes is the simplest and most widely used method for generating transgenic (Tg) mice. However, it is always associated with random integration of multiple copies of the transgene, resulting in unstable, low, or no transgene expression due to positional effects and/or repeat-induced gene silencing. In addition, random integration sometimes disrupts an endogenous gene that can affect the phenotypes of Tg mice. Our recently developed pronuclear injection-based targeted transgenesis (PITT) method enables the integration of a single-copy transgene into a predetermined genomic locus through Cre-loxP site-specific recombination. The PITT method enables stable and reliable transgene expression in Tg mice and is also applicable for generating knockdown mice. Therefore, the PITT method could represent next-generation transgenesis that overcomes the pitfalls of conventional transgenesis.

  1. [Copy number alterations in adult patients with mature B acute lymphoblastic leukemia treated with specific immunochemotherapy].

    Science.gov (United States)

    Ribera, Jordi; Zamora, Lurdes; García, Olga; Hernández-Rivas, Jesús-María; Genescà, Eulàlia; Ribera, Josep-Maria

    2016-12-02

    Unlike Burkitt lymphoma, molecular abnormalities other than C-MYC rearrangements have scarcely been studied in patients with mature B acute lymphoblastic leukemia (B-ALL). The aim of this study was to analyze the frequency and prognostic significance of copy number alterations (CNA) in genes involved in lymphoid differentiation, cell cycle and tumor suppression in adult patients with B-ALL. We have analyzed by multiplex ligation-dependent probe amplification the genetic material from bone marrow at diagnosis from 25 adult B-ALL patients treated with rituximab and specific chemotherapy. The most frequent CNA were alterations in the 14q32.33 region (11 cases, 44%) followed by alterations in the cell cycle regulator genes CDKN2A/B and RB1 (16%). No correlation between the presence of specific CNA and the clinical-biologic features or the response to therapy was found. The high frequency of CNA in the 14q32.33 region, CDKN2A/B and RB1 found in our study could contribute to the aggressiveness and invasiveness of mature B-ALL. Copyright © 2016 Elsevier España, S.L.U. All rights reserved.

  2. Recombinant Envelope-Proteins with Mutations in the Conserved Fusion Loop Allow Specific Serological Diagnosis of Dengue-Infections.

    Directory of Open Access Journals (Sweden)

    Alexandra Rockstroh

    2015-11-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus and a major international public health concern in many tropical and sub-tropical areas worldwide. DENV is divided into four major serotypes, and infection with one serotype leads to immunity against the same, but not the other serotypes. The specific diagnosis of DENV-infections via antibody-detection is problematic due to the high degree of cross-reactivity displayed by antibodies against related flaviviruses, such as West Nile virus (WNV, Yellow Fever virus (YFV or Tick-borne encephalitis virus (TBEV. Especially in areas where several flaviviruses co-circulate or in the context of vaccination e.g. against YFV or TBEV, this severely complicates diagnosis and surveillance. Most flavivirus cross-reactive antibodies are produced against the highly conserved fusion loop (FL domain in the viral envelope (E protein. We generated insect-cell derived recombinant E-proteins of the four DENV-serotypes which contain point mutations in the FL domain. By using specific mixtures of these mutant antigens, cross-reactivity against heterologous flaviviruses was strongly reduced, enabling sensitive and specific diagnosis of the DENV-infected serum samples in IgG and IgM-measurements. These results have indications for the development of serological DENV-tests with improved specificity.

  3. Chromosomal rearrangements formed by rrn recombination do not improve replichore balance in host-specific Salmonella enterica serovars.

    Directory of Open Access Journals (Sweden)

    T David Matthews

    2010-10-01

    Full Text Available Most of the ∼2,600 serovars of Salmonella enterica have a broad host range as well as a conserved gene order. In contrast, some Salmonella serovars are host-specific and frequently exhibit large chromosomal rearrangements from recombination between rrn operons. One hypothesis explaining these rearrangements suggests that replichore imbalance introduced from horizontal transfer of pathogenicity islands and prophages drives chromosomal rearrangements in an attempt to improve balance.This hypothesis was directly tested by comparing the naturally-occurring chromosomal arrangement types to the theoretically possible arrangement types, and estimating their replichore balance using a calculator. In addition to previously characterized strains belonging to host-specific serovars, the arrangement types of 22 serovar Gallinarum strains was also determined. Only 48 out of 1,440 possible arrangement types were identified in 212 host-specific strains. While the replichores of most naturally-occurring arrangement types were well-balanced, most theoretical arrangement types had imbalanced replichores. Furthermore, the most common types of rearrangements did not change replichore balance.The results did not support the hypothesis that replichore imbalance causes these rearrangements, and suggest that the rearrangements could be explained by aspects of a host-specific lifestyle.

  4. Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes

    NARCIS (Netherlands)

    Mlynarova, L.; Conner, A.J.; Nap, J.P.H.

    2006-01-01

    A major challenge for future genetically modified (GM) crops is to prevent undesired gene flow of transgenes to plant material intended for another use. Recombinase-mediated auto excision of transgenes directed by a tightly controlled microspore-specific promoter allows efficient removal of either

  5. Dissociating motivational direction and affective valence: specific emotions alter central motor processes.

    Science.gov (United States)

    Coombes, Stephen A; Cauraugh, James H; Janelle, Christopher M

    2007-11-01

    We aimed to clarify the relation between affective valence and motivational direction by specifying how central and peripheral components of extension movements are altered according to specific unpleasant affective states. As predicted, premotor reaction time was quicker for extension movements initiated during exposure to attack than for extension movements initiated during exposure to all other valence categories (mutilation, erotic couples, opposite-sex nudes, neutral humans, household objects, blank). Exposure to erotic couples and mutilations yielded greater peak force than exposure to images of attack, neutral humans, and household objects. Finally, motor reaction time and peak electromyographic amplitude were not altered by valence. These findings indicate that unpleasant states do not unilaterally prime withdrawal movements, and that the quick execution of extension movements during exposure to threatening images is due to rapid premotor, rather than motor, reaction time. Collectively, our findings support the call for dissociating motivational direction and affective valence.

  6. Quantitative X-ray - UV Line and Continuum Spectroscopy with Application to AGN: State-Specific Hydrogenic Recombination Cooling Coefficients for a Wide Range of Conditions

    Science.gov (United States)

    LaMothe, J.; Ferland, Gary J.

    2002-01-01

    Recombination cooling, in which a free electron emits light while being captured to an ion, is an important cooling process in photoionized clouds that are optically thick or have low metallicity. State specific rather than total recombination cooling rates are needed since the hydrogen atom tends to become optically thick in high-density regimes such as Active Galactic Nuclei. This paper builds upon previous work to derive the cooling rate over the full temperature range where the process can be a significant contributor in a photoionized plasma. We exploit the fact that the recombination and cooling rates are given by intrinsically similar formulae to express the cooling rate in terms of the closely related radiative recombination rate. We give an especially simple but accurate approximation that works for any high hydrogenic level and can be conveniently employed in large-scale numerical simulations.

  7. HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles

    International Nuclear Information System (INIS)

    Haffar, O.K.; Smithgall, M.D.; Moran, P.A.; Travis, B.M.; Zarling, J.M.; Hu, S.L.

    1991-01-01

    Recombinant human immunodeficiency virus type-1 (HIV-1)-like gag-env particles produced in mammalian cells were inoculated into two New Zealand white rabbits. In parallel, two control rabbits were inoculated with the homologous HIV-1 virions inactivated by ultraviolet light (uv) and psoralen treatments. The humoral and cellular immune responses to HIV-1 were evaluated for both groups of animals. Recombinant particles elicited humoral immunity that was specific for all the viral structural proteins. The antibodies recognized both denatured and nondenatured proteins. Moreover, the sera neutralized the in vitro infectivity of the homologous virus in CEM cells. Importantly, the recombinant particles also generated a T helper response by priming with the HIV proteins. Similar results were observed with inactivated virus immunization. Therefore, the authors results suggest that the recombinant HIV-like particles elicit functional humoral immunity as well as cellular immunity and represent a novel vaccine candidate for AIDS

  8. Auditory training alters the physiological detection of stimulus-specific cues in humans.

    Science.gov (United States)

    Tremblay, Kelly L; Shahin, Antoine J; Picton, Terence; Ross, Bernhard

    2009-01-01

    Auditory training alters neural activity in humans but it is unknown if these alterations are specific to the trained cue. The objective of this study was to determine if enhanced cortical activity was specific to the trained voice-onset-time (VOT) stimuli 'mba' and 'ba', or whether it generalized to the control stimulus 'a' that did not contain the trained cue. Thirteen adults were trained to identify a 10ms VOT cue that differentiated the two experimental stimuli. We recorded event-related potentials (ERPs) evoked by three different speech sounds 'ba' 'mba' and 'a' before and after six days of VOT training. The P2 wave increased in amplitude after training for both control and experimental stimuli, but the effects differed between stimulus conditions. Whereas the effects of training on P2 amplitude were greatest in the left hemisphere for the trained stimuli, enhanced P2 activity was seen in both hemispheres for the control stimulus. In addition, subjects with enhanced pre-training N1 amplitudes were more responsive to training and showed the most perceptual improvement. Both stimulus-specific and general effects of training can be measured in humans. An individual's pre-training N1 response might predict their capacity for improvement. N1 and P2 responses can be used to examine physiological correlates of human auditory perceptual learning.

  9. Disorder-specific white matter alterations in adolescent borderline personality disorder.

    Science.gov (United States)

    Maier-Hein, Klaus H; Brunner, Romuald; Lutz, Kira; Henze, Romy; Parzer, Peter; Feigl, Nina; Kramer, Jasmin; Meinzer, Hans-Peter; Resch, Franz; Stieltjes, Bram

    2014-01-01

    The pathogenesis of borderline personality disorder (BPD) is complex and not fully understood. Using diffusion tensor imaging, recent studies suggest that white matter abnormalities may occur in adult patients with BPD. However, deeper insight into the disorder-specific developmental psychobiology (e.g., analysis of adolescents with BPD; inclusion of clinical control groups) is missing. Twenty adolescent patients with BPD (aged 14-18 years), 20 healthy, and 20 clinical control subjects were assessed using diffusion tensor imaging. All subjects were right-handed girls, matched for age and IQ. Microstructural parameters were analyzed via tractography of the main bundles in the limbic system and using Tract-Based Spatial Statistics, an explorative, global approach. BPD was associated with decreased fractional anisotropy in the fornix when compared with clinical (p < .001) or healthy (nonsignificant trend) control subjects. Using Tract-Based Spatial Statistics, significant disorder-specific white matter alterations were found in the long association bundles interconnecting the heteromodal association cortex and in connections between the thalamus and hippocampus. The study strongly supports the hypothesis that white matter alterations play a key role in the pathogenesis of BPD. These disorder-specific alterations include white matter pathways involved in emotion regulation but also affect parts of the heteromodal association cortex that are related to emotion recognition. Our findings unify previously documented deficits in emotion recognition and regulation and suggest that a large-scale network of emotion processing is disrupted in BPD. Continued research is essential to evaluate the predictive value of these early disruptions in a clinical context. Copyright © 2014 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  10. Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9.

    Science.gov (United States)

    Hirano, Seiichi; Nishimasu, Hiroshi; Ishitani, Ryuichiro; Nureki, Osamu

    2016-03-17

    The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets bearing a PAM (protospacer adjacent motif) and complementarity to the guide RNA. A recent study showed that, whereas wild-type Streptococcus pyogenes Cas9 (SpCas9) recognizes the 5'-NGG-3' PAM, the engineered VQR, EQR, and VRER SpCas9 variants recognize the 5'-NGA-3', 5'-NGAG-3', and 5'-NGCG-3' PAMs, respectively, thus expanding the targetable sequences in Cas9-mediated genome editing applications. Here, we present the high-resolution crystal structures of the three SpCas9 variants in complexes with a single-guide RNA and its altered PAM-containing, partially double-stranded DNA targets. A structural comparison of the three SpCas9 variants with wild-type SpCas9 revealed that the multiple mutations synergistically induce an unexpected displacement in the phosphodiester backbone of the PAM duplex, thereby allowing the SpCas9 variants to directly recognize the altered PAM nucleotides. Our findings explain the altered PAM specificities of the SpCas9 variants and establish a framework for further rational engineering of CRISPR-Cas9. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Recombinant influenza virus expressing HIV-1 p24 capsid protein induces mucosal HIV-specific CD8 T-cell responses.

    Science.gov (United States)

    Tan, Hyon-Xhi; Gilbertson, Brad P; Jegaskanda, Sinthujan; Alcantara, Sheilajen; Amarasena, Thakshila; Stambas, John; McAuley, Julie L; Kent, Stephen J; De Rose, Robert

    2016-02-24

    Influenza viruses are promising mucosal vaccine vectors for HIV but their use has been limited by difficulties in engineering the expression of large amounts of foreign protein. We developed recombinant influenza viruses incorporating the HIV-1 p24 gag capsid into the NS-segment of PR8 (H1N1) and X31 (H3N2) influenza viruses with the use of multiple 2A ribosomal skip sequences. Despite the insertion of a sizable HIV-1 gene into the influenza genome, recombinant viruses were readily rescued to high titers. Intracellular expression of p24 capsid was confirmed by in vitro infection assays. The recombinant influenza viruses were subsequently tested as mucosal vaccines in BALB/c mice. Recombinant viruses were attenuated and safe in immunized mice. Systemic and mucosal HIV-specific CD8 T-cell responses were elicited in mice that were immunized via intranasal route with a prime-boost regimen. Isolated HIV-specific CD8 T-cells displayed polyfunctional cytokine and degranulation profiles. Mice boosted via intravaginal route induced recall responses from the distal lung mucosa and developed heightened HIV-specific CD8 T-cell responses in the vaginal mucosa. These findings demonstrate the potential utility of recombinant influenza viruses as vaccines for mucosal immunity against HIV-1 infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Novel recombinant immunotoxin of EGFR specific nanobody fused with cucurmosin, construction and antitumor efficiency in vitro.

    Science.gov (United States)

    Deng, Cuimin; Xiong, Jiani; Gu, Xiaofan; Chen, Xiaoying; Wu, Shuifa; Wang, Zhe; Wang, Duanduan; Tu, Jinjin; Xie, Jieming

    2017-06-13

    Epidermal growth factor receptor (EGFR) overexpression is related to the increased aggressiveness, metastases, and poor prognosis in various cancers. In this study, we successfully constructed a new EGFR nanobody-based immunotoxin rE/CUS containing cucurmosin (CUS), The immunotoxin was expressed by prokaryotic system and we obtained a yield of 5 mg protein per liter expression medium. The percentage of it's binding ability totumor cell lines A549, HepG2, SW116, which highly expressed EGFR was 55.6%, 79.6% and 97.1%, respectively, but SW620 was only 4.45%. rE/CUS has the ability to bind A549, HepG2, SW116 cells specifically, and the antigen binding capability was not affected because of extra part of CUS component. The rE/CUS significantly inhibited the cell viability against EGFR over expression tumor cell lines in a dose-and time-dependent manner. Moreover, rE/CUS also induced apoptosis of HepG2 and A549 mightily. Our results demonstrate that rE/CUS is a potential therapeutic strategy for treating EGFR-positive solid tumors.

  13. AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity.

    Science.gov (United States)

    Blair, Jessica M A; Bavro, Vassiliy N; Ricci, Vito; Modi, Niraj; Cacciotto, Pierpaolo; Kleinekathӧfer, Ulrich; Ruggerone, Paolo; Vargiu, Attilio V; Baylay, Alison J; Smith, Helen E; Brandon, Yvonne; Galloway, David; Piddock, Laura J V

    2015-03-17

    The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.

  14. Organization of DNA partners and strand exchange mechanisms during Flp site-specific recombination analyzed by difference topology, single molecule FRET and single molecule TPM.

    Science.gov (United States)

    Ma, Chien-Hui; Liu, Yen-Ting; Savva, Christos G; Rowley, Paul A; Cannon, Brian; Fan, Hsiu-Fang; Russell, Rick; Holzenburg, Andreas; Jayaram, Makkuni

    2014-02-20

    Flp site-specific recombination between two target sites (FRTs) harboring non-homology within the strand exchange region does not yield stable recombinant products. In negatively supercoiled plasmids containing head-to-tail sites, the reaction produces a series of knots with odd-numbered crossings. When the sites are in head-to-head orientation, the knot products contain even-numbered crossings. Both types of knots retain parental DNA configuration. By carrying out Flp recombination after first assembling the topologically well defined Tn3 resolvase synapse, it is possible to determine whether these knots arise by a processive or a dissociative mechanism. The nearly exclusive products from head-to-head and head-to-tail oriented "non-homologous" FRT partners are a 4-noded knot and a 5-noded knot, respectively. The corresponding products from a pair of native (homologous) FRT sites are a 3-noded knot and a 4-noded catenane, respectively. These results are consistent with non-homology-induced two rounds of dissociative recombination by Flp, the first to generate reciprocal recombinants containing non-complementary base pairs and the second to produce parental molecules with restored base pairing. Single molecule fluorescence resonance energy transfer (smFRET) analysis of geometrically restricted FRTs, together with single molecule tethered particle motion (smTPM) assays of unconstrained FRTs, suggests that the sites are preferentially synapsed in an anti-parallel fashion. This selectivity in synapse geometry occurs prior to the chemical steps of recombination, signifying early commitment to a productive reaction path. The cumulative topological, smFRET and smTPM results have implications for the relative orientation of DNA partners and the directionality of strand exchange during recombination mediated by tyrosine site-specific recombinases. Copyright © 2013. Published by Elsevier Ltd.

  15. Alterations in hemagglutinin receptor-binding specificity accompany the emergence of highly pathogenic avian influenza viruses.

    Science.gov (United States)

    Heider, Alla; Mochalova, Larisa; Harder, Timm; Tuzikov, Alexander; Bovin, Nicolai; Wolff, Thorsten; Matrosovich, Mikhail; Schweiger, Brunhilde

    2015-05-01

    Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3'SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLe(c)) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3'SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLe(x)), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical "poultry-like" receptor, 3'SLN, is provided by substitutions in the receptor

  16. Alterations in Hemagglutinin Receptor-Binding Specificity Accompany the Emergence of Highly Pathogenic Avian Influenza Viruses

    Science.gov (United States)

    Mochalova, Larisa; Harder, Timm; Tuzikov, Alexander; Bovin, Nicolai; Wolff, Thorsten; Matrosovich, Mikhail; Schweiger, Brunhilde

    2015-01-01

    ABSTRACT Highly pathogenic avian influenza viruses (HPAIVs) of hemagglutinin H5 and H7 subtypes emerge after introduction of low-pathogenic avian influenza viruses (LPAIVs) from wild birds into poultry flocks, followed by subsequent circulation and evolution. The acquisition of multiple basic amino acids at the endoproteolytical cleavage site of the hemagglutinin (HA) is a molecular indicator for high pathogenicity, at least for infections of gallinaceous poultry. Apart from the well-studied significance of the multibasic HA cleavage site, there is only limited knowledge on other alterations in the HA and neuraminidase (NA) molecules associated with changes in tropism during the emergence of HPAIVs from LPAIVs. We hypothesized that changes in tropism may require alterations of the sialyloligosaccharide specificities of HA and NA. To test this hypothesis, we compared a number of LPAIVs and HPAIVs for their HA-mediated binding and NA-mediated desialylation of a set of synthetic receptor analogs, namely, α2-3-sialylated oligosaccharides. NA substrate specificity correlated with structural groups of NAs and did not correlate with pathogenic potential of the virus. In contrast, all HPAIVs differed from LPAIVs by a higher HA receptor-binding affinity toward the trisaccharides Neu5Acα2-3Galβ1-4GlcNAcβ (3′SLN) and Neu5Acα2-3Galβ1-3GlcNAcβ (SiaLec) and by the ability to discriminate between the nonfucosylated and fucosylated sialyloligosaccharides 3′SLN and Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ (SiaLex), respectively. These results suggest that alteration of the receptor-binding specificity accompanies emergence of the HPAIVs from their low-pathogenic precursors. IMPORTANCE Here, we have found for the first time correlations of receptor-binding properties of the HA with a highly pathogenic phenotype of poultry viruses. Our study suggests that enhanced receptor-binding affinity of HPAIVs for a typical “poultry-like” receptor, 3′SLN, is provided by

  17. Protein-engineering of chitosanase from Bacillus sp. MN to alter its substrate specificity.

    Science.gov (United States)

    Regel, Eva K; Weikert, Tobias; Niehues, Anna; Moerschbacher, Bruno M; Singh, Ratna

    2018-04-01

    Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine, and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (-2), which is impossible for the wildtype enzyme. © 2017 Wiley Periodicals, Inc.

  18. Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

    Science.gov (United States)

    Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Alterations in regulatory T cells induced by specific oligosaccharides improve vaccine responsiveness in mice.

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    Marcel A Schijf

    Full Text Available Prophylactic vaccinations are generally performed to protect naïve individuals with or without suppressed immune responsiveness. In a mouse model for Influenza vaccinations the specific alterations of CD4(+CD25(+Foxp3(+ regulatory T-cells (Tregs in the immune modulation induced by orally supplied oligosaccharides containing scGOS/lcFOS/pAOS was assessed. This dietary intervention increased vaccine specific DTH responses. In addition, a significant increased percentage of T-bet(+ (Th1 activated CD69(+CD4(+ T cells (p<0.001 and reduced percentage of Gata-3(+ (Th2 activated CD69(+CD4(+T cells (p<0.001 was detected in the mesenteric lymph nodes (MLN of mice receiving scGOS/lcFOS/pAOS compared to control mice. Although no difference in the number or percentage of Tregs (CD4(+Foxp3(+ could be determined after scGOS/lcFOS/pAOS intervention, the percentage of CXCR3 (+ /T-bet(+ (Th1-Tregs was significantly reduced (p<0.05 in mice receiving scGOS/lcFOS/pAOS as compared to mice receiving placebo diets. Moreover, although no absolute difference in suppressive capacity could be detected, an alteration in cytokine profile suggests a regulatory T cell shift towards a reducing Th1 suppression profile, supporting an improved vaccination response.These data are indicative for improved vaccine responsiveness due to reduced Th1 suppressive capacity in the Treg population of mice fed the oligosaccharide specific diet, showing compartmentalization within the Treg population. The modulation of Tregs to control immune responses provides an additional arm of intervention using alternative strategies possibly leading to the development of improved vaccines.

  20. Deletion of the nucleotide excision repair gene Ercc1 reduces immunoglobulin class switching and alters mutations near switch recombination junctions

    NARCIS (Netherlands)

    C.E. Schrader; J. Vardo; E. Linehan; M.Z. Twarog; L.J. Niedernhofer (Laura); J. Stavnezer; J.H.J. Hoeijmakers (Jan)

    2004-01-01

    textabstractThe structure-specific endonuclease ERCC1-XPF is an essential component of the nucleotide excision DNA repair pathway. ERCC1-XPF nicks double-stranded DNA immediately adjacent to 3' single-strand regions. Substrates include DNA bubbles and flaps. Furthermore, ERCC1 interacts with Msh2, a

  1. Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

    Science.gov (United States)

    Fan, Hsiu-Fang

    2012-01-01

    Tyrosine family recombinases (YRs) are widely utilized in genome engineering systems because they can easily direct DNA rearrangement. Cre recombinases, one of the most commonly used types of YRs, catalyze site-specific recombination between two loxP sites without the need for high-energy cofactors, other accessory proteins or a specific DNA target sequence between the loxP sites. Previous structural, analytical ultracentrifuge and electrophoretic analyses have provided details of the reaction kinetics and mechanisms of Cre recombinase activity; whether there are reaction intermediates or side pathways involved has been left unaddressed. Using tethered particle motion (TPM), the Cre-mediated site-specific recombination process has been delineated, from beginning to end, at the single-molecule level, including the formation of abortive complexes and wayward complexes blocking inactive nucleoprotein complexes from entering the recombination process. Reversibility in the strand-cleavage/-ligation process and the formation of a thermally stable Holliday junction intermediate were observed within the Cre-mediated site-specific recombination process. Rate constants for each elementary step, which explain the overall reaction outcomes under various conditions, were determined. Taking the findings of this study together, they demonstrate the potential of single-molecule methodology as an alternative approach for exploring reaction mechanisms in detail. PMID:22467208

  2. Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases

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    Todo Tomoki

    2006-09-01

    Full Text Available Abstract Background Oncolytic herpes simplex virus (HSV vectors that specifically replicate in and kill tumor cells sparing normal cells are a promising cancer therapy. Traditionally, recombinant HSV vectors have been generated through homologous recombination between the HSV genome and a recombination plasmid, which usually requires laborious screening or selection and can take several months. Recent advances in bacterial artificial chromosome (BAC technology have enabled cloning of the whole HSV genome as a BAC plasmid and subsequent manipulation in E. coli. Thus, we sought a method to generate recombinant oncolytic HSV vectors more easily and quickly using BAC technology. Results We have developed an HSV-BAC system, termed the Flip-Flop HSV-BAC system, for the rapid generation of oncolytic HSV vectors. This system has the following features: (i two site-specific recombinases, Cre and FLPe, are used sequentially to integrate desired sequences and to excise the BAC sequences, respectively; and (ii the size of the HSV-BAC-insert genome exceeds the packaging limit of HSV so only correctly recombined virus grows efficiently. We applied this to the construction of an HSV-BAC plasmid that can be used for the generation of transcriptionally-targeted HSV vectors. BAC sequences were recombined into the UL39 gene of HSV ICP4-deletion mutant d120 to generate M24-BAC virus, from which HSV-BAC plasmid pM24-BAC was isolated. An ICP4 expression cassette driven by an exogenous promoter was re-introduced to pM24-BAC by Cre-mediated recombination and nearly pure preparations of recombinant virus were obtained typically in two weeks. Insertion of the ICP4 coding sequence alone did not restore viral replication and was only minimally better than an ICP4-null construct, whereas insertion of a CMVIE promoter-ICP4 transgene (bM24-CMV efficiently drove viral replication. The levels of bM24-CMV replication in tumor cells varied considerably compared to hrR3 (UL39

  3. Component-resolved diagnosis of baker's allergy based on specific IgE to recombinant wheat flour proteins.

    Science.gov (United States)

    Sander, Ingrid; Rihs, Hans-Peter; Doekes, Gert; Quirce, Santiago; Krop, Esmeralda; Rozynek, Peter; van Kampen, Vera; Merget, Rolf; Meurer, Ursula; Brüning, Thomas; Raulf, Monika

    2015-06-01

    Sensitization to wheat flour plays an important role in the development and diagnosis of baker's asthma. We evaluated wheat allergen components as sensitizers for bakers with work-related complaints, with consideration of cross-reactivity to grass pollen. Nineteen recombinant wheat flour proteins and 2 cross-reactive carbohydrate determinants were tested by using CAP-FEIA in sera of 101 bakers with wheat flour allergy (40 German, 37 Dutch, and 24 Spanish) and 29 pollen-sensitized control subjects with wheat-specific IgE but without occupational exposure. IgE binding to the single components was inhibited with wheat flour, rye flour, and grass pollen. The diagnostic efficiencies of IgE tests with single allergens and combinations were evaluated by assessing their ability to discriminate between patients with baker's allergy and control subjects based on receiver operating characteristic analyses. Eighty percent of bakers had specific IgE levels of 0.35 kUA/L or greater and 91% had specific IgE levels of 0.1 kUA/L or greater to at least one of the 21 allergens. The highest frequencies of IgE binding were found for thiol reductase (Tri a 27) and the wheat dimeric α-amylase inhibitor 0.19 (Tri a 28). Cross-reactivity to grass pollen was proved for 9 components, and cross-reactivity to rye flour was proved for 18 components. A combination of IgE tests to 5 components, Tri a 27, Tri a 28, tetrameric α-amylase inhibitor CM2 (Tri a 29.02), serine protease inhibitor-like allergen (Tri a 39), and 1-cys-peroxiredoxin (Tri a 32), produced the maximal area under the curve (AUC = 0.84) in receiver operating characteristic analyses, but this was still lower than the AUC for wheat- or rye flour-specific IgE (AUC = 0.89 or 0.88, respectively). Component-resolved diagnostics help to distinguish between sensitization caused by occupational flour exposure and wheat seropositivity based on cross-reactivity to grass pollen. For routine diagnosis of baker's allergy, however

  4. Eye-specific projections of retinogeniculate axons are altered in albino mice.

    Science.gov (United States)

    Rebsam, Alexandra; Bhansali, Punita; Mason, Carol A

    2012-04-04

    The divergence of retinal ganglion cell (RGC) axons into ipsilateral and contralateral projections at the optic chiasm and the subsequent segregation of retinal inputs into eye-specific domains in their target, the dorsal lateral geniculate nucleus (dLGN), are crucial for binocular vision. In albinism, affected individuals exhibit a lack or reduction of pigmentation in the eye and skin, a concomitant reduced ipsilateral projection, and diverse visual defects. Here we investigate how such altered decussation affects eye-specific retinogeniculate targeting in albino mice using the C57BL/6 Tyr(c-2J/c-2J) strain, in which tyrosinase, necessary for melanogenesis, is mutated. In albino mice, fewer RGCs from the ventrotemporal (VT) retina project ipsilaterally, reflected in a decrease in cells expressing ipsilateral markers. In addition, a population of RGCs from the VT retina projects contralaterally and, within the dLGN, their axons cluster into a patch separated from the contralateral termination area. Furthermore, eye-specific segregation is not complete in the albino dLGN and, upon perturbing postnatal retinal activity with epibatidine, the ipsilateral projection fragments and the aberrant contralateral patch disappears. These results suggest that the defects in afferent targeting and activity-dependent refinement in the albino dLGN arise from RGC misspecification together with potential perturbations of early activity patterns in the albino retina.

  5. Fast track antibody V-gene rescue, recombinant expression in plants and characterization of a PfMSP4-specific antibody.

    Science.gov (United States)

    Kapelski, Stephanie; Boes, Alexander; Spiegel, Holger; de Almeida, Melanie; Klockenbring, Torsten; Reimann, Andreas; Fischer, Rainer; Barth, Stefan; Fendel, Rolf

    2015-02-05

    Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequences enables not only the recombinant expression of the original antibody for further applications, but opens the road for antibody engineering towards innovative diagnostic or therapeutic applications. A time- and cost-efficient production system enabling the detailed analysis of the antibodies is an essential requirement in this context. Sequences were rescued from three hybridoma cell lines, subjected to sequence analysis, subcloned into binary expression vectors and recombinantly expressed as chimeric mAb (constant regions of human IgG1:k1) in Nicotiana benthamiana plants. The properties of the recombinant and the murine mAbs were compared using competition enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. The recognition of native PfMSP4 by the recombinant mAb was analysed by immunofluorescence staining of Pf 3D7A schizonts and by western blot analysis of merozoite extract. The rescued sequences of all three hybridoma cell lines were identical. The recombinant mAb was successfully expressed as IgG in plants at moderate levels (45 mg/kg fresh leaf weight). Preservation of the original epitope was demonstrated in a competition ELISA, using recombinant mAb and the three murine mAbs. EGF_PfMSP4-specific affinities were determined by SPR spectroscopy to 8 nM and 10 nM for the murine or recombinant mAb, respectively. Binding to parasite PfMSP4 was confirmed in an immunofluorescence assay showing a characteristic staining pattern and by western blot

  6. Oral vaccination of mice with recombinant Schistosoma japonicum proteins induces specific anti-parasite antibodies and damage to adult worms after a challenge infection.

    Science.gov (United States)

    Yang, W; Gobert, G N; McManus, D P

    1997-07-01

    Mucosal immunisation by the oral route represents a cheap and simple method for delivering protective antigens to a host against gastrointestinal and respiratory pathogens. In the case of schistosome (bloodfluke) worms, 2 life-cycle stages may be exposed to the host's mucosa; the larval schistosomulum is exposed to the respiratory mucosa and, depending on the species, the egg may come into contact with the intestinal or urinogenital mucosa. Both IgA and some Isotypes of IgG have been implicated in protective immunity against schistosomiasis in humans and in experimental animal models. We have used a novel approach to determine whether schistosome-specific antibodies and protective immunity could be generated in mice by oral administration of bacterial lysates containing recombinant Schistosoma japonicum proteins. The mice produced specific antibodies to paramyosin and GST26, 2 important vaccine candidates for schistosomiasis, but there was no significant reduction in worm burdens in groups of mice immunised with either protein. Significantly, however, transmission electron microscopy revealed damage to the teguments of adult female and male S. japonicum worms obtained from mice vaccinated with recombinant paramyosin; there was also extensive damage to the tegument of male worms recovered from mice vaccinated with recombinant GST26. Our observations that oral vaccination with bacterial lysates containing recombinant proteins induced particular classes and subclasses of circulating antibodies with resultant damage to the surface of adult worms may have important implications for the future development of oral vaccines against a systemic infection such as schistosomiasis.

  7. Does Scoliosis-Specific Exercise Treatment in Adolescence Alter Adult Quality of Life?

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    Maciej Płaszewski

    2014-01-01

    Full Text Available Objective. Health-related quality of life in adults, who in adolescence participated in a scoliosis-specific exercise program, was not previously studied. Design. Cross-sectional study, with retrospective data collection. Material and Methods. Homogenous groups of 68 persons (43 women aged 30.10 (25–39 years, with mild or moderate scoliosis, and 76 (38 women able-bodied persons, aged 30.11 (24–38 years, who 16.5 (12–26 years earlier had completed scoliosis-specific exercise or observation regimes, participated. Their respiratory characteristics did not differ from predicted values. The WHOQOL-BREF questionnaire, Oswestry Disability Questionnaire, and pain scale (VAS were applied. Results. The transformed WHOQOL-BREF scores ranged from 54.6 ± 11.19 in the physical domain in the mild scoliotic subgroup to 77.1 ± 16.05 in the social domain in the able-bodied subgroup. The ODQ values did not generally exceed 5.3 ± 7.53. Inter- and intragroup differences were nonsignificant. Age, marital status, education, and gender were significantly associated with the ODQ scores. Significant association between the ODQ and WHOQOL-BREF social relationships domain scores with the participation in exercise treatment was found. Conclusions. Participants with the history of exercise treatment generally did not differ significantly from their peers who were only under observation. This study cannot conclude that scoliosis-specific exercise treatment in adolescence alters quality of life in adulthood.

  8. Altered Cytokine Production By Specific Human Peripheral Blood Cell Subsets Immediately Following Spaceflight

    Science.gov (United States)

    Crucian, Brian E.; Cubbage, Michael L.; Sams, Clarence F.

    1999-01-01

    In this study, we have attempted to combine standard immunological assays with the cellular resolving power of the flow cytometer to positively identify the specific cell types involved in spaceflight-induced immune alterations. We have obtained whole blood samples from 27 astronauts collected at three timepoints (L-10, R+0 and R+3) surrounding four recent space shuttle missions. The duration of these missions ranged from 10 to 18 days. Assays performed included serum/urine cortisol, comprehensive subset phenotyping, assessment of cellular activation markers and intracellular cytokine production following mitogenic stimulation. Absolute levels of peripheral granulocytes were significantly elevated following spaceflight, but the levels of circulating lymphocytes and monocytes were unchanged. Lymphocyte subset analysis demonstrated trends towards a decreased percentage of T cells and an increased percentage of B cells. Nearly all of the astronauts exhibited an increased CD4:CD8 ratio, which was dramatic in some individuals. Assessment of memory (CD45RA+) vs. naive (CD45RO+) CD4+ T cell subsets was more ambiguous, with subjects tending to group more as a flight crew. All subjects from one mission demonstrated an increased CD45RA:CD45RO ratio, while all subjects from another Mission demonstrated a decreased ratio. While no significant trend was seen in the monocyte population as defined by scatter, a decreased percentage of the CD14+ CD16+ monocyte subset was seen following spaceflight in all subjects tested. In general, most of the cellular changes described above which were assessed at R+O and compared to L-10 trended to pre-flight levels by R+3. Although no significant differences were seen in the expression of the cellular activation markers CD69 and CD25 following exposure to microgravity, significant alterations were seen in cytokine production in response to mitogenic activation for specific subsets. T cell (CD3+) production of IL-2 was significantly decreased

  9. An oral recombinant Salmonella enterica serovar Typhimurium mutant elicits systemic antigen-specific CD8+ T cell cytokine responses in mice

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    Chin'ombe Nyasha

    2009-04-01

    Full Text Available Abstract Background The induction of antigen-specific CD8+ T cell cytokine responses against an attenuated, oral recombinant Salmonella enterica serovar Typhimurium vaccine expressing a green fluorescent protein (GFP model antigen was investigated. A GFP expression plasmid was constructed in which the gfp gene was fused in-frame with the 5' domain of the Escherichia coli β-galactosidase α-gene fragment with expression under the lac promoter. Groups of mice were orally immunized three times with the bacteria and systemic CD8+ T cell cytokine responses were evaluated. Results High level of the GFP model antigen was expressed by the recombinant Salmonella vaccine vector. Systemic GFP-specific CD8+ T cell cytokine (IFN-γ and IL-4 immune responses were detected after mice were orally vaccinated with the bacteria. It was shown that 226 net IFN-γ and 132 net IL-4 GFP-specific SFUs/10e6 splenocytes were formed in an ELISPOT assay. The level of IFN-γ produced by GFP peptide-stimulated cells was 65.2-fold above background (p Conclusion These results suggested that a high expressing recombinant Salmonella vaccine given orally to mice would elicit antigen-specific CD8+ T cell responses in the spleen. Salmonella bacteria may, therefore, be used as potential mucosal vaccine vectors.

  10. Functionally Distinct Tendons From Elastin Haploinsufficient Mice Exhibit Mild Stiffening and Tendon-Specific Structural Alteration.

    Science.gov (United States)

    Eekhoff, Jeremy D; Fang, Fei; Kahan, Lindsey G; Espinosa, Gabriela; Cocciolone, Austin J; Wagenseil, Jessica E; Mecham, Robert P; Lake, Spencer P

    2017-11-01

    Elastic fibers are present in low quantities in tendon, where they are located both within fascicles near tenocytes and more broadly in the interfascicular matrix (IFM). While elastic fibers have long been known to be significant in the mechanics of elastin-rich tissue (i.e., vasculature, skin, lungs), recent studies have suggested a mechanical role for elastic fibers in tendons that is dependent on specific tendon function. However, the exact contribution of elastin to properties of different types of tendons (e.g., positional, energy-storing) remains unknown. Therefore, this study purposed to evaluate the role of elastin in the mechanical properties and collagen alignment of functionally distinct supraspinatus tendons (SSTs) and Achilles tendons (ATs) from elastin haploinsufficient (HET) and wild type (WT) mice. Despite the significant decrease in elastin in HET tendons, a slight increase in linear stiffness of both tendons was the only significant mechanical effect of elastin haploinsufficiency. Additionally, there were significant changes in collagen nanostructure and subtle alteration to collagen alignment in the AT but not the SST. Hence, elastin may play only a minor role in tendon mechanical properties. Alternatively, larger changes to tendon mechanics may have been mitigated by developmental compensation of HET tendons and/or the role of elastic fibers may be less prominent in smaller mouse tendons compared to the larger bovine and human tendons evaluated in previous studies. Further research will be necessary to fully elucidate the influence of various elastic fiber components on structure-function relationships in functionally distinct tendons.

  11. Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution.

    Science.gov (United States)

    Lingen, Bettina; Kolter-Jung, Doris; Dünkelmann, Pascal; Feldmann, Ralf; Grötzinger, Joachim; Pohl, Martina; Müller, Michael

    2003-08-04

    Alteration of the substrate specificity of thiamin diphosphate (ThDP)-dependent benzoylformate decarboxylase (BFD) by error-prone PCR is described. Two mutant enzymes, L476Q and M365L-L461S, were identified that accept ortho-substituted benzaldehyde derivatives as donor substrates, which leads to the formation of 2-hydroxy ketones. Both variants, L476Q and M365L-L461S, selectively catalyze the formation of enantiopure (S)-2-hydroxy-1-(2-methylphenyl)propan-1-one with excellent yields, a reaction which is only poorly catalyzed by the wild-type enzyme. Different ortho-substituted benzaldehyde derivatives, such as 2-chloro-, 2-methoxy-, or 2-bromobenzaldehyde are accepted as donor substrates by both BFD variants as well and conversion with acetaldehyde resulted in the corresponding (S)-2-hydroxy-1-phenylpropan-1-one derivatives. As deduced from modeling studies based on the 3D structure of wild-type BFD, reduction of the side chain size at position L461 probably results in an enlarged substrate binding site and facilitates the initial binding of ortho-substituted benzaldehyde derivatives to the cofactor ThDP.

  12. Altering the sex determination pathway in Drosophila fat body modifies sex-specific stress responses

    Science.gov (United States)

    Neckameyer, Wendi S.

    2014-01-01

    The stress response in Drosophila melanogaster reveals sex differences in behavior, similar to what has been observed in mammals. However, unlike mammals, the sex determination pathway in Drosophila is well established, making this an ideal system to identify factors involved in the modulation of sex-specific responses to stress. In this study, we show that the Drosophila fat body, which has been shown to be important for energy homeostasis and sex determination, is a dynamic tissue that is altered in response to stress in a sex and time-dependent manner. We manipulated the sex determination pathway in the fat body via targeted expression of transformer and transformer-2 and analyzed these animals for changes in their response to stress. In the majority of cases, manipulation of transformer or transformer-2 was able to change the physiological output in response to starvation and oxidative stress to that of the opposite sex. Our data also uncover the possibility of additional downstream targets for transformer and transformer-2 that are separate from the sex determination pathway and can influence behavioral and physiological responses. PMID:24789992

  13. Establishment of human sperm-specific voltage-dependent anion channel 3 recombinant vector for the production of a male contraceptive vaccine

    Directory of Open Access Journals (Sweden)

    Asmarinah Asmarinah

    2012-05-01

    Full Text Available Background: The aim of this study was to construct a recombinant vector of human sperm specific VDAC3 gene for production of VDAC3 antibody, which is potential as male contraception vaccine.Methods: Target fragment sequence of VDAC3 gene was obtained through amplification of human sperm VDAC3 cDNA with primers covering exon 5 to exon 8. Its PCR product in size of 435 bp was cloned to the pET101/D-TOPO expression vector (5753 bp. E. coli bacteria were transformed with this vector. Cloning of VDAC3 fragment gene to the vector was confirmed by the using of XbaI restriction enzyme and PCR colony method with primers covering exons 5-8 of the human VDAC3 gene.Results: Alignment analysis of amplified fragment covering exon 5 to exon 8 of VDAC3 gene showed 94% homology to human VDAC3 gene from databank. After cloning to the expression vector and transformation to E. coli competent cells, twelve colonies could grow in culture media. Gel electrophoresis of sliced VDAC3 recombinant vector showed a single band in the size of 6181 bp in 8 colonies. After application of PCR colony and amplicon sequencing, the result showed a single band in the size of 435 bp and fragment sequence with 94% identity to human VDAC3 gene.Conclusion: The construction of human sperm specific VDAC3 gene recombinant vector was established in this study. In the future, this recombinant vector will be used to produce VDAC3 antibody for the development of a male contraception vaccine. (Med J Indones. 2012;21:61-5Keywords: Contraception, recombinant vector, sperm, VDAC3

  14. Establishment of tumor-specific copy number alterations from plasma DNA of patients with cancer.

    Science.gov (United States)

    Heitzer, Ellen; Auer, Martina; Hoffmann, Eva Maria; Pichler, Martin; Gasch, Christin; Ulz, Peter; Lax, Sigurd; Waldispuehl-Geigl, Julie; Mauermann, Oliver; Mohan, Sumitra; Pristauz, Gunda; Lackner, Carolin; Höfler, Gerald; Eisner, Florian; Petru, Edgar; Sill, Heinz; Samonigg, Hellmut; Pantel, Klaus; Riethdorf, Sabine; Bauernhofer, Thomas; Geigl, Jochen B; Speicher, Michael R

    2013-07-15

    With the increasing number of available predictive biomarkers, clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. We tested whether tumor-specific copy number changes can be inferred from the peripheral blood of patients with cancer. To this end, we determined the plasma DNA size distribution and the fraction of mutated plasma DNA fragments with deep sequencing and an ultrasensitive mutation-detection method, i.e., the Beads, Emulsion, Amplification, and Magnetics (BEAMing) assay. When analyzing the plasma DNA of 32 patients with Stage IV colorectal carcinoma, we found that a subset of the patients (34.4%) had a biphasic size distribution of plasma DNA fragments that was associated with increased circulating tumor cell numbers and elevated concentration of mutated plasma DNA fragments. In these cases, we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. Thus, we could analyze the current copy number status of the tumor genome, which was in some cases many years after diagnosis of the primary tumor. An unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. When we analyzed plasma DNA from 35 patients with metastatic breast cancer, we made similar observations suggesting that our approach may be applicable to a variety of tumor entities. This is the first description of such a biphasic distribution in a surprisingly high proportion of cancer patients which may have important implications for tumor diagnosis and monitoring. Copyright © 2013 UICC.

  15. Vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: Lessons from the past and novel mechanisms of action for the future.

    Science.gov (United States)

    Valenta, Rudolf; Campana, Raffaela; Focke-Tejkl, Margit; Niederberger, Verena

    2016-02-01

    In the past, the development of more effective, safe, convenient, broadly applicable, and easy to manufacture vaccines for allergen-specific immunotherapy (AIT) has been limited by the poor quality of natural allergen extracts. Progress made in the field of molecular allergen characterization has now made it possible to produce defined vaccines for AIT and eventually for preventive allergy vaccination based on recombinant DNA technology and synthetic peptide chemistry. Here we review the characteristics of recombinant and synthetic allergy vaccines that have reached clinical evaluation and discuss how molecular vaccine approaches can make AIT more safe and effective and thus more convenient. Furthermore, we discuss how new technologies can facilitate the reproducible manufacturing of vaccines of pharmaceutical grade for inhalant, food, and venom allergens. Allergy vaccines in clinical trials based on recombinant allergens, recombinant allergen derivatives, and synthetic peptides allow us to target selectively different immune mechanisms, and certain of those show features that might make them applicable not only for therapeutic but also for prophylactic vaccination. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Sensory experience alters specific branches of individual corticocortical axons during development

    NARCIS (Netherlands)

    Bruno, A.C; Hahn, T.T.; Wallace, D.J.; de Kock, C.P.J.; Sakmann, B.

    2009-01-01

    Sensory experience can, over the course of days to weeks, produce long-lasting changes in brain function. Recent studies suggest that functional plasticity is mediated by alterations of the strengths of existing synapses or dynamics of dendritic spines. Alterations of cortical axons could also

  17. QTL delineation for five fiber quality traits based on an intra-specific Gossypium hirsutum L. recombinant inbred line population.

    Science.gov (United States)

    Jia, Xiaoyun; Wang, Hantao; Pang, Chaoyou; Ma, Qifeng; Su, Junji; Wei, Hengling; Song, Meizhen; Fan, Shuli; Yu, Shuxun

    2018-02-08

    Gossypium hirsutum L. is the most important fiber crop worldwide and contributes to more than 95% of global cotton production. Marker-assisted selection (MAS) is an effective approach for improving fiber quality, and quantitative trait loci (QTL) mapping of fiber quality traits is important for cotton breeding. In this study, a permanent intra-specific recombinant inbred line (RIL) population containing 137 families was used for fiber quality testing. Based on a previously reported high-density genetic map with an average marker distance of 0.63 cM, 186 additive QTLs were obtained for five fiber quality traits over five consecutive years, including 39 for fiber length (FL), 36 for fiber strength (FS), 50 for fiber uniformity (FU), 33 for micronaire (MC) and 28 for fiber elongation (FE). Three stable QTLs, qMC-A4-1, qMC-D2-3 and qFS-D9-1, were detected in four datasets, and another eight stable QTLs, qMC-A4-2, qMC-D11-2, qFU-A9-1, qFU-A10-4, qFS-D11-1, qFL-D9-2, qFL-D11-1 and qFE-A3-2, were detected in three datasets. The annotated genes in these 11 stable QTLs were collected, and these genes included many transcription factors with functions during fiber development. 33 QTL coincidence regions were found, and these involved nearly half of the total QTLs. Four chromosome regions containing at least 6 QTLs were promising for fine mapping. In addition, 41 pairs of epistatic QTLs (e-QTLs) were screened, including 6 for FL, 30 for FS, 2 for FU and 3 for MC. The identification of stable QTLs adds valuable information for further QTL fine mapping and gene positional cloning for fiber quality genetic detection and provides useful markers for further molecular breeding in enhancing fiber quality.

  18. Recombinant in vitro assembled hepatitis C virus core particles induce strong specific immunity enhanced by formulation with an oil-based adjuvant

    Directory of Open Access Journals (Sweden)

    NELSON ACOSTA-RIVERO

    2009-01-01

    Full Text Available In the present work, immunogenicity of recombinant in vitro assembled hepatitis C virus core particles, HCcAg.120-VLPs, either alone or in combination with different adjuvants was evaluated in BALB/c mice. HCcAg.120-VLPs induced high titers of anti-HCcAg.120 antibodies and virus-specific cellular immune responses. Particularly, HCcAg.120-VLPs induced specific delayed type hypersensitivity, and generated a predominant T helper 1 cytokine pro file in immunized mice. In addition, HCcAg.120-VLPs prime splenocytes proliferate in vitro against different HCcAg.120-specific peptides, depending on either the immunization route or the adjuvant used. Remarkably, immunization with HCcAg.120-VLPs/Montanide ISA888 formulation resulted in a significant control of vaccinia virus titer in mice after challenge with a recombinant vaccinia virus expressing HCV core protein, vvCore. Animals immunized with this formulation had a marked increase in the number of IFN-γ producing spleen cells, after stimulation with P815 cells infected with vvCore. These results suggest the use of recombinant HCV core particles as components of therapeutic or preventive vaccine candidates against HCV.

  19. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone

    International Nuclear Information System (INIS)

    Balasuriya, Udeni B.R.; Dobbe, Jessika C.; Heidner, Hans W.; Smalley, Victoria L.; Navarrette, Andrea; Snijder, Eric J.; MacLachlan, N. James

    2004-01-01

    We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions

  20. Usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis.

    Science.gov (United States)

    Rastawicki, Waldemar; Smietafiska, Karolina; Chrost, Anna; Wolkowicz, Tomasz; Rokosz-Chudziak, Natalia

    Proper analysis of the human immune response is crucial in the laboratory diagnosis of many bacterial infections-The current serological diagnosis of yersiniosis often is carried out using ELISA with native antigens. However, recombinant proteins increase the specificity of the serological assays, particularly in patients with chronic, non- specific infections. The aim of the present study was to evaluate the usefulness of in-house obtained recombinant proteins Yop of Yersinia enterocolitica as highly specific antigens in ELISA and recom-dot performed in the serodiagnosis of yersiniosis. Recombinant YopD, YopB, YopE and V-Ag proteins of Y enterocolitica were expressing in E. coli BL21 (DE3) using the pET-30 Ek/LIC expression vector (Novagen). Purification was accomplished by immobilized metal (Ni2) affinity column chromatography (His-trap). The proteins were used as antigens in standard ELISA and recom-dot assay, which was performed on nitrocellulose strips. The study population, used for characterization of the humoral immune response to the recombinant proteins, consisted of 74 patients suspected for Y enterocolitica infection and 41 clinically healthy blood donors. Some of the results obtained by ELISA and recom-dot were compared with results obtained by commercial western-blot Yersinia (Virotech). In the group of patients suspected for yersiniosis in clinical investigation the most positive results were obtained in ELISA with the recombinant protein YopD (IgA respectively 25 (42.4%), IgG 41 (69.5%), IgM 24 (40.7%). The percentage ofpositive results in the group of blood donors did not exceed 10.0% in IgG and 5.0% in IgA/IgM classes of immunoglobulin. The results obtained in the recom-dot assay showed that among 74 tested serum samples obtained from individuals suspected of yersiniosis the most common IgA, IgG and IgM antibodies were found for recombinant protein YopD (respectively IgG in 60.8%, IgA in 37.8% and IgM in 33.8% of serum samples). IgG antibodies to

  1. Developmental ethanol exposure alters the morphology of mouse prefrontal neurons in a layer-specific manner.

    Science.gov (United States)

    Louth, Emma L; Luctkar, Hanna D; Heney, Kayla A; Bailey, Craig D C

    2018-01-01

    Chronic developmental exposure to ethanol can lead to a wide variety of teratogenic effects, which in humans are known as fetal alcohol spectrum disorders (FASD). Individuals affected by FASD may exhibit persistent impairments to cognitive functions such as learning, memory, and attention, which are highly dependent on medial prefrontal cortex (mPFC) circuitry. The objective of this study was to determine long-term effects of chronic developmental ethanol exposure on mPFC neuron morphology, in order to better-understand potential neuronal mechanisms underlying cognitive impairments associated with FASD. C57BL/6-strain mice were exposed to ethanol or an isocaloric/isovolumetric amount of sucrose (control) via oral gavage, administered both to the dam from gestational day 10-18 and directly to pups from postnatal day 4-14. Brains from male mice were collected at postnatal day 90 and neurons were stained using a modified Golgi-Cox method. Pyramidal neurons within layers II/III, V and VI of the mPFC were imaged, traced in three dimensions, and assessed using Sholl and branch structure analyses. Developmental ethanol exposure differentially impacted adult pyramidal neuron morphology depending on mPFC cortical layer. Neurons in layer II/III exhibited increased size and diameter of dendrite trees, whereas neurons in layer V were not affected. Layer VI neurons with long apical dendrites had trees with decreased diameter that extended farther from the soma, and layer VI neurons with short apical dendrite trees exhibited decreased tree size overall. These layer-specific alterations to mPFC neuron morphology may form a novel morphological mechanism underlying long-term mPFC dysfunction and resulting cognitive impairments in FASD. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Delayed neurogenesis leads to altered specification of ventrotemporal retinal ganglion cells in albino mice

    Science.gov (United States)

    2014-01-01

    Background Proper binocular vision depends on the routing at the optic chiasm of the correct proportion of retinal ganglion cell (RGC) axons that project to the same (ipsilateral) and opposite (contralateral) side of the brain. The ipsilateral RGC projection is reduced in mammals with albinism, a congenital disorder characterized by deficient pigmentation in the skin, hair, and eyes. Compared to the pigmented embryonic mouse retina, the albino embryonic mouse retina has fewer RGCs that express the zinc-finger transcription factor, Zic2, which is transiently expressed by RGCs fated to project ipsilaterally. Here, using Zic2 as a marker of ipsilateral RGCs, Islet2 as a marker of contralateral RGCs, and birthdating, we investigate spatiotemporal dynamics of RGC production as they relate to the phenotype of diminished ipsilateral RGC number in the albino retina. Results At embryonic day (E)15.5, fewer Zic2-positive (Zic2+) RGCs are found in the albino ventrotemporal (VT) retina compared with the pigmented VT retina, as we previously reported. However, the reduction in Zic2+ RGCs in the albino is not accompanied by a compensatory increase in Zic2-negative (Zic2−) RGCs, resulting in fewer RGCs in the VT retina at this time point. At E17.5, however, the number of RGCs in the VT region is similar in pigmented and albino retinae, implicating a shift in the timing of RGC production in the albino. Short-term birthdating assays reveal a delay in RGC production in the albino VT retina between E13 and E15. Specifically, fewer Zic2+ RGCs are born at E13 and more Zic2− RGCs are born at E15. Consistent with an increase in the production of Zic2− RGCs born at later ages, more RGCs at E17.5 express the contralateral marker, Islet2, in the albino VT retina compared with the pigmented retina. Conclusions A delay in neurogenesis in the albino retina is linked to the alteration of RGC subtype specification and consequently leads to the reduced ipsilateral projection that

  3. Altered awareness of action in schizophrenia: a specific deficit in predicting action consequences.

    Science.gov (United States)

    Voss, Martin; Moore, James; Hauser, Marta; Gallinat, Juergen; Heinz, Andreas; Haggard, Patrick

    2010-10-01

    Patients suffering from schizophrenia may report unusual experiences of their own actions. They may either feel that external forces are controlling their actions or even their thoughts, or they may feel in control of events that in fact are not caused by their actions. Most theories link these disturbances in the sense of agency to deficits in motor prediction, resulting in a mismatch between predicted and actual sensory feedback at a central comparator mechanism. Such theories therefore can account for situations in which the sense of agency is reduced. However, other experiments as well as clinical observations show an enhanced rather than reduced sense of agency in schizophrenic patients. Here, we distinguish between a predictive and a retrospective mechanism where both contribute to the experience of agency, and show that schizophrenia is associated with a specific impairment to the predictive component. We measured subjective time estimates of self-initiated voluntary action (a key press) that were followed by a sensory effect (a tone). When the voluntary actions had a high probability of causing tones, healthy volunteers showed a predictive shift of the perceptual estimate of the action towards the tone, even on occasional trials where the tone was omitted. No such shift occurred in the absence of the tone on blocks when tones were less frequent. The predictive component of action awareness was calculated as the difference between time estimates on 'action only' trials from blocks with lower and higher tone probabilities. Schizophrenic patients lacked this predictive component of action awareness, showing a shift on 'action only' trials, regardless of the probability of the tone. Importantly, the schizophrenic deficit in predicting the relation between action and effect was strongly correlated with severity of positive psychotic symptoms, specifically delusions and hallucinations. Furthermore, the patients showed an exaggerated retrospective binding between

  4. A rapid method for site-specific mutagenesis and directional subcloning by using the polymerase chain reaction to generate recombinant circles.

    Science.gov (United States)

    Jones, D H; Howard, B H

    1990-02-01

    Site-specific mutagenesis and directional subcloning were accomplished by using the polymerase chain reaction to generate products that can recombine to form circular DNA. This DNA was transfected into E. coli without phosphorylation of primers, restriction enzyme digestion or ligation. Specifically, the polymerase chain reaction was used to generate products that when combined, denatured and reannealed, form double-stranded DNA with discrete, cohesive single-stranded ends. The generation of these cohesive ends of DNA permits the formation of precise, directional DNA joints without dependence on enzyme restriction sites. The primers were designed such that these cohesive single-stranded ends annealed to form circular DNA. The recombinant of interest was generated following only 14 amplification cycles. These recombinant circles of DNA were directly transfected into E. coli. In the mutagenesis protocol, the desired mutant was obtained at 83%-100% efficiency. Unwanted mutations were not detected, indicating a less than 0.025% nucleotide misincorporation frequency. In the directional subcloning protocol, inserts were positioned precisely in the recipient plasmid and were in the correct orientation. One unwanted mutation was detected after sequencing 900 bases, indicating a 0.11% nucleotide misincorporation frequency. Each manipulation, from setting up for the DNA amplification to transfection into E. coli. can easily be accomplished in one day.

  5. Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

    Science.gov (United States)

    Du, Zhimei; Treiber, David; McCarter, John D; Fomina-Yadlin, Dina; Saleem, Ramsey A; McCoy, Rebecca E; Zhang, Yuling; Tharmalingam, Tharmala; Leith, Matthew; Follstad, Brian D; Dell, Brad; Grisim, Brent; Zupke, Craig; Heath, Carole; Morris, Arvia E; Reddy, Pranhitha

    2015-01-01

    The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in

  6. Induction of neutralizing antibodies specific for the envelope proteins of the koala retrovirus by immunization with recombinant proteins or with DNA.

    Science.gov (United States)

    Fiebig, Uwe; Dieckhoff, Britta; Wurzbacher, Christian; Möller, Annekathrin; Kurth, Reinhard; Denner, Joachim

    2015-04-30

    The koala retrovirus (KoRV) is the result of a transspecies transmission of a gammaretrovirus with fatal consequences for the new host. Like many retroviruses, KoRV induces lymphoma, leukemia and an immunodeficiency that is associated with opportunistic infections in the virus-infected animals. We recently reported the induction of neutralizing antibodies by immunization with the recombinant ectodomain of the transmembrane envelope protein p15E of KoRV. Since the neutralization titers of the p15E-specific sera were only moderate, we investigated the use of the surface envelope protein gp70 to induce neutralizing antibodies. We immunized rats and goats with the recombinant gp70 protein of the KoRV, an unglycosylated protein of 52kD (rgp70/p52) or with the corresponding DNA. In parallel we immunized with recombinant rp15E or with a combination of rp15E and rgp70/p52. In all cases binding and neutralizing antibodies were induced. The gp70-specific sera had titers of neutralizing antibodies that were 15-fold higher than the p15E-specific sera. Combining rp15E and rgp70/p52 did not significantly increase neutralizing titers compared to rgp70/p52 alone. High titers of neutralizing antibodies specific for gp70 were also induced by immunization with DNA. Since KoRV and PERV are closely related, we investigated cross-neutralization of the antisera. The antisera against p15E and gp70 of PERV and KoRV inhibited infection by both viruses. The envelope proteins of the KoRV may therefore form the basis of an effective preventive vaccine to protect uninfected koalas from infection and possibly an immunotherapeutic treatment for those already infected.

  7. Effect of differential processing of the native and recombinant α-amylase fromBacillus amyloliquefaciensJJC33M on specificity and enzyme properties.

    Science.gov (United States)

    Montor-Antonio, Juan José; Hernández-Heredia, Sarahi; Ávila-Fernández, Ángela; Olvera, Clarita; Sachman-Ruiz, Bernardo; Del Moral, Sandra

    2017-10-01

    AmyJ33, an α-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.

  8. Specific functional connectivity alterations of the dorsal striatum in young people with depression

    Directory of Open Access Journals (Sweden)

    Rebecca Kerestes

    2015-01-01

    Conclusions: The results provide evidence that alterations in corticostriatal connectivity are evident at the early stages of the illness and are not a result of antidepressant treatment. Increased connectivity between the dorsal caudate, which is usually associated with cognitive processes, and the more affectively related ventrolateral prefrontal cortex may reflect a compensatory mechanism for dysfunctional cognitive-emotional processing in youth depression.

  9. Generation of Recombinant Monoclonal Antibodies from Immunised Mice and Rabbits via Flow Cytometry and Sorting of Antigen-Specific IgG+ Memory B Cells.

    Directory of Open Access Journals (Sweden)

    Dale O Starkie

    Full Text Available Single B cell screening strategies, which avoid both hybridoma fusion and combinatorial display, have emerged as important technologies for efficiently sampling the natural antibody repertoire of immunized animals and humans. Having access to a range of methods to interrogate different B cell subsets provides an attractive option to ensure large and diverse panels of high quality antibody are produced. The generation of multiple antibodies and having the ability to find rare B cell clones producing IgG with unique and desirable characteristics facilitates the identification of fit-for-purpose molecules that can be developed into therapeutic agents or research reagents. Here, we describe a multi-parameter flow cytometry single-cell sorting technique for the generation of antigen-specific recombinant monoclonal antibodies from single IgG+ memory B cells. Both mouse splenocytes and rabbit PBMC from immunised animals were used as a source of B cells. Reagents staining both B cells and other unwanted cell types enabled efficient identification of class-switched IgG+ memory B cells. Concurrent staining with antigen labelled separately with two spectrally-distinct fluorophores enabled antigen-specific B cells to be identified, i.e. those which bind to both antigen conjugates (double-positive. These cells were then typically sorted at one cell per well using FACS directly into a 96-well plate containing reverse transcriptase reaction mix. Following production of cDNA, PCR was performed to amplify cognate heavy and light chain variable region genes and generate transcriptionally-active PCR (TAP fragments. These linear expression cassettes were then used directly in a mammalian cell transfection to generate recombinant antibody for further testing. We were able to successfully generate antigen-specific recombinant antibodies from both the rabbit and mouse IgG+ memory B cell subset within one week. This included the generation of an anti-TNFR2 blocking

  10. In vivo and in vitro characterization of site-specific recombination of a novel serine integrase from the temperate phage EFC-1

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Bohyun; Kim, Inki; Nam, Ja-Ae [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of); Chang, Hyo-Ihl [College of Life Sciences & Biotechnology, Korea University, 5-1 Anam-dong, Seongbuk-gu, Seoul 136-701 (Korea, Republic of); Ha, Chang Hoon, E-mail: chhoonha@amc.seoul.kr [Asan Institute for Life Sciences, Asan Medical Center, College of Medicine, University of Ulsan, 86 Asanbyeoungwon-gil, Songpa-gu, Seoul 138-736 (Korea, Republic of)

    2016-04-22

    EFC-1 integrase is a site-specific recombinase that belongs to the large family of serine recombinase. In previously study, we isolated the temperate phage EFC-1, and characterized its genomic sequence. Within its genome, Orf28 was predicted encode a 464 amino acid of a putative integrase gene. In this study, EFC-1 integrase was characterized in vitro and in vivo. In vitro assay was performed using purified His-tag fusion integrase. Also, to identify which serine is involved in the catalytic domain, we used site-directed mutagenesis and analyzed by a recombination assay in vitro. In vivo assay involved PCR and confocal microscopy in HEK293 cells, and determined the minimal lengths of attP and attB sites. According to our results, the EFC-1 integrase-mediated recombination was site-specific and unidirectional system in vitro and in vivo. Serine 21 of EFC-1 integrase plays a major role in the catalytic domain, and minimal sizes of attB and attP was defined 48 and 54 bp. Our finding may help develop a useful tool for gene therapy and gene delivery system. - Highlights: • EFC-1 integrase-mediated recombination was site-specific and unidirectional system. • Serine 21 of EFC-1 integrase plays a major role in the catalytic domain. • The functional minimal sizes of attB and attP was defined 48 and 54 bp.

  11. Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries - Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1.

    Science.gov (United States)

    Haka, Jaana; Niemi, Merja H; Iljin, Kristiina; Reddy, Vanga Siva; Takkinen, Kristiina; Laukkanen, Marja-Leena

    2015-05-27

    Around 3-5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen

  12. Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

    Science.gov (United States)

    Gerszberg, Aneta; Wiktorek-Smagur, Aneta; Hnatuszko-Konka, Katarzyna; Łuchniak, Piotr; Kononowicz, Andrzej K

    2012-03-01

    One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the β-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

  13. A dense SNP-based linkage map for Atlantic salmon (Salmo salar reveals extended chromosome homeologies and striking differences in sex-specific recombination patterns

    Directory of Open Access Journals (Sweden)

    Lien Sigbjørn

    2011-12-01

    Full Text Available Abstract Background The Atlantic salmon genome is in the process of returning to a diploid state after undergoing a whole genome duplication (WGD event between 25 and100 million years ago. Existing data on the proportion of paralogous sequence variants (PSVs, multisite variants (MSVs and other types of complex sequence variation suggest that the rediplodization phase is far from over. The aims of this study were to construct a high density linkage map for Atlantic salmon, to characterize the extent of rediploidization and to improve our understanding of genetic differences between sexes in this species. Results A linkage map for Atlantic salmon comprising 29 chromosomes and 5650 single nucleotide polymorphisms (SNPs was constructed using genotyping data from 3297 fish belonging to 143 families. Of these, 2696 SNPs were generated from ESTs or other gene associated sequences. Homeologous chromosomal regions were identified through the mapping of duplicated SNPs and through the investigation of syntenic relationships between Atlantic salmon and the reference genome sequence of the threespine stickleback (Gasterosteus aculeatus. The sex-specific linkage maps spanned a total of 2402.3 cM in females and 1746.2 cM in males, highlighting a difference in sex specific recombination rate (1.38:1 which is much lower than previously reported in Atlantic salmon. The sexes, however, displayed striking differences in the distribution of recombination sites within linkage groups, with males showing recombination strongly localized to telomeres. Conclusion The map presented here represents a valuable resource for addressing important questions of interest to evolution (the process of re-diploidization, aquaculture and salmonid life history biology and not least as a resource to aid the assembly of the forthcoming Atlantic salmon reference genome sequence.

  14. Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Tang, Jin-Bao [School of Pharmacy, Weifang Medical University, Weifang 261053 (China); Tang, Ying [Affiliated Hospital of Weifang Medical University, Weifang 261041 (China); Yang, Hong-Ming, E-mail: yanghongming2006@sohu.com [School of Pharmacy, Weifang Medical University, Weifang 261053 (China)

    2015-02-15

    Highlights: • An efficient signal amplification strategy for label-free EIA is proposed. • Divalent biotinylated AP and monovalent biotinylated ZZ were prepared via Avitag–BirA system. • The above site-specific biotinylated fusion proteins form complex via SA–biotin interaction. • The mechanism relies on the ZZ–Avi-B/SA/AP–(Avi-B){sub 2} complex. • The analytical signals are enhanced (32-fold) by the proposed strategy. - Abstract: Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag–BirA system. Through the high streptavidin (SA)–biotin interaction, the divalent biotinylated APs were clustered in the SA–biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ–AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable

  15. Temporal- and Location-Specific Alterations of the GABA Recycling System in Mecp2 KO Mouse Brains

    Directory of Open Access Journals (Sweden)

    Seok K. Kang

    2014-01-01

    Full Text Available Rett syndrome (RTT, associated with mutations in methyl-CpG-binding protein 2 (Mecp2, is linked to diverse neurological symptoms such as seizures, motor disabilities, and cognitive impairments. An altered GABAergic system has been proposed as one of many underlying pathologies of progressive neurodegeneration in several RTT studies. This study for the first time investigated the temporal- and location-specific alterations in the expression of γ-amino butyric acid (GABA transporter 1 (GAT-1, vesicular GABA transporter (vGAT, and glutamic acid decarboxylase 67kD (GAD67 in wild type (WT and knockout (KO mice in the Mecp2 m1.1Bird/y mouse model of RTT. Immunohistochemistry (IHC co-labeling of GAT-1 with vGAT identified GABAergic synapses that were quantitated for mid-sagittal sections in the frontal cortex (FC, hippocampal dentate gyrus (DG, and striatum (Str. An age-dependent increase in the expression of synaptic GABA transporters, GAT-1, and vGAT, was observed in the FC and DG in WT brains. Mecp2 KO mice showed a significant alteration in this temporal profile that was location-specific, only in the FC. GAD67-positive cell densities also showed an age-dependent increase in the FC, but a decrease in the DG in WT mice. However, these densities were not significantly altered in the KO mice in the regions examined in this study. Therefore, the significant location-specific downregulation of synaptic GABA transporters in Mecp2 KO brains with unaltered densities of GAD67-positive interneurons may highlight the location-specific synaptic pathophysiology in this model of RTT.

  16. Does prostate-specific antigen density alter decision making on biopsy?

    NARCIS (Netherlands)

    Vleeming, R.; de Craen, A. J.; de Reijke, T. M.; van Andel, G.; Kurth, K. H.

    1996-01-01

    The ability of prostate-specific antigen density (PSAD) to predict prostate cancer in biopsy specimens is evaluated in patients with benign digital rectal examination (DRE) and prostate-specific antigen (PSA) between 4.0 and 10.0 ng/ml. 144 referred patients with a benign DRE and PSA > 4.0 ng/ml

  17. Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

    Science.gov (United States)

    de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

    2016-06-10

    The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Recombinant N-Domain of Pregnancy-Specific Glycoprotein from E. coli Cells: Analysis of the Spectrum of Polyclonal Antibodies.

    Science.gov (United States)

    Prokopenko, P G; Shkoporov, A N; Petrenko, O Yu; Efimov, B A; Negrebetskii, V V; Terent'ev, A A

    2015-11-01

    We studied antibody spectrum in antisera to IgG-like recombinant N-domain of pregnancyspecific glycoprotein-1 (rPSG-N) from E. coli cells. In three experimental series, the fraction of IgG antibodies from anti-rPSG-N sera was immobilized on 3 immunoadsorbents: by polymerization with glutaraldehyde, on glutaraldehyde activated biogel P-300, and on commercial CNBr-activated 4B sepharose. Retroplacental serum was incubated with immobilized antibodies to rPSG1-N, protein was eluted and tested in the precipitation test in standard test systems with PSG1, IgG, and human serum albumin. Three proteins were eluted from all 3 immunoadsorbents: PSG1, IgG, and human serum albumin, which demonstrated the spectrum of antibodies to 3 proteins present also in natural serum PSG1 complex. The proportions of PSG1 and IgG obtained in these experiments were similar to those in natural serum PSG1 complex, while the level of human serum albumin was significantly higher in natural PSG1 complex. Thus, we failed to obtain PSG1 monoprotein free from IgG and human serum albumin. Antigenic mosaicism of the polypeptide chain of IgG-like rPSG1-N relative to the antigenic polyvalence of the complex of three proteins present in bioactive preparation of natural serum PSG1 was discussed.

  19. Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera.

    Science.gov (United States)

    Hoelzle, Ludwig E; Hoelzle, Katharina; Wittenbrink, Max M

    2004-10-05

    Recombinant major outer membrane proteins (rMOMP) of Chlamydophila (Ch.) abortus, Ch. pecorum, and Chlamydia (C.) suis were used as antigens to distinguish chlamydial species-specific antibodies in (i) immune sera from six rabbits and three pigs raised against native purified elementary bodies, (ii) serum samples from 25 sows vaccinated with Ch. abortus, and (iii) 40 serum samples from four heifers experimentally infected with Ch. abortus. All post-exposition sera contained chlamydial antibodies as confirmed by strong ELISA seroreactivities against the chlamydial LPS. For the rMOMP ELISA mean IgG antibody levels were at least 5.8-fold higher with the particular rMOMP homologous to the chlamydial species used for immunisation or infection than with heterologous rMOMPs (P <0.001). Preferential rMOMP ELISA reactivities of sera were confirmed by Western blotting. The results suggest that the entire chlamydial rMOMP could provide a species-specific serodiagnostic antigen.

  20. Priming with a recombinant pantothenate auxotroph of Mycobacterium bovis BCG and boosting with MVA elicits HIV-1 Gag specific CD8+ T cells.

    Directory of Open Access Journals (Sweden)

    Rosamund Chapman

    Full Text Available A safe and effective HIV vaccine is required to significantly reduce the number of people becoming infected with HIV each year. In this study wild type Mycobacterium bovis BCG Pasteur and an attenuated pantothenate auxotroph strain (BCGΔpanCD that is safe in SCID mice, have been compared as vaccine vectors for HIV-1 subtype C Gag. Genetically stable vaccines BCG[pHS400] (BCG-Gag and BCGΔpanCD[pHS400] (BCGpan-Gag were generated using the Pasteur strain of BCG, and a panothenate auxotroph of Pasteur respectively. Stability was achieved by the use of a codon optimised gag gene and deletion of the hsp60-lysA promoter-gene cassette from the episomal vector pCB119. In this vector expression of gag is driven by the mtrA promoter and the Gag protein is fused to the Mycobacterium tuberculosis 19 kDa signal sequence. Both BCG-Gag and BCGpan-Gag primed the immune system of BALB/c mice for a boost with a recombinant modified vaccinia virus Ankara expressing Gag (MVA-Gag. After the boost high frequencies of predominantly Gag-specific CD8(+ T cells were detected when BCGpan-Gag was the prime in contrast to induction of predominantly Gag-specific CD4(+ T cells when priming with BCG-Gag. The differing Gag-specific T-cell phenotype elicited by the prime-boost regimens may be related to the reduced inflammation observed with the pantothenate auxotroph strain compared to the parent strain. These features make BCGpan-Gag a more desirable HIV vaccine candidate than BCG-Gag. Although no Gag-specific cells could be detected after vaccination of BALB/c mice with either recombinant BCG vaccine alone, BCGpan-Gag protected mice against a surrogate vaccinia virus challenge.

  1. Host specific differences alter the requirement for certain Salmonella genes during swine colonization.

    Science.gov (United States)

    Bearson, Bradley L; Bearson, Shawn M D

    2011-06-02

    The pathogenic potential of Salmonella is determined during the complex interaction between pathogen and host, requiring optimal regulation of multiple bacterial genetic systems within variable in vivo environments. The mouse model of systemic disease has been an extremely productive model to investigate the pathogenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium). Although the mouse model is a widely used paradigm for studying the pathogenesis of systemic disease caused by Salmonella, investigations concerning food safety interventions should employ natural hosts to examine gastrointestinal colonization by Salmonella. Recent research has demonstrated specific differences in the attenuation of certain S. Typhimurium mutants in mice compared to swine. This variation in pathogenesis between the mouse model and pigs for the S. Typhimurium mutants is presumably dependent upon either the requirements for specific gene products during systemic disease (mouse) versus gastrointestinal colonization (pig) or host specific differences. In addition, host specific diversity in Salmonella colonization of swine has also been described in comparison to other food-producing animals, including cattle and chickens. Differences in Salmonella colonization and pathogenesis across diverse animal species highlight the importance of species-specific studies of gastrointestinal colonization for the development of Salmonella interventions to enhance pork safety. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. Dietary selenium and nutritional plane alter specific aspects of maternal endocrine status during pregnancy and lactation.

    Science.gov (United States)

    Lemley, C O; Meyer, A M; Neville, T L; Hallford, D M; Camacho, L E; Maddock-Carlin, K R; Wilmoth, T A; Wilson, M E; Perry, G A; Redmer, D A; Reynolds, L P; Caton, J S; Vonnahme, K A

    2014-01-01

    Objectives were to examine effects of selenium (Se) supply and maternal nutritional plane during gestation on placental size at term and maternal endocrine profiles throughout gestation and early lactation. Ewe lambs (n = 84) were allocated to treatments that included Se supply of adequate Se (ASe; 11.5 μg/kg BW) or high Se (HSe; 77 μg/kg BW) initiated at breeding and nutritional plane of 60% (RES), 100% (CON), or 140% (EXC) of requirements beginning on day 40 of gestation. At parturition, lambs were removed from their dams, and ewes were transitioned to a common diet that met requirements of lactation. Blood samples were taken from a subset of ewes (n = 42) throughout gestation, during parturition, and throughout lactation to determine hormone concentrations. Cotyledon number was reduced (P = 0.03) in RES and EXC ewes compared with CON ewes. Placental delivery time tended (P = 0.08) to be shorter in HSe ewes than in ASe ewes, whereas placental delivery time was longer (P = 0.02) in RES ewes than in CON and EXC ewes. During gestation, maternal progesterone, estradiol-17β, and GH were increased (P maternal cortisol, IGF-I, prolactin, triiodothyronine, and thyroxine were decreased in RES ewes and increased in EXC ewes compared with CON ewes during gestation. Selenium supply did not alter maternal hormone profiles during gestation. During parturition and lactation, maternal hormone concentrations were influenced by both Se and maternal nutritional plane. During the parturient process, HSe ewes tended to have greater (P = 0.06) concentrations of estradiol-17β than ASe ewes. Three hours after parturition a surge of GH was observed in ASe-RES ewes that was muted in HSe-RES ewes and not apparent in other ewes. Growth hormone area under the curve during the parturient process was increased (P < 0.05) in ASe-RES vs HSe-RES ewes. Ewes that were overfed during gestation had reduced (P < 0.05) estradiol-17β but greater IGF-I, triiodothyronine, and thyroxine (P < 0

  3. Multi-walled carbon nanotubes functionalized with recombinant Dengue virus 3 envelope proteins induce significant and specific immune responses in mice.

    Science.gov (United States)

    Versiani, Alice F; Astigarraga, Ruiz G; Rocha, Eliseu S O; Barboza, Ana Paula M; Kroon, Erna G; Rachid, Milene A; Souza, Daniele G; Ladeira, Luiz O; Barbosa-Stancioli, Edel F; Jorio, Ado; Da Fonseca, Flávio G

    2017-04-04

    Dengue is the most prevalent arthropod-borne viral disease in the world. In this article we present results on the development, characterization and immunogenic evaluation of an alternative vaccine candidate against Dengue. The MWNT-DENV3E nanoconjugate was developed by covalent functionalization of carboxylated multi-walled carbon nanotubes (MWNT) with recombinant dengue envelope (DENV3E) proteins. The recombinant antigens were bound to the MWNT using a diimide-activated amidation process and the immunogen was characterized by TEM, AFM and Raman Spectroscopy. Furthermore, the immunogenicity of this vaccine candidate was evaluated in a murine model. Immunization with MWNT-DENV3E induced comparable IgG responses in relation to the immunization with non-conjugated proteins; however, the inoculation of the nanoconjugate into mice generated higher titers of neutralizing antibodies. Cell-mediated responses were also evaluated, and higher dengue-specific splenocyte proliferation was observed in cell cultures derived from mice immunized with MWNT-DENV3E when compared to animals immunized with the non-conjugated DENV3E. Despite the recent licensure of the CYD-TDV dengue vaccine in some countries, results from the vaccine's phase III trial have cast doubts about its overall efficacy and global applicability. While questions about the effectiveness of the CYD-TDV vaccine still lingers, it is wise to keep at hand an array of vaccine candidates, including alternative non-classical approaches like the one presented here.

  4. A recombinant fusion protein containing a spider toxin specific for the insect voltage-gated sodium ion channel shows oral toxicity towards insects of different orders

    Science.gov (United States)

    Yang, Sheng; Pyati, Prashant; Fitches, Elaine; Gatehouse, John A.

    2014-01-01

    Recombinant fusion protein technology allows specific insecticidal protein and peptide toxins to display activity in orally-delivered biopesticides. The spider venom peptide δ-amaurobitoxin-PI1a, which targets insect voltage-gated sodium channels, was fused to the “carrier” snowdrop lectin (GNA) to confer oral toxicity. The toxin itself (PI1a) and an amaurobitoxin/GNA fusion protein (PI1a/GNA) were produced using the yeast Pichia pastoris as expression host. Although both proteins caused mortality when injected into cabbage moth (Mamestra brassicae) larvae, the PI1a/GNA fusion was approximately 6 times as effective as recombinant PI1a on a molar basis. PI1a alone was not orally active against cabbage moth larvae, but a single 30 μg dose of the PI1a/GNA fusion protein caused 100% larval mortality within 6 days when fed to 3rd instar larvae, and caused significant reductions in survival, growth and feeding in 4th – 6th instar larvae. Transport of fusion protein from gut contents to the haemolymph of cabbage moth larvae, and binding to the nerve chord, was shown by Western blotting. The PI1a/GNA fusion protein also caused mortality when delivered orally to dipteran (Musca domestica; housefly) and hemipteran (Acyrthosiphon pisum; pea aphid) insects, making it a promising candidate for development as a biopesticide. PMID:24486516

  5. Tissue-specific alterations in thyroid hormone homeostasis in combined Mct10 and Mct8 deficiency

    NARCIS (Netherlands)

    J. Müller (Julia); S. Mayerl (Steffen); T.J. Visser (Theo); V.M. Darras (Veerle); A. Boelen (Anita); L. Frappart (Lucien); L. Mariotta (Luca); F. Verrey; H. Heuer (Heike)

    2014-01-01

    textabstractThe monocarboxylate transporter Mct10 (Slc16a10; T-type amino acid transporter) facilitates the cellular transport of thyroid hormone (TH) and shows an overlapping expression with the wellestablished TH transporter Mct8. Because Mct8 deficiency is associated with distinct tissue-specific

  6. Tissue-specific alterations in thyroid hormone homeostasis in combined Mct10 and Mct8 deficiency

    NARCIS (Netherlands)

    Müller, Julia; Mayerl, Steffen; Visser, Theo J.; Darras, Veerle M.; Boelen, Anita; Frappart, Lucien; Mariotta, Luca; Verrey, Francois; Heuer, Heike

    2014-01-01

    The monocarboxylate transporter Mct10 (Slc16a10; T-type amino acid transporter) facilitates the cellular transport of thyroid hormone (TH) and shows an overlapping expression with the well-established TH transporter Mct8. Because Mct8 deficiency is associated with distinct tissue-specific

  7. Structural Alterations of the Language Connectome in Children with Specific Language Impairment

    Czech Academy of Sciences Publication Activity Database

    Vydrová, R.; Komárek, V.; Šanda, J.; Štěrbová, K.; Jahodová, A.; Maulisová, A.; Žáčková, J.; Reissigová, Jindra; Kršek, P.; Kyncl, M.

    2015-01-01

    Roč. 151, December (2015), s. 35-41 ISSN 0093-934X Institutional support: RVO:67985807 Keywords : Specific language disorder * DTI * Arcuate fascicle * IFOF * Ventral stream Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 3.038, year: 2015

  8. Association of oral yeast carriage with specific host factors and altered mouth sensation.

    Science.gov (United States)

    Shimizu, Chika; Kuriyama, Tomoari; Williams, David W; Karasawa, Tadahiro; Inoue, Katsumi; Nakagawa, Kiyomasa; Yamamoto, Etsuhide

    2008-04-01

    The aim of this study was to determine if there was a significant association between the presence of altered mouth and taste sensations with oral carriage of yeasts and to assess the factors that influence the yeast carriage. The oral and dental status including unstimulated (USFR) and stimulated (SSFR) whole salivary flow rates of a total of 509 subjects was recorded. Saliva specimens were collected for microbiologic examination. Multiple logistic regression analysis was performed to identify any factors that were significantly associated with the prevalence of oral yeasts. Old age, clinical signs of oral dryness, denture wearing, and a reduction in USFR increased the prevalence of yeasts, whereas patient gender, levels of dentition, the sensation of dry or burning mouth, taste disorders, and SSFR were not associated with increased prevalence of oral yeasts. An increased prevalence of oral yeasts was not found to relate to changes in mouth sensation alone. Other factors, most notably patient age, the wearing of dentures, clinical signs of oral dryness, and salivary flow rate under rest conditions, were, however, found to be closely associated with oral yeast carriage.

  9. Pam (Peptidylglycine α-amidating monooxygenase) heterozygosity alters brain copper handling with region specificity

    Science.gov (United States)

    Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

    2013-01-01

    Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in postsynaptic vesicular fractions. Cu followed a similar pattern, with ~20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) is selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not the hippocampus in PAM+/− mice, along with GABAB receptor mRNA levels. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. PMID:24032518

  10. Replacement of Imu-Cmu intron by NeoR gene alters Imu germ-line expression but has no effect on V(D)J recombination.

    Science.gov (United States)

    Haddad, Dania; Dougier, Hei-Lanne; Laviolette, Nathalie; Puget, Nadine; Khamlichi, Ahmed Amine

    2010-02-01

    The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3' regulatory region (3'RR). This polarized long-range effect of the 3'RR is disturbed upon insertion of NeoR gene in the IgH constant (C(H)) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Emu enhancer or Emu core enhancer (cEmu) by NeoR gene fully blocked V(D)J recombination and mu0 GL transcription which originates 5' of DQ52 and severely diminished Imu GL transcription derived from Emu/Imu promoter, suggesting a critical role for cEmu in the regulation of V(D)J recombination and of mu0 and Imu expression. Here we focus on the effect of NeoR gene on mu0 and Imu GL transcription in a mouse line in which the Imu-Cmu intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Emu. mu0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Imu transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Imu expression. Surprisingly, in stark contrast with the NeoR effect in the C(H) region, LPS-induced NeoR expression restored Imu transcript levels to normal. The data suggest that Emu enhancer may be the master control element that counteracts the down-regulatory "Neo effect" on Imu expression upon LPS

  11. Generation and characterization of recombinant human antibodies specific for native laminin epitopes. Potential application in cancer therapy. Cancer Immunol. Immunother

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Russell, Stephen J.

    2001-01-01

    Laminins are specific cellular regulators that directly and indirectly control activities such as cell attachment and migration, differentiation and polarity, proliferation and apoptosis, and protease expression. Considering the centrality of these issues to tumor progression, the generation of h...

  12. Myxoviruses do not induce non-specific alterations in membrane permeability early on in infection

    International Nuclear Information System (INIS)

    Foster, K.A.; Micklem, K.J.; Bogomolova, N.N.; Boriskin, Y.S.; Pasternak, C.A.

    1983-01-01

    The permeability characteristics of cells infected with myxoviruses have been studied by measuring the concentrative uptake of nutrients, the concentration of intracellular K + , and the maintenance of the Na + gradient across the plasma membrane. Cells either show no change at all (Sendai virus-infected BHK cells and measles virus-infected Vero cells) or they show a decreased ability to concentrate nutrients, while intracellular K + and the Na + gradient remain unchanged (Sendai and influenza virus-infected L-1210 cells, measles virus-infected lymphocytes and mumps virus-infected L-41 cells). In no case, therefore, was a change observed that resembles the non-specific increase in membrane permeability induced by haemolytic paramyxoviruses (35, 42) or the non-specific membrane leakiness postulated to take place in infected cells (8, 9). A preliminary account of some of these findings has been presented (39)

  13. Neuropathy-specific alterations in a Mexican population of diabetic patients.

    Science.gov (United States)

    Carbajal-Ramírez, Angélica; García-Macedo, Rebeca; Díaz-García, Carlos Manlio; Sanchez-Soto, Carmen; Padrón, Araceli Méndez; de la Peña, Jorge Escobedo; Cruz, Miguel; Hiriart, Marcia

    2017-08-25

    Neuropathy is one of the major complications of type 2 diabetes mellitus. Our first aim was to determine the clinical characteristics of a population of diabetic patients with different types of neuropathy. Our next goal was to characterize the cytokine profile (IL-6 and IL-10), nerve growth factor (NGF) and circulating cell-adhesion molecules in these patients. Finally, we aimed to compare the renal function among the groups of neuropathic patients. In a cross-sectional study, we included 217 diabetic patients classified in three groups: sensory polyneuropathy with hypoesthesia (DS h P) or hyperesthesia (DS H P), and motor neuropathy (DMN). Two control groups were included: one of 26 diabetic non-neuropathic patients (DNN), and the other of 375 non-diabetic (ND) healthy subjects. The participants were attending to the Mexican Institute of Social Security. The circulating levels of NGF were significantly lower in diabetic patients, compared to healthy subjects. The range of IL-6 and IL-10 levels in neuropathic patients was higher than the control groups; however, several samples yielded null measurements. Neuropathic patients also showed increased circulating levels of the adhesion molecules ICAM, VCAM, and E-Selectin, compared to the ND group. Moreover, neuropathic patients showed reduced glomerular filtration rates compared to healthy subjects (82-103 ml/min per 1.73 m 2 , data as range from 25th-75th percentiles), especially in the group with DMN (45-76 ml/min per 1.73 m 2 ). Some particular alterations in neuropathic patients included -but were not limited to- changes in circulating NGF, cell adhesion molecules, inflammation, and the worsening of the renal function. This study supports the need for further clinical surveillance and interventions considering a neuropathy-related basis.

  14. Alterations of specific biomarkers of metabolic pathways in vascular tree from patients with Type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Rosa Bernal-Lopez M

    2012-07-01

    Full Text Available Abstract The aims of this study were to check whether different biomarkers of inflammatory, apoptotic, immunological or lipid pathways had altered their expression in the occluded popliteal artery (OPA compared with the internal mammary artery (IMA and femoral vein (FV and to examine whether glycemic control influenced the expression of these genes. The study included 20 patients with advanced atherosclerosis and type 2 diabetes mellitus, 15 of whom had peripheral arterial occlusive disease (PAOD, from whom samples of OPA and FV were collected. PAOD patients were classified based on their HbA1c as well (HbA1c ≤ 6.5 or poorly (HbA1c > 6.5 controlled patients. Controls for arteries without atherosclerosis comprised 5 IMA from patients with ischemic cardiomyopathy (ICM. mRNA, protein expression and histological studies were analyzed in IMA, OPA and FV. After analyzing 46 genes, OPA showed higher expression levels than IMA or FV for genes involved in thrombosis (F3, apoptosis (MMP2, MMP9, TIMP1 and TIM3, lipid metabolism (LRP1 and NDUFA, immune response (TLR2 and monocytes adhesion (CD83. Remarkably, MMP-9 expression was lower in OPA from well-controlled patients. In FV from diabetic patients with HbA1c ≤6.5, gene expression levels of BCL2, CDKN1A, COX2, NDUFA and SREBP2 were higher than in FV from those with HbA1c >6.5. The atherosclerotic process in OPA from diabetic patients was associated with high expression levels of inflammatory, lipid metabolism and apoptotic biomarkers. The degree of glycemic control was associated with gene expression markers of apoptosis, lipid metabolism and antioxidants in FV. However, the effect of glycemic control on pro-atherosclerotic gene expression was very low in arteries with established atherosclerosis.

  15. Specific genomic regions are differentially affected by copy number alterations across distinct cancer types, in aggregated cytogenetic data.

    Science.gov (United States)

    Kumar, Nitin; Cai, Haoyang; von Mering, Christian; Baudis, Michael

    2012-01-01

    Regional genomic copy number alterations (CNA) are observed in the vast majority of cancers. Besides specifically targeting well-known, canonical oncogenes, CNAs may also play more subtle roles in terms of modulating genetic potential and broad gene expression patterns of developing tumors. Any significant differences in the overall CNA patterns between different cancer types may thus point towards specific biological mechanisms acting in those cancers. In addition, differences among CNA profiles may prove valuable for cancer classifications beyond existing annotation systems. We have analyzed molecular-cytogenetic data from 25579 tumors samples, which were classified into 160 cancer types according to the International Classification of Disease (ICD) coding system. When correcting for differences in the overall CNA frequencies between cancer types, related cancers were often found to cluster together according to similarities in their CNA profiles. Based on a randomization approach, distance measures from the cluster dendrograms were used to identify those specific genomic regions that contributed significantly to this signal. This approach identified 43 non-neutral genomic regions whose propensity for the occurrence of copy number alterations varied with the type of cancer at hand. Only a subset of these identified loci overlapped with previously implied, highly recurrent (hot-spot) cytogenetic imbalance regions. Thus, for many genomic regions, a simple null-hypothesis of independence between cancer type and relative copy number alteration frequency can be rejected. Since a subset of these regions display relatively low overall CNA frequencies, they may point towards second-tier genomic targets that are adaptively relevant but not necessarily essential for cancer development.

  16. Alteration of substrate specificity of leucine dehydrogenase by site-directed mutagenesis

    OpenAIRE

    片岡, 邦重; Kataoka, Kunishige; Tanizawa, Katsuyuki

    2003-01-01

    The residues L40, A113, V291, and V294, in leucine dehydrogenase (LeuDH), predicted to be involved in recognition of the substrate side chain, have been mutated on the basis of the molecular modeling to mimic the substrate specificities of phenylalanine (PheDH), glutamate (GluDH), and lysine dehydrogenases (LysDH). The A113G and A113G/V291L mutants, imitating the PheDH active site, displayed activities toward -phenylalanine and phenylpyruvate with 1.6 and 7.8% of kcat values of the wild-type ...

  17. Efficient Culture Adaptation of Hepatitis C Virus Recombinants with Genotype-Specific Core-NS2 by Using Previously Identified Mutations

    DEFF Research Database (Denmark)

    Scheel, Troels Kasper Høyer; Gottwein, Judith M; Carlsen, Thomas H R

    2011-01-01

    Hepatitis C virus (HCV) is an important cause of chronic liver disease, and interferon-based therapy cures only 40 to 80% of patients, depending on HCV genotype. Research was accelerated by genotype 2a (strain JFH1) infectious cell culture systems. We previously developed viable JFH1-based...... mutations did not adapt to culture. Universal adaptive effects of mutations in NS3 (Q1247L, I1312V, K1398Q, R1408W, and Q1496L) and NS5A (V2418L) were investigated for JFH1-based genotype 1 to 5 core-NS2 recombinants; several mutations conferred adaptation to H77C (1a), J4 (1b), S52 (3a), and SA13 (5a......-specific patterns in HCV disease and control....

  18. Expression of recombinant HA1 protein for specific detection of influenza A/H1N1/2009 antibodies in human serum.

    Science.gov (United States)

    Luo, Lizhong; Nishi, Krista; Macleod, Erin; Sabara, Marta I; Coleman, Brenda L; Gubbay, Jonathan B; Li, Yan

    2013-01-01

    The hemagglutinin genes (HA1 subunit) from human and animal 2009 pandemic H1N1 virus isolates were expressed with a baculovirus vector. Recombinant HA1 (rHA1) protein-based ELISA was evaluated for detection of specific influenza A(H1N1)pdm09 antibodies in serum samples from vaccinated humans. It was found that rHA1 ELISA consistently differentiated between antibodies recognizing the seasonal influenza H1N1 and pdm09 viruses, with a concordance of 94% as compared to the hemagglutination inhibition test. This study suggests the utility of rHA1 ELISA in serosurveillance. © 2012 The Societies and Wiley Publishing Asia Pty Ltd.

  19. Learning to see the difference specifically alters the most informative V4 neurons.

    Science.gov (United States)

    Raiguel, Steven; Vogels, Rufin; Mysore, Santosh G; Orban, Guy A

    2006-06-14

    Perceptual learning is an instance of adult plasticity whereby training in a sensory (e.g., a visual task) results in neuronal changes leading to an improved ability to perform the task. Yet studies in primary visual cortex have found that changes in neuronal response properties were relatively modest. The present study examines the effects of training in an orientation discrimination task on the response properties of V4 neurons in awake rhesus monkeys. Results indicate that the changes induced in V4 are indeed larger than those in V1. Nonspecific effects of training included a decrease in response variance, and an increase in overall orientation selectivity in V4. The orientation-specific changes involved a local steepening in the orientation tuning curve around the trained orientation that selectively improved orientation discriminability at the trained orientation. Moreover, these changes were largely confined to the population of neurons whose orientation tuning was optimal for signaling small differences in orientation at the trained orientation. Finally, the modifications were restricted to the part of the tuning curve close to the trained orientation. Thus, we conclude that it is the most informative V4 neurons, those most directly involved in the discrimination, that are specifically modified by perceptual learning.

  20. Fear of predation alters clone-specific performance in phloem-feeding prey.

    Science.gov (United States)

    Khudr, Mouhammad Shadi; Buzhdygan, Oksana Y; Petermann, Jana S; Wurst, Susanne

    2017-08-09

    Fear of predation has been shown to affect prey fitness and behaviour, however, to date little is known about the underlying genetics of responses to predator-associated risk. In an effort to fill this gap we exposed four naïve clones of green peach aphid (Myzus persicae), maintained on the model crop Brassica oleracea, to different types of cues from aphid lion (Chrysoperla carnea). The respective predation risks, we termed Fear Factors, were either lethal (consumption by predator), or non-lethal (non-consumptive predator-associated cues: plant-tethered predator cadavers and homogenised shoot-sprayed or soil-infused blends of predator remains). Our results show that the non-lethal risk cues differentially impeded prey reproductive success that varied by clone, suggesting genotype-specific response to fear of predation. Furthermore, whether plants were perceived as being safe or risky influenced prey responses as avoidance behaviour in prey depended on clone type. Our findings highlight that intra-specific genetic variation underlies prey responses to consumptive and non-consumptive effects of predation. This allows selection to act on anti-predator responses to fear of predation that may ramify and influence higher trophic levels in model agroecosystems.

  1. The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence.

    Science.gov (United States)

    Gautam, Shweta D; Chen, Jon K; Murray, Vincent

    2017-08-01

    Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, we constructed a plasmid clone to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, we were able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. We observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and, in fact, the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.

  2. Calcific Aortic Valve Disease Is Associated with Layer-Specific Alterations in Collagen Architecture.

    Directory of Open Access Journals (Sweden)

    Heather N Hutson

    Full Text Available Disorganization of the valve extracellular matrix (ECM is a hallmark of calcific aortic valve disease (CAVD. However, while microarchitectural features of the ECM can strongly influence the biological and mechanical behavior of tissues, little is known about the ECM microarchitecture in CAVD. In this work, we apply advanced imaging techniques to quantify spatially heterogeneous changes in collagen microarchitecture in CAVD. Human aortic valves were obtained from individuals between 50 and 75 years old with no evidence of valvular disease (healthy and individuals who underwent valve replacement surgery due to severe stenosis (diseased. Second Harmonic Generation microscopy and subsequent image quantification revealed layer-specific changes in fiber characteristics in healthy and diseased valves. Specifically, the majority of collagen fiber changes in CAVD were found to occur in the spongiosa, where collagen fiber number increased by over 2-fold, and fiber width and density also significantly increased. Relatively few fibrillar changes occurred in the fibrosa in CAVD, where fibers became significantly shorter, but did not otherwise change in terms of number, width, density, or alignment. Immunohistochemical staining for lysyl oxidase showed localized increased expression in the diseased fibrosa. These findings reveal a more complex picture of valvular collagen enrichment and arrangement in CAVD than has previously been described using traditional analysis methods. Changes in fiber architecture may play a role in regulating the pathobiological events and mechanical properties of valves during CAVD. Additionally, characterization of the ECM microarchitecture can inform the design of fibrous scaffolds for heart valve tissue engineering.

  3. Bacterial production of site specific {sup 13}C labeled phenylalanine and methodology for high level incorporation into bacterially expressed recombinant proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L., E-mail: robert.mcfeeters@uah.edu [University of Alabama in Huntsville, Department of Chemistry (United States)

    2017-01-15

    Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific {sup 13}C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct {sup 13}C chemical shifts and multiple magnetically equivalent {sup 1}H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with {sup 13}C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated {sup 1}H-{sup 13}C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.

  4. Site-specific recombination for the modification of transgenic strains of the Mediterranean fruit fly Ceratitis capitata

    Science.gov (United States)

    Insect transgenesis is mainly based on the random genomic integration of DNA fragments embedded into non-autonomous transposable elements. Once a random insertion into a specific location of the genome has been identified as particularly useful with respect to transgene expression, the ability to ma...

  5. Induction of HIV-1-specific mucosal immune responses following intramuscular recombinant adenovirus serotype 26 HIV-1 vaccination of humans.

    Science.gov (United States)

    Baden, Lindsey R; Liu, Jinyan; Li, Hualin; Johnson, Jennifer A; Walsh, Stephen R; Kleinjan, Jane A; Engelson, Brian A; Peter, Lauren; Abbink, Peter; Milner, Danny A; Golden, Kevin L; Viani, Kyle L; Stachler, Matthew D; Chen, Benjamin J; Pau, Maria G; Weijtens, Mo; Carey, Brittany R; Miller, Caroline A; Swann, Edith M; Wolff, Mark; Loblein, Hayley; Seaman, Michael S; Dolin, Raphael; Barouch, Dan H

    2015-02-15

    Defining mucosal immune responses and inflammation to candidate human immunodeficiency virus type 1 (HIV-1) vaccines represents a current research priority for the HIV-1 vaccine field. In particular, it is unclear whether intramuscular immunization can elicit immune responses at mucosal surfaces in humans. In this double-blind, randomized, placebo-controlled clinical trial, we evaluated systemic and mucosal immune responses to a candidate adenovirus serotype 26 (Ad26) vectored HIV-1 envelop (Env) vaccine in baseline Ad26-seronegative and Ad26-seropositive healthy volunteers. Systematic mucosal sampling with rectal Weck-Cel sponges and rectal biopsies were performed. Intramuscular immunization elicited both systemic and mucosal Env-specific humoral and cellular immune responses in the majority of subjects. Individuals with preexisting Ad26-specific neutralizing antibodies had vaccine-elicited immune responses comparable to those of subjects who were Ad26 seronegative. We also observed no increase in activated total or vector-specific mucosal CD4+ T lymphocytes following vaccination by either histopathology or flow cytometry. These data demonstrate that a single intramuscular administration of this Ad26-vectored HIV-1 Env vaccine elicited both systemic and mucosal immune responses in humans. Induction of antigen-specific humoral and cellular mucosal immunity was not accompanied by a detectable increase in mucosal inflammation. NCT01103687. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Specific inflammatory response of Anemonia sulcata (Cnidaria) after bacterial injection causes tissue reaction and enzymatic activity alteration.

    Science.gov (United States)

    Trapani, M R; Parisi, M G; Parrinello, D; Sanfratello, M A; Benenati, G; Palla, F; Cammarata, M

    2016-03-01

    The evolution of multicellular organisms was marked by adaptations to protect against pathogens. The mechanisms for discriminating the ''self'' from ''non-self" have evolved into a long history of cellular and molecular strategies, from damage repair to the co-evolution of host-pathogen interactions. We investigated the inflammatory response in Anemonia sulcata (Cnidaria: Anthozoa) following injection of substances that varied in type and dimension, and observed clear, strong and specific reactions, especially after injection of Escherichia coli and Vibrio alginolyticus. Moreover, we analyzed enzymatic activity of protease, phosphatase and esterase, showing how the injection of different bacterial strains alters the expression of these enzymes and suggesting a correlation between the appearance of the inflammatory reaction and the modification of enzymatic activities. Our study shows for the first time, a specific reaction and enzymatic responses following injection of bacteria in a cnidarian. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Genetic Recombination

    Science.gov (United States)

    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)

  8. Altered consolidation of extinction-like inhibitory learning in genotype-specific dysfunctional coping fostered by chronic stress in mice.

    Science.gov (United States)

    Campus, P; Maiolati, M; Orsini, C; Cabib, S

    2016-12-15

    Genetic and stress-related factors interact to foster mental disorders, possibly through dysfunctional learning. In a previous study we reported that a temporary experience of reduced food availability increases forced swim (FS)-induced helplessness tested 14days after a first experience in mice of the standard inbred C57BL/6(B6) strain but reduces it in mice of the genetically unrelated DBA/2J (D2) strain. Because persistence of FS-induced helplessness influences adaptive coping with stress challenge and involve learning processes the present study tested whether the behavioral effects of restricted feeding involved altered consolidation of FS-related learning. First, we demonstrated that restricted feeding does not influence behavior expressed on the first FS experience, supporting a specific effect on persistence rather then development of helplessness. Second, we found that FS-induced c-fos expression in the infralimbic cortex (IL) was selectively enhanced in food-restricted (FR) B6 mice and reduced in FR D2 mice, supporting opposite alterations of consolidation processes involving this brain area. Third, we demonstrated that immediate post-FS inactivation of IL prevents 24h retention of acquired helplessness by continuously free-fed mice of both strains, indicating the requirement of a functioning IL for consolidation of FS-related learning in either mouse strain. Finally, in line with the known role of IL in consolidation of extinction memories, we found that restricted feeding selectively facilitated 24h retention of an acquired extinction in B6 mice whereas impairing it in D2 mice. These findings support the conclusion that an experience of reduced food availability strain-specifically affects persistence of newly acquired passive coping strategies by altering consolidation of extinction-like inhibitory learning. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Fear conditioning leads to alteration in specific genes expression in cortical and thalamic neurons that project to the lateral amygdala.

    Science.gov (United States)

    Katz, Ira K; Lamprecht, Raphael

    2015-02-01

    RNA transcription is needed for memory formation. However, the ability to identify genes whose expression is altered by learning is greatly impaired because of methodological difficulties in profiling gene expression in specific neurons involved in memory formation. Here, we report a novel approach to monitor the expression of genes after learning in neurons in specific brain pathways needed for memory formation. In this study, we aimed to monitor gene expression after fear learning. We retrogradely labeled discrete thalamic neurons that project to the lateral amygdala (LA) of rats. The labeled neurons were dissected, using laser microdissection microscopy, after fear conditioning learning or unpaired training. The RNAs from the dissected neurons were subjected to microarray analysis. The levels of selected RNAs detected by the microarray analysis to be altered by fear conditioning were also assessed by nanostring analysis. We observed that the expression of genes involved in the regulation of translation, maturation and degradation of proteins was increased 6 h after fear conditioning compared to unpaired or naïve trained rats. These genes were not expressed 24 h after training or in cortical neurons that project to the LA. The expression of genes involved in transcription regulation and neuronal development was altered after fear conditioning learning in the cortical-LA pathway. The present study provides key information on the identity of genes expressed in discrete thalamic and cortical neurons that project to the LA after fear conditioning. Such an approach could also serve to identify gene products as targets for the development of a new generation of therapeutic agents that could be aimed to functionally identified brain circuits to treat memory-related disorders. © 2014 International Society for Neurochemistry.

  10. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen thi Man; Morris, G.E. (North East Wales Inst., Clwyd (United Kingdom))

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  11. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E.; Lamers, Wouter H.; Chaudhry, Farrukh A.; Verlander, Jill W.

    2016-01-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4+ with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na+-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression. PMID:27009341

  12. Histone peptide AKRHRK enhances H2O2-induced DNA damage and alters its site specificity

    International Nuclear Information System (INIS)

    Midorikawa, Kaoru; Murata, Mariko; Kawanishi, Shosuke

    2005-01-01

    Histone proteins are involved in compaction of DNA and the protection of cells from oxygen toxicity. However, several studies have demonstrated that the metal-binding histone reacts with H 2 O 2 , leading to oxidative damage to a nucleobase. We investigated whether histone can accelerate oxidative DNA damage, using a minimal model for the N-terminal tail of histone H4, CH 3 CO-AKRHRK-CONH 2 , which has a metal-binding site. This histone peptide enhanced DNA damage induced by H 2 O 2 and Cu(II), especially at cytosine residues, and induced additional DNA cleavage at the 5'-guanine of GGG sequences. The peptide also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine and ESR spin-trapping signal from H 2 O 2 and Cu(II). Cyclic redox reactions involving histone-bound Cu(II) and H 2 O 2 , may give rise to multiple production of radicals leading to multiple hits in DNA. It is noteworthy that the histone H4 peptide with specific sequence AKRHRK can cause DNA damage rather than protection under metal-overloaded condition

  13. Specific gut commensal flora locally alters T cell tuning to endogenous ligands.

    Science.gov (United States)

    Chappert, Pascal; Bouladoux, Nicolas; Naik, Shruti; Schwartz, Ronald H

    2013-06-27

    Differences in gut commensal flora can dramatically influence autoimmune responses, but the mechanisms behind this are still unclear. We report, in a Th1-cell-driven murine model of autoimmune arthritis, that specific gut commensals, such as segmented filamentous bacteria, have the ability to modulate the activation threshold of self-reactive T cells. In the local microenvironment of gut-associated lymphoid tissues, inflammatory cytokines elicited by the commensal flora dynamically enhanced the antigen responsiveness of T cells that were otherwise tuned down to a systemic self-antigen. Together with subtle differences in early lineage differentiation, this ultimately led to an enhanced recruitment of pathogenic Th1 cells and the development of a more severe form of autoimmune arthritis. These findings define a key role for the gut commensal flora in sustaining ongoing autoimmune responses through the local fine tuning of T-cell-receptor-proximal activation events in autoreactive T cells. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism.

    Science.gov (United States)

    Lee, Hyun-Wook; Osis, Gunars; Handlogten, Mary E; Lamers, Wouter H; Chaudhry, Farrukh A; Verlander, Jill W; Weiner, I David

    2016-06-01

    Glutamine synthetase (GS) catalyzes the recycling of NH4 (+) with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and Na(+)-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

  15. Alteration of sequence specificity of the type IIS restriction endonuclease BtsI.

    Directory of Open Access Journals (Sweden)

    Shengxi Guan

    Full Text Available The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0. It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0, which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0. This is the first successful case of a specificity change among this restriction endonuclease type.

  16. Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus.

    Science.gov (United States)

    Witherden, Elizabeth A; Bajanca-Lavado, Maria Paula; Tristram, Stephen G; Nunes, Alexandra

    2014-06-01

    To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. The ftsI genes of 100 Haemophilus isolates comprising genetically defined β-lactamase-negative ampicillin-susceptible (gBLNAS), β-lactamase-positive ampicillin-resistant (gBLPAR), β-lactamase-negative ampicillin-resistant (gBLNAR) and β-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All

  17. Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

    International Nuclear Information System (INIS)

    Song, Y.R.; Wu, B.; Yang, Y.T.; Chen, J.; Zhang, L.J.; Zhang, Z.W.; Shi, H.Y.; Huang, C.L.; Pan, J.X.; Xie, P.

    2015-01-01

    Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes

  18. Assessment of altered binding specificity of bacteriophage for ciprofloxacin-induced antibiotic-resistant Salmonella Typhimurium.

    Science.gov (United States)

    Kim, Jeongjin; Jo, Ara; Ding, Tian; Lee, Hyeon-Yong; Ahn, Juhee

    2016-08-01

    This study describes a new effort toward understanding the interaction mechanisms between antibiotic-resistant Salmonella Typhimurium and phages. The antibiotic susceptibility, β-lactamase activity, bacterial motility, gene expression, and lytic activity were evaluated in ciprofloxacin-induced antibiotic-sensitive Salmonella Typhimurium (ASST(CIP)) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium (ARST(CIP)), which were compared to the wild-type strains (ASST(WT) and ARST(WT)). The MIC values of ampicillin, norfloxacin, chloramphenicol, and tetracycline were significantly increased to > 512, 16, 16, and 256 μg/ml, respectively, in the ARST(CIP). The lowest and highest extracellular lactamase activities were observed in ASST(WT) (6.85 μmol/min/ml) and ARST(CIP) (48.83 μmol/min/ml), respectively. The acrA, lpfE, and hilA genes were significantly upregulated by more than tenfold in both ASST(CIP) and ARST(CIP). The induction of multiple antibiotic resistance resulted from the increased efflux pump activity (AcrAB-TolC). The highest phage adsorption rates were more than 95 % for ASST(WT), ASST(CIP), and ARST(WT), while the lowest adsorption rate was 52 % for ARST(CIP) at 15 min of infection. The least lytic activity of phage was 20 % against the ARST(CIP), followed by ASST(CIP) (30 %). The adsorption rate of phage against ARST(CIP) was 52 % at 15 min of infection, which resulted in the decrease in lytic activity (12 %). Understanding the interaction of phage and bacteria is essential for the practical application of phage to control and detect antibiotic-resistant bacteria. The results provide useful information for understanding the binding specificity of phages for multiple antibiotic-resistant pathogens.

  19. Specific alterations in plasma proteins during depressed, manic, and euthymic states of bipolar disorder

    Energy Technology Data Exchange (ETDEWEB)

    Song, Y.R. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Wu, B. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Yang, Y.T.; Chen, J. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Zhang, L.J.; Zhang, Z.W. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Shi, H.Y. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Huang, C.L.; Pan, J.X. [Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Xie, P. [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China)

    2015-09-08

    Bipolar disorder (BD) is a common psychiatric mood disorder affecting more than 1-2% of the general population of different European countries. Unfortunately, there is no objective laboratory-based test to aid BD diagnosis or monitor its progression, and little is known about the molecular basis of BD. Here, we performed a comparative proteomic study to identify differentially expressed plasma proteins in various BD mood states (depressed BD, manic BD, and euthymic BD) relative to healthy controls. A total of 10 euthymic BD, 20 depressed BD, 15 manic BD, and 20 demographically matched healthy control subjects were recruited. Seven high-abundance proteins were immunodepleted in plasma samples from the 4 experimental groups, which were then subjected to proteome-wide expression profiling by two-dimensional electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight tandem mass spectrometry. Proteomic results were validated by immunoblotting and bioinformatically analyzed using MetaCore. From a total of 32 proteins identified with 1.5-fold changes in expression compared with healthy controls, 16 proteins were perturbed in BD independent of mood state, while 16 proteins were specifically associated with particular BD mood states. Two mood-independent differential proteins, apolipoprotein (Apo) A1 and Apo L1, suggest that BD pathophysiology may be associated with early perturbations in lipid metabolism. Moreover, down-regulation of one mood-dependent protein, carbonic anhydrase 1 (CA-1), suggests it may be involved in the pathophysiology of depressive episodes in BD. Thus, BD pathophysiology may be associated with early perturbations in lipid metabolism that are independent of mood state, while CA-1 may be involved in the pathophysiology of depressive episodes.

  20. Nailfold Videocapillaroscopy Alterations in Dermatomyositis and Systemic Sclerosis: Toward Identification of a Specific Pattern.

    Science.gov (United States)

    Manfredi, Andreina; Sebastiani, Marco; Campomori, Federica; Pipitone, Nicolò; Giuggioli, Dilia; Colaci, Michele; Praino, Emanuela; Ferri, Clodoveo

    2016-08-01

    The term scleroderma pattern typically defines capillary abnormalities of scleroderma spectrum disorders, mainly systemic sclerosis (SSc) and dermatomyositis (DM). Our study aimed to investigate differences in nailfold videocapillaroscopy (NVC) between DM and SSc, with a cross-sectional and longitudinal evaluation. NVC features of 29 consecutive patients with DM were compared with 90 patients with SSc categorized into the 3 subsets of scleroderma pattern: early, active, and late. Twenty patients with DM and all with SSc were also longitudinally reevaluated after 30 months of followup. At baseline, all SSc groups showed giant capillaries, with significant differences with DM only for early and active pattern. Ramified capillaries were significantly more frequent and severe in DM than in early and active patterns, while DM showed an opposite trend compared with late pattern. Capillary loss was lower in early pattern and higher in active and late, compared with DM. Finally, giant-ramified capillaries were almost exclusive of DM. During followup, NVC showed a different evolution in DM and SSc. In DM we recorded a reduction of giant capillaries, while ramified capillaries increased both in DM and in early and active SSc pattern. The number of capillaries recovered in DM; conversely, capillary loss slightly worsened in all SSc patterns. Giant-ramified capillaries significantly decreased in patients with DM, remaining rare in patients with SSc. Our study strengthens the specificity of DM and SSc microangiopathy and points out the need for large prospective studies to confirm our results and possibly to revise current terminology by distinguishing between "scleroderma" and "dermatomyositis" patterns.

  1. Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

    Science.gov (United States)

    Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

    2014-01-01

    MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair

  2. Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

    Directory of Open Access Journals (Sweden)

    Fumihito Noguchi

    Full Text Available MED1 (Mediator complex subunit 1 is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/- that develop epidermal hyperplasia. Herein, to investigate the function(s of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/- and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/- mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/- mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/- keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/- keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/- keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/- keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/- mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/- mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/- mice, indicating a decreased contribution of hair

  3. A strain-specific multiplex RT-PCR for Australian rabbit haemorrhagic disease viruses uncovers a new recombinant virus variant in rabbits and hares.

    Science.gov (United States)

    Hall, R N; Mahar, J E; Read, A J; Mourant, R; Piper, M; Huang, N; Strive, T

    2018-04-01

    Rabbit haemorrhagic disease virus (RHDV, or GI.1) is a calicivirus in the genus Lagovirus that has been widely utilized in Australia as a biological control agent for the management of overabundant wild European rabbit (Oryctolagus cuniculus) populations since 1996. Recently, two exotic incursions of pathogenic lagoviruses have been reported in Australia; GI.1a-Aus, previously called RHDVa-Aus, is a GI.1a virus detected in January 2014, and the novel lagovirus GI.2 (previously known as RHDV2). Furthermore, an additional GI.1a strain, GI.1a-K5 (also known as 08Q712), was released nationwide in March 2017 as a supplementary tool for wild rabbit management. To discriminate between these lagoviruses, a highly sensitive strain-specific multiplex RT-PCR assay was developed, which allows fast, cost-effective and sensitive detection of the four pathogenic lagoviruses currently known to be circulating in Australia. In addition, we developed a universal RT-qPCR assay to be used in conjunction with the multiplex assay that broadly detects all four viruses and facilitates quantification of viral RNA load in samples. These assays enable rapid detection, identification and quantification of pathogenic lagoviruses in the Australian context. Using these assays, a novel recombinant lagovirus was detected in rabbit tissue samples, which contained the non-structural genes of GI.1a-Aus and the structural genes of GI.2. This variant was also recovered from the liver of a European brown hare (Lepus europaeus). The impact of this novel recombinant on Australian wild lagomorph populations and its competitiveness in relation to circulating field strains, particularly GI.2, requires further studies. © 2017 Blackwell Verlag GmbH.

  4. Using QCM and SPR for the Kinetic Evaluation of the Binding Between A New Recombinant Chimeric Protein and Specific Antibodies of the Visceral Leishmaniasis.

    Science.gov (United States)

    Souto, Denio E P; Faria, Angelica R; de Andrade, Helida M; Kubota, Lauro T

    2015-01-01

    In the present study, the surface plasmon resonance (SPR) and quartz crystal microbalance (QCM) techniques were employed to kinetically evaluate the binding affinity of a new recombinant chimeric protein (CP10) toward anti-Leishmania infantum antibodies for the immunodiagnostics of the visceral leishmaniasis (VL). This chimeric protein was formed by the union in a same artificial coding DNA of ten different peptides, which showed themselves reactive toward positive canine serum for VL. Using the CP10 in enzyme-linked immunosorbent assays (ELISA), it was possible to detect 80% of the asymptomatic infected dogs. After this, SPR and QCM immunosensors were constructed by the covalent immobilization of the CP10 on a self-assembled monolayer (SAM) formed by adsorption of alkanethiol on gold substrates. The thickness (6.80 nm) and the refractive index (1.475) of the protein on the SAM were simultaneously determined through SPR curves measured in different wavelengths (670 and 785 nm). Interactions between the CP10 and its specific IgGs (anti-CP10 antibodies) were characterized by the electrochemical impedance spectroscopy, SPR and QCM techniques. The equilibrium dissociation constant obtained by SPR (K(D) = 8.27 x 10(-10) mol.L(-1)) and QCM (K(D) = 2.42 x 10(- 10) mol.L(-1)) demonstrated high binding affinity of the CP10 toward anti-CP10 antibodies. In this sense, this work quantitatively proves the strong antigenic character of a new recombinant chimeric protein, giving evidence to potential contribution for the use of this protein in programs of control of the VL.

  5. Utilisation of antibody microarrays for the selection of specific and informative antibodies from recombinant library binders of unknown quality

    DEFF Research Database (Denmark)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J

    2016-01-01

    reducing the effort of antibody characterisation by concentrating on relevant molecules. In a pilot scheme, a library of 456 single-chain variable fragment (scFv) binders to 134 antigens was used. They were arranged in a microarray format and incubated with the protein content of clinical tissue samples...... isolated from pancreatic ductal adenocarcinoma and healthy pancreas, as well as recurrent and non-recurrent bladder tumours. We observed significant variation in the expression of the E3 ubiquitin-protein ligase (CHFR) as well as the glutamate receptor interacting protein 2 (GRIP2), for example, always...... with more than one of the scFvs binding to these targets. Only the relevant antibodies were then characterised further on antigen microarrays and by surface plasmon resonance experiments so as to select the most specific and highest affinity antibodies. These binders were in turn used to confirm...

  6. Generation and characterization of recombinant human antibodies specific for native laminin epitopes. Potential application in cancer therapy. Cancer Immunol. Immunother

    DEFF Research Database (Denmark)

    Sanz, Laura; Kristensen, Peter; Russell, Stephen J.

    2001-01-01

    indicated that these antibodies recognize the native protein through conformational epitopes. All of them reduced tumor cell attachment to laminin, suggesting that domains of the laminin molecule that are recognized by these antibodies likely bind to cell-surface receptors. The antibody fragments bind...... to mouse, rat and human laminin. and show strong immunohistochemical reactivity with basement membranes in human and murine tissue sections. Their properties make them ideal candidates for in vivo applications....... of single chain antibodies was selected using intact mouse laminin, and five different clones were identified. All the antibodies were specific for their cognate antigen, as revealed by lack of cross-reactivity with other components of the basement membranes. A more extensive characterization of the panel...

  7. Increasing the ex vivo antigen-specific IFN-γ production in subpopulations of T cells and NKp46+ cells by anti-CD28, anti-CD49d and recombinant IL-12 costimulation in cattle vaccinated with recombinant proteins from Mycobacterium avium subspecies paratuberculosis

    DEFF Research Database (Denmark)

    Thakur, Aneesh; Riber, Ulla; Davis, William C.

    2013-01-01

    -γ secretion by CD4, CD8, γδ T cells and NK cells. Age matched male jersey calves, experimentally infected with Mycobacterium avium subsp. paratuberculosis (MAP), were vaccinated with a cocktail of recombinant MAP proteins or left unvaccinated. Vaccine induced ex vivo recall responses were measured through Ag......T cells, which encounter specific antigen (Ag), require additional signals to mount a functional immune response. Here, we demonstrate activation of signal 2, by anti-CD28 mAb (aCD28) and other costimulatory molecules (aCD49d, aCD5), and signal 3, by recombinant IL-12, enhance Ag-specific IFN...

  8. Phloem-specific expression of a melon Aux/IAA in tomato plants alters auxin sensitivity and plant development

    Directory of Open Access Journals (Sweden)

    Guy eGolan

    2013-08-01

    Full Text Available Phloem sap contains a large repertoire of macromolecules in addition to sugars, amino acids, growth substances and ions. The transcription profile of melon phloem sap contains over 1,000 mRNA molecules, most of them associated with signal transduction, transcriptional control, and stress and defense responses. Heterografting experiments have established the long-distance trafficking of numerous mRNA molecules. Interestingly, several trafficking transcripts are involved in the auxin response, including two molecules coding for auxin/indole acetic acid (Aux/IAA. To further explore the biological role of the melon Aux/IAA transcript CmF-308 in the vascular tissue, a cassette containing the coding sequence of this gene under a phloem-specific promoter was introduced into tomato plants. The number of lateral roots was significantly higher in transgenic plants expressing CmF-308 under the AtSUC2 promoter than in controls. A similar effect on root development was obtained after transient expression of CmF-308 in source leaves of N. benthamiana plants. An auxin-response assay showed that CmF-308-transgenic roots are more sensitive to auxin than control roots. In addition to the altered root development, phloem-specific expression of CmF-308 resulted in shorter plants, a higher number of lateral shoots and delayed flowering, a phenotype resembling reduced apical dominance. In contrast to the root response, cotyledons of the transgenic plants were less sensitive to auxin than control cotyledons. The reduced auxin sensitivity in the shoot tissue was confirmed by lower relative expression of several Aux/IAA genes in leaves and an increase in the relative expression of a cytokinin-response regulator, TRR8/9b. The accumulated data suggest that expression of Aux/IAA in the phloem modifies auxin sensitivity in a tissue-specific manner, thereby altering plant development.

  9. Gender-specific alteration of energy balance and circadian locomotor activity in the Crtc1 knockout mouse model of depression

    KAUST Repository

    Rossetti, Clara

    2017-12-06

    Obesity and depression are major public health concerns, and there is increasing evidence that they share etiological mechanisms. CREB-regulated transcription coactivator 1 (CRTC1) participates in neurobiological pathways involved in both mood and energy balance regulation. Crtc1 -/- mice rapidly develop a depressive-like and obese phenotype in early adulthood, and are therefore a relevant animal model to explore possible common mechanisms underlying mood disorders and obesity. Here, the obese phenotype of male and female Crtc1 -/- mice was further characterized by investigating CRTC1\\'s role in the homeostatic and hedonic regulation of food intake, as well as its influence on daily locomotor activity. Crtc1 -/- mice showed a strong gender difference in the homeostatic regulation of energy balance. Mutant males were hyperphagic and rapidly developed obesity on normal chow diet, whereas Crtc1 -/- females exhibited mild late-onset obesity without hyperphagia. Overeating of mutant males was accompanied by alterations in the expression of several orexigenic and anorexigenic hypothalamic genes, thus confirming a key role of CRTC1 in the central regulation of food intake. No alteration in preference and conditioned response for saccharine was observed in Crtc1 -/- mice, suggesting that mutant males\\' hyperphagia was not due to an altered hedonic regulation of food intake. Intriguingly, mutant males exhibited a hyperphagic behavior only during the resting (diurnal) phase of the light cycle. This abnormal feeding behavior was associated with a higher diurnal locomotor activity indicating that the lack of CRTC1 may affect circadian rhythmicity. Collectively, these findings highlight the male-specific involvement of CRTC1 in the central control of energy balance and circadian locomotor activity.

  10. Resting-state Network-specific Breakdown of Functional Connectivity during Ketamine Alteration of Consciousness in Volunteers.

    Science.gov (United States)

    Bonhomme, Vincent; Vanhaudenhuyse, Audrey; Demertzi, Athena; Bruno, Marie-Aurélie; Jaquet, Oceane; Bahri, Mohamed Ali; Plenevaux, Alain; Boly, Melanie; Boveroux, Pierre; Soddu, Andrea; Brichant, Jean François; Maquet, Pierre; Laureys, Steven

    2016-11-01

    Consciousness-altering anesthetic agents disturb connectivity between brain regions composing the resting-state consciousness networks (RSNs). The default mode network (DMn), executive control network, salience network (SALn), auditory network, sensorimotor network (SMn), and visual network sustain mentation. Ketamine modifies consciousness differently from other agents, producing psychedelic dreaming and no apparent interaction with the environment. The authors used functional magnetic resonance imaging to explore ketamine-induced changes in RSNs connectivity. Fourteen healthy volunteers received stepwise intravenous infusions of ketamine up to loss of responsiveness. Because of agitation, data from six subjects were excluded from analysis. RSNs connectivity was compared between absence of ketamine (wake state [W1]), light ketamine sedation, and ketamine-induced unresponsiveness (deep sedation [S2]). Increasing the depth of ketamine sedation from W1 to S2 altered DMn and SALn connectivity and suppressed the anticorrelated activity between DMn and other brain regions. During S2, DMn connectivity, particularly between the medial prefrontal cortex and the remaining network (effect size β [95% CI]: W1 = 0.20 [0.18 to 0.22]; S2 = 0.07 [0.04 to 0.09]), and DMn anticorrelated activity (e.g., right sensory cortex: W1 = -0.07 [-0.09 to -0.04]; S2 = 0.04 [0.01 to 0.06]) were broken down. SALn connectivity was nonuniformly suppressed (e.g., left parietal operculum: W1 = 0.08 [0.06 to 0.09]; S2 = 0.05 [0.02 to 0.07]). Executive control networks, auditory network, SMn, and visual network were minimally affected. Ketamine induces specific changes in connectivity within and between RSNs. Breakdown of frontoparietal DMn connectivity and DMn anticorrelation and sensory and SMn connectivity preservation are common to ketamine and propofol-induced alterations of consciousness.

  11. Grapevine Plasticity in Response to an Altered Microclimate: Sauvignon Blanc Modulates Specific Metabolites in Response to Increased Berry Exposure.

    Science.gov (United States)

    Young, Philip R; Eyeghe-Bickong, Hans A; du Plessis, Kari; Alexandersson, Erik; Jacobson, Dan A; Coetzee, Zelmari; Deloire, Alain; Vivier, Melané A

    2016-03-01

    In this study, the metabolic and physiological impacts of an altered microclimate on quality-associated primary and secondary metabolites in grape (Vitis vinifera) 'Sauvignon Blanc' berries was determined in a high-altitude vineyard. The leaf and lateral shoot removal in the bunch zones altered the microclimate by increasing the exposure of the berries. The physical parameters (berry diameter and weight), primary metabolites (sugars and organic acids), as well as bunch temperature and leaf water potential were predominantly not affected by the treatment. The increased exposure led to higher levels of specific carotenoids and volatile terpenoids in the exposed berries, with earlier berry stages reacting distinctly from the later developmental stages. Plastic/nonplastic metabolite responses could be further classified to identify metabolites that were developmentally controlled and/or responded to the treatment in a predictable fashion (assessed over two consecutive vintages). The study demonstrates that grapevine berries exhibit a degree of plasticity within their secondary metabolites and respond physiologically to the increased exposure by increasing metabolites with potential antioxidant activity. Taken together, the data provide evidence that the underlying physiological responses relate to the maintenance of stress pathways by modulating antioxidant molecules in the berries. © 2016 American Society of Plant Biologists. All Rights Reserved.

  12. Grapevine Plasticity in Response to an Altered Microclimate: Sauvignon Blanc Modulates Specific Metabolites in Response to Increased Berry Exposure1

    Science.gov (United States)

    du Plessis, Kari; Jacobson, Dan A.

    2016-01-01

    In this study, the metabolic and physiological impacts of an altered microclimate on quality-associated primary and secondary metabolites in grape (Vitis vinifera) ‘Sauvignon Blanc’ berries was determined in a high-altitude vineyard. The leaf and lateral shoot removal in the bunch zones altered the microclimate by increasing the exposure of the berries. The physical parameters (berry diameter and weight), primary metabolites (sugars and organic acids), as well as bunch temperature and leaf water potential were predominantly not affected by the treatment. The increased exposure led to higher levels of specific carotenoids and volatile terpenoids in the exposed berries, with earlier berry stages reacting distinctly from the later developmental stages. Plastic/nonplastic metabolite responses could be further classified to identify metabolites that were developmentally controlled and/or responded to the treatment in a predictable fashion (assessed over two consecutive vintages). The study demonstrates that grapevine berries exhibit a degree of plasticity within their secondary metabolites and respond physiologically to the increased exposure by increasing metabolites with potential antioxidant activity. Taken together, the data provide evidence that the underlying physiological responses relate to the maintenance of stress pathways by modulating antioxidant molecules in the berries. PMID:26628747

  13. Highly Pathogenic Influenza A(H5Nx) Viruses with Altered H5 Receptor-Binding Specificity.

    Science.gov (United States)

    Guo, Hongbo; de Vries, Erik; McBride, Ryan; Dekkers, Jojanneke; Peng, Wenjie; Bouwman, Kim M; Nycholat, Corwin; Verheije, M Helene; Paulson, James C; van Kuppeveld, Frank J M; de Haan, Cornelis A M

    2017-02-01

    Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses.

  14. Human immunodeficiency virus type 1 specific cytotoxic T lymphocyte responses in Chinese infected with HIV-1 B'/C Recombinant (CRF07_BC

    Directory of Open Access Journals (Sweden)

    Yu Xu G

    2007-08-01

    Full Text Available Abstract Background The characterization of HIV-1-specific T cell responses in people infected with locally circulating HIV-1 strain will facilitate the development of HIV-1 vaccine. Sixty intravenous drug users infected with HIV-1 circulating recombinant form 07_BC (CRF07_BC, which has been spreading rapidly in western China from north to south, were recruited from Xinjiang, China to assess the HIV-1-specific T cell responses at single peptide level with overlapping peptides (OLP covering the whole concensus clades B and C proteome. Results The median of the total magnitude and total number of OLPs recognized by CTL responses were 10925 SFC/million PBMC and 25 OLPs, respectively, when tested by clade C peptides, which was significantly higher than when tested by clade B peptides. The immunodominant regions, which cover 14% (58/413 of the HIV-1 proteome, are widely distributed throughout the HIV-1 proteome except in Tat, Vpu and Pol-PR, with Gag, Pol-RT, Pol-Int and Nef being most frequently targeted. The subdominant epitopes are mostly located in p24, Nef, integrase, Vpr and Vif. Of the responses directed to clade C OLPs, 61.75% (972/1574 can be observed when tested with corresponding clade B OLPs. However, Pol-PR and Vpu tend to be targeted in the clade B sequence rather than the clade C sequence, which is in line with the recombinant pattern of CRF07_BC. Stronger and broader CTL responses in subjects with CD4 cell counts ranging from 200 to 400/mm3 were observed when compared to those with less than 200/mm3 or more than 400/mm3, though there have been no significant correlations identified between the accumulative CTL responses or overall breadth and CD4 cell count or plasma viral load. Conclusion This is the first study conducted to comprehensively address T cell responses in Chinese subjects infected with HIV-1 CRF07_BC in which subtle differences in cross-reactivity were observed, though similar patterns of overall immune responses were

  15. Recombinant mammaglobin A adenovirus-infected dendritic cells induce mammaglobin A-specific CD8+ cytotoxic T lymphocytes against breast cancer cells in vitro.

    Directory of Open Access Journals (Sweden)

    Huixia Cui

    Full Text Available Mammaglobin A (MGBA is a novel breast cancer-associated antigen almost exclusively over-expressed in primary and metastatic human breast cancers, making it a potential therapeutic target for breast cancer. The development of dendritic cell (DC-induced tumor antigen specific CD8(+ cytotoxic T lymphocytes (CTLs may hold promise in cancer immunotherapy. In this study we constructed recombinant replication-defective adenoviral (Ad vectors encoding MGBA and evaluated their ability to trigger anti-tumor immunity in vitro. DCs were isolated from the human peripheral blood monocyte cells (PBMCs of two HLA-A33(+ healthy female volunteers, and infected with adenovirus carrying MGBA cDNA (Ad-MGBA. After that, the Ad-MGBA-infected DCs were used to stimulate CD8(+ CTLs in vitro and the latter was used for co-culture with breast cancer cell lines. The data revealed that infection with Ad-MGBA improved DC maturation and up-regulated the expression of co-stimulatory molecules and the secretion of interleukin-12 (IL-12, but down-regulated interleukin-10 (IL-10 secretion from DCs. Ad-MGBA-infected DC-stimulated CD8(+CTLs displayed the highest cytotoxicity towards HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells compared with other CD8(+CTL populations, and compared with the cytotoxicity towards HLA-A33(-/MGBA(+ breast cancer HBL-100 cells and HLA-A33(-/MGBA(- breast cancer MDA-MB 231 cells. In addition, Ad-MGBA-infected DC-stimulated CD8(+ CTLs showed a high level of IFNγ secretion when stimulated with HLA-A33(+/MGBA(+ breast cancer MDA-MB-415 cells, but not when stimulated with HLA-A33(-/MGBA(+ HBL-100 and HLA-A33(-/MGBA(-MDA-MB-231 cells. In addition, killing of CD8(+CTLs against breast cancer was in a major histocompability complex (MHC-limited pattern. Finally, the data also determined the importance of TNF-α in activating DCs and T cells. These data together suggest that MGBA recombinant adenovirus-infected DCs could induce specific anti-tumor immunity

  16. High-efficiency transduction and specific expression of ChR2opt for optogenetic manipulation of primary cortical neurons mediated by recombinant adeno-associated viruses.

    Science.gov (United States)

    Jin, Lei; Lange, Wienke; Kempmann, Annika; Maybeck, Vanessa; Günther, Anne; Gruteser, Nadine; Baumann, Arnd; Offenhäusser, Andreas

    2016-09-10

    In recent years, optogenetic approaches have significantly advanced the experimental repertoire of cellular and functional neuroscience. Yet, precise and reliable methods for specific expression of optogenetic tools remain challenging. In this work, we studied the transduction efficiency of seven different adeno-associated virus (AAV) serotypes in primary cortical neurons and revealed recombinant (r) AAV6 to be the most efficient for constructs under control of the cytomegalovirus (CMV) promoter. To further specify expression of the transgene, we exchanged the CMV promoter for the human synapsin (hSyn) promoter. In primary cortical-glial mixed cultures transduced with hSyn promoter-containing rAAVs, expression of ChR2opt (a Channelrhodopsin-2 variant) was limited to neurons. In these neurons action potentials could be reliably elicited upon laser stimulation (473nm). The use of rAAV serotype alone to restrict expression to neurons results in a lower transduction efficiency than the use of a broader transducing serotype with specificity conferred via a restrictive promoter. Cells transduced with the hSyn driven gene expression were able to elicit action potentials with more spatially and temporally accurate illumination than neurons electrofected with the CMV driven construct. The hSyn promoter is particularly suited to use in AAVs due to its small size. These results demonstrate that rAAVs are versatile tools to mediate specific and efficient transduction as well as functional and stable expression of transgenes in primary cortical neurons. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Fungal Infection Induces Sex-Specific Transcriptional Changes and Alters Sexual Dimorphism in the Dioecious Plant Silene latifolia.

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    Niklaus Zemp

    2015-10-01

    Full Text Available Sexual dimorphism, including differences in morphology, behavior and physiology between females and males, is widespread in animals and plants and is shaped by gene expression differences between the sexes. Such expression differences may also underlie sex-specific responses of hosts to pathogen infections, most notably when pathogens induce partial sex reversal in infected hosts. The genetic changes associated with sex-specific responses to pathogen infections on the one hand, and sexual dimorphism on the other hand, remain poorly understood. The dioecious White Campion (Silene latifolia displays sexual dimorphism in floral traits and infection with the smut fungus Micobrotryum lychnidis-dioicae induces a partial sex reversal in females. We find strong sex-specific responses to pathogen infection and reduced sexual dimorphism in infected S. latifolia. This provides a direct link between pathogen-mediated changes in sex-biased gene expression and altered sexual dimorphism in the host. Expression changes following infection affected mainly genes with male-biased expression in healthy plants. In females, these genes were up-regulated, leading to a masculinization of the transcriptome. In contrast, infection in males was associated with down-regulation of these genes, leading to a demasculinization of the transcriptome. To a lesser extent, genes with female-biased expression in healthy plants were also affected in opposite directions in the two sexes. These genes were overall down-regulated in females and up-regulated in males, causing, respectively, a defeminization in infected females and a feminization of the transcriptome in infected males. Our results reveal strong sex-specific responses to pathogen infection in a dioecious plant and provide a link between pathogen-induced changes in sex-biased gene expression and sexual dimorphism.

  18. Patient-Specific Alterations in CO2 Cerebrovascular Responsiveness in Acute and Sub-Acute Sports-Related Concussion

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    W. Alan C. Mutch

    2018-01-01

    Full Text Available BackgroundPreliminary studies suggest that sports-related concussion (SRC is associated with alterations in cerebral blood flow (CBF regulation. Here, we use advanced magnetic resonance imaging (MRI techniques to measure CBF and cerebrovascular responsiveness (CVR in individual SRC patients and healthy control subjects.Methods15 SRC patients (mean age = 16.3, range 14–20 years and 27 healthy control subjects (mean age = 17.6, range 13–21 years underwent anatomical MRI, pseudo-continuous arterial spin labeling (pCASL MRI and model-based prospective end-tidal targeting (MPET of CO2 during blood oxygenation level-dependent (BOLD MRI. Group differences in global mean resting CBF were examined. Voxel-by-voxel group and individual differences in regional CVR were examined using statistical parametric mapping (SPM. Leave-one-out receiver operating characteristic curve analysis was used to evaluate the utility of brain MRI CO2 stress testing biomarkers to correctly discriminate between SRC patients and healthy control subjects.ResultsAll studies were tolerated with no complications. Traumatic structural findings were identified in one SRC patient. No significant group differences in global mean resting CBF were observed. There were no significant differences in the CO2 stimulus and O2 targeting during BOLD MRI. Significant group and patient-specific differences in CVR were observed with SRC patients demonstrating a predominant pattern of increased CVR. Leave-one-out ROC analysis for voxels demonstrating a significant increase in CVR was found to reliably discriminate between SRC patients and healthy control subjects (AUC of 0.879, p = 0.0001. The optimal cutoff for increased CVR declarative for SRC was 1,899 voxels resulting in a sensitivity of 0.867 and a specificity of 0.778 for this specific ROC analysis. There was no correlation between abnormal voxel counts and Postconcussion Symptom Scale scores among SRC patients

  19. An amphipathic sequence in the cytoplasmic tail of HIV-1 Env alters cell tropism and modulates viral receptor specificity.

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    Vzorov, A N; Yang, C; Compans, R W

    2015-09-01

    The human immunodeficiency virus type 1 (HIV-1) 92UG046 Env protein, obtained from a CD4-independent HIV-1 primary isolate (Zerhouni et al., 2004), has the ability to initiate an infection in HeLa cells expressing CD4 when carrying the full-length (FL) Env, but uses CD8 molecules for receptor-mediated entry when carrying a truncated Env (CT84). To determine whether a specific length or structure in the cytoplasmic tail (CT) is responsible for this alteration of tropism, we compared a series of Env constructs with different CT truncations and the presence or absence of an amphipathic alpha- helical sequence. We found that truncated constructs containing the alpha-helical LLP-2 structure in their CT domains conferred a switch from CD4 to CD8 tropism. The results support the conclusion that the structure of the CT domain can play an important role in determining receptor specificity.

  20. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance.

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    Emily Xie

    Full Text Available The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases.

  1. Oral Delivery of a Novel Recombinant Streptococcus mitis Vector Elicits Robust Vaccine Antigen-Specific Oral Mucosal and Systemic Antibody Responses and T Cell Tolerance

    Science.gov (United States)

    Xie, Emily; Kotha, Abhiroop; Biaco, Tracy; Sedani, Nikita; Zou, Jonathan; Stashenko, Phillip; Duncan, Margaret J.; Campos-Neto, Antonio; Cayabyab, Mark J.

    2015-01-01

    The pioneer human oral commensal bacterium Streptococcus mitis has unique biologic features that make it an attractive mucosal vaccine or therapeutic delivery vector. S. mitis is safe as a natural persistent colonizer of the mouth, throat and nasopharynx and the oral commensal bacterium is capable of inducing mucosal antibody responses. A recombinant S. mitis (rS. mitis) that stably expresses HIV envelope protein was generated and tested in the germ-free mouse model to evaluate the potential usefulness of this vector as a mucosal vaccine against HIV. Oral vaccination led to the efficient and persistent bacterial colonization of the mouth and the induction of both salivary and systemic antibody responses. Interestingly, persistently colonized animals developed antigen-specific systemic T cell tolerance. Based on these findings we propose the use of rS. mitis vaccine vector for the induction of mucosal antibodies that will prevent the penetration of the mucosa by pathogens such as HIV. Moreover, the first demonstration of rS. mitis having the ability to elicit T cell tolerance suggest the potential use of rS. mitis as an immunotherapeutic vector to treat inflammatory, allergic and autoimmune diseases. PMID:26618634

  2. Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara

    International Nuclear Information System (INIS)

    Sharpe, Sally; Hanke, Tomas; Tinsley-Bown, Anne; Dennis, Mike; Dowall, Stuart; McMichael, Andrew; Cranage, Martin

    2003-01-01

    Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.HW, encoding a multi-CTL epitope gene, was incorporated into poly(D,L-lactic-co-glycolic acid) microparticles of less than 10 μm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01 + rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals

  3. Nicotine Alters the Gut Microbiome and Metabolites of Gut-Brain Interactions in a Sex-Specific Manner.

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    Chi, Liang; Mahbub, Ridwan; Gao, Bei; Bian, Xiaoming; Tu, Pengcheng; Ru, Hongyu; Lu, Kun

    2017-12-18

    As the primary active substance in tobacco, nicotine affects the activity of the central nervous system, and its effects are sex-dependent. There are complex interactions between the gut and brain, and the gut microbiome can influence neuronal activity and host behavior, with diverse chemical signaling being involved. However, it is unclear whether nicotine can affect the normal gut microbiome and associated chemical signaling of the gut-brain axis. Sex is an important factor that shapes the gut microbiome, but the role of sex in the interaction among nicotine, gut bacteria, and related metabolites remains unknown. In this study, we applied high-throughput sequencing and gas chromatography-mass spectrometry (GC-MS) to explore how nicotine exposure affects the gut microbiome and its metabolism in female and male C57BL/6J mice, with a focus on the chemical signaling involved in gut-brain interactions. 16S sequencing results indicated that the community composition of the gut microbiome was differentially perturbed by nicotine in females and males. Differential alterations of bacterial carbohydrate metabolic pathways are consistent with lower body weight gain in nicotine-treated males. Oxidative stress response and DNA repair genes were also specifically enriched in the nicotine-treated male gut microbiome. The fecal metabolome indicated that multiple neurotransmitters, such as glutamate, gamma-aminobutyric acid (GABA), and glycine, were differentially altered in female and male mice. Some neuroactive metabolites, including leucine and uric acid, were also changed. This study demonstrates a sex-dependent effect of nicotine on gut microbiome community composition, functional bacterial genes, and the fecal metabolome.

  4. Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

    Science.gov (United States)

    Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

    2014-03-01

    Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

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    Tullila, Antti; Nevanen, Tarja K.

    2017-01-01

    Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

  6. Altered behavioral performance and live imaging of circuit-specific neural deficiencies in a zebrafish model for psychomotor retardation.

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    David Zada

    2014-09-01

    Full Text Available The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2 gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8-/- zebrafish using zinc-finger nuclease (ZFN-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8-/- larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8-/- larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8-/- larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8-/- larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit

  7. Stage-specific heat effects: timing and duration of heat waves alter demographic rates of a global insect pest.

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    Zhang, Wei; Rudolf, Volker H W; Ma, Chun-Sen

    2015-12-01

    The frequency and duration of periods with high temperatures are expected to increase under global warming. Thus, even short-lived organisms are increasingly likely to experience periods of hot temperatures at some point of their life-cycle. Despite recent progress, it remains unclear how various temperature experiences during the life-cycle of organisms affect demographic traits. We simulated hot days (daily mean temperature of 30 °C) increasingly experienced under field conditions and investigated how the timing and duration of such hot days during the life cycle of Plutella xylostella affects adult traits. We show that hot days experienced during some life stages (but not all) altered adult lifespan, fecundity, and oviposition patterns. Importantly, the effects of hot days were contingent on which stage was affected, and these stage-specific effects were not always additive. Thus, adults that experience different temporal patterns of hot periods (i.e., changes in timing and duration) during their life-cycle often had different demographic rates and reproductive patterns. These results indicate that we cannot predict the effects of current and future climate on natural populations by simply focusing on changes in the mean temperature. Instead, we need to incorporate the temporal patterns of heat events relative to the life-cycle of organisms to describe population dynamics and how they will respond to future climate change.

  8. Isoform-Specific Na,K-ATPase Alterations Precede Disuse-Induced Atrophy of Rat Soleus Muscle

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    Violetta V. Kravtsova

    2015-01-01

    Full Text Available This study examines the isoform-specific effects of short-term hindlimb suspension (HS on the Na,K-ATPase in rat soleus muscle. Rats were exposed to 24–72 h of HS and we analyzed the consequences on soleus muscle mass and contractile parameters; excitability and the resting membrane potential (RMP of muscle fibers; the electrogenic activity, protein, and mRNA content of the α1 and α2 Na,K-ATPase; the functional activity and plasma membrane localization of the α2 Na,K-ATPase. Our results indicate that 24–72 h of HS specifically decreases the electrogenic activity of the Na,K-ATPase α2 isozyme and the RMP of soleus muscle fibers. This decrease occurs prior to muscle atrophy or any change in contractile parameters. The α2 mRNA and protein content increased after 24 h of HS and returned to initial levels at 72 h; however, even the increased content was not able to restore α2 enzyme activity in the disused soleus muscle. There was no change in the membrane localization of α2 Na,K-ATPase. The α1 Na,K-ATPase electrogenic activity, protein and mRNA content did not change. Our findings suggest that skeletal muscle use is absolutely required for α2 Na,K-ATPase transport activity and provide the first evidence that Na,K-ATPase alterations precede HS-induced muscle atrophy.

  9. Gateway Recombinational Cloning.

    Science.gov (United States)

    Reece-Hoyes, John S; Walhout, Albertha J M

    2018-01-02

    The Gateway recombinatorial cloning system was developed for cloning multiple DNA fragments in parallel (e.g., in 96-well formats) in a standardized manner using the same enzymes. Gateway cloning is based on the highly specific integration and excision reactions of bacteriophage λ into and out of the Escherichia coli genome. Because the sites of recombination (" att " sites) are much longer (25-242 bp) than restriction sites, they are extremely unlikely to occur by chance in DNA fragments. Therefore, the same recombination enzyme can be used to robustly clone many different fragments of variable size in parallel reactions. © 2018 Cold Spring Harbor Laboratory Press.

  10. Specific genes involved in synthesis and editing of heparan sulfate proteoglycans show altered expression patterns in breast cancer

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    Fernández-Vega Iván

    2013-01-01

    Full Text Available Abstract Background The expression of a specific set of genes controls the different structures of heparan sulfate proteoglycans (HSPGs, which are involved in the growth, invasion and metastatic properties of cancerous cells. The purpose of this study is to increase knowledge of HSPG alterations in breast cancer. Methods Twenty-three infiltrating ductal adenocarcinomas (IDCs, both metastatic and non-metastatic were studied. A transcriptomic approach to the structure of heparan sulfate (HS chains was used, employing qPCR to analyze both the expression of the enzymes involved in their biosynthesis and editing, as well as the proteoglycan core proteins. Since some of these proteoglycans can also carry chondroitin sulfate chains, we extended the study to include the genes involved in the biosynthesis of these glycosaminoglycans. Histochemical techniques were also used to analyze tissular expression of particular genes showing significant expression differences, of potential interest. Results No significant change in transcription was detected in approximately 70% of analyzed genes. However, 13 demonstrated changes in both tumor types (40% showing more intense deregulation in the metastatic, while 5 genes showed changes only in non-metastatic tumors. Changes were related to 3 core proteins: overexpression of syndecan-1 and underexpression of glypican-3 and perlecan. HS synthesis was affected by lower levels of some 3-O-sulfotransferase transcripts, the expression of NDST4 and, only in non metastatic tumors, higher levels of extracellular sulfatases. Furthermore, the expression of chondroitin sulfate also was considerably affected, involving both the synthesis of the saccharidic chains and sulfations at all locations. However, the pro-metastatic enzyme heparanase did not exhibit significant changes in mRNA expression, although in metastatic tumors it appeared related to increased levels of the most stable form of mRNA. Finally, the expression of

  11. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

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    Johannes S Gach

    Full Text Available Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5 boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART. Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC. We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

  12. Comparative genomic analysis reveals multiple long terminal repeats, lineage-specific amplification, and frequent interelement recombination for Cassandra retrotransposon in pear (Pyrus bretschneideri Rehd.).

    Science.gov (United States)

    Yin, Hao; Du, Jianchang; Li, Leiting; Jin, Cong; Fan, Lian; Li, Meng; Wu, Jun; Zhang, Shaoling

    2014-06-04

    Cassandra transposable elements belong to a specific group of terminal-repeat retrotransposons in miniature (TRIM). Although Cassandra TRIM elements have been found in almost all vascular plants, detailed investigations on the nature, abundance, amplification timeframe, and evolution have not been performed in an individual genome. We therefore conducted a comprehensive analysis of Cassandra retrotransposons using the newly sequenced pear genome along with four other Rosaceae species, including apple, peach, mei, and woodland strawberry. Our data reveal several interesting findings for this particular retrotransposon family: 1) A large number of the intact copies contain three, four, or five long terminal repeats (LTRs) (∼20% in pear); 2) intact copies and solo LTRs with or without target site duplications are both common (∼80% vs. 20%) in each genome; 3) the elements exhibit an overall unbiased distribution among the chromosomes; 4) the elements are most successfully amplified in pear (5,032 copies); and 5) the evolutionary relationships of these elements vary among different lineages, species, and evolutionary time. These results indicate that Cassandra retrotransposons contain more complex structures (elements with multiple LTRs) than what we have known previously, and that frequent interelement unequal recombination followed by transposition may play a critical role in shaping and reshaping host genomes. Thus this study provides insights into the property, propensity, and molecular mechanisms governing the formation and amplification of Cassandra retrotransposons, and enhances our understanding of the structural variation, evolutionary history, and transposition process of LTR retrotransposons in plants. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  13. Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.

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    Nicolas Grandchamp

    Full Text Available Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.

  14. Gender-specific behavioral and immunological alterations in an animal model of autism induced by prenatal exposure to valproic acid.

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    Schneider, Tomasz; Roman, Adam; Basta-Kaim, Agnieszka; Kubera, Marta; Budziszewska, Bogusława; Schneider, Karolina; Przewłocki, Ryszard

    2008-07-01

    Autism is a severe behavioral disorder characterized by pervasive impairments in social interactions, deficits in verbal and non-verbal communication, and stereotyped behaviors, with a four times higher incidence in boys than in girls. The core symptoms are frequently accompanied by a spectrum of neurobehavioral and immunological derangements, including: aberrant sensitivity to sensory stimulation, anxiety, and decreased cellular immune capacity. Recently, a new potential rodent model of autism induced by prenatal exposure to valproic acid (VPA rats) has been proposed. In order to determine if gender has an influence on alterations observed in VPA rats, male and female rats have been evaluated in a battery of behavioral, immunological, and endocrinological tests. A plethora of aberrations has been found in male VPA rats: lower sensitivity to pain, increased repetitive/stereotypic-like activity, higher anxiety, decreased level of social interaction, increased basal level of corticosterone, decreased weight of the thymus, decreased splenocytes proliferative response to concanavaline A, lower IFN-gamma/IL-10 ratio, and increased production of NO by peritoneal macrophages. Female VPA rats exhibited only increased repetitive/stereotypic-like activity and decreased IFN-gamma/IL-10 ratio. Sexual dimorphism characteristics for measured parameters have been observed in both groups of animals, except social interaction in VPA rats. Our results confirm existence of similarities between the observed pattern of aberrations in VPA rats and features of disturbed behavior and immune function in autistic patients, and suggest that they are gender-specific, which is intriguing in light of disproportion in boys to girls ratio in autism.

  15. Potential of Diffusion Tensor Imaging and Relaxometry for the Detection of Specific Pathological Alterations in Parkinson's Disease (PD.

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    Regina Esterhammer

    Full Text Available The purpose of the present study was to evaluate the potential of multimodal MR imaging including mean diffusivity (MD, fractional anisotropy (FA, relaxation rates R2 and R2* to detect disease specific alterations in Parkinson's Disease (PD. We enrolled 82 PD patients (PD-all with varying disease durations (≤5 years: PD≤5, n = 43; >5 years: PD>5, n = 39 and 38 matched healthy controls (HC, receiving diffusion tensor imaging as well as R2 and R2* relaxometry calculated from multi-echo T2*-weighted and dual-echo TSE imaging, respectively. ROIs were drawn to delineate caudate nucleus (CN, putamen (PU, globus pallidus (GP and substantia nigra (SN on the co-registered maps. The SN was divided in 3 descending levels (SL 1-3. The most significant parameters were used for a flexible discrimination analysis (FDA in a training collective consisting of 25 randomized subjects from each group in order to predict the classification of remaining subjects. PD-all showed significant increases in MD, R2 and R2* within SN and its subregions as well as in MD and R2* within different basal ganglia regions. Compared to the HC group, the PD≤5 and the PD>5 group showed significant MD increases within the SN and its lower two subregions, while the PD≤5 group exhibited significant increases in R2 and R2* within SN and its subregions, and tended to elevation within the basal ganglia. The PD>5 group had significantly increased MD in PU and GP, whereas the PD≤5 group presented normal MD within the basal ganglia. FDA achieved right classification in 84% of study participants. Micro-structural damage affects primarily the SN of PD patients and in later disease stages the basal ganglia. Iron contents of PU, GP and SN are increased at early disease stages of PD.

  16. Perinatal Lead Exposure Alters Gut Microbiota Composition and Results in Sex-specific Bodyweight Increases in Adult Mice.

    Science.gov (United States)

    Wu, Jianfeng; Wen, Xiaoquan William; Faulk, Christopher; Boehnke, Kevin; Zhang, Huapeng; Dolinoy, Dana C; Xi, Chuanwu

    2016-06-01

    Heavy metal pollution is a principle source of environmental contamination. Epidemiological and animal data suggest that early life lead (Pb) exposure results in critical effects on epigenetic gene regulation and child and adult weight trajectories. Using a mouse model of human-relevant exposure, we investigated the effects of perinatal Pb exposure on gut microbiota in adult mice, and the link between gut microbiota and bodyweight changes. Following Pb exposure during gestation and lactation via maternal drinking water, bodyweight in A(vy) strain wild-type non-agouti (a/a) offspring was tracked through adulthood. Gut microbiota of adult mice were characterized by deep DNA sequencing of bacterial 16S ribosomal RNA genes. Data analyses were stratified by sex and adjusted for litter effects. A Bayesian variable selection algorithm was used to analyze associations between bacterial operational taxonomic units and offspring adult bodyweight. Perinatal Pb exposure was associated with increased adult bodyweight in male (P compositions were significantly different (analysis of molecular variance, P gut microbiota were highly associated with adult bodyweight (P = .028; effect size = 2.59). Thus, perinatal Pb exposure results in altered adult gut microbiota regardless of sex, and these changes are highly correlated with increased bodyweight in males. Adult gut microbiota can be shaped by early exposures and may contribute to disease risks in a sex-specific manner. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  17. Compartment-specific distribution of human intestinal innate lymphoid cells is altered in HIV patients under effective therapy.

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    Benjamin Krämer

    2017-05-01

    Full Text Available Innate lymphocyte cells (ILCs, a novel family of innate immune cells are considered to function as key orchestrators of immune defences at mucosal surfaces and to be crucial for maintaining an intact intestinal barrier. Accordingly, first data suggest depletion of ILCs to be involved in human immunodeficiency virus (HIV-associated damage of the intestinal mucosa and subsequent microbial translocation. However, although ILCs are preferentially localized at mucosal surfaces, only little is known regarding distribution and function of ILCs in the human gastrointestinal tract. Here, we show that in HIV(- individuals composition and functional capacity of intestinal ILCs is compartment-specific with group 1 ILCs representing the major fraction in the upper gastrointestinal (GI tract, whereas ILC3 are the predominant population in ileum and colon, respectively. In addition, we present first data indicating that local cytokine concentrations, especially that of IL-7, might modulate composition of gut ILCs. Distribution of intestinal ILCs was significantly altered in HIV patients, who displayed decreased frequency of total ILCs in ileum and colon owing to reduced numbers of both CD127(+ILC1 and ILC3. Of note, frequency of colonic ILC3 was inversely correlated with serum levels of I-FABP and sCD14, surrogate markers for loss of gut barrier integrity and microbial translocation, respectively. Both expression of the IL-7 receptor CD127 on ILCs as well as mucosal IL-7 mRNA levels were decreased in HIV(+ patients, especially in those parts of the GI tract with reduced ILC frequencies, suggesting that impaired IL-7 responses of ILCs might contribute to incomplete reconstitution of ILCs under effective anti-retroviral therapy. This is the first report comparing distribution and function of ILCs along the intestinal mucosa of the entire human gastrointestinal tract in HIV(+ and HIV(- individuals.

  18. WaveCNV: allele-specific copy number alterations in primary tumors and xenograft models from next-generation sequencing.

    Science.gov (United States)

    Holt, Carson; Losic, Bojan; Pai, Deepa; Zhao, Zhen; Trinh, Quang; Syam, Sujata; Arshadi, Niloofar; Jang, Gun Ho; Ali, Johar; Beck, Tim; McPherson, John; Muthuswamy, Lakshmi B

    2014-03-15

    Copy number variations (CNVs) are a major source of genomic variability and are especially significant in cancer. Until recently microarray technologies have been used to characterize CNVs in genomes. However, advances in next-generation sequencing technology offer significant opportunities to deduce copy number directly from genome sequencing data. Unfortunately cancer genomes differ from normal genomes in several aspects that make them far less amenable to copy number detection. For example, cancer genomes are often aneuploid and an admixture of diploid/non-tumor cell fractions. Also patient-derived xenograft models can be laden with mouse contamination that strongly affects accurate assignment of copy number. Hence, there is a need to develop analytical tools that can take into account cancer-specific parameters for detecting CNVs directly from genome sequencing data. We have developed WaveCNV, a software package to identify copy number alterations by detecting breakpoints of CNVs using translation-invariant discrete wavelet transforms and assign digitized copy numbers to each event using next-generation sequencing data. We also assign alleles specifying the chromosomal ratio following duplication/loss. We verified copy number calls using both microarray (correlation coefficient 0.97) and quantitative polymerase chain reaction (correlation coefficient 0.94) and found them to be highly concordant. We demonstrate its utility in pancreatic primary and xenograft sequencing data. Source code and executables are available at https://github.com/WaveCNV. The segmentation algorithm is implemented in MATLAB, and copy number assignment is implemented Perl. lakshmi.muthuswamy@gmail.com Supplementary data are available at Bioinformatics online.

  19. Recombinant Helicoverpa armigera nucleopolyhedrovirus with arthropod-specific neurotoxin gene RjAa17f from Rhopalurus junceus enhances the virulence against the host larvae.

    Science.gov (United States)

    Yu, Huan; Zhou, Bin; Meng, Jiao; Xu, Jian; Liu, Tong-Xian; Wang, Dun

    2017-06-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV) expressing the insect-selective neurotoxin (RjAa17f) from Cuban scorpion Rhopalurus junceus was constructed by replacing the UDP-glucosyltransferase gene (egt) using λ-red homologous recombination system. Another egt deleted control HearNPV was constructed in a similar way by inserting egfp gene into the egt locus. One-step viral growth curve and viral DNA replication curve analysis confirmed that the recombination did not affect the viral growth and DNA replication in host cells. There is no discernable difference in occlusion-body morphogenesis between RjAa17f-HearNPV, Egfp-HearNPV and HZ8-HearNPV, which was confirmed by transmission electron microscopy analysis. However, the insecticidal activity of RjAa17f-HearNPV is enhanced against the third instar H. armigera larvae according to the bioassay on virulence comparison. There is a dramatic reduction (56.9%) in median lethal dose (LD 50 ) and also a reduction (13.4%) in median survival time (ST 50 ) for the recombinant RjAa17f-HearNPV compared to the HZ8-HearNPV, but only a 27.5% reduction in LD 50 and 10.1% reduction in ST 50 value when Egfp-HearNPV is compared with HZ8-HearNPV. The daily diet consumption analysis showed that the RjAa17f-HearNPV was able to inhibit the infected larvae feeding compared with the egt minus HearNPV. These results demonstrated that this novel recombinant RjAa17f-HearNPV could improve the insecticidal effect against its host insects and RjAa17f could be a considerable candidate for other recombinant baculovirus constructions. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  20. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    for assaying recombination using tetrad analysis in a higher eukaryotic system (6). This system enabled the measurement of the frequency and distribution of recombination events at a genome wide level in wild type Arabidopsis (7), construction of genetic linkage maps which include positions for each centromere (8), and modeling of the strength and pattern of interference (9). This proposal extends the use of tetrad analysis in Arabidopsis by using it as the basis for assessing the phenotypes of mutants in genes important for recombination and the regulation of crossover interference and performing a novel genetic screen. In addition to broadening our knowledge of a classic genetic problem - the regulation of recombination by crossover interference - this proposal also provides broader impact by: generating pedagogical tools for use in hands-on classroom experience with genetics, building interdisciplinary collegial partnerships, and creating a platform for participation by junior scientists from underrepresented groups. There are three specific aims: (1) Isolate mutants in Arabidopsis MUS81 homologs using T-DNA and TILLING (2) Characterize recombination levels and interference in mus81 mutants (3) Execute a novel genetic screen, based on tetrad analysis, for genes that regulate meiotic recombination

  1. Recombinant renewable polyclonal antibodies.

    Science.gov (United States)

    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.

  2. Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases.

    Science.gov (United States)

    Niehrs, C; Huttner, W B; Carvallo, D; Degryse, E

    1990-06-05

    Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.

  3. Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A Case Study for the Site-Specific Attachment of Polyethylene Glycol (PEGylation) of Recombinant Human Ciliary Neurotrophic Factor.

    Science.gov (United States)

    Wang, Qi; Zhang, Chun; Guo, Fangxia; Li, Zenglan; Liu, Yongdong; Su, Zhiguo

    2017-11-15

    In this paper, we reported a novel strategy for the site-specific attachment of polyethylene glycol (PEGylation) of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was monomethoxy-polyethylene glycol-maleimide (mPEG-MAL). PEGylation with mPEG (40 kDa)-MAL at pH 7.0 under normal pressure for 5 h achieved a less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: (1) high-hydrostatic-pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG-MAL), while the conventional process needs a much-higher ratio. (2) Short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. (3) The reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by a factor of three from pH 7.0 to 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software, and we found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was the facilitation of target-site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated that the HHPP product was comparable to the PEGylated rhCNTF prepared through a conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules.

  4. High-Mannose Specific Lectin and Its Recombinants from a Carrageenophyta Kappaphycus alvarezii Represent a Potent Anti-HIV Activity Through High-Affinity Binding to the Viral Envelope Glycoprotein gp120.

    Science.gov (United States)

    Hirayama, Makoto; Shibata, Hiromi; Imamura, Koji; Sakaguchi, Takemasa; Hori, Kanji

    2016-02-01

    We previously reported that a high-mannose binding lectin KAA-2 from the red alga Kappaphycus alvarezii, which is an economically important species and widely cultivated as a source of carrageenans, had a potent anti-influenza virus activity. In this study, the full-length sequences of two KAA isoforms, KAA-1 and KAA-2, were elucidated by a combination of peptide mapping and complementary DNA (cDNA) cloning. They consisted of four internal tandem-repeated domains, which are conserved in high-mannose specific lectins from lower organisms, including a cyanobacterium Oscillatoria agardhii and a red alga Eucheuma serra. Using an Escherichia coli expression system, an active recombinant form of KAA-1 (His-tagged rKAA-1) was successfully generated in the yield of 115 mg per liter of culture. In a detailed oligosaccharide binding analysis by a centrifugal ultrafiltration-HPLC method with 27 pyridylaminated oligosaccharides, His-tagged rKAA-1 and rKAA-1 specifically bound to high-mannose N-glycans with an exposed α1-3 mannose in the D2 arm as the native lectin did. Predicted from oligosaccharide binding specificity, a surface plasmon resonance analysis revealed that the recombinants exhibit strong interaction with gp120, a heavily glycosylated envelope glycoprotein of HIV with high association constants (1.48 - 1.61 × 10(9) M(-1)). Native KAAs and the recombinants inhibited the HIV-1 entry at IC50s of low nanomolar levels (7.3-12.9 nM). Thus, the recombinant proteins would be useful as antiviral reagents targeting the viral surface glycoproteins with high-mannose N-glycans, and the cultivated alga K. alvarezii could also be a good source of not only carrageenans but also this functional lectin(s).

  5. Mineralogical characterization of pristine, bio-eroded and fossil bivalve shell material for the evaluation of a species-specific alteration potential

    Science.gov (United States)

    Hippler, Dorothee; Goetschl, Katja Elisabeth; Gerstmann, Brigitte Simone; Rafael Garcia-March, Jose; Dietzel, Martin

    2015-04-01

    Biogenic carbonates of marine calcifiers can provide a wealth of information for the reconstruction of modern and palaeo-environments. However, their composite carbonate shells are often prone to different alteration processes that might occur during their lifetime, post-mortem or during early diagenesis. In order to use these calcifiers as palaeo-archives or proxy carriers, it is thus of crucial importance to assess their alteration potential. Here, we present the mineral phase composition of four different Mediterranean bivalve species (Spondylus, Lithophaga, Arca, Glycymeris) using spatially selected, powder XRD analysis, as well as in-situ high-resolution Raman spectroscopy. The sample set thereby comprises pristine-modern, bioeroded-modern, Holocene and Pleistocene specimens of the same bivalve species in order to characterize and evaluate the species-specific susceptibility to bioerosion and diagenetic alteration. We reveal species-specific shell compositions that are validated by both analytical methods. Differences in shell mineralogy occur between the outermost (periostracum), the outer (ostracum) and inner (hypostracum) layer, with the outer layer mainly composed of calcite and the inner layers of aragonite with variable portions of calcite. Considerable species-specific changes in mineralogy of the respective shell layers with increasing geological age are not found. Our results indicate that the original shell mineralogy (calcite, aragonite and carbonate fluorapatite) as well as the composition, structure and thickness of the respective shell layers are important factors favouring or preventing alteration to occur. Moreover, our findings highlight the effect of bioerosion during the alteration process. The analysis of distinct areas of the shells hinting at microbial activity reveals slight changes in shell mineralogy. We thus postulate that processes related to shell taphonomy are crucial for the shell's alteration/preservational potential and thus

  6. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    Science.gov (United States)

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  7. Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer's Disease.

    Science.gov (United States)

    Zafar, Saima; Younas, Neelam; Correia, Susana; Shafiq, Mohsin; Tahir, Waqas; Schmitz, Matthias; Ferrer, Isidre; Andréoletti, Olivier; Zerr, Inga

    2017-01-01

    There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrP C and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical

  8. Distinguishing nuclei-specific benzo[a]pyrene-induced effects from whole-cell alterations in MCF-7 cells using Fourier-transform infrared spectroscopy.

    Science.gov (United States)

    Obinaju, Blessing E; Fullwood, Nigel J; Martin, Francis L

    2015-09-01

    Exposure to chemicals such as benzo[a]pyrene (B[a]P) can generate intracellular toxic mechanisms. Fourier-transform infrared (FTIR) spectroscopy is a novel approach that allows the non-destructive analysis of underlying chemical bond alterations in patho-physiological processes. This study set out to examine whether B[a]P-induced whole cell alterations could be distinguished from effects on nuclei of exposed cells. Using attenuated total reflection FTIR (ATR-FTIR) spectroscopy, alterations in nuclei isolated from B[a]P-treated MCF-7 cells concentrated either in G0/G1- or S-phase were observed. B[a]P-induced effects in whole-cells included alterations to lipids, DNA and protein spectral regions. Absorbance areas for protein and DNA/RNA regions in B[a]P-treated whole cells differed significantly (PFTIR spectroscopy has the ability to identify specific chemical-induced alterations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Habitat-specific differences alter traditional biogeographic patterns of life history in a climate-change induced range expansion

    OpenAIRE

    Riley, Megan E.; Griffen, Blaine D.

    2017-01-01

    Range shifts and expansions resulting from global climate change have the potential to create novel communities with unique plant-animal interactions. Organisms expanding their range into novel biotic and abiotic environments may encounter selection pressures that alter traditional biogeographic patterns of life history traits. Here, we used field surveys to examine latitudinal patterns of life history traits in a broadly distributed ectotherm (mangrove tree crab Aratus pisonii) that has rece...

  10. MicroRNA Expression Is Altered in an Ovalbumin-Induced Asthma Model and Targeting miR-155 with Antagomirs Reveals Cellular Specificity.

    Directory of Open Access Journals (Sweden)

    Maximilian W Plank

    Full Text Available MicroRNAs are post-transcriptional regulators of gene expression that are differentially regulated during development and in inflammatory diseases. A role for miRNAs in allergic asthma is emerging and further investigation is required to determine whether they may serve as potential therapeutic targets. We profiled miRNA expression in murine lungs from an ovalbumin-induced allergic airways disease model, and compared expression to animals receiving dexamethasone treatment and non-allergic controls. Our analysis identified 29 miRNAs that were significantly altered during allergic inflammation. Target prediction analysis revealed novel genes with altered expression in allergic airways disease and suggests synergistic miRNA regulation of target mRNAs. To assess the impacts of one induced miRNA on pathology, we targeted miR-155-5p using a specific antagomir. Antagomir administration successfully reduced miR-155-5p expression with high specificity, but failed to alter the disease phenotype. Interestingly, further investigation revealed that antagomir delivery has variable efficacy across different immune cell types, effectively targeting myeloid cell populations, but exhibiting poor uptake in lymphocytes. Our findings demonstrate that antagomir-based targeting of miRNA function in the lung is highly specific, but highlights cell-specificity as a key limitation to be considered for antagomir-based strategies as therapeutics.

  11. 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

    Science.gov (United States)

    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. PMID:24086379

  12. 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.

    Directory of Open Access Journals (Sweden)

    Avinash Kollipara

    Full Text Available 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.

  13. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination.

    Science.gov (United States)

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-03-31

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of 'non-productive', 'pre-synaptic' and 'synaptic' complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination

    Science.gov (United States)

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-01-01

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. PMID:25765648

  15. Hemodynamics in Transplant Renal Artery Stenosis and its Alteration after Stent Implantation Based on a Patient-specific Computational Fluid Dynamics Model

    OpenAIRE

    Wang, Hong-Yang; Liu, Long-Shan; Cao, Hai-Ming; Li, Jun; Deng, Rong-Hai; Fu, Qian; Zhang, Huan-Xi; Fei, Ji-Guang; Wang, Chang-Xi

    2017-01-01

    Background: Accumulating studies on computational fluid dynamics (CFD) support the involvement of hemodynamic factors in artery stenosis. Based on a patient-specific CFD model, the present study aimed to investigate the hemodynamic characteristics of transplant renal artery stenosis (TRAS) and its alteration after stent treatment. Methods: Computed tomography angiography (CTA) data of kidney transplant recipients in a single transplant center from April 2013 to November 2014 were reviewed. Th...

  16. Site directed recombination

    Science.gov (United States)

    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.

  17. A demographic transition altered the strength of selection for fitness and age-specific survival and fertility in a 19th century American population.

    Science.gov (United States)

    Moorad, Jacob A

    2013-06-01

    Modernization has increased longevity and decreased fertility in many human populations, but it is not well understood how or to what extent these demographic transitions have altered patterns of natural selection. I integrate individual-based multivariate phenotypic selection approaches with evolutionary demographic methods to demonstrate how a demographic transition in 19th century female populations of Utah altered relationships between fitness and age-specific survival and fertility. Coincident with this demographic transition, natural selection for fitness, as measured by the opportunity for selection, increased by 13% to 20% over 65 years. Proportional contributions of age-specific survival to total selection (the complement to age-specific fertility) diminished from approximately one third to one seventh following a marked increase in infant survival. Despite dramatic reductions in age-specific fertility variance at all ages, the absolute magnitude of selection for fitness explained by age-specific fertility increased by approximately 45%. I show that increases in the adaptive potential of fertility traits followed directly from decreased population growth rates. These results suggest that this demographic transition has increased the adaptive potential of the Utah population, intensified selection for reproductive traits, and de-emphasized selection for survival-related traits. © 2013 The Author(s). Evolution © 2013 The Society for the Study of Evolution.

  18. Controlled Release from Recombinant Polymers

    Science.gov (United States)

    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

  19. Specifically altered brain responses to threat in generalized anxiety disorder relative to social anxiety disorder and panic disorder

    Directory of Open Access Journals (Sweden)

    Christine Buff

    2016-01-01

    Conclusion: The findings present the first evidence for GAD-specific neural correlates of hyper-responsive threat processing, possibly reflecting exaggerated threat sensitivity, maladaptive appraisal and attention-allocation processes.

  20. Recombinant Collagenlike Proteins

    Science.gov (United States)

    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.

  1. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines.

    Science.gov (United States)

    Khurana, Surender; Larkin, Christopher; Verma, Swati; Joshi, Manju B; Fontana, Juan; Steven, Alasdair C; King, Lisa R; Manischewitz, Jody; McCormick, William; Gupta, Rajesh K; Golding, Hana

    2011-08-05

    Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009-2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified. Published by Elsevier Ltd.

  2. Specifically altered brain responses to threat in generalized anxiety disorder relative to social anxiety disorder and panic disorder.

    Science.gov (United States)

    Buff, Christine; Brinkmann, Leonie; Neumeister, Paula; Feldker, Katharina; Heitmann, Carina; Gathmann, Bettina; Andor, Tanja; Straube, Thomas

    2016-01-01

    Despite considerable effort, the neurobiological underpinnings of hyper-responsive threat processing specific to patients suffering from generalized anxiety disorder (GAD) remain poorly understood. The current functional magnetic resonance imaging (fMRI) study aims to delineate GAD-specific brain activity during immediate threat processing by comparing GAD patients to healthy controls (HC), to social anxiety disorder (SAD) and to panic disorder (PD) patients. Brain activation and functional connectivity patterns to threat vs. neutral pictures were investigated using event-related fMRI. The sample consisted of 21 GAD, 21 PD, 21 SAD and 21 HC. GAD-specific elevated activity to threat vs. neutral pictures was found in cingulate cortex, dorsal anterior insula/frontal operculum (daI/FO) and posterior dorsolateral prefrontal cortex (dlPFC). Defining these effects as seed regions, we detected GAD-specific increased functional connectivity to threat vs. neutral pictures between posterior dlPFC and ventrolateral prefrontal cortex, between cingulate cortex and amygdala, between cingulate cortex and anterior insula, as well as decreased functional connectivity between daI/FO and mid-dlPFC. The findings present the first evidence for GAD-specific neural correlates of hyper-responsive threat processing, possibly reflecting exaggerated threat sensitivity, maladaptive appraisal and attention-allocation processes.

  3. Hip strategy alterations in patients with history of low disc herniation and non-specific low back pain measured by surface electromyography and balance platform.

    Science.gov (United States)

    Ciesielska, Jagoda; Lisiński, Przemysław; Bandosz, Agata; Huber, Juliusz; Kulczyk, Aleksandra; Lipiec, Joanna

    2015-01-01

    The appearance of pathology in the lumbar spine, such as a previous episode of low disc herniation or non-specific low back pain contributes to improper activation of the hip muscles. The aim of the study was to detect alterations in hip strategy manifested by differences in balance parameters and rectus femoris and gluteus maximus activity in people with previous episode of pain radiation to one lower limb caused by low disc herniation or non-specific low back pain. We studied 11 patients with history of low-disc herniation, 9 patients with history of non-specific low back pain and 10 healthy subjects. Hip strategy alterations were detected by measuring rectus femoris and gluteus maximus activity in bilateral surface polyelectromyographic recordings and by stability measurements on a balance platform. In the surface polyelectromyography study, in both patients' group the value of the average amplitude was higher and the amount of the fluctuations was lower than in healthy subjects. There were no significant differences in stability parameters. A changed pattern of hip muscles activity was detected in the patients without changes in stability parameters. Greater disorder occurs in people in with previous episode of pain radiation to one lower limb caused by low disc herniation than in people with non-specific low back pain.

  4. Nondisjunction of chromosome 15: Origin and recombination

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, W.P.; Bernasconi, F.; Schinzel, A.A.; Mutirangura, A.; Ledbetter, D.H. (Baylor College of Medicine, Houston, TX (United States)); Langlois, S. (Univ. of Britisch Columbia, Vancouver (Canada)); 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.

  5. Perinatal BPA exposure alters body weight and composition in a dose specific and sex specific manner: The addition of peripubertal exposure exacerbates adverse effects in female mice.

    Science.gov (United States)

    Rubin, Beverly S; Paranjpe, Maneesha; DaFonte, Tracey; Schaeberle, Cheryl; Soto, Ana M; Obin, Martin; Greenberg, Andrew S

    2017-03-01

    Body weight (BW) and body composition were examined in CD-1 mice exposed perinatally or perinatally and peripubertally to 0, 0.25, 2.5, 25, or 250μg BPA/kg BW/day. Our goal was to identify the BPA dose (s) and the exposure window(s) that increased BW and adiposity, and to assess potential sex differences in this response. Both perinatal exposure alone and perinatal plus peripubertal exposure to environmentally relevant levels of BPA resulted in lasting effects on body weight and body composition. The effects were dose specific and sex specific and were influenced by the precise window of BPA exposure. The addition of peripubertal BPA exposure following the initial perinatal exposure exacerbated adverse effects in the females but appeared to reduce differences in body weight and body composition between control and BPA exposed males. Some effects of BPA on body weight and body composition showed a non-linear dose response. Copyright © 2016. Published by Elsevier Inc.

  6. Various lamin A/C mutations alter expression profile of mesenchymal stem cells in mutation specific manner.

    Science.gov (United States)

    Malashicheva, Anna; Bogdanova, Maria; Zabirnyk, Arsenii; Smolina, Natalia; Ignatieva, Elena; Freilikhman, Olga; Fedorov, Anton; Dmitrieva, Renata; Sjöberg, Gunnar; Sejersen, Thomas; Kostareva, Anna

    2015-01-01

    Various mutations in LMNA gene, encoding for nuclear lamin A/C protein, lead to laminopathies and contribute to over ten human disorders, mostly affecting tissues of mesenchymal origin such as fat tissue, muscle tissue, and bones. Recently it was demonstrated that lamins not only play a structural role providing communication between extra-nuclear structures and components of cell nucleus but also control cell fate and differentiation. In our study we assessed the effect of various LMNA mutations on the expression profile of mesenchymal multipotent stem cells (MMSC) during adipogenic and osteogenic differentiation. We used lentiviral approach to modify human MMSC with LMNA-constructs bearing mutations associated with different laminopathies--G465D, R482L, G232E, R527C, and R471C. The impact of various mutations on MMSC differentiation properties and expression profile was assessed by colony-forming unit analysis, histological staining, expression of the key differentiation markers promoting adipogenesis and osteogenesis followed by the analysis of the whole set of genes involved in lineage-specific differentiation using PCR expression arrays. We demonstrate that various LMNA mutations influence the differentiation efficacy of MMSC in mutation-specific manner. Each LMNA mutation promotes a unique expression pattern of genes involved in a lineage-specific differentiation and this pattern is shared by the phenotype-specific mutations. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Obesity Induces Artery-Specific Alterations: Evaluation of Vascular Function and Inflammatory and Smooth Muscle Phenotypic Markers

    Directory of Open Access Journals (Sweden)

    Antonio Garcia Soares

    2017-01-01

    Full Text Available Vascular alterations are expected to occur in obese individuals but the impact of obesity could be different depending on the artery type. We aimed to evaluate the obesity effects on the relaxing and contractile responses and inflammatory and smooth muscle (SM phenotypic markers in two vascular beds. Obesity was induced in C57Bl/6 mice by 16-week high-fat diet and vascular reactivity, mRNA expression of inflammatory and SM phenotypic markers, and collagen deposition were evaluated in small mesenteric arteries (SMA and thoracic aorta (TA. Endothelium-dependent relaxation in SMA and TA was not modified by obesity. In contrast, contraction induced by depolarization and contractile agonists was reduced in SMA, whereas only contraction induced by adrenergic agonist was reduced in TA of obese mice. Obesity increased the mRNA expression of pro- and anti-inflammatory cytokines in SMA and TA. The expression of genes necessary for maintaining contractile ability was increased by obesity, but the increase was more pronounced in TA. Collagen deposition was increased in SMA, but not in TA, of obese mice. Although the endothelial function was still preserved, the SM of the two artery types was impaired by obesity, but the impairment was higher in SMA, which could be associated with SM phenotypic changes.

  8. Oral vaccination with a recombinant Salmonella vaccine vector provokes systemic HIV-1 subtype C Gag-specific CD4+ Th1 and Th2 cell immune responses in mice

    Directory of Open Access Journals (Sweden)

    Williamson Anna-Lise

    2009-06-01

    Full Text Available Abstract Background Recombinant Salmonella vaccine vectors may potentially be used to induce specific CD4+ T cell responses against foreign viral antigens. Such immune responses are required features of vaccines against pathogens such as human immunodeficiency virus type 1 (HIV-1. The aim of this study was to investigate the induction of systemic HIV-1-specific CD4+ T helper (Th responses in mice after oral immunization with a live attenuated Salmonella vaccine vector that expressed HIV-1 subtype C Gag. Groups of BALB/c mice were vaccinated orally three times (4 weeks apart with this recombinant Salmonella. At sacrifice, 28 days after the last immunization, systemic CD4+ Th1 and Th2 cytokine responses were evaluated by enzyme-linked immunospot assay and cytometric bead array. HIV-1 Gag-specific IgG1 and IgG2a humoral responses in the serum were determined by enzyme-linked immunosorbent assay. Results Mice vaccinated with the recombinant Salmonella elicited both HIV-1-specific Th1 (interferon-gamma (IFN-γ and tumour necrosis factor-alpha (TNF-α and Th2 (interleukin-4 (IL-4 and interleukin-5 (IL-5 cytokine responses. The vaccine induced 70 (IFN-γ spot-forming units (SFUs/10e6 splenocytes and 238 IL-4 SFUs/10e6 splenocytes. Splenocytes from vaccinated mice also produced high levels of Th1 and Th2 cytokines upon stimulation with a Gag CD4 peptide. The levels of IFN-γ, TNF-α, IL-4 and IL-5 were 7.5-, 29.1-, 26.2- and 89.3-fold above the background, respectively. Both HIV-1 Gag-specific IgG1 and IgG2a antibodies were detected in the sera of vaccinated mice. Conclusion The study highlights the potential of orally-delivered attenuated Salmonella as mucosal vaccine vectors for HIV-1 Subtype C Gag to induce Gag-specific CD4+ Th1 and Th2 cellular immune responses and antibodies which may be important characteristics required for protection against HIV-1 infection.

  9. Plasmid-Chromosome Recombination of Irradiated Shuttle Vector DNA in African Green Monkey Kidney Cells.

    Science.gov (United States)

    Mudgett, John Stuart

    1987-09-01

    An autonomously replicating shuttle vector was used to investigate the enhancement of plasmid-chromosome recombination in mammalian host cells by ultraviolet light and gamma radiation. Sequences homologous to the shuttle vector were stably inserted into the genome of African Green Monkey kidney cells to act as the target substrate for these recombination events. The SV40- and pBR322-derived plasmid DNA was irradiated with various doses of radiation before transfection into the transformed mammalian host cells. The successful homologous transfer of the bacterial ampicillin resistance (amp^{rm r}) gene from the inserted sequences to replace a mutant amp^->=ne on the shuttle vector was identified by plasmid extraction and transformation into E. coli host cells. Ultraviolet light (UV) was found not to induce homologous plasmid-chromosome recombination, while gamma radiation increased the frequency of recombinant plasmids detected. The introduction of specific double -strand breaks in the plasmid or prolonging the time of plasmid residence in the mammalian host cells also enhanced plasmid-chromosome recombination. In contrast, plasmid mutagenesis was found to be increased by plasmid UV irradiation, but not to change with time. Plasmid survival, recombination, and mutagenesis were not affected by treating the mammalian host cells with UV light prior to plasmid transfection. The amp^{rm r} recombinant plasmid molecules analyzed were found to be mostly the result of nonconservative exchanges which appeared to involve both homologous and possibly nonhomologous interactions with the host chromosome. The observation that these recombinant structures were obtained from all of the plasmid alterations investigated suggests a common mechanistic origin for plasmid -chromosome recombination in these mammalian cells.

  10. Hypertensive patients exhibit an altered metabolism. A specific metabolite signature in urine is able to predict albuminuria progression.

    Science.gov (United States)

    Gonzalez-Calero, Laura; Martin-Lorenzo, Marta; Martínez, Paula J; Baldan-Martin, Montserrat; Ruiz-Hurtado, Gema; Segura, Julian; de la Cuesta, Fernando; Barderas, Maria G; Ruilope, Luis M; Vivanco, Fernando; Alvarez-Llamas, Gloria

    2016-12-01

    Hypertension (HTN) is increasing in prevalence, and albuminuria is a strong indicator of cardiovascular risk and renal damage progression. Despite blood pressure control with chronic treatment, a relevant subgroup of patients develop albuminuria. However, the biological factors responsible for albuminuria development and progression are underexplored. We aimed to identify key metabolic targets and biological pathways involved in the negative progression of cardiovascular and renal damage in hypertensives undergoing chronic treatment. A series of 1533 patients were followed for 5 years to investigate the evolution of albuminuria. Patients were classified as: (1) patients with persistent normoalbuminuria; (2) patients developing de novo albuminuria; and (3) patients with maintained albuminuria. At the end of follow-up, urine from 30 nonhypertensive subjects (control group) and a representative cohort of 118 patients was collected for metabolomic analysis. Metabolic patterns of interest were identified in a first discovery phase by nuclear magnetic resonance and further confirmed by liquid chromatography-mass spectrometry. Metabolites corresponding to HTN or albuminuria were measured in a prospective study carried out in 35 individuals still in normoalbuminuria, to evaluate their potential as predictors of albuminuria development. Nine metabolites were significantly altered, linking β-alanine metabolism, arginine and proline metabolism, and tricarboxylic acid cycle. The prospective study revealed a panel composed of guanidinoacetate, glutamate, and pantothenate, which was able to predict development of albuminuria. These metabolic signatures open new possibilities in hypertensive therapy and cardiovascular risk control, providing prompt and more efficient intervention, particularly in patients with worse cardiovascular prognosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Accumulation of DNA damage-induced chromatin alterations in tissue-specific stem cells: the driving force of aging?

    Directory of Open Access Journals (Sweden)

    Nadine Schuler

    Full Text Available Accumulation of DNA damage leading to stem cell exhaustion has been proposed to be a principal mechanism of aging. Using 53BP1-foci as a marker for DNA double-strand breaks (DSBs, hair follicle stem cells (HFSCs in mouse epidermis were analyzed for age-related DNA damage response (DDR. We observed increasing amounts of 53BP1-foci during the natural aging process independent of telomere shortening and after protracted low-dose radiation, suggesting substantial accumulation of DSBs in HFSCs. Electron microscopy combined with immunogold-labeling showed multiple small 53BP1 clusters diffusely distributed throughout the highly compacted heterochromatin of aged HFSCs, but single large 53BP1 clusters in irradiated HFSCs. These remaining 53BP1 clusters did not colocalize with core components of non-homologous end-joining, but with heterochromatic histone modifications. Based on these results we hypothesize that these lesions were not persistently unrepaired DSBs, but may reflect chromatin rearrangements caused by the repair or misrepair of DSBs. Flow cytometry showed increased activation of repair proteins and damage-induced chromatin modifications, triggering apoptosis and cellular senescence in irradiated, but not in aged HFSCs. These results suggest that accumulation of DNA damage-induced chromatin alterations, whose structural dimensions reflect the complexity of the initial genotoxic insult, may lead to different DDR events, ultimately determining the biological outcome of HFSCs. Collectively, our findings support the hypothesis that aging might be largely the remit of structural changes to chromatin potentially leading to epigenetically induced transcriptional deregulation.

  12. Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity.

    Science.gov (United States)

    Morelli, A E; Larregina, A T; Smith-Arica, J; Dewey, R A; Southgate, T D; Ambar, B; Fontana, A; Castro, M G; Lowenstein, P R

    1999-03-01

    Gene therapy using Fas ligand (FasL) for treatment of tumours and protection of transplant rejection is hampered because of the systemic toxicity of FasL. In the present study, recombinant replication-defective adenovirus vectors (RAds) encoding FasL under the control of either the neuronal-specific neuronal-specific enolase (NSE) promoter or the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter have been constructed. The cell type-specific expression of FasL in both neurons and glial cells in primary cultures, and in neuronal and glial cell lines is demonstrated. Furthermore, transgene expression driven by the neuronal and glial promoter was not detected in fibroblastic or epithelial cell lines. Expression of FasL driven by a major immediate early human cytomegalovirus promoter (MIEhCMV) was, however, achieved in all cells tested. As a final test of the stringency of transgene-specific expression, the RAds were injected directly into the bloodstream of mice. The RAds encoding FasL under the control of the non-cell type-specific MIEhCMV promoter induced acute generalized liver haemorrhage with hepatocyte apoptosis, while the RAds containing the NSE or GFAP promoter sequences were completely non-toxic. This demonstrates the specificity of transgene expression, enhanced safety during systemic administration, and tightly regulated control of transgene expression of highly cytotoxic gene products, encoded within transcriptionally targeted RAds.

  13. Disturbed Placental Imprinting in Preeclampsia Leads to Altered Expression of DLX5, a Human-Specific Early Trophoblast Marker.

    Science.gov (United States)

    Zadora, Julianna; Singh, Manvendra; Herse, Florian; Przybyl, Lukasz; Haase, Nadine; Golic, Michaela; Yung, Hong Wa; Huppertz, Berthold; Cartwright, Judith E; Whitley, Guy; Johnsen, Guro M; Levi, Giovanni; Isbruch, Annette; Schulz, Herbert; Luft, Friedrich C; Müller, Dominik N; Staff, Anne Cathrine; Hurst, Laurence D; Dechend, Ralf; Izsvák, Zsuzsanna

    2017-11-07

    Preeclampsia is a complex and common human-specific pregnancy syndrome associated with placental pathology. The human specificity provides both intellectual and methodological challenges, lacking a robust model system. Given the role of imprinted genes in human placentation and the vulnerability of imprinted genes to loss of imprinting changes, there has been extensive speculation, but no robust evidence, that imprinted genes are involved in preeclampsia. Our study aims to investigate whether disturbed imprinting contributes to preeclampsia. We first aimed to confirm that preeclampsia is a disease of the placenta by generating and analyzing genome-wide molecular data on well-characterized patient material. We performed high-throughput transcriptome analyses of multiple placenta samples from healthy controls and patients with preeclampsia. Next, we identified differentially expressed genes in preeclamptic placentas and intersected them with the list of human imprinted genes. We used bioinformatics/statistical analyses to confirm association between imprinting and preeclampsia and to predict biological processes affected in preeclampsia. Validation included epigenetic and cellular assays. In terms of human specificity, we established an in vitro invasion-differentiation trophoblast model. Our comparative phylogenetic analysis involved single-cell transcriptome data of human, macaque, and mouse preimplantation embryogenesis. We found disturbed placental imprinting in preeclampsia and revealed potential candidates, including GATA3 and DLX5 , with poorly explored imprinted status and no prior association with preeclampsia. As a result of loss of imprinting, DLX5 was upregulated in 69% of preeclamptic placentas. Levels of DLX5 correlated with classic preeclampsia markers. DLX5 is expressed in human but not in murine trophoblast. The DLX5 high phenotype resulted in reduced proliferation, increased metabolism, and endoplasmic reticulum stress-response activation in

  14. The carboxy-terminal αN helix of the archaeal XerA tyrosine recombinase is a molecular switch to control site-specific recombination.

    Directory of Open Access Journals (Sweden)

    Marie-Claude Serre

    Full Text Available Tyrosine recombinases are conserved in the three kingdoms of life. Here we present the first crystal structure of a full-length archaeal tyrosine recombinase, XerA from Pyrococcus abyssi, at 3.0 Å resolution. In the absence of DNA substrate XerA crystallizes as a dimer where each monomer displays a tertiary structure similar to that of DNA-bound Tyr-recombinases. Active sites are assembled in the absence of dif except for the catalytic Tyr, which is extruded and located equidistant from each active site within the dimer. Using XerA active site mutants we demonstrate that XerA follows the classical cis-cleavage reaction, suggesting rearrangements of the C-terminal domain upon DNA binding. Surprisingly, XerA C-terminal αN helices dock in cis in a groove that, in bacterial tyrosine recombinases, accommodates in trans αN helices of neighbour monomers in the Holliday junction intermediates. Deletion of the XerA C-terminal αN helix does not impair cleavage of suicide substrates but prevents recombination catalysis. We propose that the enzymatic cycle of XerA involves the switch of the αN helix from cis to trans packing, leading to (i repositioning of the catalytic Tyr in the active site in cis and (ii dimer stabilisation via αN contacts in trans between monomers.

  15. Analysis of nuclear and organellar genomes of Plasmodium knowlesi in humans reveals ancient population structure and recent recombination among host-specific subpopulations

    KAUST Repository

    Diez Benavente, Ernest

    2017-09-18

    The macaque parasite Plasmodium knowlesi is a significant concern in Malaysia where cases of human infection are increasing. Parasites infecting humans originate from genetically distinct subpopulations associated with the long-tailed (Macaca fascicularis (Mf)) or pig-tailed macaques (Macaca nemestrina (Mn)). We used a new high-quality reference genome to re-evaluate previously described subpopulations among human and macaque isolates from Malaysian-Borneo and Peninsular-Malaysia. Nuclear genomes were dimorphic, as expected, but new evidence of chromosomal-segment exchanges between subpopulations was found. A large segment on chromosome 8 originating from the Mn subpopulation and containing genes encoding proteins expressed in mosquito-borne parasite stages, was found in Mf genotypes. By contrast, non-recombining organelle genomes partitioned into 3 deeply branched lineages, unlinked with nuclear genomic dimorphism. Subpopulations which diverged in isolation have re-connected, possibly due to deforestation and disruption of wild macaque habitats. The resulting genomic mosaics reveal traits selected by host-vector-parasite interactions in a setting of ecological transition.

  16. Altered heart rate variability in spontaneously hypertensive rats is associated with specific particulate matter components in Detroit, Michigan.

    Science.gov (United States)

    Rohr, Annette C; Kamal, Ali; Morishita, Masako; Mukherjee, Bhramar; Keeler, Gerald J; Harkema, Jack R; Wagner, James G

    2011-04-01

    Exposure to fine particulate matter [aerodynamic diameter ≤ 2.5 μm (PM2.5)] is linked to adverse cardiopulmonary health effects; however, the responsible constituents are not well defined. We used a rat model to investigate linkages between cardiac effects of concentrated ambient particle (CAP) constituents and source factors using a unique, highly time-resolved data set. Spontaneously hypertensive rats inhaled Detroit Michigan, CAPs during summer or winter (2005-2006) for 13 consecutive days. Electrocardiogram data were recorded continuously, and heart rate (HR) and heart rate variability (HRV) metrics were derived. Extensive CAP characterization, including use of a Semicontinuous Elements in Aerosol Sampler (SEAS), was performed, and positive matrix factorization was applied to investigate source factors. Mean CAP exposure concentrations were 518 μg/m(3) and 357 μg/m(3) in the summer and winter, respectively. Significant reductions in the standard deviation of the normal-to-normal intervals (SDNN) in the summer were strongly associated with cement/lime, iron/steel, and gasoline/diesel factors, whereas associations with the sludge factor and components were less consistent. In winter, increases in HR were associated with a refinery factor and its components. CAP-associated HR decreases in winter were linked to sludge incineration, cement/lime, and coal/secondary sulfate factors and most of their associated components. Specific relationships for increased root mean square of the standard deviation of successive normal-to-normal intervals (RMSSD) in winter were difficult to determine because of lack of consistency between factors and associated constituents. Our results indicate that specific modulation of cardiac function in Detroit was most strongly linked to local industrial sources. Findings also highlight the need to consider both factor analytical results and component-specific results when interpreting findings.

  17. Loss of abdominal muscle in Pitx2 mutants associated with altered axial specification of lateral plate mesoderm.

    Directory of Open Access Journals (Sweden)

    Diana Eng

    Full Text Available Sequence specific transcription factors (SSTFs combinatorially define cell types during development by forming recursively linked network kernels. Pitx2 expression begins during gastrulation, together with Hox genes, and becomes localized to the abdominal lateral plate mesoderm (LPM before the onset of myogenesis in somites. The somatopleure of Pitx2 null embryos begins to grow abnormally outward before muscle regulatory factors (MRFs or Pitx2 begin expression in the dermomyotome/myotome. Abdominal somites become deformed and stunted as they elongate into the mutant body wall, but maintain normal MRF expression domains. Subsequent loss of abdominal muscles is therefore not due to defects in specification, determination, or commitment of the myogenic lineage. Microarray analysis was used to identify SSTF families whose expression levels change in E10.5 interlimb body wall biopsies. All Hox9-11 paralogs had lower RNA levels in mutants, whereas genes expressed selectively in the hypaxial dermomyotome/myotome and sclerotome had higher RNA levels in mutants. In situ hybridization analyses indicate that Hox gene expression was reduced in parts of the LPM and intermediate mesoderm of mutants. Chromatin occupancy studies conducted on E10.5 interlimb body wall biopsies showed that Pitx2 protein occupied chromatin sites containing conserved bicoid core motifs in the vicinity of Hox 9-11 and MRF genes. Taken together, the data indicate that Pitx2 protein in LPM cells acts, presumably in combination with other SSTFs, to repress gene expression, that are normally expressed in physically adjoining cell types. Pitx2 thereby prevents cells in the interlimb LPM from adopting the stable network kernels that define sclerotomal, dermomyotomal, or myotomal mesenchymal cell types. This mechanism may be viewed either as lineage restriction or specification.

  18. Loss of abdominal muscle in Pitx2 mutants associated with altered axial specification of lateral plate mesoderm.

    Science.gov (United States)

    Eng, Diana; Ma, Hsiao-Yen; Xu, Jun; Shih, Hung-Ping; Gross, Michael K; Kioussi, Chrissa; Kiouss, Chrissa

    2012-01-01

    Sequence specific transcription factors (SSTFs) combinatorially define cell types during development by forming recursively linked network kernels. Pitx2 expression begins during gastrulation, together with Hox genes, and becomes localized to the abdominal lateral plate mesoderm (LPM) before the onset of myogenesis in somites. The somatopleure of Pitx2 null embryos begins to grow abnormally outward before muscle regulatory factors (MRFs) or Pitx2 begin expression in the dermomyotome/myotome. Abdominal somites become deformed and stunted as they elongate into the mutant body wall, but maintain normal MRF expression domains. Subsequent loss of abdominal muscles is therefore not due to defects in specification, determination, or commitment of the myogenic lineage. Microarray analysis was used to identify SSTF families whose expression levels change in E10.5 interlimb body wall biopsies. All Hox9-11 paralogs had lower RNA levels in mutants, whereas genes expressed selectively in the hypaxial dermomyotome/myotome and sclerotome had higher RNA levels in mutants. In situ hybridization analyses indicate that Hox gene expression was reduced in parts of the LPM and intermediate mesoderm of mutants. Chromatin occupancy studies conducted on E10.5 interlimb body wall biopsies showed that Pitx2 protein occupied chromatin sites containing conserved bicoid core motifs in the vicinity of Hox 9-11 and MRF genes. Taken together, the data indicate that Pitx2 protein in LPM cells acts, presumably in combination with other SSTFs, to repress gene expression, that are normally expressed in physically adjoining cell types. Pitx2 thereby prevents cells in the interlimb LPM from adopting the stable network kernels that define sclerotomal, dermomyotomal, or myotomal mesenchymal cell types. This mechanism may be viewed either as lineage restriction or specification.

  19. Antenatal glucocorticoid treatment alters Na+ uptake in renal proximal tubule cells from adult offspring in a sex-specific manner

    OpenAIRE

    Su, Yixin; Bi, Jianli; Pulgar, Victor M.; Figueroa, Jorge; Chappell, Mark; Rose, James C.

    2015-01-01

    We have shown a sex-specific effect of fetal programming on Na+ excretion in adult sheep. The site of this effect in the kidney is unknown. Therefore, we tested the hypothesis that renal proximal tubule cells (RPTCs) from adult male sheep exposed to betamethasone (Beta) before birth have greater Na+ uptake than do RPTCs from vehicle-exposed male sheep and that RPTCs from female sheep similarly exposed are not influenced by antenatal Beta. In isolated RPTCs from 1- to 1.5-yr-old male and femal...

  20. Disease Type- and Status-Specific Alteration of CSF Metabolome Coordinated with Clinical Parameters in Inflammatory Demyelinating Diseases of CNS.

    Directory of Open Access Journals (Sweden)

    Soo Jin Park

    Full Text Available Central nervous system (CNS inflammatory demyelinating diseases (IDDs are a group of disorders with different aetiologies, characterized by inflammatory lesions. These disorders include multiple sclerosis (MS, neuromyelitis optica spectrum disorder (NMOSD, and idiopathic transverse myelitis (ITM. Differential diagnosis of the CNS IDDs still remains challenging due to frequent overlap of clinical and radiological manifestation, leading to increased demands for new biomarker discovery. Since cerebrospinal fluid (CSF metabolites may reflect the status of CNS tissues and provide an interfacial linkage between blood and CNS tissues, we explored multi-component biomarker for different IDDs from CSF samples using gas chromatography mass spectrometry-based metabolite profiling coupled to multiplex bioinformatics approach. We successfully constructed the single model with multiple metabolite variables in coordinated regression with clinical characteristics, expanded disability status scale, oligoclonal bands, and protein levels. The multi-composite biomarker simultaneously discriminated four different immune statuses (a total of 145 samples; 54 MS, 49 NMOSD, 30 ITM, and 12 normal controls. Furthermore, systematic characterization of transitional metabolic modulation identified relapse-associated metabolites and proposed insights into the disease network underlying type-specific metabolic dysfunctionality. The comparative analysis revealed the lipids, 1-monopalmitin and 1-monostearin were common indicative for MS, NMOSD, and ITM whereas fatty acids were specific for the relapse identified in all types of IDDs.

  1. Muscle and liver-specific alterations in lipid and acylcarnitine metabolism after a single bout of exercise in mice.

    Science.gov (United States)

    Hoene, Miriam; Li, Jia; Li, Yanjie; Runge, Heike; Zhao, Xinjie; Häring, Hans-Ulrich; Lehmann, Rainer; Xu, Guowang; Weigert, Cora

    2016-02-26

    Intracellular lipid pools are highly dynamic and tissue-specific. Physical exercise is a strong physiologic modulator of lipid metabolism, but most studies focus on changes induced by long-term training. To assess the acute effects of endurance exercise, mice were subjected to one hour of treadmill running, and (13)C16-palmitate was applied to trace fatty acid incorporation in soleus and gastrocnemius muscle and liver. The amounts of carnitine, FFA, lysophospholipids and diacylglycerol and the post-exercise increase in acetylcarnitine were pronouncedly higher in soleus than in gastrocnemius. In the liver, exercise increased the content of lysophospholipids, plasmalogens and carnitine as well as transcript levels of the carnitine transporter. (13)C16-palmitate was detectable in several lipid and acylcarnitine species, with pronounced levels of tracer-derived palmitoylcarnitine in both muscles and a strikingly high incorporation into triacylglycerol and phosphatidylcholine in the liver. These data illustrate the high lipid storing activity of the liver immediately after exercise whereas in muscle, fatty acids are directed towards oxidation. The observed muscle-specific differences accentuate the need for single-muscle analyses as well as careful consideration of the particular muscle employed when studying lipid metabolism in mice. In addition, our results reveal that lysophospholipids and plasmalogens, potential lipid signalling molecules, are acutely regulated by physical exercise.

  2. Pilot Study on the Use of DNA Priming Immunization to Enhance Y. pestis LcrV-Specific B Cell Responses Elicited by a Recombinant LcrV Protein Vaccine

    Directory of Open Access Journals (Sweden)

    Wei Li

    2013-12-01

    Full Text Available Recent studies indicate that DNA immunization is powerful in eliciting antigen-specific antibody responses in both animal and human studies. However, there is limited information on the mechanism of this effect. In particular, it is not known whether DNA immunization can also enhance the development of antigen-specific B cell development. In this report, a pilot study was conducted using plague LcrV immunogen as a model system to determine whether DNA immunization is able to enhance LcrV-specific B cell development in mice. Plague is an acute and often fatal infectious disease caused by Yersinia pestis (Y. pestis. Humoral immune responses provide critical protective immunity against plague. Previously, we demonstrated that a DNA vaccine expressing LcrV antigen can protect mice from lethal mucosal challenge. In the current study, we further evaluated whether the use of a DNA priming immunization is able to enhance the immunogenicity of a recombinant LcrV protein vaccine, and in particular, the development of LcrV-specific B cells. Our data indicate that DNA immunization was able to elicit high-level LcrV antibody responses when used alone or as part of a prime-boost immunization approach. Most significantly, DNA immunization was also able to increase the levels of LcrV-specific B cell development. The finding that DNA immunization can enhance antigen-specific B cell responses is highly significant and will help guide similar studies in other model antigen systems.

  3. Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins.

    Science.gov (United States)

    Peracaula, Rosa; Tabarés, Glòria; Royle, Louise; Harvey, David J; Dwek, Raymond A; Rudd, Pauline M; de Llorens, Rafael

    2003-06-01

    Prostate-specific antigen (PSA) is a glycoprotein secreted by prostate epithelial cells. PSA is currently used as a marker of prostate carcinoma because high levels of PSA are indicative of a tumor situation. However, PSA tests still suffer from a lack of specificity to distinguish between benign prostate hyperplasia and prostate cancer. To determine whether PSA glycosylation could provide a means of differentiating between PSA from normal and tumor origins, N-glycan characterization of PSA from seminal fluid and prostate cancer cells (LNCaP cell line) by sequencing analysis and mass spectrometry was carried out. Glycans from normal PSA (that correspond to low and high pI PSA fractions) were sialylated biantennary complex structures, half of them being disialylated in the low pI PSA fraction and mostly monosialylated in the high pI PSA. PSA from LNCaP cells was purified to homogeneity, and its glycan analysis showed a significantly different pattern, especially in the outer ends of the biantennary complex structures. In contrast to normal PSA glycans, which were sialylated, LNCaP PSA oligosaccharides were all neutral and contained a higher fucose content. In 10-15% of the structures fucose was linked alpha1-2 to galactose, forming the H2 epitope absent in normal PSA. GalNAc was increased in LNCaP glycans to 65%, whereas in normal PSA it was only present in 25% of the structures. These carbohydrate differences allow a distinction to be made between PSA from normal and tumor origins and suggest a valuable biochemical tool for diagnosis and follow-up purposes.

  4. Molecular In Vivo Imaging Using a Noninvasive Cardiac-Specific MLC-2v Promoter Driven Dual-Gene Recombinant Lentivirus Monitoring System.

    Directory of Open Access Journals (Sweden)

    Miao Zhang

    Full Text Available Our study aimed to demonstrate the feasibility of using the sodium/iodide symporter (NIS to monitor vascular endothelial growth factor (VEGF165 expression in vivo.We constructed a recombinant lentivirus plasmid with the MLC-2v promoter driving the sodium/iodide symporter (NIS reporter gene linked to the VEGF165 gene. Expression of NIS and VEGF gene were identified by Western blot. On days 2 and 54, 99mTc-MIBI imaging was used to evaluate changes in myocardial ischemia. Noninvasive 125I micro-SPECT/CT imaging was used to assess the expression of NIS reporter gene dynamically over the next 2 months.Western blot analysis showed that both NIS and VEGF165 were highly expressed in rat cardiomyoblast H9C2 cells transduced with Lenti-MLC-2v-NIS--VEGF165. 125I micro-SPECT/CT reporter imaging showed higher uptake in mouse myocardium transduced with Lenti-MLC-2v-VEGF165-IRES-NIS. NIS expression peaked on day 1 after transduction followed by a progressive decline to negligible levels by day 21. On day 1, mean 125I activity value in group 1 was higher than that in group 2 (P 0.05. In group 1 (test group, 99mTc-MIBI SPECT/CT revealed improvements in perfusion and wall thickening in the apical anterior wall. Mean IOD values of NIS and CD34 were significantly higher in group 1 than group 3 (P<0.05. Our study proved mean I-125 uptake was significantly correlated with mean IOD value of NIS and CD34 (P<0.05.This study demonstrates the feasibility of using the NIS gene to monitor VEGF165 expression in a mouse myocardial ischemia model.

  5. Single-cell genetic expression of mutant GABAA receptors causing Human genetic epilepsy alters dendritic spine and GABAergic bouton formation in a mutation-specific manner

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    Pamela eLachance-Touchette

    2014-10-01

    Full Text Available Mutations in genes encoding for GABAA receptor subunits is a well-established cause of genetic generalized epilepsy. GABA neurotransmission is implicated in several developmental processes including neurite outgrowth and synapse formation. Alteration in excitatory/inhibitory synaptic activities plays a critical role in epilepsy, thus here we investigated whether mutations in α1 subunit of GABAA receptor may affect dendritic spine and GABAergic bouton formation. In particular, we examined the effects of three mutations of the GABRA1 gene (D219N, A322D and K353delins18X that were found in a cohort of families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1flox/flox mice and simultaneously inactivating endogenous GABRA1 and transfecting mutant α1 subunits in single glutamatergic pyramidal cells and basket GABAergic interneurons by biolistic transfection. We found that GABRA1-/- GABAergic cells showed reduced innervation field, which was rescued by co-expressing α1-A322D and α1-WT but not α1-D219N. We further found that the expression of the most severe GABRA1 missense mutation (α1-A322D induced a striking increase of spine density in pyramidal cells along with an increase in the number of mushroom-like spines. In addition, α1-A322D expression in GABAergic cells slightly increased perisomatic bouton density, whereas other mutations did not alter bouton formation. All together, these results suggest that the effects of different GABAAR mutations on GABAergic bouton and dendritic spine formation are specific to the mutation and cannot be always explained by a simple loss-of-function gene model. The use of single cell genetic manipulation in organotypic cultures may provide a better understanding of the specific and distinct neural circuit alterations caused by different GABAA receptor subunit mutations and will help define the pathophysiology of genetic

  6. Site-specific keloid fibroblasts alter the behaviour of normal skin and normal scar fibroblasts through paracrine signalling.

    Directory of Open Access Journals (Sweden)

    Kevin J Ashcroft

    Full Text Available Keloid disease (KD is an abnormal cutaneous fibroproliferative disorder of unknown aetiopathogenesis. Keloid fibroblasts (KF are implicated as mediators of elevated extracellular matrix deposition. Aberrant secretory behaviour by KF relative to normal skin fibroblasts (NF may influence the disease state. To date, no previous reports exist on the ability of site-specific KF to induce fibrotic-like phenotypic changes in NF or normal scar fibroblasts (NS by paracrine mechanisms. Therefore, the aim of this study was to investigate the influence of conditioned media from site-specific KF on the cellular and molecular behaviour of both NF and NS enabled by paracrine mechanisms. Conditioned media was collected from cultured primary fibroblasts during a proliferative log phase of growth including: NF, NS, peri-lesional keloid fibroblasts (PKF and intra-lesional keloid fibroblasts (IKF. Conditioned media was used to grow NF, NS, PKF and IKF cells over 240 hrs. Cellular behavior was monitored through real time cell analysis (RTCA, proliferation rates and migration in a scratch wound assay. Fibrosis-associated marker expression was determined at both protein and gene level. PKF conditioned media treatment of both NF and NS elicited enhanced cell proliferation, spreading and viability as measured in real time over 240 hrs versus control conditioned media. Following PKF and IKF media treatments up to 240 hrs, both NF and NS showed significantly elevated proliferation rates (p<0.03 and migration in a scratch wound assay (p<0.04. Concomitant up-regulation of collagen I, fibronectin, α-SMA, PAI-1, TGF-β and CTGF (p<0.03 protein expression were also observed. Corresponding qRT-PCR analysis supported these findings (P<0.03. In all cases, conditioned media from growing marginal PKF elicited the strongest effects. In conclusion, primary NF and NS cells treated with PKF or IKF conditioned media exhibit enhanced expression of fibrosis-associated molecular markers

  7. High Gestational Folic Acid Supplementation Alters Expression of Imprinted and Candidate Autism Susceptibility Genes in a sex-Specific Manner in Mouse Offspring.

    Science.gov (United States)

    Barua, Subit; Kuizon, Salomon; Brown, W Ted; Junaid, Mohammed A

    2016-02-01

    Maternal nutrients play critical roles in modulating epigenetic events and exert long-term influences on the progeny's health. Folic acid (FA) supplementation during pregnancy has decreased the incidence of neural tube defects in newborns, but the influence of high doses of maternal FA supplementation on infants' brain development is unclear. The present study was aimed at investigating the effects of a high dose of gestational FA on the expression of genes in the cerebral hemispheres (CHs) of 1-day-old pups. One week prior to mating and throughout the entire period of gestation, female C57BL/6J mice were fed a diet, containing FA at either 2 mg/kg (control diet (CD)) or 20 mg/kg (high maternal folic acid (HMFA)). At postnatal day 1, pups from different dams were sacrificed and CH tissues were collected. Quantitative RT-PCR and Western blot analysis confirmed sex-specific alterations in the expression of several genes that modulate various cellular functions (P < 0.05) in pups from the HMFA group. Genomic DNA methylation analysis showed no difference in the level of overall methylation in pups from the HMFA group. These findings demonstrate that HMFA supplementation alters offsprings' CH gene expression in a sex-specific manner. These changes may influence infants' brain development.

  8. Embryo-specific expression of soybean oleosin altered oil body morphogenesis and increased lipid content in transgenic rice seeds.

    Science.gov (United States)

    Liu, Wen Xian; Liu, Hua Liang; Qu, Le Qing

    2013-09-01

    Oleosin is the most abundant protein in the oil bodies of plant seeds, playing an important role in regulating oil body formation and lipid accumulation. To investigate whether lipid accumulation in transgenic rice seeds depends on the expression level of oleosin, we introduced two soybean oleosin genes encoding 24 kDa proteins into rice under the control of an embryo-specific rice promoter REG-2. Overexpression of soybean oleosin in transgenic rice leads to an increase of seed lipid content up to 36.93 and 46.06 % higher than that of the non-transgenic control, respectively, while the overall fatty acid profiles of triacylglycerols remained unchanged. The overexpression of soybean oleosin in transgenic rice seeds resulted in more numerous and smaller oil bodies compared with wild type, suggesting that an inverse relationship exists between oil body size and the total oleosin level. The increase in lipid content is accompanied by a reduction in the accumulation of total seed protein. Our results suggest that it is possible to increase rice seed oil content for food use and for use as a low-cost feedstock for biodiesel by overexpressing oleosin in rice seeds.

  9. High Fat Diet Administration during Specific Periods of Pregnancy Alters Maternal Fatty Acid Profiles in the Near-Term Rat

    Directory of Open Access Journals (Sweden)

    Marlon E. Cerf

    2016-01-01

    Full Text Available Excessive fat intake is a global health concern as women of childbearing age increasingly ingest high fat diets (HFDs. We therefore determined the maternal fatty acid (FA profiles in metabolic organs after HFD administration during specific periods of gestation. Rats were fed a HFD for the first (HF1, second (HF2, or third (HF3 week, or for all three weeks (HFG of gestation. Total maternal plasma non-esterified fatty acid (NEFA concentrations were monitored throughout pregnancy. At day 20 of gestation, maternal plasma, liver, adipose tissue, and placenta FA profiles were determined. In HF3 mothers, plasma myristic and stearic acid concentrations were elevated, whereas docosahexaenoic acid (DHA was reduced in both HF3 and HFG mothers. In HF3 and HFG mothers, hepatic stearic and oleic acid proportions were elevated; conversely, DHA and linoleic acid (LA proportions were reduced. In adipose tissue, myristic acid was elevated, whereas DHA and LA proportions were reduced in all mothers. Further, adipose tissue stearic acid proportions were elevated in HF2, HF3, and HFG mothers; with oleic acid increased in HF1 and HFG mothers. In HF3 and HFG mothers, placental neutral myristic acid proportions were elevated, whereas DHA was reduced. Further, placental phospholipid DHA proportions were reduced in HF3 and HFG mothers. Maintenance on a diet, high in saturated fat, but low in DHA and LA proportions, during late or throughout gestation, perpetuated reduced DHA across metabolic organs that adapt during pregnancy. Therefore a diet, with normal DHA proportions during gestation, may be important for balancing maternal FA status.

  10. L-kynurenine treatment alters contextual fear conditioning and context discrimination but not cue-specific fear conditioning.

    Science.gov (United States)

    Chess, Amy C; Landers, Allison M; Bucci, David J

    2009-08-12

    The glia-derived molecule kynurenic acid (KYNA) is an antagonist of alpha7 nicotinic acetylcholine receptors and the glycine(B) binding site on NMDA receptors. KYNA levels are elevated in the brain and cerebrospinal fluid of persons with schizophrenia and Alzheimer's disease, both of which are characterized by deficits in contextual learning and memory. The present study tested the hypothesis that increases in KYNA concentration would impair contextual fear conditioning but spare cue-specific fear conditioning. Rats in each experiment received injections of vehicle solution or l-kynurenine (L-KYN, 100mg/kg), the precursor for KYNA. Administration of L-KYN has been shown to produce clinically relevant increases in KYNA concentration. In Experiment 1, L-KYN-treated rats exhibited impaired contextual fear memory compared to control rats, while fear conditioning to a discrete auditory cue was unaffected. In Experiment 2, rats were trained to discriminate between two different training environments, one in which foot shock was delivered and one that was not paired with foot shock. Although both groups of rats eventually learned the discrimination, learning was slower in L-KYN-treated rats. The results of Experiment 3 demonstrated that the deficits in context discrimination could not be explained by the preferential use of an elemental learning strategy by L-KYN-treated rats. Together, these findings indicate that elevated concentration of endogenous KYNA interferes with contextual learning and memory and support the notion that increased concentration of KYNA may contribute to cognitive dysfunction. In addition, these data provide new insight into how novel 'gliotransmitters' may modulate neuronal function and behavior.

  11. High Fat Diet Administration during Specific Periods of Pregnancy Alters Maternal Fatty Acid Profiles in the Near-Term Rat.

    Science.gov (United States)

    Cerf, Marlon E; Herrera, Emilio

    2016-01-04

    Excessive fat intake is a global health concern as women of childbearing age increasingly ingest high fat diets (HFDs). We therefore determined the maternal fatty acid (FA) profiles in metabolic organs after HFD administration during specific periods of gestation. Rats were fed a HFD for the first (HF1), second (HF2), or third (HF3) week, or for all three weeks (HFG) of gestation. Total maternal plasma non-esterified fatty acid (NEFA) concentrations were monitored throughout pregnancy. At day 20 of gestation, maternal plasma, liver, adipose tissue, and placenta FA profiles were determined. In HF3 mothers, plasma myristic and stearic acid concentrations were elevated, whereas docosahexaenoic acid (DHA) was reduced in both HF3 and HFG mothers. In HF3 and HFG mothers, hepatic stearic and oleic acid proportions were elevated; conversely, DHA and linoleic acid (LA) proportions were reduced. In adipose tissue, myristic acid was elevated, whereas DHA and LA proportions were reduced in all mothers. Further, adipose tissue stearic acid proportions were elevated in HF2, HF3, and HFG mothers; with oleic acid increased in HF1 and HFG mothers. In HF3 and HFG mothers, placental neutral myristic acid proportions were elevated, whereas DHA was reduced. Further, placental phospholipid DHA proportions were reduced in HF3 and HFG mothers. Maintenance on a diet, high in saturated fat, but low in DHA and LA proportions, during late or throughout gestation, perpetuated reduced DHA across metabolic organs that adapt during pregnancy. Therefore a diet, with normal DHA proportions during gestation, may be important for balancing maternal FA status.

  12. Embedding the Ni-SOD mimetic Ni-NCC within a polypeptide sequence alters specificity of the reaction pathway

    Science.gov (United States)

    Krause, Mary E.; Glass, Amanda M.; Jackson, Timothy A.; Laurence, Jennifer S.

    2013-01-01

    The unique metal abstracting peptide (MAP) asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to DLD-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion. Characterization of the NCC sequence within longer peptides with absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies and mass spectrometry (MS) shows that the geometry of metal coordination is maintained, though the electronic properties of the complex are varied to a small extent due to bis-amide, rather than amine/amide, coordination. In addition, both the Ni-tripeptides and Ni-pentapeptides have a −2 charge. The study here demonstrates that the chiral inversion chemistry does not occur when NCC is embedded in a longer polypeptide sequence. Nonetheless, the superoxide scavenging reactivity of the embedded Ni-NCC module is similar to that of the chirally inverted tripeptide complex, which is consistent with a minor change in reduction potential for the Ni-pentapeptide. Together, this suggests that the charge of the complex could affect the SOD activity as much as a change in primary coordination sphere. In Ni-NCC and other Ni-SOD mimics, changes in chirality, superoxide scavenging activity, and oxidation of the peptide itself all depend on the presence of dioxygen or its reduced derivatives (e.g., superoxide), and the extent to which each of these distinct reactions occurs is ruled by electronic and steric effects that emenate from the organization of ligands around the metal center. PMID:23214928

  13. Recombinant protein production technology

    Science.gov (United States)

    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...

  14. Enzyme-linked immunosorbent assays using recombinant TgSAG2 and NcSAG1 to detect Toxoplasma gondii and Neospora caninum-specific antibodies in domestic animals in Turkey

    Science.gov (United States)

    ZHOU, Mo; CAO, Shinuo; SEVINC, Ferda; SEVINC, Mutlu; CEYLAN, Onur; LIU, Mingming; WANG, Guanbo; MOUMOUNI, Paul Franck Adjou; JIRAPATTHARASATE, Charoonluk; SUZUKI, Hiroshi; NISHIKAWA, Yoshifumi; XUAN, Xuenan

    2016-01-01

    Considering the scarce information on occurrences of Toxoplasma gondii and Neospora caninum in domestic animals from Turkey, the aim of this study was to investigate the seroprevalence of these parasite infections in cattle, horses, sheep, goats and dogs in Turkey. The specific antibodies against T. gondii and N. caninum were detected by iELISAs based on the recombinant TgSAG2 or NcSAG1 in a total of 2,039 serum samples from eleven provinces. The seroprevalence of T. gondii infections was 46.3%, 4.0%, 20.0%, 12.9% and 19.8%, that of N. caninum infections was 0.3%, 7.4%, 2.1%, 3.2% and 16.6% in the horses, cattle, sheep, goats and dogs, respectively. These results indicated that T. gondii and N. caninum infections are prevalent in Turkish domestic animals. PMID:27523504

  15. Enzyme-linked immunosorbent assays using recombinant TgSAG2 and NcSAG1 to detect Toxoplasma gondii and Neospora caninum-specific antibodies in domestic animals in Turkey.

    Science.gov (United States)

    Zhou, Mo; Cao, Shinuo; Sevinc, Ferda; Sevinc, Mutlu; Ceylan, Onur; Liu, Mingming; Wang, Guanbo; Moumouni, Paul Franck Adjou; Jirapattharasate, Charoonluk; Suzuki, Hiroshi; Nishikawa, Yoshifumi; Xuan, Xuenan

    2017-01-10

    Considering the scarce information on occurrences of Toxoplasma gondii and Neospora caninum in domestic animals from Turkey, the aim of this study was to investigate the seroprevalence of these parasite infections in cattle, horses, sheep, goats and dogs in Turkey. The specific antibodies against T. gondii and N. caninum were detected by iELISAs based on the recombinant TgSAG2 or NcSAG1 in a total of 2,039 serum samples from eleven provinces. The seroprevalence of T. gondii infections was 46.3%, 4.0%, 20.0%, 12.9% and 19.8%, that of N. caninum infections was 0.3%, 7.4%, 2.1%, 3.2% and 16.6% in the horses, cattle, sheep, goats and dogs, respectively. These results indicated that T. gondii and N. caninum infections are prevalent in Turkish domestic animals.

  16. Effects of UV radiation on genetic recombination

    International Nuclear Information System (INIS)

    Vlahovic, K.; Zahradka, D.; Petranovic, M.; Petranovic, D.

    1996-01-01

    We have used the model consisting of Escherichia coli cells and l phage to study the effects of UV radiation on genetic recombination. We found two radiation induced processes that reduce or inhibit genetic recombination. One such process leads to the inability of prophage to excise itself from the irradiated bacterial chromosome by the site-specific recombination. The other process was shown to inhibit a type of general recombination by which the prophage transfers one of its genetic markers to the infecting homologous phage. Loss of the prophage ability to take part in both site-specific and general recombination was shown to develop in recB + but not in recB cells. From this we infer that the loss of prophage recombinogenicity in irradiated cells is a consequence of one process in which RecBCD enzyme (the product of recB, recC and recD genes) plays an essential role. (author)

  17. Induction of HIV-1–Specific Mucosal Immune Responses Following Intramuscular Recombinant Adenovirus Serotype 26 HIV-1 Vaccination of Humans

    Science.gov (United States)

    Baden, Lindsey R.; Liu, Jinyan; Li, Hualin; Johnson, Jennifer A.; Walsh, Stephen R.; Kleinjan, Jane A.; Engelson, Brian A.; Peter, Lauren; Abbink, Peter; Milner, Danny A.; Golden, Kevin L.; Viani, Kyle L.; Stachler, Matthew D.; Chen, Benjamin J.; Pau, Maria G.; Weijtens, Mo; Carey, Brittany R.; Miller, Caroline A.; Swann, Edith M.; Wolff, Mark; Loblein, Hayley; Seaman, Michael S.; Dolin, Raphael; Barouch, Dan H.

    2015-01-01

    Background Defining mucosal immune responses and inflammation to candidate human immunodeficiency virus type 1 (HIV-1) vaccines represents a current research priority for the HIV-1 vaccine field. In particular, it is unclear whether intramuscular immunization can elicit immune responses at mucosal surfaces in humans. Methods In this double-blind, randomized, placebo-controlled clinical trial, we evaluated systemic and mucosal immune responses to a candidate adenovirus serotype 26 (Ad26) vectored HIV-1 envelop (Env) vaccine in baseline Ad26-seronegative and Ad26-seropositive healthy volunteers. Systematic mucosal sampling with rectal Weck-Cel sponges and rectal biopsies were performed. Results Intramuscular immunization elicited both systemic and mucosal Env-specific humoral and cellular immune responses in the majority of subjects. Individuals with preexisting Ad26-specific neutralizing antibodies had vaccine-elicited immune responses comparable to those of subjects who were Ad26 seronegative. We also observed no increase in activated total or vector-specific mucosal CD4+ T lymphocytes following vaccination by either histopathology or flow cytometry. Conclusions These data demonstrate that a single intramuscular administration of this Ad26-vectored HIV-1 Env vaccine elicited both systemic and mucosal immune responses in humans. Induction of antigen-specific humoral and cellular mucosal immunity was not accompanied by a detectable increase in mucosal inflammation. Clinical Trials Registration NCT01103687. PMID:25165165

  18. Cross-Linking of a CD4-Mimetic Miniprotein with HIV-1 Env gp140 Alters Kinetics and Specificities of Antibody Responses against HIV-1 Env in Macaques.

    Science.gov (United States)

    Shen, Xiaoying; Bogers, Willy M; Yates, Nicole L; Ferrari, Guido; Dey, Antu K; Williams, William T; Jaeger, Frederick H; Wiehe, Kevin; Sawant, Sheetal; Alam, S Munir; LaBranche, Celia C; Montefiori, David C; Martin, Loic; Srivastava, Indresh; Heeney, Jonathan; Barnett, Susan W; Tomaras, Georgia D

    2017-10-01

    Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions. IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen designs in

  19. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay, E-mail: sgupta@actrec.gov.in

    2015-03-15

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair.

  20. Dynamic alteration in H3 serine 10 phosphorylation is G1-phase specific during ionization radiation induced DNA damage response in human cells

    International Nuclear Information System (INIS)

    Sharma, Ajit K.; Bhattacharya, Saikat; Khan, Shafqat A.; Khade, Bharat; Gupta, Sanjay

    2015-01-01

    Highlights: • Loss of H3S10P in response to DNA damage is a universal phenomenon from G1 cells. • The loss happens predominantly from histone H3.3, a transcription activation mark. • Compaction of chromatin occurs during repair stage of DDR. • The alteration of H3S10P shows an inverse correlation with γH2AX. - Abstract: Chromatin acts as a natural barrier in DNA-damage recognition and repair. Histones undergo differential post-translational modification(s) to facilitate DNA damage response (DDR). Importance of modifications like phosphorylation of histone variant H2A.X in DNA repair is very well understood, however, ambiguous results exist in literature regarding the levels of certain histone modifications and their possible role in repair. In the present study, we have investigated in depth the alteration in the level of the highly dynamic histone mark H3S10P as it plays a dual role in different phases of the cell cycle. We show here that H3S10P decreases specifically from irradiated G1-enriched cells irrespective of the damaging agent or the cell line used in the study. Interestingly, the loss occurs predominantly from H3.3 variant which is a transcription activation mark like H3S10P itself, suggesting that the alteration might be implicated in transcription repression. The decrease in other transcription marks like H3K9Ac, H3K14Ac, H3K56Ac and H3S28P along with the occurrence of chromatin condensation in response to DNA damage in G1 phase strengthens the hypothesis. In addition, the alteration in the level of H3S10P shows an inverse correlation with that of γH2AX in a dose-dependent manner and probably occurs from the same mononucleosome. We propose that the drop in the levels of histone H3S10 phosphorylation is a universal phenomenon in response to DNA damage and is a trigger to induce transcription repressive state to facilitate repair

  1. Osbpl8 deficiency in mouse causes an elevation of high-density lipoproteins and gender-specific alterations of lipid metabolism.

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    Olivier Béaslas

    Full Text Available OSBP-related protein 8 (ORP8 encoded by Osbpl8 is an endoplasmic reticulum sterol sensor implicated in cellular lipid metabolism. We generated an Osbpl8(-/- (KO C57Bl/6 mouse strain. Wild-type and Osbpl8KO animals at the age of 13-weeks were fed for 5 weeks either chow or high-fat diet, and their plasma lipids/lipoproteins and hepatic lipids were analyzed. The chow-fed Osbpl8KO male mice showed a marked elevation of high-density lipoprotein (HDL cholesterol (+79% and phospholipids (+35%, while only minor increase of apolipoprotein A-I (apoA-I was detected. In chow-fed female KO mice a less prominent increase of HDL cholesterol (+27% was observed, while on western diet the HDL increment was prominent in both genders. The HDL increase was accompanied by an elevated level of HDL-associated apolipoprotein E in male, but not female KO animals. No differences between genotypes were observed in lecithin:cholesterol acyltransferase (LCAT or hepatic lipase (HL activity, or in the fractional catabolic rate of fluorescently labeled mouse HDL injected in chow-diet fed animals. The Osbpl8KO mice of both genders displayed reduced phospholipid transfer protein (PLTP activity, but only on chow diet. These findings are consistent with a model in which Osbpl8 deficiency results in altered biosynthesis of HDL. Consistent with this hypothesis, ORP8 depleted mouse hepatocytes secreted an increased amount of nascent HDL into the culture medium. In addition to the HDL phenotype, distinct gender-specific alterations in lipid metabolism were detected: Female KO animals on chow diet showed reduced lipoprotein lipase (LPL activity and increased plasma triglycerides, while the male KO mice displayed elevated plasma cholesterol biosynthetic markers cholestenol, desmosterol, and lathosterol. Moreover, modest gender-specific alterations in the hepatic expression of lipid homeostatic genes were observed. In conclusion, we report the first viable OsbplKO mouse model

  2. Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-Specific Monoclonal Antibody and Recombinant F(ab

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    Lawrence K. Silbart

    2013-03-01

    Full Text Available Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs such as 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK. These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers.

  3. Blood Interferon Signatures Putatively Link Lack of Protection Conferred by the RTS,S Recombinant Malaria Vaccine to an Antigen-specific IgE Response [version 2; referees: 2 approved

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    Darawan Rinchai

    2017-07-01

    Full Text Available Malaria remains a major cause of mortality and morbidity worldwide. Progress has been made in recent years with the development of vaccines that could pave the way towards protection of hundreds of millions of exposed individuals. Here we used a modular repertoire approach to re-analyze a publically available microarray blood transcriptome dataset monitoring the response to malaria vaccination. We report the seminal identification of interferon signatures in the blood of subjects on days 1, 3 and 14 following administration of the third dose of the RTS,S recombinant malaria vaccine. These signatures at day 1 correlate with protection, and at days 3 and 14 to susceptibility to subsequent challenge of study subjects with live parasites. In addition we putatively link the decreased abundance of interferon-inducible transcripts observed at days 3 and 14 post-vaccination with the elicitation of an antigen-specific IgE response in a subset of vaccine recipients that failed to be protected by the RTS,S vaccine. Furthermore, profiling of antigen-specific levels of IgE in a Mozambican cohort of malaria-exposed children vaccinated with RTS,S identified an association between elevated baseline IgE levels and subsequent development of naturally acquired malaria infection during follow up. Taken together these findings warrant further investigation of the role of antigen-specific IgE in conferring susceptibility to malaria infection.

  4. 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.

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    Wenping Gong

    Full Text Available 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.

  5. The development of a highly specific radiochemical compound based on labeled 99mtc recombinant molecules for targeted imaging of cells with the overexpression of Her-2 / neu

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    Olga D. Bragina

    2017-01-01

    Full Text Available Currently, there is a urgent need to search for new diagnostic methods that allow us to reveal malignant tumors with the overexpression of Her-2/neu with high accuracy. In recent years radioisotope methods have been actively developing to identify specific tumor targets, with antibodies being the “targeting” module.The purpose of the study. Creation of a chemically stable radiochemical compound for the imaging of cells with the overexpression of Her-2/neu.Materials and methods. The study was conducted using two human adenocarcinoma cell lines with expression (BT-474 and without expression (MCF-7 Her-2/neu. The specificity of the binding of the test complex with Her-2/neu receptor was determined by direct radiometric and planar scintigraphy. To evaluate the differences in quantitative characteristics between the groups a non-parametric Mann – Whitney test was used.Results. The yield of the labeled complex was more than 91% and the radiochemical frequency was more than 94%. When performing a visual scintigraphic evaluation, a much higher accumulation rate of the studied radiopharmaceutical preparation (RFP was observed in the culture of cells with overexpression of the surface Her-2/neu receptor. Direct radiometric results also demonstrated a higher accumulation of RFPs in the human BT-474 mammary adenocarcinoma cell line with Her-2/neu overexpression in comparison with the control group.Conclusion. Preclinical studies demonstrated high stability of the test compound, as well as its accumulation in the group of cells with Her-2/neu overexpression

  6. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    International Nuclear Information System (INIS)

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro

  7. Hepatitis B virus (HBV)-specific T-cell responses to recombinant HBV core protein in patients with normal liver function and co-infected with chronic HBV and human immunodeficiency virus 1 (HIV-1)

    Science.gov (United States)

    2013-01-01

    Background Little is known about HBV-specific T-cell responses in chronic Hepatitis B patients (HBV) that are co-infected with Human immunodeficiency virus type 1 (HIV-1), especially those with normal alanine aminotransferase (ALT) levels. Methods Twenty-five patients with chronic HBV (11 hepatitis B e antigen [HBeAg]-positive, 14 HBeAg-negative) were enrolled in a cross-sectional study. A longitudinal study as also conducted in which follow-up was done at 3, 12, and 24 months, after acute HIV-1 infection, in 11 individuals who also had chronic HBV. Peripheral blood mononuclear cells were stimulated with recombinant HBV surface protein (S protein), core protein (C protein) or gag peptide. IFN-γ-secreting T cells were identified by ELISPOT assay. Results In the cross-sectional study, co-infected chronic HBV patients had lower C protein-specific T-cell responses compared with mono-infected individuals, though the difference was not significant. In co-infected, chronic HBV patients, the magnitude of C protein-specific T-cell responses was significantly greater in HBeAg-positive subjects compared to HBeAg-negative subjects (p = 0.011). C protein-specific T-cell responses were positively correlated with HBV viral load (rs = 0.40, p = 0.046). However, gag-specific T-cell responses were negatively correlated with HIV viral load (rs = −0.44, p = 0.026) and positively correlated with CD4+ count (rs = 0.46, p = 0.021). The results were different in mono-infected individuals. PBMCs from co-infected HBeAg-positive patients secreted more specific-IFN-γ in cultured supernatants compared with PBMCs from co-infected HBeAg-negative patients (p = 0.019). In the longitudinal study, S protein- and C protein-specific T-cell responses were decreased as the length of follow-up increased (p = 0.034, for S protein; p = 0.105, for C protein). Additionally, the S protein- and C protein-specific T-cell responses were significantly higher in HBe

  8. Enhanced target-specific signal detection using an Escherichia coli lysate in multiplex microbead immunoassays with E. coli-derived recombinant antigens.

    Science.gov (United States)

    Crestani, Sandra; Leitolis, Amanda; Lima, Lucianna Freitas Oliveira; Krieger, Marco A; Foti, Leonardo

    2016-08-01

    Diverse techniques have been developed to analyze antibody-mediated responses to infections. However, the most common tests, i.e., enzyme-linked immunosorbent assays, require separate reactions for each antigen and consequently necessitate large sample volumes. Luminex technology allows the detection of multiple antibodies in a single experiment, but nonspecific binding can impair the results. Therefore, we examined the use of Escherichia coli lysates to reduce nonspecific binding and improve the results of liquid microarrays based on Luminex technology. Anti-bacteria antibodies were detected in human serum samples, as evidenced by high median fluorescence intensity (MFI) in assays performed with paramagnetic microspheres coupled with E. coli lysates. Moreover, the addition of an E. coli lysate as a blocker reduced the nonspecific binding of antigens produced by E. coli in a concentration-dependent manner. Tris-HCl reduced MFI values in negative samples, but did not affect MFI for positive samples. For microspheres coupled with different antigens, an E. coli lysate blocker significantly improved the fluorescence signals from positive samples. The addition of Tris-HCl and the E. coli lysate induced antigen-specific differences in MFI. This combination of the E. coli lysate blocker and Tris-HCl yielded a statistically significant improvement in MFI in the assays for Chagas disease and hepatitis C virus samples. However, for the Treponema pallidum p47 antigen improvement in MFI was only observed for the preparation with the E. coli blocker at a concentration of 3%. In conclusion, the addition of an E. coli lysate and Tris-HCl to the microarray assay reduced the nonspecific binding of human anti-bacteria antibodies and, therefore, increased the specific MFI. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease

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    Marina de Oliveira Paro

    2013-01-01

    Full Text Available Von Willebrand disease (VWD is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF. The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides among which three were representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1 assessment of VWF: antigen interaction, (2 VWF multimer analysis, and (3 production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura.

  10. Antigenic Peptides Capable of Inducing Specific Antibodies for Detection of the Major Alterations Found in Type 2B Von Willebrand Disease

    Science.gov (United States)

    Paro, Marina de Oliveira; Ferreira, Cyntia Silva; Vieira, Fernanda Silva; de Santana, Marcos Aurélio; Namen-Lopes, Maria Sueli Silva; Leclercq, Sophie Yvette; Velloso-Rodrigues, Cibele; Guerra de Andrade, Milton Hércules

    2013-01-01

    Von Willebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of the von Willebrand factor (VWF). The disease represents the most common human coagulopathy afflicting 1.3% of the population. Qualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF. The differential diagnosis of the VWD is a difficult task, relying on a panel of tests aimed to assess the plasma levels and function of the VWF. Here, we propose biochemical approaches for the identification of structural variants of the VWF. A bioinformatic analysis was conducted to design seven peptides among which three were representatives of specific amino acid sequences belonging to normal VWF and four encompassed sequences found in the most common VWD subtype 2B. These peptides were used to immunize mice, after which, peptide-specific immunoglobulins were purified. This resulted in four Ig preparations capable of detecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF. The panel of antibodies could serve many applications among them (1) assessment of VWF: antigen interaction, (2) VWF multimer analysis, and (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura. PMID:23970904

  11. Human muscle-specific A-kinase anchoring protein (mAKAP) polymorphisms modulate the susceptibility to cardiovascular diseases by altering cAMP/ PKA signaling.

    Science.gov (United States)

    Suryavanshi, Santosh V; Jadhav, Shweta M; Anderson, Kody L; Katsonis, Panagiotis; Lichtarge, Olivier; McConnell, Bradley K

    2018-03-30

    One of the crucial cardiac signaling pathways is cAMP-mediated PKA signal transduction which is regulated by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). Muscle-specific AKAP (mAKAP) partly regulates cardiac cAMP/PKA signaling by binding to PKA and phosphodiesterase4D3 (PDE4D3) among other proteins and plays a central role in modulating cardiac remodeling. Moreover, genetics plays an incomparable role in modifying the risk of cardiovascular diseases (CVDs). Especially, single nucleotide polymorphisms (SNPs) in various proteins have been shown to predispose individuals to CVDs. Hence, we hypothesized that human mAKAP polymorphisms found in humans with CVDs alter cAMP/PKA pathway influencing the susceptibility of individuals to CVDs. Our computational analyses revealed two mAKAP SNPs found in cardiac disease related patients with highest predicted deleterious effects, Ser(S) 1653 Arg(R) and Glu(E) 2124 Gly(G). Co-immunoprecipitation data in HEK293T cells showed that S1653R SNP, present in the PDE4D3 binding domain of mAKAP, changed the binding of PDE4D3 to mAKAP and E2124G SNP, flanking the 3'-PKA binding domain, changed the binding of PKA before and after stimulation with isoproterenol. These SNPs significantly altered intracellular cAMP levels, global PKA activity and cytosolic PDE activity when compared with the wild-type (WT) before and after isoproterenol stimulation. PKA-mediated phosphorylation of pathological markers was found to be up-regulated after cell stimulation in both mutants. In conclusion, human mAKAP polymorphisms may influence the propensity of developing CVDs by affecting cAMP/PKA signaling supporting the clinical significance of PKA-mAKAP-PDE4D3 interactions.

  12. Transfection of siRNAs can alter miRNA levels and trigger non-specific protein degradation in mammalian cells.

    Science.gov (United States)

    Liang, Xue-Hai; Hart, Christopher E; Crooke, Stanley T

    2013-05-01

    Sequence-non-specific effects of siRNAs that alter the expression of non-targeted genes have been reported, including competition of siRNAs with endogenous RISC components. However, the detailed mechanisms and subsequent effects of such competition are not well documented. Here we analyze the competition of miRNAs in mammalian cells with low concentrations of siRNAs, and found that: 1) transfection of different siRNAs in the low nanomolar range used to deplete target RNAs can reduce the levels of miRNAs in different cell types, 2) siRNA transfection results in rapid reduction of Ago2-associated miRNAs concurrent with accumulation of Ago2-bound siRNAs and a significant change in the expression levels of many miRNAs, 3) competition largely depends on Ago2 and not Dicer, 4) microarray analysis showed that the majority of highly expressed miRNAs are reduced, in a siRNA concentration dependent manner, and low abundant miRNAs may be unchanged or repressed and a few miRNAs appear to have increased levels, and 5) consistent with previous studies, the expression levels of mRNAs that are targeted by highly repressed miRNAs are preferentially increased. As a consequence of such competition, we observed that α-tubulin, a substrate of two up-regulated proteases, granzyme B and granzyme M, was rapidly degraded at the protein level upon siRNA transfection. Our results support a model in which transfection of siRNAs can change the levels of many miRNAs by competition for Ago2, leading to altered expression of many miRNA target genes, which can in turn affect downstream gene expression even at the protein level. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. The fibrous form of intracellular inclusion bodies in recombinant variant fibrinogen-producing cells is specific to the hepatic fibrinogen storage disease-inducible variant fibrinogen.

    Science.gov (United States)

    Arai, Shinpei; Ogiwara, Naoko; Mukai, Saki; Takezawa, Yuka; Sugano, Mitsutoshi; Honda, Takayuki; Okumura, Nobuo

    2017-06-01

    Fibrinogen storage disease (FSD) is a rare disorder that is characterized by the accumulation of fibrinogen in hepatocytes and induces liver injury. Six mutations in the γC domain (γG284R, γT314P, γD316N, the deletion of γG346-Q350, γG366S, and γR375W) have been identified for FSD. Our group previously established γ375W fibrinogen-producing Chinese hamster ovary (CHO) cells and observed aberrant large granular and fibrous forms of intracellular inclusion bodies. The aim of this study was to investigate whether fibrous intracellular inclusion bodies are specific to FSD-inducible variant fibrinogen. Thirteen expression vectors encoding the variant γ-chain were stably or transiently transfected into CHO cells expressing normal fibrinogen Aα- and Bβ-chains or HuH-7 cells, which were then immunofluorescently stained. Six CHO and HuH-7 cell lines that transiently produced FSD-inducible variant fibrinogen presented the fibrous (3.2-22.7 and 2.1-24.5%, respectively) and large granular (5.4-25.5 and 7.7-23.9%) forms of intracellular inclusion bodies. Seven CHO and HuH-7 cell lines that transiently produced FSD-non-inducible variant fibrinogen only exhibit the large granular form. These results demonstrate that transiently transfected variant fibrinogen-producing CHO cells and inclusion bodies of the fibrous form may be useful in non-invasive screening for FSD risk factors for FSD before its onset.

  14. Use of recombinant nucleoproteins in enzyme-linked immunosorbent assays for detection of virus-specific immunoglobulin A (IgA) and IgG antibodies in influenza virus A- or B-infected patients

    NARCIS (Netherlands)

    J. Groen (Jan); D. van Alphen; E.C.J. Claas (Eric); R. de Groot (Ronald); G.F. Rimmelzwaan (Guus); J.T.M. Voeten; A.D.M.E. Osterhaus (Albert)

    1998-01-01

    textabstractThe nucleoprotein genes of influenza virus A/Netherlands/018/94 (H3N2) and influenza virus B/Harbin/7/94 were cloned into the bacterial expression vector pMalC to yield highly purified recombinant influenza virus A and B nucleoproteins. With these recombinant influenza

  15. Recombinant clotting factors.

    Science.gov (United States)

    Pipe, Steven W

    2008-05-01

    The recombinant era for haemophilia began in the early 1980s with the cloning and subsequent expression of functional proteins for both factors VIII and IX. Efficient production of recombinant clotting factors in mammalian cell culture systems required overcoming significant challenges due to the complex post-translational modifications that were integral to their pro-coagulant function. The quick development and commercialization of recombinant clotting factors was, in part, facilitated by the catastrophic impact of viral contamination of plasma-derived clotting factor concentrates at the time. Since their transition into the clinic, the recombinant versions of both factor VIII and IX have proven to be remarkable facsimiles of their plasma-derived counterparts. The broad adoption of recombinant therapy throughout the developed world has significantly increased the supply of clotting factor concentrates and helped advance aggressive therapeutic interventions such as prophylaxis. The development of recombinant VIIa was a further advance bringing a recombinant option to haemophilia patients with inhibitors. Recombinant DNA technology remains the platform to address ongoing challenges in haemophilia care such as reducing the costs of therapy, increasing the availability to the developing world, and improving the functional properties of these proteins. In turn, the ongoing development of new recombinant clotting factor concentrates is providing alternatives for patients with other inherited bleeding disorders.

  16. NY-ESO-1 specific antibody and cellular responses in melanoma patients primed with NY-ESO-1 protein in ISCOMATRIX and boosted with recombinant NY-ESO-1 fowlpox virus.

    Science.gov (United States)

    Chen, Ji-Li; Dawoodji, Amina; Tarlton, Andrea; Gnjatic, Sacha; Tajar, Abdelouahid; Karydis, Ioannis; Browning, Judy; Pratap, Sarah; Verfaille, Christian; Venhaus, Ralph R; Pan, Linda; Altman, Douglas G; Cebon, Jonathan S; Old, Lloyd L; Nathan, Paul; Ottensmeier, Christian; Middleton, Mark; Cerundolo, Vincenzo

    2015-03-15

    Vaccination strategies based on repeated injections of NY-ESO-1 protein formulated in ISCOMATRIX particles (NY-ESO-1 ISCOMATRIX) have shown to elicit combined NY-ESO-1 specific antibody and T cell responses. However, it remains unclear whether heterologous prime-boost strategies based on the combination with NY-ESO-1 ISCOMATRIX with different NY-ESO-1 boosting reagents could be used to increase NY-ESO-1 CD8(+) or CD4(+) T cell responses. To address this question, we carried out a randomized clinical trial in 39 high-risk, resected melanoma patients vaccinated with NY-ESO-1 ISCOMATRIX, and then boosted with repeated injections of either recombinant fowlpox virus encoding full length NY-ESO-1 (rF-NY-ESO-1) (Arm A) or NY-ESO-1 ISCOMATRIX alone (Arm B). We have comprehensively analyzed NY-ESO-1 specific T cells and B cells response in all patients before and after vaccination for a total of seven time points per patient. NY-ESO-1 ISCOMATRIX alone elicited a strong NY-ESO-1 specific CD4(+) T cell and antibody response, which was maintained by both regiments at similar levels. However, CD8(+) T cell responses were significantly boosted in 3 out of 18 patients in Arm A after the first rF-NY-ESO-1 injection and such responses were maintained until the end of the trial, while no patients in Arm B showed similar CD8(+) T cell responses. In addition, our results clearly identified immunodominant regions in the NY-ESO-1 protein: NY-ESO-179-102 and NY-ESO-1115-138 for CD4+ T cells and NY-ESO-185-108 for CD8+ T cells in a large proportion of vaccinated patients. These regions of NY-ESO-1 protein should be considered in future clinical trials as immunodominant epitopes. © 2014 UICC.

  17. The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice.

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    Michal Krupka

    Full Text Available Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag OspC (rOspC could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b with free rOspC and Montanide PET GEL A; (c with free rOspC and alum; or (d with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.

  18. Distinct pattern of cerebral blood flow alterations specific to schizophrenics experiencing auditory verbal hallucinations with and without insight: a pilot study.

    Science.gov (United States)

    Jing, Rixing; Huang, Jiangjie; Jiang, Deguo; Lin, Xiaodong; Ma, Xiaolei; Tian, Hongjun; Li, Jie; Zhuo, Chuanjun

    2018-01-23

    Schizophrenia is associated with widespread and complex cerebral blood flow (CBF) disturbance. Auditory verbal hallucinations (AVH) and insight are the core symptoms of schizophrenia. However, to the best of our knowledge, very few studies have assessed the CBF characteristics of the AVH suffered by schizophrenic patients with and without insight. Based on our previous findings, Using a 3D pseudo-continuous ASL (pcASL) technique, we investigated the differences in AVH-related CBF alterations in schizophrenia patients with and without insight. We used statistical parametric mapping (SPM8) and statistical non-parametric mapping (SnPM13) to perform the fMRI analysis. We found that AVH-schizophrenia patients without insight showed an increased CBF in the left temporal pole and a decreased CBF in the right middle frontal gyrus when compared to AVH-schizophrenia patients with insight. Our novel findings suggest that AVH-schizophrenia patients without insight possess a more complex CBF disturbance. Simultaneously, our findings also incline to support the idea that the CBF aberrant in some specific brain regions may be the common neural basis of insight and AVH. Our findings support the mostly current hypotheses regarding AVH to some extent. Although our findings come from a small sample, it provide the evidence that indicate us to conduct a larger study to thoroughly explore the mechanisms of schizophrenia, especially the core symptoms of AVHs and insight.

  19. Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy.

    Science.gov (United States)

    Woodall, Benjamin P; Woodall, Meryl C; Luongo, Timothy S; Grisanti, Laurel A; Tilley, Douglas G; Elrod, John W; Koch, Walter J

    2016-10-14

    GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2 fl/fl ) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2 fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β 2 -adrenergic receptor (β 2 AR) agonist, was significantly enhanced in MLC-Cre:GRK2 fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β 2 AR-induced hypertrophy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Embryonic GABA(B) receptor blockade alters cell migration, adult hypothalamic structure, and anxiety- and depression-like behaviors sex specifically in mice.

    Science.gov (United States)

    Stratton, Matthew S; Staros, Michelle; Budefeld, Tomaz; Searcy, Brian T; Nash, Connor; Eitel, Chad; Carbone, David; Handa, Robert J; Majdic, Gregor; Tobet, Stuart A

    2014-01-01

    Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA(B) receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA(B) receptor to a 7-day critical period (E11-E17) during embryonic development. Experiments tested the role of GABA(B) receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA(B) receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA(B) receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA(B) receptor antagonist. Embryonic exposure to GABA(B) receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA(B) receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.

  1. Embryonic GABA(B receptor blockade alters cell migration, adult hypothalamic structure, and anxiety- and depression-like behaviors sex specifically in mice.

    Directory of Open Access Journals (Sweden)

    Matthew S Stratton

    Full Text Available Neurons of the paraventricular nucleus of the hypothalamus (PVN regulate the hypothalamic- pituitary-adrenal (HPA axis and the autonomic nervous system. Females lacking functional GABA(B receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA(B receptor to a 7-day critical period (E11-E17 during embryonic development. Experiments tested the role of GABA(B receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA(B receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA(B receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA(B receptor antagonist. Embryonic exposure to GABA(B receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA(B receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.

  2. Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy*

    Science.gov (United States)

    Woodall, Benjamin P.; Woodall, Meryl C.; Luongo, Timothy S.; Grisanti, Laurel A.; Tilley, Douglas G.; Elrod, John W.; Koch, Walter J.

    2016-01-01

    GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy. PMID:27566547

  3. Containment air circulation for optimal hydrogen recombination

    International Nuclear Information System (INIS)

    Spinks, N.; Krause, M.

    1997-01-01

    An accepted first-line defense for hydrogen mitigation is to design for the hydrogen to be rapidly mixed with the containment atmosphere and diluted to below flammability concentrations. Then, as hydrogen continues to be produced in the longer term, recombiners can be used to remove hydrogen: recombiners can be located in forced-air ducts or passive recombiners can be distributed within containment and the heat of recombination used to promote local air circulation. However, this principle does not eliminate the possibility of high hydrogen concentrations at locations removed from the recombiners. An improvement on this strategy is to arrange for a specific, buoyancy-driven, overall circulation of the containment atmosphere such that the recombiners can be located within the recirculation flow, immediately downstream of the hydrogen source. This would make the mixing process more predictable and solve the mass-transfer problem associated with distributed recombiners. Ideally, the recombiners would be located just above the hydrogen source so that the heat of recombination would assist the overall circulation. In this way, the hydrogen would be removed as close as possible to the source, thereby minimizing the amount of hydrogen immediately downstream of the source and reducing the hydrogen concentration to acceptable levels at other locations. Such a strategy requires the containment volume to be divided into an upflow path, past the hydrogen source and the recombiner, and a downflow path to complete the circuit. The flow could be generated actively using fans or passively using buoyancy forces arising from the difference in density of gases in the upfiow and downflow paths; the gases in the downflow path being cooled at an elevated heat sink. (author)

  4. The recombinational anatomy of a mouse chromosome.

    Directory of Open Access Journals (Sweden)

    Kenneth Paigen

    2008-07-01

    Full Text Available Among mammals, genetic recombination occurs at highly delimited sites known as recombination hotspots. They are typically 1-2 kb long and vary as much as a 1,000-fold or more in recombination activity. Although much is known about the molecular details of the recombination process itself, the factors determining the location and relative activity of hotspots are poorly understood. To further our understanding, we have collected and mapped the locations of 5,472 crossover events along mouse Chromosome 1 arising in 6,028 meioses of male and female reciprocal F1 hybrids of C57BL/6J and CAST/EiJ mice. Crossovers were mapped to a minimum resolution of 225 kb, and those in the telomere-proximal 24.7 Mb were further mapped to resolve individual hotspots. Recombination rates were evolutionarily conserved on a regional scale, but not at the local level. There was a clear negative-exponential relationship between the relative activity and abundance of hotspot activity classes, such that a small number of the most active hotspots account for the majority of recombination. Females had 1.2x higher overall recombination than males did, although the sex ratio showed considerable regional variation. Locally, entirely sex-specific hotspots were rare. The initiation of recombination at the most active hotspot was regulated independently on the two parental chromatids, and analysis of reciprocal crosses indicated that parental imprinting has subtle effects on recombination rates. It appears that the regulation of mammalian recombination is a complex, dynamic process involving multiple factors reflecting species, sex, individual variation within species, and the properties of individual hotspots.

  5. Strain-specific and recessive QTLs involved in the control of partial resistance to Fusarium oxysporum f. sp. melonis race 1.2 in a recombinant inbred line population of melon.

    Science.gov (United States)

    Perchepied, L; Dogimont, C; Pitrat, M

    2005-06-01

    Fusarium oxysporum f. sp. melonis (FOM) causes serious economic losses in melon (Cucumis melo L.). Two dominant resistance genes have been identified, Fom-1 and Fom-2, which provide resistance to races 0 and 2 and races 0 and 1, respectively, however FOM race 1.2 overcomes these resistance genes. A partial resistance to FOM race 1.2 that has been found in some Far East accessions is under polygenic control. A genetic map of melon was constructed to tag FOM race 1.2 resistance with DNA markers on a recombinant inbred line population derived from a cross between resistant (Isabelle) and susceptible (cv. Védrantais) lines. Artificial root inoculations on plantlets of this population using two strains, one that causes wilting (FOM 1.2w) and one that causes yellowing (FOM 1.2y), resulted in phenotypic and genotypic data that enabled the identification of nine quantitative trait loci (QTLs). These QTLs were detected on five linkage groups by composite interval mapping and explained between 41.9% and 66.4% of the total variation. Four digenic epistatic interactions involving seven loci were detected and increased the total phenotypic variation that was explained. Co-localizations between QTLs and resistance gene homologs or resistance genes, such as Fom-2 and Vat, were observed. A strain-specific QTL was detected, and some QTLs appeared to be recessive.

  6. Hemodynamics in Transplant Renal Artery Stenosis and its Alteration after Stent Implantation Based on a Patient-specific Computational Fluid Dynamics Model.

    Science.gov (United States)

    Wang, Hong-Yang; Liu, Long-Shan; Cao, Hai-Ming; Li, Jun; Deng, Rong-Hai; Fu, Qian; Zhang, Huan-Xi; Fei, Ji-Guang; Wang, Chang-Xi

    Accumulating studies on computational fluid dynamics (CFD) support the involvement of hemodynamic factors in artery stenosis. Based on a patient-specific CFD model, the present study aimed to investigate the hemodynamic characteristics of transplant renal artery stenosis (TRAS) and its alteration after stent treatment. Computed tomography angiography (CTA) data of kidney transplant recipients in a single transplant center from April 2013 to November 2014 were reviewed. The three-dimensional geometry of transplant renal artery (TRA) was reconstructed from the qualified CTA images and categorized into three groups: the normal, stenotic, and stented groups. Hemodynamic parameters including pressure distribution, velocity, wall shear stress (WSS), and mass flow rate (MFR) were extracted. The data of hemodynamic parameters were expressed as median (interquartile range), and Mann-Whitney U-test was used for analysis. Totally, 6 normal, 12 stenotic, and 6 stented TRAs were included in the analysis. TRAS presented nonuniform pressure distribution, adverse pressure gradient across stenosis throat, flow vortex, and a separation zone at downstream stenosis. Stenotic arteries had higher maximal velocity and maximal WSS (2.94 [2.14, 3.30] vs. 1.06 [0.89, 1.15] m/s, 256.5 [149.8, 349.4] vs. 41.7 [37.8, 45.3] Pa at end diastole, P= 0.001; 3.25 [2.67, 3.56] vs. 1.65 [1.18, 1.72] m/s, 281.3 [184.3, 364.7] vs. 65.8 [61.2, 71.9] Pa at peak systole, P= 0.001) and lower minimal WSS and MFRs (0.07 [0.03, 0.13] vs. 0.52 [0.45, 0.67] Pa, 1.5 [1.0, 3.0] vs. 11.0 [8.0, 11.3] g/s at end diastole, P= 0.001; 0.08 [0.03, 0.19] vs. 0.70 [0.60, 0.81] Pa, 2.0 [1.3, 3.3] vs. 16.5 [13.0, 20.3] g/s at peak systole, P= 0.001) as compared to normal arteries. Stent implantation ameliorated all the alterations of the above hemodynamic factors except low WSS. Hemodynamic factors were significantly changed in severe TRAS. Stent implantation can restore or ameliorate deleterious change of hemodynamic

  7. Hemodynamics in Transplant Renal Artery Stenosis and its Alteration after Stent Implantation Based on a Patient-specific Computational Fluid Dynamics Model

    Science.gov (United States)

    Wang, Hong-Yang; Liu, Long-Shan; Cao, Hai-Ming; Li, Jun; Deng, Rong-Hai; Fu, Qian; Zhang, Huan-Xi; Fei, Ji-Guang; Wang, Chang-Xi

    2017-01-01

    Background: Accumulating studies on computational fluid dynamics (CFD) support the involvement of hemodynamic factors in artery stenosis. Based on a patient-specific CFD model, the present study aimed to investigate the hemodynamic characteristics of transplant renal artery stenosis (TRAS) and its alteration after stent treatment. Methods: Computed tomography angiography (CTA) data of kidney transplant recipients in a single transplant center from April 2013 to November 2014 were reviewed. The three-dimensional geometry of transplant renal artery (TRA) was reconstructed from the qualified CTA images and categorized into three groups: the normal, stenotic, and stented groups. Hemodynamic parameters including pressure distribution, velocity, wall shear stress (WSS), and mass flow rate (MFR) were extracted. The data of hemodynamic parameters were expressed as median (interquartile range), and Mann–Whitney U-test was used for analysis. Results: Totally, 6 normal, 12 stenotic, and 6 stented TRAs were included in the analysis. TRAS presented nonuniform pressure distribution, adverse pressure gradient across stenosis throat, flow vortex, and a separation zone at downstream stenosis. Stenotic arteries had higher maximal velocity and maximal WSS (2.94 [2.14, 3.30] vs. 1.06 [0.89, 1.15] m/s, 256.5 [149.8, 349.4] vs. 41.7 [37.8, 45.3] Pa at end diastole, P = 0.001; 3.25 [2.67, 3.56] vs. 1.65 [1.18, 1.72] m/s, 281.3 [184.3, 364.7] vs. 65.8 [61.2, 71.9] Pa at peak systole, P = 0.001) and lower minimal WSS and MFRs (0.07 [0.03, 0.13] vs. 0.52 [0.45, 0.67] Pa, 1.5 [1.0, 3.0] vs. 11.0 [8.0, 11.3] g/s at end diastole, P = 0.001; 0.08 [0.03, 0.19] vs. 0.70 [0.60, 0.81] Pa, 2.0 [1.3, 3.3] vs. 16.5 [13.0, 20.3] g/s at peak systole, P = 0.001) as compared to normal arteries. Stent implantation ameliorated all the alterations of the above hemodynamic factors except low WSS. Conclusions: Hemodynamic factors were significantly changed in severe TRAS. Stent implantation can restore or

  8. Identifying Neurofibromin-Specific Regulatory Nodes for Therapeutic Targeting in NF1

    Science.gov (United States)

    2016-10-01

    analysis and electron microscopy, as shown in Figure 9. Figure 9. Biophysical characterization of recombinant , full length neurofibromin. Left panel...libraries targeting cancer specific genetic alterations; ii) Identify recurrently mutated genes that regulate oncogenic pathways and drug responses...iii) Produce genetic interaction maps to uncover pathway relationships between candidate drivers. Overlap: None Funding Number: N/A PI

  9. Recombinant gene expression protocols

    National Research Council Canada - National Science Library

    Tuan, Rocky S

    1997-01-01

    .... A fundamental requirement for successful recombinant gene expression is the design of the cloning vector and the choice of the host organism for expression. Recombinant Gene Expression Protocols grows out of the need for a laboratory manual that provides the reader the background and rationale, as well as the practical protocols for the preparation of...

  10. FASEB Summer Research Conference. Genetic Recombination and Chromosome Rearrangements

    Energy Technology Data Exchange (ETDEWEB)

    Jinks-Robertson, Sue

    2002-02-01

    The 2001 meeting entitled ''Genetic Recombination and Genome Rearrangements'' was held July 21-26 in Snowmass, Colorado. The goal of the meeting was to bring together scientists using diverse approaches to study all aspects of genetic recombination. This goal was achieved by integrating talks covering the genetics, biochemistry and structural biology of homologous recombination, site-specific recombination, and nonhomologous recombination. The format of the meeting consisted of a keynote address on the opening evening, two formal plenary sessions on each of the four full meeting days, a single afternoon workshop consisting of short talks chosen from among submitted abstracts, and afternoon poster sessions on each of the four full meeting days. The eight plenary session were entitled: (1) Recombination Mechanisms, (2) Prokaryotic Recombination, (3) Repair and Recombination, (4) Site-specific Recombination and Transposition, (5) Eukaryotic Recombination I, (6) Genome Rearrangements, (7) Meiosis, and (8) Eukaryotic Recombination II. Each session included a mix of genetic, biochemical and structural talks; talks were limited to 20 minutes, followed by 10 minutes of very lively, general discussion. Much of the data presented in the plenary sessions was unpublished, thus providing attendees with the most up-to-date knowledge of this rapidly-moving field.

  11. Perinatal maternal high-fat diet induces early obesity and sex-specific alterations of the endocannabinoid system in white and brown adipose tissue of weanling rat offspring.

    Science.gov (United States)

    Almeida, Mariana M; Dias-Rocha, Camilla P; Souza, André S; Muros, Mariana F; Mendonca, Leonardo S; Pazos-Moura, Carmen C; Trevenzoli, Isis H

    2017-11-01

    Perinatal maternal high-fat (HF) diet programmes offspring obesity. Obesity is associated with overactivation of the endocannabinoid system (ECS) in adult subjects, but the role of the ECS in the developmental origins of obesity is mostly unknown. The ECS consists of endocannabinoids, cannabinoid receptors (cannabinoid type-1 receptor (CB1) and cannabinoid type-2 receptor (CB2)) and metabolising enzymes. We hypothesised that perinatal maternal HF diet would alter the ECS in a sex-dependent manner in white and brown adipose tissue of rat offspring at weaning in parallel to obesity development. Female rats received standard diet (9 % energy content from fat) or HF diet (29 % energy content from fat) before mating, during pregnancy and lactation. At weaning, male and female offspring were killed for tissue harvest. Maternal HF diet induced early obesity, white adipocyte hypertrophy and increased lipid accumulation in brown adipose tissue associated with sex-specific changes of the ECS's components in weanling rats. In male pups, maternal HF diet decreased CB1 and CB2 protein in subcutaneous adipose tissue. In female pups, maternal HF diet increased visceral and decreased subcutaneous CB1. In brown adipose tissue, maternal HF diet increased CB1 regardless of pup sex. In addition, maternal HF diet differentially changed oestrogen receptor across the adipose depots in male and female pups. The ECS and oestrogen signalling play an important role in lipogenesis, adipogenesis and thermogenesis, and we observed early changes in their targets in adipose depots of the offspring. The present findings provide insights into the involvement of the ECS in the developmental origins of metabolic disease induced by inadequate maternal nutrition in early life.

  12. Recombinant human erythropoietin in sports: a review

    Directory of Open Access Journals (Sweden)

    Rafael Maia de Almeida Bento

    2003-06-01

    Full Text Available Erythropoietin is an endogenous hormone of glicoproteic nature secreted by the kidneys and is the main regulator of the erythropoiesis. An alteration in its production generates a disturbance in the plasmatic concentration giving rise to several types of pathologies related to the hematopoietic system. The recombinant forms of erythropoietin have indiscriminately been used by athletes, mainly in endurance sports, by increasing the erythrocytes concentration, generating a better delivery of oxygen to the muscle tissue. The administration of recombinant erythropoietin was prohibited by the International Olympic Committee and its use considered as doping. This review has the intention to describe the physical, biological and pharmacokinetic properties of the endogenous erythropoietin, as well as its recombinant form, describing also its use in sports and the process of searching methodologies for its detection in doping control.

  13. Test tube systems with cutting/recombination operations

    Energy Technology Data Exchange (ETDEWEB)

    Freund, R. [Technische Universitaet Wien (Austria); Csuhaj-Varju, E. [Computer and Automation Institute, Budapest (Hungary); Wachtler, F. [Universitaet Wien (Austria)

    1996-12-31

    We introduce test tube systems based on operations that are closely related to the splicing operations, i.e. we consider the operations of cutting a string at a specific site into two pieces with marking them at the cut ends and of recombining two strings with specifically marked endings. Whereas in the splicing of two strings these strings are cut at specific sites and the cut pieces are recombined immediately in a crosswise way, in CR(cutting/recombination)-schemes cutting can happen independently from recombining the cut pieces. Test tube systems based on these operations of cutting and recombination turn out to have maximal generative power even if only very restricted types of input filters for the test tubes are used for the redistribution of the contents of the test tubes after a period of cuttings and recombinations in the test tubes. 10 refs.

  14. Natural type 3/type 2 intertypic vaccine-related poliovirus recombinants with the first crossover sites within the VP1 capsid coding region.

    Science.gov (United States)

    Zhang, Yong; Zhu, Shuangli; Yan, Dongmei; Liu, Guiyan; Bai, Ruyin; Wang, Dongyan; Chen, Li; Zhu, Hui; An, Hongqiu; Kew, Olen; Xu, Wenbo

    2010-12-21

    Ten uncommon natural type 3/type 2 intertypic poliovirus recombinants were isolated from stool specimens from nine acute flaccid paralysis case patients and one healthy vaccinee in China from 2001 to 2008. Complete genomic sequences revealed their vaccine-related genomic features and showed that their first crossover sites were randomly distributed in the 3' end of the VP1 coding region. The length of donor Sabin 2 sequences ranged from 55 to 136 nucleotides, which is the longest donor sequence reported in the literature for this type of poliovirus recombination. The recombination resulted in the introduction of Sabin 2 neutralizing antigenic site 3a (NAg3a) into a Sabin 3 genomic background in the VP1 coding region, which may have been altered by some of the type 3-specific antigenic properties, but had not acquired any type 2-specific characterizations. NAg3a of the Sabin 3 strain seems atypical; other wild-type poliovirus isolates that have circulated in recent years have sequences of NAg3a more like the Sabin 2 strain. 10 natural type 3/type 2 intertypic VP1 capsid-recombinant polioviruses, in which the first crossover sites were found to be in the VP1 coding region, were isolated and characterized. In spite of the complete replacement of NAg3a by type 2-specific amino acids, the serotypes of the recombinants were not altered, and they were totally neutralized by polyclonal type 3 antisera but not at all by type 2 antisera. It is possible that recent type 3 wild poliovirus isolates may be a recombinant having NAg3a sequences derived from another strain during between 1967 and 1980, and the type 3/type 2 recombination events in the 3' end of the VP1 coding region may result in a higher fitness.

  15. Structural and Mechanistic Basis for Extended-Spectrum Drug-Resistance Mutations in Altering the Specificity of TEM, CTX-M, and KPC β-lactamases

    Directory of Open Access Journals (Sweden)

    Timothy Palzkill

    2018-02-01

    Full Text Available The most common mechanism of resistance to β-lactam antibiotics in Gram-negative bacteria is the production of β-lactamases that hydrolyze the drugs. Class A β-lactamases are serine active-site hydrolases that include the common TEM, CTX-M, and KPC enzymes. The TEM enzymes readily hydrolyze penicillins and older cephalosporins. Oxyimino-cephalosporins, such as cefotaxime and ceftazidime, however, are poor substrates for TEM-1 and were introduced, in part, to circumvent β-lactamase-mediated resistance. Nevertheless, the use of these antibiotics has lead to evolution of numerous variants of TEM with mutations that significantly increase the hydrolysis of the newer cephalosporins. The CTX-M enzymes emerged in the late 1980s and hydrolyze penicillins and older cephalosporins and derive their name from the ability to also hydrolyze cefotaxime. The CTX-M enzymes, however, do not efficiently hydrolyze ceftazidime. Variants of CTX-M enzymes, however, have evolved that exhibit increased hydrolysis of ceftazidime. Finally, the KPC enzyme emerged in the 1990s and is characterized by its broad specificity that includes penicillins, most cephalosporins, and carbapenems. The KPC enzyme, however, does not efficiently hydrolyze ceftazidime. As with the TEM and CTX-M enzymes, variants have recently evolved that extend the spectrum of KPC β-lactamase to include ceftazidime. This review discusses the structural and mechanistic basis for the expanded substrate specificity of each of these enzymes that result from natural mutations that confer oxyimino-cephalosporin resistance. For the TEM enzyme, extended-spectrum mutations act by establishing new interactions with the cephalosporin. These mutations increase the conformational heterogeneity of the active site to create sub-states that better accommodate the larger drugs. The mutations expanding the spectrum of CTX-M enzymes also affect the flexibility and conformation of the active site to accommodate

  16. Mechanisms and factors that influence high frequency retroviral recombination

    DEFF Research Database (Denmark)

    Delviks-Frankenberry, Krista; Galli, Andrea; Nikolaitchik, Olga

    2011-01-01

    With constantly changing environmental selection pressures, retroviruses rely upon recombination to reassort polymorphisms in their genomes and increase genetic diversity, which improves the chances for the survival of their population. Recombination occurs during DNA synthesis, whereby reverse...... transcriptase undergoes template switching events between the two copackaged RNAs, resulting in a viral recombinant with portions of the genetic information from each parental RNA. This review summarizes our current understanding of the factors and mechanisms influencing retroviral recombination, fidelity...... of the recombination process, and evaluates the subsequent viral diversity and fitness of the progeny recombinant. Specifically, the high mutation rates and high recombination frequencies of HIV-1 will be analyzed for their roles in influencing HIV-1 global diversity, as well as HIV-1 diagnosis, drug treatment...

  17. Recombinant vaccines: experimental and applied aspects

    DEFF Research Database (Denmark)

    Lorenzen, Niels

    1999-01-01

    Development of vaccines for aquaculture fish represent an important applied functional aspect of fish immunology research. Particularly in the case of recombinant vaccines, where a single antigen is usually expected to induce immunity to a specific pathogen, knowledge of mechanisms involved...... in induction of a protective immune response may become vital. The few recombinant vaccines licensd so far, despite much research during the last decade, illustrate that this is not a straightforward matter. However, as vaccine technology as well as our knowledge of the fish immune system is steadily improved......, these fields will open up a number of interesting research objectives of mutual benefit. Recent aspects of recombinant protein vaccines, live recombinant vaccines and DNA vaccines are discussed....

  18. A recombinant Bacille Calmette-Guérin construct expressing the Plasmodium falciparum circumsporozoite protein enhances dendritic cell activation and primes for circumsporozoite-specific memory cells in BALB/c mice.

    Science.gov (United States)

    Arama, Charles; Waseem, Shahid; Fernández, Carmen; Assefaw-Redda, Yohannes; You, Liya; Rodriguez, Ariane; Radošević, Katarina; Goudsmit, Jaap; Kaufmann, Stefan H E; Reece, Stephen T; Troye-Blomberg, Marita

    2012-08-10

    A protective malaria vaccine may induce both high levels of neutralising antibodies and strong T-cell responses. The Plasmodium falciparum circumsporozoite protein (CSp) is a leading pre-erythrocytic vaccine candidate. CSp is a week immunogen per se, but Mycobacterium bovis Bacille Calmette-Guérin (BCG) has excellent adjuvant activity and has been utilized as a vector to deliver heterologous vaccine candidate antigens. It is safe in immunocompetent individuals and inexpensive to produce. We assessed in vitro and in vivo a recombinant BCG-expressing CSp (BCG-CS) as malaria vaccine candidate. Immunisation of BALB/c mice with BCG-CS augmented numbers of dendritic cells (DCs) in draining lymph nodes and in the spleen. The activation markers MHC-class-II, CD40, CD80 and CD86 on DCs were significantly upregulated by BCG-CS as compared to wild-type BCG (wt-BCG). In vitro stimulation of bone marrow-derived DCs and macrophages with BCG-CS induced IL-12 and TNF-α production. BCG-CS induced higher phagocytic activity in macrophages as compared to wt-BCG. Immunogenicity studies show that BCG-CS induced CS-specific antibodies and IFN-γ-producing memory cells. In conclusion, BCG-CS is highly efficient in activating antigen-presenting cells (APCs) for priming of adaptive immunity. Implications for the rational design of novel vaccines against malaria and TB, the two major devastating poverty-related diseases, are discussed. Copyright © 2012. Published by Elsevier Ltd.

  19. Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution

    Directory of Open Access Journals (Sweden)

    Monjane Adérito L

    2011-12-01

    Full Text Available Abstract Background Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae, the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Results Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots

  20. Recombination hotspots and host susceptibility modulate the adaptive value of recombination during maize streak virus evolution.

    Science.gov (United States)

    Monjane, Adérito L; van der Walt, Eric; Varsani, Arvind; Rybicki, Edward P; Martin, Darren P

    2011-12-02

    Maize streak virus -strain A (MSV-A; Genus Mastrevirus, Family Geminiviridae), the maize-adapted strain of MSV that causes maize streak disease throughout sub-Saharan Africa, probably arose between 100 and 200 years ago via homologous recombination between two MSV strains adapted to wild grasses. MSV recombination experiments and analyses of natural MSV recombination patterns have revealed that this recombination event entailed the exchange of the movement protein - coat protein gene cassette, bounded by the two genomic regions most prone to recombination in mastrevirus genomes; the first surrounding the virion-strand origin of replication, and the second around the interface between the coat protein gene and the short intergenic region. Therefore, aside from the likely adaptive advantages presented by a modular exchange of this cassette, these specific breakpoints may have been largely predetermined by the underlying mechanisms of mastrevirus recombination. To investigate this hypothesis, we constructed artificial, low-fitness, reciprocal chimaeric MSV genomes using alternating genomic segments from two MSV strains; a grass-adapted MSV-B, and a maize-adapted MSV-A. Between them, each pair of reciprocal chimaeric genomes represented all of the genetic material required to reconstruct - via recombination - the highly maize-adapted MSV-A genotype, MSV-MatA. We then co-infected a selection of differentially MSV-resistant maize genotypes with pairs of reciprocal chimaeras to determine the efficiency with which recombination would give rise to high-fitness progeny genomes resembling MSV-MatA. Recombinants resembling MSV-MatA invariably arose in all of our experiments. However, the accuracy and efficiency with which the MSV-MatA genotype was recovered across all replicates of each experiment depended on the MSV susceptibility of the maize genotypes used and the precise positions - in relation to known recombination hotspots - of the breakpoints required to re-create MSV

  1. Specific gene expression signatures induced by the multiple oncogenic alterations that occur within the PTEN/PI3K/AKT pathway in lung cancer.

    Directory of Open Access Journals (Sweden)

    Carmela De Marco

    Full Text Available Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B expressing active mutant AKT1 (AKT1-E17K, active mutant PIK3CA (PIK3CA-E545K or that are silenced for PTEN. We found that, altogether, aberrant PI3K/AKT signalling in lung epithelial cells regulated the expression of 1,960/20,436 genes (9%, though only 30 differentially expressed genes (DEGs (15 up-regulated, 12 down-regulated and 3 discordant out of 20,436 that were common among BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells (0.1%. Conversely, DEGs specific for mutant AKT1 were 133 (85 up-regulated; 48 down-regulated, DEGs specific for mutant PIK3CA were 502 (280 up-regulated; 222 down-regulated and DEGs specific for PTEN loss were 1549 (799 up-regulated, 750 down-regulated. The results obtained from array analysis were confirmed by quantitative RT-PCR on selected up- and down-regulated genes (n = 10. Treatment of BEAS-C cells and the corresponding derivatives with pharmacological inhibitors of AKT (MK2206 or PI3K (LY294002 further validated the significance of our findings. Moreover, mRNA expression of selected DEGs (SGK1, IGFBP3, PEG10, GDF15, PTGES, S100P, respectively correlated with the activation status of the PI3K/AKT pathway assessed by S473 phosphorylation in NSCLC cell lines (n = 6. Finally, we made use of Ingenuity Pathway Analysis (IPA to investigate the relevant Bio

  2. Hadron correlations from recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    Quark recombination is a successful model to describe the hadronization of a deconfined quark gluon plasma. Jet-like dihadron correlations measured at RHIC provide a challenge for this picture. We discuss how correlations between hadrons can arise from correlations between partons before hadronization. An enhancement of correlations through the recombination process, similar to the enhancement of elliptic flow is found. Hot spots from completely or partially quenched jets are a likely source of such parton correlations.

  3. Sex speeds adaptation by altering the dynamics of molecular evolution.

    Science.gov (United States)

    McDonald, Michael J; Rice, Daniel P; Desai, Michael M

    2016-03-10

    Sex and recombination are pervasive throughout nature despite their substantial costs. Understanding the evolutionary forces that maintain these phenomena is a central challenge in biology. One longstanding hypothesis argues that sex is beneficial because recombination speeds adaptation. Theory has proposed several distinct population genetic mechanisms that could underlie this advantage. For example, sex can promote the fixation of beneficial mutations either by alleviating interference competition (the Fisher-Muller effect) or by separating them from deleterious load (the ruby in the rubbish effect). Previous experiments confirm that sex can increase the rate of adaptation, but these studies did not observe the evolutionary dynamics that drive this effect at the genomic level. Here we present the first, to our knowledge, comparison between the sequence-level dynamics of adaptation in experimental sexual and asexual Saccharomyces cerevisiae populations, which allows us to identify the specific mechanisms by which sex speeds adaptation. We find that sex alters the molecular signatures of evolution by changing the spectrum of mutations that fix, and confirm theoretical predictions that it does so by alleviating clonal interference. We also show that substantially deleterious mutations hitchhike to fixation in adapting asexual populations. In contrast, recombination prevents such mutations from fixing. Our results demonstrate that sex both speeds adaptation and alters its molecular signature by allowing natural selection to more efficiently sort beneficial from deleterious mutations.

  4. Late replicating domains are highly recombining in females but have low male recombination rates: implications for isochore evolution.

    Directory of Open Access Journals (Sweden)

    Catherine J Pink

    Full Text Available In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in

  5. Late Replicating Domains Are Highly Recombining in Females but Have Low Male Recombination Rates: Implications for Isochore Evolution

    Science.gov (United States)

    Pink, Catherine J.; Hurst, Laurence D.

    2011-01-01

    In mammals sequences that are either late replicating or highly recombining have high rates of evolution at putatively neutral sites. As early replicating domains and highly recombining domains both tend to be GC rich we a priori expect these two variables to covary. If so, the relative contribution of either of these variables to the local neutral substitution rate might have been wrongly estimated owing to covariance with the other. Against our expectations, we find that sex-averaged recombination rates show little or no correlation with replication timing, suggesting that they are independent determinants of substitution rates. However, this result masks significant sex-specific complexity: late replicating domains tend to have high recombination rates in females but low recombination rates in males. That these trends are antagonistic explains why sex-averaged recombination is not correlated with replication timing. This unexpected result has several important implications. First, although both male and female recombination rates covary significantly with intronic substitution rates, the magnitude of this correlation is moderately underestimated for male recombination and slightly overestimated for female recombination, owing to covariance with replicating timing. Second, the result could explain why male recombination is strongly correlated with GC content but female recombination is not. If to explain the correlation between GC content and replication timing we suppose that late replication forces reduced GC content, then GC promotion by biased gene conversion during female recombination is partly countered by the antagonistic effect of later replicating sequence tending increase AT content. Indeed, the strength of the correlation between female recombination rate and local GC content is more than doubled by control for replication timing. Our results underpin the need to consider sex-specific recombination rates and potential covariates in analysis of GC

  6. Intranasal Delivery of Recombinant NT4-NAP/AAV Exerts Potential Antidepressant Effect.

    Science.gov (United States)

    Ma, Xian-Cang; Chu, Zheng; Zhang, Xiao-Ling; Jiang, Wen-Hui; Jia, Min; Dang, Yong-Hui; Gao, Cheng-Ge

    2016-06-01

    The present study was designed to construct a recombinant adeno-associated virus (rAAV) which can express NAP in the brain and examine whether this virus can produce antidepressant effects on C57 BL/6 mice that had been subjected to open field test and forced swimming test, via nose-to-brain pathway. When the recombinant plasmid pGEM-T Easy/NT4-NAP was digested by EcoRI, 297 bp fragments can be obtained and NT4-NAP sequence was consistent with the designed sequence confirmed by DNA sequencing. When the recombinant plasmid pSSCMV/NT4-NAP was digested by EcoRI, 297 bp fragments is visible. Immunohistochemical staining of fibroblasts revealed that expression of NAP was detected in NT4-NAP/AAV group. Intranasal delivery of NT4-NAP/AAV significantly reduced immobility time when the FST was performed after 1 day from the last administration. The effects observed in the FST could not be attributed to non-specific increases in activity since intranasal delivery of NT4-NAP/AAV did not alter the behavior of the mice during the open field test. The results indicated that a recombinant AAV vector which could express NAP in cells was successfully constructed and NAP may be a potential target for therapeutic action of antidepressant treatment.

  7. Metabolomic Analyses of Brain Tissue in Sepsis Induced by Cecal Ligation Reveal Specific Redox Alterations-Protective Effects of the Oxygen Radical Scavenger Edaravone

    DEFF Research Database (Denmark)

    Hara, Naomi; Chijiiwa, Miyuki; Yara, Miki

    2015-01-01

    at analyzing the preventive effect of the free radical scavenger edaravone on sepsis-induced brain alterations. Sepsis was induced by cecal ligation and puncture (CLP) and the mice were divided into three groups-CLP vehicle (CLPV), CLP and edaravone (MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one) (CLPE...

  8. Copy Number Alterations and Neoplasia-Specific Mutations in MELK, PDCD1LG2, TLN1, and PAX5 at 9p in Different Neoplasias

    NARCIS (Netherlands)

    Sarhadi, V.K.; Lahti, L.M.; Scheinin, I.; Ellonen, P.; Kettunen, E.; Serra, M.; Scotlandi, K.; Picci, P.; Knuutila, S.

    2014-01-01

    Genetic alterations affecting 9p are commonly present in many cancer types and many cancer-related genes are located in this chromosomal region. We sequenced all of the genes located in a 32Mb region of 9p by targeted next generation sequencing (NGS) in 96 patients with different cancer types,

  9. Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1

    DEFF Research Database (Denmark)

    Thaysen-Andersen, Morten; Thøgersen, Ida B.; Nielsen, Hans Jørgen

    2007-01-01

    A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling...... glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases....

  10. Anomalous Abundances in Gaseous Nebulae From Recombination and Collisional Lines: Improved Photoionization and Recombination Studies

    Science.gov (United States)

    Pradhan, Anil Kumar; Nahar, S. N.; Eissner, W. B.; Montenegro, M.

    2011-01-01

    A perplexing anomaly arises in the determination of abundances of common elements in gaseous nebulae, as derived from collisionally excited lines (CEL) as opposed to those from Recombination Lines (RCL). The "abundance discrepancy factors" can range from a factor of 2 to an order of magnitude or more. That has led to quite different interpretation of the physical structure and processes in gaseous nebulae, such as temperature fluctuations across the object, or metal-rich concentrations leading to a dual-abundnace scenario. We show that the problem may lie in inaccuracies in photoionization and recombination models neglecting low-energy resonance phenomena due to fine structure. Whereas the atomic physics of electron impact excitation of forbidden lines is well understood, and accurate collision strengths have long been available, that is not generally the case for electron-ion recombination cross sections. A major problem is the inclusion of relativisitic effects as it pertains to the existence of very low-energy fine structure resonances in photoionization cross sections. We carry out new relativistic calculations for photoionization and recombination cross sections using a recently extended version of the Breit-Pauli R-matrix codes, and the unified electron-ion recombination method that subsumes both the radiative and the dielectronic recombination (RR and DR) processes in an ab initio and self-consistent manner. We find that near-thresold resonances manifest themselves within fine structure levels of the ground state of ions, enhancing low-temperature recombination rate coefficients at 1000-10,000 K. The resulting enahncement in level-specific and total recombination rate coefficients should therefore lead to reduced abundances derived from RCL, and in accordance with those from CEL. We present results for photoionization of O II into, and recombination from, O III. Theoretical cross sections are benchmarked against high-resolution measurements from synchrotron

  11. RNA recombination in Hepatitis delta virus: Identification of a novel naturally occurring recombinant

    Directory of Open Access Journals (Sweden)

    Chia-Chi Lin

    2017-12-01

    Full Text Available Background/Purpose: Hepatitis delta virus (HDV is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity. It replicates in the nucleus by host RNA polymerase via a rolling circle mechanism. Similar to many RNA viruses encoding their own RNA-dependent RNA polymerases, homologous recombination of HDV occurs in mixed-genotype infections and in cultured cells cotransfected with two HDV sequences, as demonstrated by molecular analyses. Methods: Among 237 published complete genomic sequences, 34 sequences were reported from the small and isolated Miyako Island, Japan, and belonged to the Asia-specific genotypes, HDV-2 and HDV-4 (the majority of them belonged to the known Miyako Island-specific subgroup, HDV-4M. We investigated the presence of naturally occurring HDV recombinant in Miyako Island using phylogenetic and recombination analyses. Results: We identified a two-switch HDV-4/4M intersubtype recombinant with an unbranched rod-like RNA genome. Conclusion: Our data suggest that RNA recombination plays an important role in the rapid evolution of HDV, allowing the production of new HDV strains with correct genomic structures. Keywords: hepatitis delta virus, RNA recombination

  12. Chronic low-level arsenic exposure causes gender-specific alterations in locomotor activity, dopaminergic systems, and thioredoxin expression in mice

    International Nuclear Information System (INIS)

    Bardullas, U.; Limon-Pacheco, J.H.; Giordano, M.; Carrizales, L.; Mendoza-Trejo, M.S.; Rodriguez, V.M.

    2009-01-01

    Arsenic (As) is a toxic metalloid widely present in the environment. Human exposure to As has been associated with the development of skin and internal organ cancers and cardiovascular disorders, among other diseases. A few studies report decreases in intelligence quotient (IQ), and sensory and motor alterations after chronic As exposure in humans. On the other hand, studies of rodents exposed to high doses of As have found alterations in locomotor activity, brain neurochemistry, behavioral tasks, and oxidative stress. In the present study both male and female C57Bl/6J mice were exposed to environmentally relevant doses of As such as 0.05, 0.5, 5.0, or 50 mg As/L of drinking water for 4 months, and locomotor activity was assessed every month. Male mice presented hyperactivity in the group exposed to 0.5 mg As/L and hypoactivity in the group exposed to 50 mg As/L after 4 months of As exposure, whereas female mice exposed to 0.05, 0.5, and 5.0 mg As/L exhibited hyperactivity in every monthly test during As exposure. Furthermore, striatal and hypothalamic dopamine content was decreased only in female mice. Also decreases in tyrosine hydroxylase (TH) and cytosolic thioredoxin (Trx-1) mRNA expression in striatum and nucleus accumbens were observed in male and female mice, respectively. These results indicate that chronic As exposure leads to gender-dependent alterations in dopaminergic markers and spontaneous locomotor activity, and down-regulation of the antioxidant capacity of the brain.

  13. Copy number alterations and neoplasia-specific mutations in MELK, PDCD1LG2, TLN1, and PAX5 at 9p in different neoplasias.

    Science.gov (United States)

    Sarhadi, Virinder Kaur; Lahti, Leo; Scheinin, Ilari; Ellonen, Pekka; Kettunen, Eeva; Serra, Massimo; Scotlandi, Katia; Picci, Piero; Knuutila, Sakari

    2014-07-01

    Genetic alterations affecting 9p are commonly present in many cancer types and many cancer-related genes are located in this chromosomal region. We sequenced all of the genes located in a 32Mb region of 9p by targeted next generation sequencing (NGS) in 96 patients with different cancer types, including acute lymphoblastic leukemia, bone malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma, fibrosarcoma, Ewing's sarcoma, and lung carcinoma. Copy number alterations (CNA), and mutations were studied from the NGS data. We detected a deletion at the CDKN2A locus as being the most frequent genetic alteration in all cancer types. In addition to this locus, NGS also identified other small regions of copy number loss and gain. However, different cancer types did not reveal any statistically significant differences with regard to CNA frequency or type. Of the 191 genes within the target region, two novel recurrent mutations were found in the MELK and PDCD1LG2 genes. The most commonly mutated gene in sarcomas was TLN1 (8%) and PAX5 in ALL (9%). Mutations in PAX5, and RUSC2, were seen exclusively in ALL patients and those in KIAA1432, CA9, TLN1, and MELK only in sarcomas (MFH, FS, EFT). Thus using targeted NGS of the 9p region, in addition to commonly deleted CDKN2A locus, we were able to identify a number of small deletions and gains, as well as novel recurrent mutations in different cancer types. © 2014 Wiley Periodicals, Inc. Copyright © 2014 Wiley Periodicals, Inc.

  14. Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Serre, Stéphanie B N

    2013-01-01

    (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A...

  15. Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

    Directory of Open Access Journals (Sweden)

    Nathalie Kin

    2015-05-01

    Full Text Available Human coronavirus OC43 (HCoV-OC43 is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses.

  16. Analysis of chickens for recombination within the MHC (B-complex)

    DEFF Research Database (Denmark)

    Skjødt, K; Koch, C; Crone, M

    1985-01-01

    In an attempt to further map the chicken MHC (the B complex), a systematic search for genetic recombinants within the B complex was performed by serotyping the progeny from F2 crosses of chickens by means of specific anti-class I, anti-class II, and anti-class IV alloantisera. Two recombinant B...... confirmed the original serological typing of the two recombinant B haplotypes. No recombination between B-F (class I) and B-L (class II) loci was found. This very low frequency of recombination within the B complex as compared with recombination frequencies found in mammalian MHC's is discussed...

  17. Recombinant respiratory syncytial virus from which the entire SH gene has been deleted grows efficiently in cell culture and exhibits site-specific attenuation in the respiratory tract of the mouse.

    Science.gov (United States)

    Bukreyev, A; Whitehead, S S; Murphy, B R; Collins, P L

    1997-12-01

    The small hydrophobic protein SH of human respiratory syncytial virus (RSV) is a short transmembrane surface protein of unknown function. A full-length cDNA of RSV strain A2 (subgroup A) antigenomic RNA was modified such that the entire SH gene, including the transcription signals and the complete mRNA-encoding sequence, was deleted and replaced by a synthetic intergenic region. This reduced the length of the antigenome by 398 nucleotides and ablated expression of 1 of the 10 RSV mRNAs. Recombinant virus containing this engineered deletion was recovered, and the absence of the SH gene was confirmed by reverse transcription in conjunction with PCR. Northern blot analysis of intracellular RNAs and gel electrophoresis of labeled intracellular proteins confirmed the lack of expression of the SH mRNA and protein. The absence of the SH gene did not noticeably affect RNA replication, but two effects on transcription were noted. First, synthesis of the G, F, and M2 mRNAs was increased, presumably due to their being one position closer to the promoter in the gene order. Second, transcription of genes downstream of the engineered site exhibited a steeper gradient of polarity. On monolayers of HEp-2 cells, the SH-minus virus produced syncytia which were at least equivalent in size to those of the wild type and produced plaques which were 70% larger. Furthermore, the SH-minus virus grew somewhat better (up to 12.6-fold) than wild-type recombinant RSV in certain cell lines. While the function of the SH protein remains to be determined, it seems to be completely dispensable for growth in tissue culture and fusion function. When inoculated intranasally into mice, the SH-minus virus resembled the wild-type recombinant virus in its efficiency of replication in the lungs, whereas it replicated 10-fold less efficiently in the upper respiratory tract. In mice, the SH-minus and wild-type recombinant viruses were similarly immunogenic and effective in inducing resistance to virus

  18. Nonradiative recombination in semiconductors

    CERN Document Server

    Abakumov, VN; Yassievich, IN

    1991-01-01

    In recent years, great progress has been made in the understandingof recombination processes controlling the number of excessfree carriers in semiconductors under nonequilibrium conditions. As a result, it is now possible to give a comprehensivetheoretical description of these processes. The authors haveselected a number of experimental results which elucidate theunderlying physical problems and enable a test of theoreticalmodels. The following topics are dealt with: phenomenological theory ofrecombination, theoretical models of shallow and deep localizedstates, cascade model of carrier captu

  19. Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination.

    Directory of Open Access Journals (Sweden)

    Joseph A Ross

    2011-07-01

    Full Text Available The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.

  20. Recombinant bacterial hemoglobin alters metabolism of Aspergillus niger

    DEFF Research Database (Denmark)

    Hofmann, Gerald; Diano, Audrey; Nielsen, Jens

    2009-01-01

    The filamentous fungus Aspergillus niger is used extensively for the production of enzymes and organic acids. A major problem in industrial fermentations with this fungus is to ensure sufficient supply of oxygen required for respiratory metabolism of the fungus. In case of oxygen limitation......, the fungus will produce various by-products like organic acids and polyols. In order to circumvent this problem we here study the effects of the expression of a bacterial hemoglobin protein on the metabolism of A. niger. We integrated the vgb gene from Vitreoscilla sp. into the genome at the pyrA locus...... behind the strong gpdA promoter from Aspergillus nidulans. Analysis of secreted metabolites, oxygen uptake, CO2 evolution and biomass formation points towards a relief of stress in the mutant expressing VHB when it is exposed to oxygen limitation. Our findings therefore point to an interesting strategy...

  1. In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity

    NARCIS (Netherlands)

    S. Chutinimitkul (Salin); D.A.J. van Riel (Debby); V.J. Munster (Vincent); J.M.A. van den Brand (Judith); G.F. Rimmelzwaan (Guus); T. Kuiken (Thijs); A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); E. de Wit (Emmie)

    2010-01-01

    textabstractThe continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, α2,6-linked sialic acids (SA), and being able to transmit efficiently

  2. Neonatal colonisation expands a specific intestinal antigen-presenting cell subset prior to CD4 T-cell expansion, without altering T-cell repertoire.

    Directory of Open Access Journals (Sweden)

    Charlotte F Inman

    Full Text Available Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα(+ antigen-presenting cell subset, whilst SIRPα(-CD11R1(+ antigen-presenting cells (APCs are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα(+ antigen-presenting cells as orchestrators of early-life mucosal immune development.

  3. A GCH1 haplotype confers sex-specific susceptibility to pain crises and altered endothelial function in adults with sickle cell anemia

    Science.gov (United States)

    Belfer, Inna; Youngblood, Victoria; Darbari, Deepika S.; Wang, Zhengyuan; Diaw, Lena; Freeman, Lita; Desai, Krupa; Dizon, Michael; Allen, Darlene; Cunnington, Colin; Channon, Keith M.; Milton, Jacqueline; Hartley, Stephen W.; Nolan, Vikki; Kato, Gregory J.; Steinberg, Martin H.; Goldman, David; Taylor, James G.

    2014-01-01

    GTP cyclohydrolase (GCH1) is rate limiting for tetrahydrobiopterin (BH4) synthesis, where BH4 is a cofactor for nitric oxide (NO) synthases and aromatic hydroxylases. GCH1 polymorphisms are implicated in the pathophysiology of pain, but have not been investigated in African populations. We examined GCH1 and pain in sickle cell anemia where GCH1 rs8007267 was a risk factor for pain crises in discovery (n = 228; odds ratio [OR] 2.26; P = 0.009) and replication (n = 513; OR 2.23; P = 0.004) cohorts. In vitro, cells from sickle cell anemia subjects homozygous for the risk allele produced higher BH4. In vivo physiological studies of traits likely to be modulated by GCH1 showed rs8007267 is associated with altered endothelial dependent blood flow in females with SCA (8.42% of variation; P = 0.002). The GCH1 pain association is attributable to an African haplotype with where its sickle cell anemia pain association is limited to females (OR 2.69; 95% CI 1.21–5.94; P = 0.01) and has the opposite directional association described in Europeans independent of global admixture. The presence of a GCH1 haplotype with high BH4 in populations of African ancestry could explain the association of rs8007267 with sickle cell anemia pain crises. The vascular effects of GCH1 and BH4 may also have broader implications for cardiovascular disease in populations of African ancestry. PMID:24136375

  4. Alteration of the cell adhesion molecule L1 expression in a specific subset of primary afferent neurons contributes to neuropathic pain.

    Science.gov (United States)

    Yamanaka, Hiroki; Obata, Koichi; Kobayashi, Kimiko; Dai, Yi; Fukuoka, Tetsuo; Noguchi, Koichi

    2007-02-01

    The cell adhesion molecule L1 (L1-CAM) plays important functional roles in the developing and adult nervous systems. Here we show that peripheral nerve injury induced dynamic post-transcriptional alteration of L1-CAM in the rat dorsal root ganglia (DRGs) and spinal cord. Sciatic nerve transection (SCNT) changed the expression of L1-CAM protein but not L1-CAM mRNA. In DRGs, SCNT induced accumulation of the L1-CAM into the surface of somata, which resulted in the formation of immunoreactive ring structures in a number of unmyelinated C-fiber neurons. These neurons with L1-CAM-immunoreactive ring structures were heavily colocalized with phosphorylated p38 MAPK. Western blot analysis revealed the increase of full-length L1-CAM and decrease of fragments of L1-CAM after SCNT in DRGs. Following SCNT, L1-CAM-immunoreactive profiles in the dorsal horn showed an increase mainly in pre-synaptic areas of laminae I-II with a delayed onset and colocalized with growth-associated protein 43. In contrast to DRGs, SCNT increased the proteolytic 80-kDa fragment of L1-CAM and decreased full-length L1-CAM in the spinal cord. The intrathecal injection of L1-CAM antibody for the extracellular domain of L1-CAM inhibited activation of p38 MAPK and emergence of ring structures of L1-CAM immunoreactivity in injured DRG neurons. Moreover, inhibition of extracellular L1-CAM binding by intrathecal administration of antibody suppressed the mechanical allodynia and thermal hyperalgesia induced by partial SCNT. Collectively, these data suggest that the modification of L1-CAM in nociceptive pathways might be an important pathomechanism of neuropathic pain.

  5. Gestational Exposure to Air Pollution Alters Cortical Volume, Microglial Morphology, and Microglia-Neuron Interactions in a Sex-Specific Manner

    Directory of Open Access Journals (Sweden)

    Jessica L. Bolton

    2017-05-01

    Full Text Available Microglia are the resident immune cells of the brain, important for normal neural development in addition to host defense in response to inflammatory stimuli. Air pollution is one of the most pervasive and harmful environmental toxicants in the modern world, and several large scale epidemiological studies have recently linked prenatal air pollution exposure with an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD. Diesel exhaust particles (DEP are a primary toxic component of air pollution, and markedly activate microglia in vitro and in vivo in adult rodents. We have demonstrated that prenatal exposure to DEP in mice, i.e., to the pregnant dams throughout gestation, results in a persistent vulnerability to behavioral deficits in adult offspring, especially in males, which is intriguing given the greater incidence of ASD in males to females (∼4:1. Moreover, there is a striking upregulation of toll-like receptor (TLR 4 gene expression within the brains of the same mice, and this expression is primarily in microglia. Here we explored the impact of gestational exposure to DEP or vehicle on microglial morphology in the developing brains of male and female mice. DEP exposure increased inflammatory cytokine protein and altered the morphology of microglia, consistent with activation or a delay in maturation, only within the embryonic brains of male mice; and these effects were dependent on TLR4. DEP exposure also increased cortical volume at embryonic day (E18, which switched to decreased volume by post-natal day (P30 in males, suggesting an impact on the developing neural stem cell niche. Consistent with this hypothesis, we found increased microglial-neuronal interactions in male offspring that received DEP compared to all other groups. Taken together, these data suggest a mechanism by which prenatal exposure to environmental toxins may affect microglial development and long-term function, and thereby contribute

  6. Male-specific alteration in excitatory post-synaptic development and social interaction in pre-natal valproic acid exposure model of autism spectrum disorder.

    Science.gov (United States)

    Kim, Ki Chan; Kim, Pitna; Go, Hyo Sang; Choi, Chang Soon; Park, Jin Hee; Kim, Hee Jin; Jeon, Se Jin; Dela Pena, Ike Campomayor; Han, Seol-Heui; Cheong, Jae Hoon; Ryu, Jong Hoon; Shin, Chan Young

    2013-03-01

    Autism spectrum disorder (ASD) is a pervasive developmental disorder characterized by three main behavioral symptoms including social deficits, impaired communication, and stereotyped and repetitive behaviors. ASD prevalence shows gender bias to male. Prenatal exposure to valproic acid (VPA), a drug used in epilepsy and bipolar disorder, induces autistic symptoms in both human and rodents. As we reported previously, prenatally VPA-exposed animals at E12 showed impairment in social behavior without any overt reproductive toxicity. Social interactions were not significantly different between male and female rats in control condition. However, VPA-exposed male offspring showed significantly impaired social interaction while female offspring showed only marginal deficits in social interaction. Similar male inclination was observed in hyperactivity behavior induced by VPA. In addition to the ASD-like behavioral phenotype, prenatally VPA-exposed rat offspring shows crooked tail phenotype, which was not different between male and female groups. Both male and female rat showed reduced GABAergic neuronal marker GAD and increased glutamatergic neuronal marker vGluT1 expression. Interestingly, despite of the similar increased expression of vGluT1, post-synaptic marker proteins such as PSD-95 and α-CAMKII expression was significantly elevated only in male offspring. Electron microscopy showed increased number of post-synapse in male but not in female at 4 weeks of age. These results might suggest that the altered glutamatergic neuronal differentiation leads to deranged post-synaptic maturation only in male offspring prenatally exposed to VPA. Consistent with the increased post-synaptic compartment, VPA-exposed male rats showed higher sensitivity to electric shock than VPA-exposed female rats. These results suggest that prenatally VPA-exposed rats show the male preponderance of ASD-like behaviors including defective social interaction similar to human autistic patients, which

  7. The ATP/ADP substrate specificity switch between Toxoplasma gondii NTPDase1 and NTPDase3 is caused by an altered mode of binding of the substrate base.

    Science.gov (United States)

    Krug, Ulrike; Totzauer, Robert; Zebisch, Matthias; Sträter, Norbert

    2013-11-25

    Two nucleoside triphosphate diphosphohydrolase isoforms (NTPDase1 and NTPDase3) are responsible for the hydrolysis of nucleotides by the intracellular protozoan Toxoplasma gondii. They constitute about 3 % of the total parasite protein. Despite sharing 97 % sequence identity they exhibit opposite ATP versus ADP substrate discrimination ratios. Here we show by mutagenesis that the residues G492/G493 in NTPDase3 and R492/E493 in NTPDase1 are predominantly responsible for the differences in substrate specificity. Crystal structures of NTPDase1 in complexation with analogues of ATP and ADP reveal that the inverted substrate specificity of NTPDase1 relative to NTPDase3 is achieved by switching from the canonical substrate binding mode to a very different alternative one. Instead of being stacked on top of a helix of the C-terminal domain the nucleotide base is positioned in the interdomain space between the side chains of R108 and R492, recruited from both domains. Furthermore, we show that the NTPDase1 substrate specificity is mainly dependent on the presence of the side chain of E493, which causes repositioning of the ribose component of the nucleotide. All in all, binding by the flexible side chains in the alternative binding mode in NTPDase1 allows for equally good positioning of ATP and ADP with increased activity toward ADP relative to what is seen in the case of NTPDase3. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Sex recombination, and reproductive fitness: an experimental study using Paramecium

    Energy Technology Data Exchange (ETDEWEB)

    Nyberg, D.

    1982-08-01

    The effect of sex and recombination on reproductive fitness are measured using five wild stocks of Paramecium primaurelia. Among the wild stocks there were highly significant differences in growth rates. No hybrid had as low a fitness as the least fit parental stock. Recombination produced genotypes of higher fitness than those of either parent only in the cross between the two stocks of lowest fitness. The increase in variance of fitness as a result of recombination was almost exclusively attributable to the generation lines with low fitness. The fitness consequences of sexuality and mate choice were stock specific; some individuals leaving the most descendants by inbreeding, others by outcrossing. For most crosses the short-term advantage of sex, if any, accrue from the fusion of different gametes (hybrid vigor) and not from recombination. Since the homozygous genotype with the highest fitnes left the most progeny by inbreeding (no recombination), the persistence of conjugation in P. primaurelia is paradoxical. (JMT)

  9. Identification of Molecular Markers Associated with Alteration of Receptor-Binding Specificity in a Novel Genotype of Highly Pathogenic Avian Influenza A(H5N1) Viruses Detected in Cambodia in 2013

    Science.gov (United States)

    Rith, Sareth; Davis, C. Todd; Duong, Veasna; Sar, Borann; Horm, Srey Viseth; Chin, Savuth; Ly, Sovann; Laurent, Denis; Richner, Beat; Oboho, Ikwo; Jang, Yunho; Davis, William; Thor, Sharmi; Balish, Amanda; Iuliano, A. Danielle; Sorn, San; Holl, Davun; Sok, Touch; Seng, Heng; Tarantola, Arnaud; Tsuyuoka, Reiko; Parry, Amy; Chea, Nora; Allal, Lotfi; Kitsutani, Paul; Warren, Dora; Prouty, Michael; Horwood, Paul; Widdowson, Marc-Alain; Lindstrom, Stephen; Villanueva, Julie; Donis, Ruben; Cox, Nancy

    2014-01-01

    Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further. PMID:25210193

  10. In vitro assessment of attachment pattern and replication efficiency of H5N1 influenza A viruses with altered receptor specificity.

    Science.gov (United States)

    Chutinimitkul, Salin; van Riel, Debby; Munster, Vincent J; van den Brand, Judith M A; Rimmelzwaan, Guus F; Kuiken, Thijs; Osterhaus, Albert D M E; Fouchier, Ron A M; de Wit, Emmie

    2010-07-01

    The continuous circulation of the highly pathogenic avian influenza (HPAI) H5N1 virus has been a cause of great concern. The possibility of this virus acquiring specificity for the human influenza A virus receptor, alpha2,6-linked sialic acids (SA), and being able to transmit efficiently among humans is a constant threat to human health. Different studies have described amino acid substitutions in hemagglutinin (HA) of clinical HPAI H5N1 isolates or that were introduced experimentally that resulted in an increased, but not exclusive, binding of these virus strains to alpha2,6-linked SA. We introduced all previously described amino acid substitutions and combinations thereof into a single genetic background, influenza virus A/Indonesia/5/05 HA, and tested the receptor specificity of these 27 mutant viruses. The attachment pattern to ferret and human tissues of the upper and lower respiratory tract of viruses with alpha2,6-linked SA receptor preference was then determined and compared to the attachment pattern of a human influenza A virus (H3N2). At least three mutant viruses showed an attachment pattern to the human respiratory tract similar to that of the human H3N2 virus. Next, the replication efficiencies of these mutant viruses and the effects of three different neuraminidases on virus replication were determined. These data show that influenza virus A/Indonesia/5/05 potentially requires only a single amino acid substitution to acquire human receptor specificity, while at the same time remaining replication competent, thus suggesting that the pandemic threat posed by HPAI H5N1 is far from diminished.

  11. The N2-Src neuronal splice variant of C-Src has altered SH3 domain ligand specificity and a higher constitutive activity than N1-Src

    OpenAIRE

    Keenan, Sarah; Lewis, Philip A.; Wetherill, Sarah J.; Dunning, Christopher J.R.; Evans, Gareth J.O.

    2015-01-01

    N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also...

  12. Maternal stress during pregnancy causes sex-specific alterations in offspring memory performance, social interactions, indices of anxiety, and body mass

    Science.gov (United States)

    Schulz, Kalynn M.; Pearson, Jennifer N.; Neeley, Eric W.; Berger, Ralph; Leonard, Sherry; Adams, Catherine E.; Stevens, Karen E.

    2015-01-01

    Prenatal stress (PS) impairs memory function; however, it is not clear whether PS-induced memory deficits are specific to spatial memory, or whether memory is more generally compromised by PS. Here we sought to distinguish between these possibilities by assessing spatial, recognition and contextual memory function in PS and nonstressed (NS) rodents. We also measured anxiety-related and social behaviors to determine whether our unpredictable PS paradigm generates a behavioral phenotype comparable to previous studies. Female Sprague-Dawley rats were exposed to daily random stress during the last gestational week and behavior tested in adulthood. In males but not females, PS decreased memory for novel objects and novel spatial locations, and facilitated memory for novel object/context pairings. In the elevated zero maze, PS increased anxiety-related behavior only in females. Social behaviors also varied with sex and PS condition. Females showed more anogenital sniffing regardless of stress condition. In contrast, prenatal stress eliminated a male-biased sex difference in nonspecific bodily sniffing by decreasing sniffing in males, and increasing sniffing in females. Finally, PS males but not females gained significantly more weight across adulthood than did NS controls. In summary, these data indicate that PS differentially impacts males and females resulting in sex-specific adult behavioral and bodily phenotypes. PMID:21334352

  13. The complex and specific pMHC interactions with diverse HIV-1 TCR clonotypes reveal a structural basis for alterations in CTL function

    Science.gov (United States)

    Xia, Zhen; Chen, Huabiao; Kang, Seung-Gu; Huynh, Tien; Fang, Justin W.; Lamothe, Pedro A.; Walker, Bruce D.; Zhou, Ruhong

    2014-02-01

    Immune control of viral infections is modulated by diverse T cell receptor (TCR) clonotypes engaging peptide-MHC class I complexes on infected cells, but the relationship between TCR structure and antiviral function is unclear. Here we apply in silico molecular modeling with in vivo mutagenesis studies to investigate TCR-pMHC interactions from multiple CTL clonotypes specific for a well-defined HIV-1 epitope. Our molecular dynamics simulations of viral peptide-HLA-TCR complexes, based on two independent co-crystal structure templates, reveal that effective and ineffective clonotypes bind to the terminal portions of the peptide-MHC through similar salt bridges, but their hydrophobic side-chain packings can be very different, which accounts for the major part of the differences among these clonotypes. Non-specific hydrogen bonding to viral peptide also accommodates greater epitope variants. Furthermore, free energy perturbation calculations for point mutations on the viral peptide KK10 show excellent agreement with in vivo mutagenesis assays, with new predictions confirmed by additional experiments. These findings indicate a direct structural basis for heterogeneous CTL antiviral function.

  14. Genetic linkage map of a wild genome: genomic structure, recombination and sexual dimorphism in bighorn sheep

    Science.gov (United States)

    2010-01-01

    elevated mean recombination rate observed in domestic sheep, our results suggest that it is a characteristic of Ovis species. However, domestication may have played a role in altering patterns of heterochiasmy. Finally, we found that interval-specific patterns of sexual dimorphism were preserved among closely related Ovis species, possibly due to the conserved position of these intervals relative to the centromeres and telomeres. This study exemplifies how transferring genomic resources from domesticated species to close wild relative can benefit evolutionary ecologists while providing insights into the evolution of genomic structure and recombination rates of domesticated species. PMID:20920197

  15. A moderate diet restriction during pregnancy alters the levels of endocannabinoids and endocannabinoid-related lipids in the hypothalamus, hippocampus and olfactory bulb of rat offspring in a sex-specific manner.

    Science.gov (United States)

    Ramírez-López, María Teresa; Vázquez, Mariam; Lomazzo, Ermelinda; Hofmann, Clementine; Blanco, Rosario Noemi; Alén, Francisco; Antón, María; Decara, Juan; Arco, Rocío; Orio, Laura; Suárez, Juan; Lutz, Beat; Gómez de Heras, Raquel; Bindila, Laura; Rodríguez de Fonseca, Fernando

    2017-01-01

    Undernutrition during pregnancy has been associated to increased vulnerability to develop metabolic and behavior alterations later in life. The endocannabinoid system might play an important role in these processes. Therefore, we investigated the effects of a moderate maternal calorie-restricted diet on the levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG), arachidonic acid (AA) and the N-acylethanolamines (NAEs) anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in the brain of newborn rat offspring. We focused on brain structures involved in metabolism, feeding behavior, as well as emotional and cognitive responses. Female Wistar rats were assigned during the entire pregnancy to either control diet (C) or restriction diet (R), consisting of a 20% calorie-restricted diet. Weight gain and caloric intake of rat dams were monitored and birth outcomes were assessed. 2-AG, AA and NAE levels were measured in hypothalamus, hippocampus and olfactory bulb of the offspring. R dams displayed lower gain weight from the middle pregnancy and consumed less calories during the entire pregnancy. Offspring from R dams were underweight at birth, but litter size was unaffected. In hypothalamus, R male offspring displayed decreased levels of AA and OEA, with no change in the levels of the endocannabinoids 2-AG and AEA. R female exhibited decreased 2-AG and PEA levels. The opposite was found in the hippocampus, where R male displayed increased 2-AG and AA levels, and R female exhibited elevated levels of AEA, AA and PEA. In the olfactory bulb, only R female presented decreased levels of AEA, AA and PEA. Therefore, a moderate diet restriction during the entire pregnancy alters differentially the endocannabinoids and/or endocannabinoid-related lipids in hypothalamus and hippocampus of the underweight offspring, similarly in both sexes, whereas sex-specific alterations occur in the olfactory bulb. Consequently, endocannabinoid and endocannabinoid

  16. Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

    Science.gov (United States)

    2015-01-01

    Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations

  17. Specific gene mutations induced by heavy ions

    International Nuclear Information System (INIS)

    Freeling, M.; Karoly, C.W.; Cheng, D.S.K.

    1980-01-01

    This report summarizes our heavy-ion research rationale, progress, and plans for the near future. The major project involves selecting a group of maize Adh1 mutants induced by heavy ions and correlating their altered behavior with altered DNA nucleotide sequences and sequence arrangements. This research requires merging the techniques of classical genetics and recombinant DNA technology. Our secondary projects involve (1) the use of the Adh gene in the fruit fly, Drosophila melanogaster, as a second system with which to quantify the sort of specific gene mutants induced by heavy ions as compared to x rays, and (2) the development of a maize Adh1 pollen in situ monitor for environmental mutagens

  18. Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Wellner, R.B.; Ghosh, P.C.; Roecklein, B.; Wu, H.C.

    1987-01-01

    Increased [ 3 H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary and yeast mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [ 3 H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [ 3 H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [ 3 H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [ 3 H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [ 3 H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [ 3 H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [ 3 H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [ 3 H]palmitate incorporation into the 20,000 molecular weight species

  19. Is Alteration of Tuning Property in Cervical Vestibular-Evoked Myogenic Potential Specific for Ménière’s Disease?

    Directory of Open Access Journals (Sweden)

    Toshihisa Murofushi

    2017-05-01

    Full Text Available ObjectiveThe aim of this study is to show sensitivity and specificity of cervical vestibular-evoked myogenic potential (cVEMP tuning property test to Ménière’s disease (MD in comparison with healthy controls (HC and patients with other vestibular diseases.SubjectsTotally 92 subjects (50 women and 42 men, 20–77 years of age were enrolled in this study. Subjects were composed of 38 definite unilateral MD patients, 11 unilateral benign paroxysmal positional vertigo patients, 14 vestibular migraine patients, 19 unilateral vestibular neuritis patients, and 10 HC.MethodsThe subjects underwent cVEMP testing to 500 and 1,000 Hz short tone bursts (125 dBSPL. The corrected amplitudes of the first biphasic responses (p13–n23 (cVEMP were measured. Then, a tuning property index (the 500–1,000 Hz cVEMP slope was calculated.ResultsThe area of under the ROC curve (AUC was 0.75 in comparison with other vestibular disease patients, while AUC was 0.77 in comparison with other vestibular disease patients plus HC. The best cutoff point of the 500–1,000 Hz cVEMP slope was −19.9. Sensitivity of the tuning property test to MD was 0.74, while specificity was 0.76 to other vestibular disease patients.ConclusionThe tuning property test of cVEMP is useful as a screening test of MD.

  20. Catalytic hydrogen recombination for nuclear containments

    International Nuclear Information System (INIS)

    Koroll, G.W.; Lau, D.W.P.; Dewit, W.A.; Graham, W.R.C.

    1994-01-01

    Catalytic recombiners appear to be a credible option for hydrogen mitigation in nuclear containments. The passive operation, versatility and ease of back fitting are appealing for existing stations and new designs. Recently, a generation of wet-proofed catalyst materials have been developed at AECL which are highly specific to H 2 -O 2 , are active at ambient temperatures and are being evaluated for containment applications. Two types of catalytic recombiners were evaluated for hydrogen removal in containments based on the AECL catalyst. The first is a catalytic combustor for application in existing air streams such as provided by fans or ventilation systems. The second is an autocatalytic recombiner which uses the enthalpy of reaction to produce natural convective flow over the catalyst elements. Intermediate-scale results obtained in 6 m 3 and 10 m 3 spherical and cylindrical vessels are given to demonstrate self-starting limits, operating limits, removal capacity, scaling parameters, flow resistance, mixing behaviour in the vicinity of an operating recombiner and sensitivity to poisoning, fouling and radiation. (author). 13 refs., 10 figs

  1. Analysis of DNA polymerase ν function in meiotic recombination, immunoglobulin class-switching, and DNA damage tolerance.

    Directory of Open Access Journals (Sweden)

    Kei-Ichi Takata

    2017-06-01

    Full Text Available DNA polymerase ν (pol ν, encoded by the POLN gene, is an A-family DNA polymerase in vertebrates and some other animal lineages. Here we report an in-depth analysis of pol ν-defective mice and human cells. POLN is very weakly expressed in most tissues, with the highest relative expression in testis. We constructed multiple mouse models for Poln disruption and detected no anatomic abnormalities, alterations in lifespan, or changed causes of mortality. Mice with inactive Poln are fertile and have normal testis morphology. However, pol ν-disrupted mice have a modestly reduced crossover frequency at a meiotic recombination hot spot harboring insertion/deletion polymorphisms. These polymorphisms are suggested to generate a looped-out primer and a hairpin structure during recombination, substrates on which pol ν can operate. Pol ν-defective mice had no alteration in DNA end-joining during immunoglobulin class-switching, in contrast to animals defective in the related DNA polymerase θ (pol θ. We examined the response to DNA crosslinking agents, as purified pol ν has some ability to bypass major groove peptide adducts and residues of DNA crosslink repair. Inactivation of Poln in mouse embryonic fibroblasts did not alter cellular sensitivity to mitomycin C, cisplatin, or aldehydes. Depletion of POLN from human cells with shRNA or siRNA did not change cellular sensitivity to mitomycin C or alter the frequency of mitomycin C-induced radial chromosomes. Our results suggest a function of pol ν in meiotic homologous recombination in processing specific substrates. The restricted and more recent evolutionary appearance of pol ν (in comparison to pol θ supports such a specialized role.

  2. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W. [Univ. of Georgia, Athens, GA (United States)

    2014-01-25

    This research addressed the long term goals of understanding the assembly and organization of hydrogenase enzymes, of reducing them in size and complexity, of determining structure/function relationships, including energy conservation via charge separation across membranes, and in screening for novel H2 catalysts. A key overall goal of the proposed research was to define and characterize minimal hydrogenases that are produced in high yields and are oxygen-resistant. Remarkably, in spite of decades of research carried out on hydrogenases, it is not possible to readily manipulate or design the enzyme using molecular biology approaches since a recombinant form produced in a suitable host is not available. Such resources are essential if we are to understand what constitutes a “minimal” hydrogenase and design such catalysts with certain properties, such as resistance to oxygen, extreme stability and specificity for a given electron donor. The model system for our studies is Pyrococcus furiosus, a hyperthermophile that grows optimally at 100°C, which contains three different nickel-iron [NiFe-] containing hydrogenases. Hydrogenases I and II are cytoplasmic while the other, MBH, is an integral membrane protein that functions to both evolve H2 and pump protons. Three important breakthroughs were made during the funding period with P. furiosus soluble hydrogenase I (SHI). First, we produced an active recombinant form of SHI in E. coli by the co-expression of sixteen genes using anaerobically-induced promoters. Second, we genetically-engineered P. furiosus to overexpress SHI by an order of magnitude compared to the wild type strain. Third, we generated the first ‘minimal’ form of SHI, one that contained two rather than four subunits. This dimeric form was stable and active, and directly interacted with a pyruvate-oxidizing enzyme with any intermediate electron carrier. The research resulted in five peer-reviewed publications.

  3. Cell biology of mitotic recombination

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney

    2015-01-01

    Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules...... as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics...

  4. Promoter hypermethylation of the SFRP2 gene is a high-frequent alteration and tumor-specific epigenetic marker in human breast cancer

    Directory of Open Access Journals (Sweden)

    Knüchel Ruth

    2008-11-01

    Full Text Available Abstract Background We have previously reported that expression of the Wnt antagonist genes SFRP1 and SFRP5 is frequently silenced by promoter hypermethylation in breast cancer. SFRP2 is a further Wnt inhibitor whose expression was recently found being downregulated in various malignancies. Here we investigated whether SFRP2 is also implicated in human breast cancer, and if so whether SFRP2 promoter methylation might serve as a potential tumor biomarker. Methods We analyzed SFRP2 mRNA expression and SFRP2 promoter methylation in 10 breast cell lines, 199 primary breast carcinomas, 20 matched normal breast tissues and 17 cancer-unrelated normal breast tissues using RT-PCR, realtime PCR, methylation-specific PCR and Pyrosequencing, respectively. SFRP2 protein expression was assessed by immunohistochemistry on a tissue microarray. Proliferation assays after transfection with an SFRP2 expression vector were performed with mammary MCF10A cells. Statistical evaluations were accomplished with SPSS 14.0 software. Results Of the cancerous breast cell lines, 7/8 (88% lacked SFRP2 mRNA expression due to SFRP2 promoter methylation (P SFRP2 expression was substantially restored in most breast cell lines after treatment with 5-aza-2'-deoxycytidine and trichostatin A. In primary breast carcinomas SFRP2 protein expression was strongly reduced in 93 of 125 specimens (74%. SFRP2 promoter methylation was detected in 165/199 primary carcinomas (83% whereas all cancer-related and unrelated normal breast tissues were not affected by SFRP2 methylation. SFRP2 methylation was not associated with clinicopathological factors or clinical patient outcome. However, loss of SFRP2 protein expression showed a weak association with unfavorable patient overall survival (P = 0.071. Forced expression of SFRP2 in mammary MCF10A cells substantially inhibited proliferation rates (P = 0.045. Conclusion The SFRP2 gene is a high-frequent target of epigenetic inactivation in human breast

  5. Hypothyroidism Induces Hypophagia Associated with Alterations in Protein Expression of Neuropeptide Y and Proopiomelanocortin in the Arcuate Nucleus, Independently of Hypothalamic Nuclei-Specific Changes in Leptin Signaling.

    Science.gov (United States)

    Calvino, Camila; Império, Güínever Eustáquio; Wilieman, Marianna; Costa-E-Sousa, Ricardo Henrique; Souza, Luana Lopes; Trevenzoli, Isis Hara; Pazos-Moura, Carmen Cabanelas

    2016-01-01

    Thyroid hormone and leptin are essential regulators of energy homeostasis. Both hormones stimulate energy expenditure but have opposite effects on appetite. The mechanisms behind food intake regulation in thyroid dysfunctions are poorly understood. It has been shown that hypothyroid rats exhibited impaired leptin anorexigenic effect and signaling in total hypothalamus, even though they were hypophagic. It was hypothesized that hypothyroidism modulates the expression of neuropeptides: orexigenic neuropeptide Y (NPY) and anorexigenic proopiomelanocortin (POMC), independently of inducing nuclei-specific changes in hypothalamic leptin signaling. Adult male rats were rendered hypothyroid by administration of 0.03% methimazole in the drinking water for 21 days. Protein content of NPY, POMC, and leptin signaling (the signal transducer and activator of transcription 3 [STAT3] pathway) were evaluated by Western blot, and mRNA levels by real time reverse transcription polymerase chain reaction in arcuate (ARC), ventromedial (VMN), and paraventricular (PVN) hypothalamic nuclei isolated from euthyroid (eu) and hypothyroid (hypo) rats. Leptin anorexigenic effect was tested by recording food intake for two hours after intracerebroventricular (i.c.v.) administration of leptin. Statistical differences were considered significant at p ≤ 0.05. Hypothyroidism was confirmed by decreased serum triiodothyronine, thyroxine, and increased thyrotropin, in addition to increased levels of pro-TRH mRNA in PVN and Dio2 mRNA in the ARC of hypo rats. Hypothyroidism decreased body weight and food intake associated with decreased protein content of NPY and increased content of POMC in the ARC. Conversely, hypothyroidism induced central resistance to the acute anorexigenic effect of leptin, since while euthyroid rats displayed reduced food intake after leptin i.c.v. injection, hypothyroid rats showed no response. Hypothyroid rats exhibited decreased leptin receptor (ObRb) protein content in

  6. Zinc effect on the structure of recombinant human growth hormone

    International Nuclear Information System (INIS)

    El Nabarwy, F.S.

    2002-01-01

    A number of protein substances are known to undergo chemical decomposition when stored under non-ideal conditions. Metal induced precipitation of protein is being used and further developed as a processing step in protein formulation and may have utility in protein purification bulk storage. In such processes, it is important that native protein structure is maintained and the metal complexation is reversible. Series of dialysis experiments, treatment with chelating agents and divalent metal ions showed that zinc was required for such process. In this study, the effect of zinc induced precipitation recombinant human growth hormone (rh GH) was investigated. Ethylene diamine tetra acetic acid (EDTA) was added and the precipitate was dissolved yielding complete recovery of native protein. The protein has specific metal binding sites and requires specific molar ratios of zinc to protein to initiate precipitation (zinc: rh GH=2:1). Furthermore, the secondary structure of the protein was unperturbed in soluble zinc complexes and zinc induced precipitates, as measured by infrared and spectroscopy. The soluble zinc complex of rh GH had minor tertiary structural alterations. This study indicated that metal induced precipitation provides a good method maintain proteins in their native state in precipitates, which may be useful for purification storage and formulation. In addition, formation of a Zn (2+)-rh GH dimeric complex may be important for storage of rh GH in secretory granules

  7. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    Directory of Open Access Journals (Sweden)

    Steven W Paugh

    2016-02-01

    Full Text Available MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16 for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

  8. Histocompatibility leukocyte antigen-D related expression is specifically altered and predicts mortality in septic shock but not in other causes of shock.

    Science.gov (United States)

    Caille, Vincent; Chiche, Jean-Daniel; Nciri, Noureddine; Berton, Christine; Gibot, Sébastien; Boval, Bernadette; Payen, Didier; Mira, Jean-Paul; Mebazaa, Alexandre

    2004-12-01

    Although the expression of monocyte histocompatibility leukocyte antigen (HLA)-DR has been shown to be decreased during human sepsis, its level of expression in other nonseptic critical conditions is unclear. The aim of this study was to compare the level of HLA-DR expression on circulating monocytes among patients with septic, hemorrhagic, and cardiogenic shocks and severe sepsis without shock. At admission, HLA-DR expression was exclusively decreased in patients with septic shock (n = 30; P shock (n = 16), hemorrhagic shock (n = 11), severe sepsis without shock (n = 18), and healthy volunteers (n = 8). HLA-DR expression was not predictive for overall mortality, but at day 1, an HLA-DR expression of less than 14 of mean fluorescence intensity (that corresponds to 40% labeled monocytes) was predictive of mortality exclusively in patients with septic shock (odds ratio, 11.4 and 95% confidence interval, 1.7; 78.4; P culture, and number of units of blood or plasma transfused did not correlate with decreased HLA-DR expression. Thus, the decrease in HLA-DR expression is specific to septic shock and is associated, in septic shock patients, with increased mortality risk.

  9. Skin-specific deletion of stearoyl-CoA desaturase-1 alters skin lipid composition and protects mice from high fat diet-induced obesity.

    Science.gov (United States)

    Sampath, Harini; Flowers, Matthew T; Liu, Xueqing; Paton, Chad M; Sullivan, Ruth; Chu, Kiki; Zhao, Minghui; Ntambi, James M

    2009-07-24

    Stearoyl-CoA desaturase-1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids and is an important regulator of whole body energy homeostasis. Severe cutaneous changes in mice globally deficient in SCD1 also indicate a role for SCD1 in maintaining skin lipids. We have generated mice with a skin-specific deletion of SCD1 (SKO) and report here that SKO mice display marked sebaceous gland hypoplasia and depletion of sebaceous lipids. In addition, SKO mice have significantly increased energy expenditure and are protected from high fat diet-induced obesity, thereby recapitulating the hypermetabolic phenotype of global SCD1 deficiency. Genes of fat oxidation, lipolysis, and thermogenesis, including uncoupling proteins and peroxisome proliferator-activated receptor-gamma co-activator-1alpha, are up-regulated in peripheral tissues of SKO mice. However, unlike mice globally deficient in SCD1, SKO mice have an intact hepatic lipogenic response to acute high carbohydrate feeding. Despite increased basal thermogenesis, SKO mice display severe cold intolerance because of rapid depletion of fuel substrates, including hepatic glycogen, to maintain core body temperature. These data collectively indicate that SKO mice have increased cold perception because of loss of insulating factors in the skin. This results in up-regulation of thermogenic processes for temperature maintenance at the expense of fuel economy, illustrating cross-talk between the skin and peripheral tissues in maintaining energy homeostasis.

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

    Science.gov (United States)

    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.

  11. Wheel running from a juvenile age delays onset of specific motor deficits but does not alter protein aggregate density in a mouse model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    Spires Tara L

    2008-04-01

    . Comparison of different environmental manipulations, performed in specific time windows, can identify critical periods for the induction of neuroprotective 'brain reserve' in animal models of HD and related neurodegenerative diseases.

  12. Analysis and prediction of the physiological effects of altered coenzyme specificity in xylose reductase and xylitol dehydrogenase during xylose fermentation by Saccharomyces cerevisiae

    Science.gov (United States)

    Krahulec, Stefan; Klimacek, Mario; Nidetzky, Bernd

    2012-01-01

    An advanced strategy of Saccharomyces cerevisiae strain development for fermentation of xylose applies tailored enzymes in the process of metabolic engineering. The coenzyme specificities of the NADPH-preferring xylose reductase (XR) and the NAD+-dependent xylitol dehydrogenase (XDH) have been targeted in previous studies by protein design or evolution with the aim of improving the recycling of NADH or NADPH in their two-step pathway, converting xylose to xylulose. Yeast strains expressing variant pairs of XR and XDH that according to in vitro kinetic data were suggested to be much better matched in coenzyme usage than the corresponding pair of wild-type enzymes, exhibit widely varying capabilities for xylose fermentation. To achieve coherence between enzyme properties and the observed strain performance during fermentation, we explored the published kinetic parameters for wild-type and engineered forms of XR and XDH as possible predictors of xylitol by-product formation (Yxylitol) in yeast physiology. We found that the ratio of enzymatic reaction rates using NADP(H) and NAD(H) that was calculated by applying intracellular reactant concentrations to rate equations derived from bi-substrate kinetic analysis, succeeded in giving a statistically reliable forecast of the trend effect on Yxylitol. Prediction based solely on catalytic efficiencies with or without binding affinities for NADP(H) and NAD(H) were not dependable, and we define a minimum demand on the enzyme kinetic characterization to be performed for this purpose. An immediate explanation is provided for the typically lower Yxylitol in the current strains harboring XR engineered for utilization of NADH as compared to strains harboring XDH engineered for utilization of NADP+. The known XDH enzymes all exhibit a relatively high Km for NADP+ so that physiological boundary conditions are somewhat unfavorable for xylitol oxidation by NADP+. A criterion of physiological fitness is developed for engineered XR

  13. Bacteria in the vaginal microbiome alter the innate immune response and barrier properties of the human vaginal epithelia in a species-specific manner.

    Science.gov (United States)

    Doerflinger, Sylvie Y; Throop, Andrea L; Herbst-Kralovetz, Melissa M

    2014-06-15

    Bacterial vaginosis increases the susceptibility to sexually transmitted infections and negatively affects women's reproductive health. To investigate host-vaginal microbiota interactions and the impact on immune barrier function, we colonized 3-dimensional (3-D) human vaginal epithelial cells with 2 predominant species of vaginal microbiota (Lactobacillus iners and Lactobacillus crispatus) or 2 prevalent bacteria associated with bacterial vaginosis (Atopobium vaginae and Prevotella bivia). Colonization of 3-D vaginal epithelial cell aggregates with vaginal microbiota was observed with direct attachment to host cell surface with no cytotoxicity. A. vaginae infection yielded increased expression membrane-associated mucins and evoked a robust proinflammatory, immune response in 3-D vaginal epithelial cells (ie, expression of CCL20, hBD-2, interleukin 1β, interleukin 6, interleukin 8, and tumor necrosis factor α) that can negatively affect barrier function. However, P. bivia and L. crispatus did not significantly upregulate pattern-recognition receptor-signaling, mucin expression, antimicrobial peptides/defensins, or proinflammatory cytokines in 3-D vaginal epithelial cell aggregates. Notably, L. iners induced pattern-recognition receptor-signaling activity, but no change was observed in mucin expression or secretion of interleukin 6 and interleukin 8. We identified unique species-specific immune signatures from vaginal epithelial cells elicited by colonization with commensal and bacterial vaginosis-associated bacteria. A. vaginae elicited a signature that is consistent with significant disruption of immune barrier properties, potentially resulting in enhanced susceptibility to sexually transmitted infections during bacterial vaginosis. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Specific deletion of AMP-activated protein kinase (α1AMPK in murine oocytes alters junctional protein expression and mitochondrial physiology.

    Directory of Open Access Journals (Sweden)

    Michael J Bertoldo

    Full Text Available Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK, an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK-/- (ZP3-KO female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues. Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.

  15. Antisense repression of the Medicago truncatula nodule-enhanced sucrose synthase leads to a handicapped nitrogen fixation mirrored by specific alterations in the symbiotic transcriptome and metabolome.

    Science.gov (United States)

    Baier, Markus C; Barsch, Aiko; Küster, Helge; Hohnjec, Natalija

    2007-12-01

    We analyzed the role of the sucrose (Suc) synthase MtSucS1 during nodulation of the model legume Medicago truncatula, integrating data for the developmental, transcriptional, and metabolic processes affected downstream of an impaired Suc cleavage in root nodules. To reduce carbohydrate supply to nodule tissues, transgenic plants expressing a p35S-driven MtSucS1-antisense fusion were constructed. These plants displayed an up to 90% reduction of MtSucS1 proteins in roots and nodules. Phenotypic studies of two independent MtSucS1-reduced lines demonstrated that only under conditions depending on nodulation, these plants appeared to be impaired in above-ground growth. Specifically plant height, shoot weight, leaf development, flowering, as well as seed maturation were reduced, and the efficiency of photosynthesis was affected. Concomitantly, a significantly enhanced root to shoot ratio with a marked increase in root tip numbers was observed. Root nodule formation was found retarded and the impaired nodulation was accompanied by a less efficient nitrogen (N) acquisition. The decreased total N content of MtSucS1-antisense lines and an enhanced carbon to N ratio in roots, nodules, and shoots correlated with the extent of MtSucS1 knockdown. On the level of transcription, effects of an MtSucS1 reduction were evident for genes representing important nodes of the nodule carbon and N metabolism, while metabolite profiling revealed significantly lower levels of amino acids and their derivatives particularly in strongly MtSucS1-reduced nodules. Our results support the model that nodule-enhanced Suc synthase 1 of the model legume M. truncatula is required for the establishment and maintenance of an efficient N-fixing symbiosis.

  16. Altered postural sway in patients suffering from non-specific neck pain and whiplash associated disorder - A systematic review of the literature

    Science.gov (United States)

    2011-01-01

    Study design Systematic literature review. Objectives To assess differences in center of pressure (COP) measures in patients suffering from non-specific neck pain (NSNP) or whiplash-associated disorder (WAD) compared to healthy controls and any relationship between changes in postural sway and the presence of pain, its intensity, previous pain duration and the perceived level of disability. Summary of Background data Over the past 20 years, the center of pressure (COP) has been commonly used as an index of postural stability in standing. While several studies investigated COP excursions in neck pain and WAD patients and compared these to healthy individuals, no comprehensive analysis of the reported differences in postural sway pattern exists. Search methods Six online databases were systematically searched followed by a manual search of the retrieved papers. Selection Criteria Papers comparing COP measures derived from bipedal static task conditions on a force plate of people with NSNP and WAD to those of healthy controls. Data collection and analysis Two reviewers independently screened titles and abstracts for relevance. Screening for final inclusion, data extraction and quality assessment were carried out with a third reviewer to reconcile differences. Results Ten papers met the inclusion criteria. Heterogeneity in study designs prevented pooling of the data and no direct comparison of data across the studies was possible. Instead, a qualitative data analysis was conducted. There was broad consensus that patients with either type of neck pain have increased COP excursions compared to healthy individuals, a difference that was more pronounced in people with WAD. An increased sway in antero-posterior direction was observed in both groups. Conclusions Patients with neck pain (due to either NSNP or WAD) exhibit greater postural instability than healthy controls, signified by greater COP excursions irrespective of the COP parameter chosen. Further, the decreased

  17. Specific hemosiderin deposition in spleen induced by a low dose of cisplatin: altered iron metabolism and its implication as an acute hemosiderin formation model.

    Science.gov (United States)

    Wang, Yingze; Juan, L V; Ma, Xiaowei; Wang, Dongliang; Ma, Huili; Chang, Yanzhong; Nie, Guangjun; Jia, Lee; Duan, Xianglin; Liang, Xing-Jie

    2010-07-01

    Cisplatin is one of the commonly-used chemotherapeutic drugs to efficiently treat malignant tumors in clinic, however, the adverse effects of cisplatin such as nephrotoxicity, neurotoxicity, and hemolytic uremic syndrome are often observed at its clinical doses (approximately 60 mg/m(2)), which limit its broader application. In earlier studies, little attention was paid to the subtle changes in the architecture of lymphatic organs after low doses of cisplatin treatment. This paper reviews current understanding of cisplatin-induced erythrocyte injury, and presents our latest finding that a low dose of cisplatin (3.6 mg/m(2)/day, 14 days) could induce specific hemosiderin deposition in spleen of both normal and hepatoma-22 (H22) inoculated Balb/C mice. This dose of cisplatin significantly inhibited H22-induced acute ascites development. No significant toxicity was induced by this dose of cisplatin to tissues except for hemosiderin accumulation in the spleen of both normal and H22 tumor-bearing mice. Increased splenic iron content and erythrocyte injury were observed after treatment with the low dose of cisplatin. The mRNA levels of ferroportin (FPN1) and ferritin were upregulated by 25 and 5-fold in spleen, respectively. Overexpression of FPN1 and ferritin protein were also been observed at protein levels by Western blotting analysis. In addition, the mRNA expression of hepcidin was also increased, suggesting blockage of iron recycling through FPN1 in spleen with cisplatin treatment. In conclusion, cisplatin treatment damages the erythrocytes which accumulate in the red pulp of spleen with defective recycling of FPN1 and ferritin protein. Hepcidin inhibits the function of FPN1 as iron-exporter leading to iron overloaded inside ferritins of splenic cells, which are stained with abnormal hemosiderin accumulation. These results demonstrate that cisplatin-caused hemosiderin deposition in spleen provides a valuable clue for understanding the molecular basis of toxicity of

  18. Conditional FKBP12.6 overexpression in mouse cardiac myocytes prevents triggered ventricular tachycardia through specific alterations in excitation-contraction coupling.

    Science.gov (United States)

    Gellen, Barnabas; Fernández-Velasco, María; Briec, François; Vinet, Laurent; LeQuang, Khai; Rouet-Benzineb, Patricia; Bénitah, Jean-Pierre; Pezet, Mylène; Palais, Gael; Pellegrin, Noémie; Zhang, Andy; Perrier, Romain; Escoubet, Brigitte; Marniquet, Xavier; Richard, Sylvain; Jaisser, Fréderic; Gómez, Ana María; Charpentier, Flavien; Mercadier, Jean-Jacques

    2008-04-08

    Ca(2+) release from the sarcoplasmic reticulum via the ryanodine receptor (RyR2) activates cardiac myocyte contraction. An important regulator of RyR2 function is FKBP12.6, which stabilizes RyR2 in the closed state during diastole. Beta-adrenergic stimulation has been suggested to dissociate FKBP12.6 from RyR2, leading to diastolic sarcoplasmic reticulum Ca(2+) leakage and ventricular tachycardia (VT). We tested the hypothesis that FKBP12.6 overexpression in cardiac myocytes can reduce susceptibility to VT in stress conditions. We developed a mouse model with conditional cardiac-specific overexpression of FKBP12.6. Transgenic mouse hearts showed a marked increase in FKBP12.6 binding to RyR2 compared with controls both at baseline and on isoproterenol stimulation (0.2 mg/kg i.p.). After pretreatment with isoproterenol, burst pacing induced VT in 10 of 23 control mice but in only 1 of 14 transgenic mice (P<0.05). In isolated transgenic myocytes, Ca(2+) spark frequency was reduced by 50% (P<0.01), a reduction that persisted under isoproterenol stimulation, whereas the sarcoplasmic reticulum Ca(2+) load remained unchanged. In parallel, peak I(Ca,L) density decreased by 15% (P<0.01), and the Ca(2+) transient peak amplitude decreased by 30% (P<0.001). A 33.5% prolongation of the caffeine-evoked Ca(2+) transient decay was associated with an 18% reduction in the Na(+)-Ca(2+) exchanger protein level (P<0.05). Increased FKBP12.6 binding to RyR2 prevents triggered VT in normal hearts in stress conditions, probably by reducing diastolic sarcoplasmic reticulum Ca(2+) leak. This indicates that the FKBP12.6-RyR2 complex is an important candidate target for pharmacological prevention of VT.

  19. MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

    DEFF Research Database (Denmark)

    Bonde, Mads; Klausen, Michael Schantz; Anderson, Mads Valdemar

    2014-01-01

    as combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo......Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well......, which confers the corresponding genetic change, is performed manually. To address these challenges, we have developed the MAGE Oligo Design Tool (MODEST). This web-based tool allows designing of MAGE oligos for (i) tuning translation rates by modifying the ribosomal binding site, (ii) generating...

  20. Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

    Science.gov (United States)

    Throop, Andrea L.; LaBaer, Joshua

    2015-01-01

    The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

  1. Exposure to altered gravity during specific developmental periods differentially affects growth, development, the cerebellum and motor functions in male and female rats

    Science.gov (United States)

    Nguon, K.; Ladd, B.; Sajdel-Sulkowska, E. M.

    2006-01-01

    We previously reported that perinatal exposure to hypergravity affects cerebellar structure and motor coordination in rat neonates. In the present study, we explored the hypothesis that neonatal cerebellar structure and motor coordination may be particularly vulnerable to the effects of hypergravity during specific developmental stages. To test this hypothesis, we compared neurodevelopment, motor behavior and cerebellar structure in rat neonates exposed to 1.65 G on a 24-ft centrifuge during discrete periods of time: the 2nd week of pregnancy [gestational day (G) 8 through G15; group A], the 3rd week of pregnancy (G15 through birth on G22/G23; group B), the 1st week of nursing [birth through postnatal day (P) 6; group C], the 2nd and 3rd weeks of nursing (P6 through P21; group D), the combined 2nd and 3rd weeks of pregnancy and nursing (G8 through P21; group E) and stationary control (SC) neonates (group F). Prenatal exposure to hypergravity resulted in intrauterine growth retardation as reflected by a decrease in the number of pups in a litter and lower average mass at birth. Exposure to hypergravity immediately after birth impaired the righting response on P3, while the startle response in both males and females was most affected by exposure during the 2nd and 3rd weeks after birth. Hypergravity exposure also impaired motor functions, as evidenced by poorer performance on a rotarod; while both males and females exposed to hypergravity during the 2nd and 3rd weeks after birth performed poorly on P21, male neonates were most dramatically affected by exposure to hypergravity during the second week of gestation, when the duration of their recorded stay on the rotarod was one half that of SC males. Cerebellar mass was most reduced by later postnatal exposure. Thus, for the developing rat cerebellum, the postnatal period that overlaps the brain growth spurt is the most vulnerable to hypergravity. However, male motor behavior is also affected by midpregnancy exposure to

  2. Inversion of Moraxella lacunata type 4 pilin gene sequences by a Neisseria gonorrhoeae site-specific recombinase.

    Science.gov (United States)

    Rozsa, F W; Meyer, T F; Fussenegger, M

    1997-01-01

    A plasmid library of Neisseria gonorrhoeae sequences was screened for the ability to mediate recombinations on a sequence containing the Moraxella lacunata type 4 pilin gene invertible region in Escherichia coli. A plasmid containing the N. gonorrhoeae sequence encoding the putative recombinase (gcr) was identified and sequenced. Plasmids containing gcr were able to mediate site-specific recombinations despite a weak amino acid homology to Piv, the native M. lacunata pilin gene invertase. The gcr gene is present only in pathogenic strains of Neisseria tested; however, in our assays gene knockouts of gcr did not alter the variation of surface features that play a role in the pathogenesis of N. gonorrhoeae. PMID:9079926

  3. Hadron Correlations and Parton Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Fries, R.J. [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)]. E-mail: rjfries@comp.tamu.edu

    2007-02-15

    Parton recombination has been found to be an extremely useful model to understand hadron production at the Relativistic Heavy Ion Collider. It is particularly important to explore its connections with hard processes. This article reviews some of the aspects of the quark recombination model and places particular emphasis on hadron correlations.

  4. Smectite alteration

    International Nuclear Information System (INIS)

    Anderson, D.M.

    1984-11-01

    This report contains the proceedings of a second workshop in Washington DC December 8-9, 1983 on the alteration of smectites intended for use as buffer materials in the long-term containment of nuclear wastes. It includes extended summaries of all presentations and a transcript of the detailed scientific discussion. The discussions centered on three main questions: What is the prerequisite for and what is the precise mechanism by which smectite clays may be altered to illite. What are likly sources of potassium with respect to the KBS project. Is it likely that the conversion of smectite to illite will be of importance in the 10 5 to the 10 6 year time frame. The workshop was convened to review considerations and conclusions in connection to these questions and also to broaden the discussion to consider the use of smectite clays as buffer materials for similar applications in different geographical and geological settings. SKBF/KBS technical report 83-03 contains the proceedings from the first workshop on these matters that was held at the State University of New York, Buffalo May 26-27, 1982. (Author)

  5. Limiting factors in Escherichia colifed-batch production of recombinant proteins

    DEFF Research Database (Denmark)

    Sanden, A.M.; Prytz, I.; Tubelekas, I.

    2003-01-01

    recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation......recombinant protein production, fed-batch, specific growth rate, feed profile, induction, mRNA, transcription, translation, acetic acid formation...

  6. Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii.

    Science.gov (United States)

    Egan, Marisa; Ramirez, Jasmine; Xander, Christian; Upreti, Chirag; Bhatt, Shantanu

    2016-01-01

    The ability to introduce site-specific mutations in bacterial pathogens is essential towards understanding their molecular mechanisms of pathogenicity. This has been greatly facilitated by the genetic engineering technique of recombineering. In recombineering, linear double- or single-stranded DNA molecules with two terminal homology arms are electroporated into hyperrecombinogenic bacteria that express a phage-encoded recombinase. The recombinase catalyzes the replacement of the endogenous allele with the exogenous allele to generate selectable or screenable recombinants. In particular, lambda red recombinase has been instrumental in engineering mutations to characterize the virulence arsenal of the attaching and effacing (A/E) pathogens enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. Escherichia albertii is another member of this taxon; however, the virulence of E. albertii remains cryptic despite accumulating evidence that E. albertii is an emerging pathogen. Multiple retrospective studies have reported that a substantial number of EPEC and EHEC isolates (~15 %) that were previously incriminated in human outbreaks actually belong to the E. albertii lineage. Thus, there is increased urgency to reliably identify and rapidly engineer mutations in E. albertii to systematically characterize its virulence determinants. To the best of our knowledge not a single chromosomal gene has been altered by targeted mutagenesis in E. albertii since it was first isolated almost 25 years ago. This is disconcerting because an E. albertii outbreak could cause significant morbidity and mortality owing to our inadequate understanding of its virulence program. In this report we describe a modified lambda red recombineering protocol to mutagenize E. albertii. As proof of principle, we successfully deleted three distinct virulence-associated genetic loci - ler, grlRA, and hfq - and replaced each wild type allele by a mutant allele

  7. A new take on V(DJ recombination: transcription driven nuclear and chromatin reorganization in RAG–mediated cleavage.

    Directory of Open Access Journals (Sweden)

    Julie eChaumeil

    2013-12-01

    Full Text Available It is nearly thirty years since the Alt lab first put forward the accessibility model, which proposes that cleavage of the various loci is controlled by lineage and stage specific factors that regulate RAG access to the different loci. Numerous labs have since demonstrated that locus opening is regulated at multiple levels that include sterile transcription, changes in chromatin packaging and alterations in locus conformation. Here we focus on the interplay between transcription and RAG binding in facilitating targeted cleavage. We discuss the results of recent studies that implicate transcription in regulating nuclear organization and altering the composition of resident nucleosomes to promote regional access to the recombinase machinery. Additionally we include new data that provide insight into the role of the RAG proteins in defining nuclear organization in recombining T cells.

  8. The potential of shifting recombination hotspots to increase genetic gain in livestock breeding.

    Science.gov (United States)

    Gonen, Serap; Battagin, Mara; Johnston, Susan E; Gorjanc, Gregor; Hickey, John M

    2017-07-04

    This study uses simulation to explore and quantify the potential effect of shifting recombination hotspots on genetic gain in livestock breeding programs. We simulated three scenarios that differed in the locations of quantitative trait nucleotides (QTN) and recombination hotspots in the genome. In scenario 1, QTN were randomly distributed along the chromosomes and recombination was restricted to occur within specific genomic regions (i.e. recombination hotspots). In the other two scenarios, both QTN and recombination hotspots were located in specific regions, but differed in whether the QTN occurred outside of (scenario 2) or inside (scenario 3) recombination hotspots. We split each chromosome into 250, 500 or 1000 regions per chromosome of which 10% were recombination hotspots and/or contained QTN. The breeding program was run for 21 generations of selection, after which recombination hotspot regions were kept the same or were shifted to adjacent regions for a further 80 generations of selection. We evaluated the effect of shifting recombination hotspots on genetic gain, genetic variance and genic variance. Our results show that shifting recombination hotspots reduced the decline of genetic and genic variance by releasing standing allelic variation in the form of new allele combinations. This in turn resulted in larger increases in genetic gain. However, the benefit of shifting recombination hotspots for increased genetic gain was only observed when QTN were initially outside recombination hotspots. If QTN were initially inside recombination hotspots then shifting them decreased genetic gain. Shifting recombination hotspots to regions of the genome where recombination had not occurred for 21 generations of selection (i.e. recombination deserts) released more of the standing allelic variation available in each generation and thus increased genetic gain. However, whether and how much increase in genetic gain was achieved by shifting recombination hotspots depended

  9. Molecular mechanisms of recombination restriction in the envelope gene of the human immunodeficiency virus.

    Directory of Open Access Journals (Sweden)

    Etienne Simon-Loriere

    2009-05-01

    Full Text Available The ability of pathogens to escape the host's immune response is crucial for the establishment of persistent infections and can influence virulence. Recombination has been observed to contribute to this process by generating novel genetic variants. Although distinctive recombination patterns have been described in many viral pathogens, little is known about the influence of biases in the recombination process itself relative to selective forces acting on newly formed recombinants. Understanding these influences is important for determining how recombination contributes to pathogen genome and proteome evolution. Most previous research on recombination-driven protein evolution has focused on relatively simple proteins, usually in the context of directed evolution experiments. Here, we study recombination in the envelope gene of HIV-1 between primary isolates belonging to subtypes that recombine naturally in the HIV/AIDS pandemic. By characterizing the early steps in the generation of recombinants, we provide novel insights into the evolutionary forces that shape recombination patterns within viral populations. Specifically, we show that the combined effects of mechanistic processes that determine the locations of recombination breakpoints across the HIV-1 envelope gene, and purifying selection acting against dysfunctional recombinants, can explain almost the entire distribution of breakpoints found within this gene in nature. These constraints account for the surprising paucity of recombination breakpoints found in infected individuals within this highly variable gene. Thus, the apparent randomness of HIV evolution via recombination may in fact be relatively more predictable than anticipated. In addition, the dominance of purifying selection in localized areas of the HIV genome defines regions where functional constraints on recombinants appear particularly strong, pointing to vulnerable aspects of HIV biology.

  10. Identifying recombinants in human and primate immunodeficiency virus sequence alignments using quartet scanning

    Directory of Open Access Journals (Sweden)

    Martin Darren P

    2009-04-01

    Full Text Available Abstract Background Recombination has a profound impact on the evolution of viruses, but characterizing recombination patterns in molecular sequences remains a challenging endeavor. Despite its importance in molecular evolutionary studies, identifying the sequences that exhibit such patterns has received comparatively less attention in the recombination detection framework. Here, we extend a quartet-mapping based recombination detection method to enable identification of recombinant sequences without prior specifications of either query and reference sequences. Through simulations we evaluate different recombinant identification statistics and significance tests. We compare the quartet approach with triplet-based methods that employ additional heuristic tests to identify parental and recombinant sequences. Results Analysis of phylogenetic simulations reveal that identifying the descendents of relatively old recombination events is a challenging task for all methods available, and that quartet scanning performs relatively well compared to the triplet based methods. The use of quartet scanning is further demonstrated by analyzing both well-established and putative HIV-1 recombinant strains. In agreement with recent findings, we provide evidence that the presumed circulating recombinant CRF02_AG is a 'pure' lineage, whereas the presumed parental lineage subtype G has a recombinant origin. We also demonstrate HIV-1 intrasubtype recombination, confirm the hybrid origin of SIV in chimpanzees and further disentangle the recombinant history of SIV lineages in a primate immunodeficiency virus data set. Conclusion Quartet scanning makes a valuable addition to triplet-based methods for identifying recombinant sequences without prior specifications of either query and reference sequences. The new method is available in the VisRD v.3.0 package http://www.cmp.uea.ac.uk/~vlm/visrd.

  11. The relationship of recombination rate, genome structure, and patterns of molecular evolution across angiosperms.

    Science.gov (United States)

    Tiley, George P; Burleigh, J Gordon; Burleigh, Gordon

    2015-09-16

    Although homologous recombination affects the efficacy of selection in populations, the pattern of recombination rate evolution and its effects on genome evolution across plants are largely unknown. Recombination can reduce genome size by enabling the removal of LTR retrotransposons, alter codon usage by GC biased gene conversion, contribute to complex histories of gene duplication and loss through tandem duplication, and enhance purifying selection on genes. Therefore, variation in recombination rate across species may explain some of the variation in genomic architecture as well as rates of molecular evolution. We used phylogenetic comparative methods to investigate the evolution of global meiotic recombination rate in angiosperms and its effects on genome architecture and selection at the molecular level using genetic maps and genome sequences from thirty angiosperm species. Recombination rate is negatively correlated with genome size, which is likely caused by the removal of LTR retrotransposons. After correcting recombination rates for euchromatin content, we also found an association between global recombination rate and average gene family size. This suggests a role for recombination in the preservation of duplicate genes or expansion of gene families. An analysis of the correlation between the ratio of nonsynonymous to synonymous substitution rates (dN/dS) and recombination rate in 3748 genes indicates that higher recombination rates are associated with an increased efficacy of purifying selection, suggesting that global recombination rates affect variation in rates of molecular evolution across distantly related angiosperm species, not just between populations. We also identified shifts in dN/dS for recombination proteins that are associated with shifts in global recombination rate across our sample of angiosperms. Although our analyses only reveal correlations, not mechanisms, and do not include potential covariates of recombination rate, like effective

  12. Hydrogen recombiner development at AECL

    International Nuclear Information System (INIS)

    Dewit, W.A.; Koroll, G.W.; Loesel Sitar, J.; Graham, W.R.C.

    1997-01-01

    Catalytic recombiners have been developed at AECL for the purpose of hydrogen removal in post-accident nuclear containment buildings. The recombiners are based on a particular catalyst designed by AECL which has extraordinary resistance to fouling from water and water vapour and a large thermodynamic range of operation. The catalysts were developed, originally, for the purpose of heavy water manufacturing by way of a catalytic exchange process. Application of these catalyst materials in recombiners for containment applications began in the late 1980's. The first application was a passive recombiner, qualified for use in control of radiolytic hydrogen in the headspace of a pool-type experimental reactor of AECL design in 1988. The passive, or natural convection recombiner concept has continued development to commercial stage for application in power reactor containments. This paper reviews the AECL recombiner development, describes the current model and shows results from tests of full-scale recombiners in the Large Scale Vented Combustion Test Facility at AECL-WL. The AECL recombiner is designed for compactness and ease of engineering into containment. The design is a simple, open-ended rectangular enclosure with catalyst elements arranged inside to promote optimum convective flow driven by heat of recombination at the catalyst surface. Self start, as evidenced by catalyst heating and initiation of flow, is achieved in less than 1% hydrogen, with available oxygen, at room temperature and 100% relative humidity. This low temperature start-up in condensing atmospheres is viewed as the most challenging condition for wet-proofing effectiveness. Cold start-up is a vital performance requirement in containments, such as CANDU, where engineered air-cooling systems are operating and where long-term hydrogen control is required, after containment atmospheres have cooled. Once started, the removal capacity scales linearly with the inlet cross-section area and the partial

  13. Review of Parton Recombination Models

    International Nuclear Information System (INIS)

    Bass, Steffen A

    2006-01-01

    Parton recombination models have been very successful in explaining data taken at RHIC on hadron spectra and emission patterns in Au+Au collisions at transverse momenta above 2 GeV/c, which have exhibited features which could not be understood in the framework of basic perturbative QCD. In this article I will review the current status on recombination models and outline which future challenges need to be addressed by this class of models

  14. Recombinant snake venom prothrombin activators

    OpenAIRE

    L?vgren, Ann

    2012-01-01

    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 ...

  15. Influence of the 3D-conformation, glycan component and microheterogeneity on the epitope structure of Ole e 1, the major olive allergen - Use of recombinant isoforms and specific monoclonal antibodies as immunological tools

    NARCIS (Netherlands)

    González, Eva M.; Villalba, Mayte; Lombardero, Manuel; Aalbers, Marja; van Ree, Ronald; Rodríguez, Rosalía

    2002-01-01

    Ole e 1 is the main allergen of olive pollen, which is a major cause of pollinosis in countries of the Mediterranean area. Nine Ole e 1-specific murine monoclonal antibodies (mAbs), as well as two Ole e 1-isoforms and two Ole e 1-like allergens from lilac and privet, all of them obtained in Pichia

  16. Identification of specific antigenic epitope at N-terminal segment of enterovirus 71 (EV-71) VP1 protein and characterization of its use in recombinant form for early diagnosis of EV-71 infection.

    Science.gov (United States)

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A; Meng, Jihong

    2014-08-30

    Human enterovirus 71 (EV-71) is the main etiologic agent of hand, foot and mouth disease (HFMD). We sought to identify EV-71 specific antigens and develop serologic assays for acute-phase EV-71 infection. A series of truncated proteins within the N-terminal 100 amino acids (aa) of EV-71 VP1 was expressed in Escherichia coli. Western blot (WB) analysis showed that positions around 11-21 aa contain EV-71-specific antigenic sites, whereas positions 1-5 and 51-100 contain epitopes shared with human coxsackievirus A16 (CV-A16) and human echovirus 6 (E-6). The N-terminal truncated protein of VP1, VP₁₆₋₄₃, exhibited good stability and was recognized by anti-EV-71 specific rabbit sera. Alignment analysis showed that VP₁₆₋₄₃ is highly conserved among EV-71 strains from different genotypes but was heterologous among other enteroviruses. When the GST-VP₁₆₋₄₃ fusion protein was incorporated as antibody-capture agent in a WB assay and an ELISA for detecting anti-EV-71 IgM in human sera, sensitivities of 91.7% and 77.8% were achieved, respectively, with 100% specificity for both. The characterized EV-71 VP1 protein truncated to positions 6-43 aa has potential as an antigen for detection of anti-EV-71 IgM for early diagnosis of EV-71 infection in a WB format. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    Full Text Available Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated VH/VL interfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1. Results Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either Discosoma or Aequorea in-between the variable regions of anti-p185HER2-ECD antibody 4D5-8 resulted in optimal VH/VL interface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly4Ser3 linker precipitated at physiological pH 7.4. Conclusions This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative in situ, in vivo and ex vivo imaging, cell sorting and cell

  18. Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

    Directory of Open Access Journals (Sweden)

    Kaku Yoshihiro

    2012-09-01

    Full Text Available Abstract Background In 2009, a novel influenza A/H1N1 virus (H1N1pdm quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1. Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009–2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs. Findings Human single-fold scFv libraries (Tomlinson I + J underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA. After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. Discussion Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display

  19. Cis- and trans-acting elements regulate the mouse Psmb9 meiotic recombination hotspot.

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    Frédéric Baudat

    2007-06-01

    Full Text Available In most eukaryotes, the prophase of the first meiotic division is characterized by a high level of homologous recombination between homologous chromosomes. Recombination events are not distributed evenly within the genome, but vary both locally and at large scale. Locally, most recombination events are clustered in short intervals (a few kilobases called hotspots, separated by large intervening regions with no or very little recombination. Despite the importance of regulating both the frequency and the distribution of recombination events, the genetic factors controlling the activity of the recombination hotspots in mammals are still poorly understood. We previously characterized a recombination hotspot located close to the Psmb9 gene in the mouse major histocompatibility complex by sperm typing, demonstrating that it is a site of recombination initiation. With the goal of uncovering some of the genetic factors controlling the activity of this initiation site, we analyzed this hotspot in both male and female germ lines and compared the level of recombination in different hybrid mice. We show that a haplotype-specific element acts at distance and in trans to activate about 2,000-fold the recombination activity at Psmb9. Another haplotype-specific element acts in cis to repress initiation of recombination, and we propose this control to be due to polymorphisms located within the initiation zone. In addition, we describe subtle variations in the frequency and distribution of recombination events related to strain and sex differences. These findings show that most regulations observed act at the level of initiation and provide the first analysis of the control of the activity of a meiotic recombination hotspot in the mouse genome that reveals the interactions of elements located both in and outside the hotspot.

  20. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

    Science.gov (United States)

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-06-16

    Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  1. FOXP3-specific immunity

    OpenAIRE

    Andersen, Mads Hald

    2013-01-01

    Forkhead box P3 (FOXP3)-specific cytotoxic CD8+ T cells are present among human peripheral blood mononuclear cells (PBMCs), especially in cancer patients. Such T lymphocytes are able not only to specifically recognize dendritic cells (DCs) that have been exposed to recombinant FOXP3 and regulatory T cells, but also to kill FOXP3+ malignant T cells. The natural occurrence of FOXP3-specific cytotoxic T lymphocytes among human PBMCs suggests a general role for these cells in the complex network ...

  2. Specific memory B cell response and participation of CD4+central and effector memory T cells in mice immunized with liposome encapsulated recombinant NE protein based Hepatitis E vaccine candidate.

    Science.gov (United States)

    Kulkarni, Shruti P; Thanapati, Subrat; Arankalle, Vidya A; Tripathy, Anuradha S

    2016-11-21

    Liposome encapsulated neutralizing epitope protein of Hepatitis E virus (HEV), rNEp, our Hepatitis E vaccine candidate, was shown to be immunogenic and safe in pregnant and non-pregnant mice and yielded sterilizing immunity in rhesus monkeys. The current study in Balb/c mice assessed the levels and persistence of anti-HEV IgG antibodies by ELISA, frequencies of B, memory B, T and memory T cells by flow cytometry and HEV-specific IgG secreting memory B cells by ELISPOT till 420days post immunization (PI) with 5?g rNEp encapsulated in liposome based adjuvant (2 doses, 4weeks apart). Mice immunized with a lower dose (1?g) were assessed only for anamnestic response post booster dose. Vaccine candidate immunized mice (5?g dose) elicited strong anti-HEV IgG response that was estimated to persist for lifetime. At day 120 PI, frequency of memory B cells was higher in immunized mice than those receiving adjuvant alone. Anti-HEV IgG titers were lower in mice immunized with 1?g dose. A booster dose yielded a heightened antibody response in mice with both high (>800GMT, 5?g) and low (?100GMT, 1?g) anti-HEV IgG titers. At day 6th post booster dose, HEV-specific antibody secreting plasma cells (ASCs) were detected in 100% and 50% of mice with high and low anti-HEV IgG titers, respectively, whereas the frequencies of CD4 + central and effector memory T cells were high in mice with high anti-HEV IgG titers only. Taken together, the vaccine candidate effectively generates persistent and anamnestic antibody response, elicits participation of CD4 + memory T cells and triggers memory B cells to differentiate into ASCs upon boosting. This approach of assessing the immunogenicity of vaccine candidate could be useful to explore the longevity of HEV-specific memory response in future HEV vaccine trials in human. Copyright © 2016. Published by Elsevier Ltd.

  3. Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system

    Directory of Open Access Journals (Sweden)

    Tsuyoshi Ikehara

    2016-09-01

    Full Text Available Protein phosphatase 2A (PP2A is an enzyme useful for detecting several natural toxins represented by okadaic acid and microcystins. We found that the production of the recombinant human PP2A catalytic subunit (rhPP2Ac in High Five insect cells could markedly increase when the cells were cultured at 19 °C instead of 27 °C used under conventional conditions. The yield and purity of the enzyme increased four- and three-folds, respectively. The benefit of the altered culturing temperature was observed with the recombinant human protein phosphatase 2B but not 2Cα. The different responses among the enzymes suggest the involvement of an enzyme-specific mechanism that leads to the catalytic subunit overexpression. This is the first report to produce rhPP2Ac at a temperature lower than that used under conventional culture conditions (27 °C used in the baculovirus expression system with High Five insect cells.

  4. Oral therapeutic vaccination with Streptococcus sobrinus recombinant enolase confers protection against dental caries in rats.

    Science.gov (United States)

    Dinis, Márcia; Tavares, Delfina; Veiga-Malta, Isabel; Fonseca, António J M M; Andrade, Elva Bonifácio; Trigo, Gabriela; Ribeiro, Adília; Videira, Arnaldo; Cabrita, António M Silvério; Ferreira, Paula

    2009-01-01

    Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

  5. 9-Phenanthrol inhibits recombinant and arterial myocyte TMEM16A channels

    Science.gov (United States)

    Burris, Sarah K; Wang, Qian; Bulley, Simon; Neeb, Zachary P; Jaggar, Jonathan H

    2015-01-01

    Background and Purpose In arterial smooth muscle cells (myocytes), intravascular pressure stimulates membrane depolarization and vasoconstriction (the myogenic response). Ion channels proposed to mediate pressure-induced depolarization include several transient receptor potential (TRP) channels, including TRPM4, and transmembrane protein 16A (TMEM16A), a Ca2+-activated Cl− channel (CaCC). 9-Phenanthrol, a putative selective TRPM4 channel inhibitor, abolishes myogenic tone in cerebral arteries, suggesting that either TRPM4 is essential for pressure-induced depolarization, upstream of activation of other ion channels or that 9-phenanthrol is non-selective. Here, we tested the hypothesis that 9-phenanthrol is also a TMEM16A channel blocker, an ion channel for which few inhibitors have been identified. Experimental Approach Patch clamp electrophysiology was used to measure rat cerebral artery myocyte and human recombinant TMEM16A (rTMEM16A) currents or currents generated by recombinant bestrophin-1, another Ca2+-activated Cl− channel, expressed in HEK293 cells. Key Results 9-Phenanthrol blocked myocyte TMEM16A currents activated by either intracellular Ca2+ or Eact, a TMEM16A channel activator. In contrast, 9-phenanthrol did not alter recombinant bestrophin-1 currents. 9-Phenanthrol reduced arterial myocyte TMEM16A currents with an IC50 of ∼12 μM. Cell-attached patch recordings indicated that 9-phenanthrol reduced single rTMEM16A channel open probability and mean open time, and increased mean closed time without affecting the amplitude. Conclusions and Implications These data identify 9-phenanthrol as a novel TMEM16A channel blocker and provide an explanation for the previous observation that 9-phenanthrol abolishes myogenic tone when both TRPM4 and TMEM16A channels contribute to this response. 9-Phenanthrol may be a promising candidate from which to develop TMEM16A channel-specific inhibitors. PMID:25573456

  6. Electric hydrogen recombiner special tests

    International Nuclear Information System (INIS)

    Wilson, J.F.

    1975-12-01

    Westinghouse has produced an electric hydrogen recombiner to control hydrogen levels in reactor containments following a postulated loss-of-coolant accident. The recombiner underwent extensive testing for NRC qualification (see WCAP 7709-L and Supplements 1, 2, 3, 4). As a result, WCAP 7709-L and Supplements 1, 2, 3, and 4 have been accepted by the NRC for reference in applications not committed to IEEE-323-1974. Supplement 5 and the next supplement will demonstrate conformance to IEEE-323-1974. This supplement describes additional tests, beyond those necessary to qualify the system, which will be referenced in supplement 6. Each test has demonstrated a considerable margin of safety over required performance. Concurrently, the test results increased the fund of technical information on the electric hydrogen recombiner

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

    Science.gov (United States)

    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'. © 2016 The Author(s).

  8. The effect of a single recombination event

    DEFF Research Database (Denmark)

    Schierup, Mikkel Heide; Jensen, Thomas Mailund; Wiuf, Carsten

    We investigate the variance in how visible a single recombination event is in a SNP data set as a function of the type of recombination event and its age. Data is simulated under the coalescent with recombination and inference is by the popular composite likelihood methods. The major determinant...... of the effect of a recombination event is the genealogical type of the event and whether SNP variation is present that can reveal the genealogical consequences of the recombination event. Recombination events that only change some branch lengths in the genealogy have a very small, but detectable, effect....... The more lineages left when the recombination event occurs, the larger effect it has, implying that it is mainly young recombination events that we detect when estimating the rate. If the population is growing, though, more lineages are present back in time and relatively more ancient recombination events...

  9. Epigenetic silencing of V(DJ recombination is a major determinant for selective differentiation of mucosal-associated invariant t cells from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Yutaka Saito

    Full Text Available Mucosal-associated invariant T cells (MAITs are innate-like T cells that play a pivotal role in the host defense against infectious diseases, and are also implicated in autoimmune diseases, metabolic diseases, and cancer. Recent studies have shown that induced pluripotent stem cells (iPSCs derived from MAITs selectively redifferentiate into MAITs without altering their antigen specificity. Such a selective differentiation is a prerequisite for the use of MAITs in cell therapy and/or regenerative medicine. However, the molecular mechanisms underlying this phenomenon remain unclear. Here, we performed methylome and transcriptome analyses of MAITs during the course of differentiation from iPSCs. Our multi-omics analyses revealed that recombination-activating genes (RAG1 and RAG2 and DNA nucleotidylexotransferase (DNTT were highly methylated with their expression being repressed throughout differentiation. Since these genes are essential for V(DJ recombination of the T cell receptor (TCR locus, this indicates that nascent MAITs are kept from further rearrangement that may alter their antigen specificity. Importantly, we found that the repression of RAGs was assured in two layers: one by the modulation of transcription factors for RAGs, and the other by DNA methylation at the RAG loci. Together, our study provides a possible explanation for the unaltered antigen specificity in the selective differentiation of MAITs from iPSCs.

  10. A trans-Complementing Recombination Trap Demonstrates a Low Propensity of Flaviviruses for Intermolecular Recombination▿

    Science.gov (United States)

    Taucher, Christian; Berger, Angelika; Mandl, Christian W.

    2010-01-01

    Intermolecular recombination between the genomes of closely related RNA viruses can result in the emergence of novel strains with altered pathogenic potential and antigenicity. Although recombination between flavivirus genomes has never been demonstrated experimentally, the potential risk of generating undesirable recombinants has nevertheless been a matter of concern and controversy with respect to the development of live flavivirus vaccines. As an experimental system for investigating the ability of flavivirus genomes to recombine, we developed a “recombination trap,” which was designed to allow the products of rare recombination events to be selected and amplified. To do this, we established reciprocal packaging systems consisting of pairs of self-replicating subgenomic RNAs (replicons) derived from tick-borne encephalitis virus (TBEV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) that could complement each other in trans and thus be propagated together in cell culture over multiple passages. Any infectious viruses with intact, full-length genomes that were generated by recombination of the two replicons would be selected and enriched by end point dilution passage, as was demonstrated in a spiking experiment in which a small amount of wild-type virus was mixed with the packaged replicons. Using the recombination trap and the JEV system, we detected two aberrant recombination events, both of which yielded unnatural genomes containing duplications. Infectious clones of both of these genomes yielded viruses with impaired growth properties. Despite the fact that the replicon pairs shared approximately 600 nucleotides of identical sequence where a precise homologous crossover event would have yielded a wild-type genome, this was not observed in any of these systems, and the TBEV and WNV systems did not yield any viable recombinant genomes at all. Our results show that intergenomic recombination can occur in the structural region of flaviviruses

  11. Protein trafficking, ergosterol biosynthesis and membrane physics impact recombinant protein secretion in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Mattanovich Diethard

    2011-11-01

    Full Text Available Abstract Background The increasing availability of 'omics' databases provide important platforms for yeast engineering strategies since they offer a lot of information on the physiology of the cells under diverse growth conditions, including environmental stresses. Notably, only a few of these approaches have considered a performance under recombinant protein production conditions. Recently, we have identified a beneficial effect of low oxygen availability on the expression of a human Fab fragment in Pichia pastoris. Transcriptional analysis and data mining allowed for the selection of potential targets for strain improvement. A first selection of these candidates has been evaluated as recombinant protein secretion enhancers. Results Based on previous transcriptomics analyses, we selected 8 genes for co-expression in the P. pastoris strain already secreting a recombinant Fab fragment. Notably, WSC4 (which is involved in trafficking through the ER has been identified as a novel potential target gene for strain improvement, with up to a 1.2-fold increase of product yield in shake flask cultures. A further transcriptomics-based strategy to modify the yeast secretion system was focused on the ergosterol pathway, an aerobic process strongly affected by oxygen depletion. By specifically partially inhibiting ergosterol synthesis with the antifungal agent fluconazole (inhibiting Erg11p, we tried to mimic the hypoxic conditions, in which the cellular ergosterol content was significantly decreased. This strategy led to an improved Fab yield (2-fold without impairing cellular growth. Since ergosterol shortage provokes alterations in the plasma membrane composition, an important role of this cellular structure in protein secretion is suggested. This hypothesis was additionally supported by the fact that the addition of non-ionic surfactants also enhanced Fab secretion. Conclusions The current study presents a systems biotechnology-based strategy for the

  12. Relationship among the repair and genetic recombination mechanisms. II. Effect of gamma radiation on the lambda recombination in E. coli AB1157 and AB1886

    International Nuclear Information System (INIS)

    Alcantara D, D.

    1988-08-01

    The objective of the present work is to determine if the radiation gamma that is a good inductor of the answer SOS of Escherichia Coli but that it produces alterations in the DNA very different to those taken place by the light UV, it is able to stimulate the viral recombination. (Author)

  13. Cloning, recombinant expression and characterization of a new ...

    African Journals Online (AJOL)

    A new amylase gene APGA1 was cloned from Aureobasidium pullulans NRRL 12974 and expressed in Pichia pastoris. This is the first report on cloning and expression of amylolytic gene from the industrially important microorganism A. pullulans. The purified recombinant protein with MW of 66 kDa and specific activity of ...

  14. Review of literature on catalytic recombination of hydrogen--oxygen

    International Nuclear Information System (INIS)

    Homsy, R.V.; Glatron, C.A.

    1968-01-01

    The results are reported of a literature search for information concerning the heterogeneous, gas phase, catalytic hydrogen-oxygen recombination. Laboratory scale experiments to test the performance of specific metal oxide catalysts under conditions simulating the atmosphere within a nuclear reactor containment vessel following a loss-of-coolant blowdown accident are suggested

  15. Monitoring of protein profiles for the optimization of recombinant fermentation processes using public domain databases.

    Science.gov (United States)

    Dürrschmid, Karin; Marzban, Gorji; Dürrschmid, Eberhard; Striedner, Gerald; Clementschitsch, Franz; Cserjan-Puschmann, Monika; Bayer, Karl

    2003-01-01

    The expression of human superoxide dismutase in fed-batch fermentation of E. coli HMS174(DE3)(pET3ahSOD) was studied as model system. Due to the frequently used strong T7 promoter system a high metabolic load is exerted, which triggers stress response mechanisms and finally leads to the differentiation of the host cell. As a consequence, host cell metabolism is partly shifted from growth to survival accompanied by significant alterations of the protein pattern. In terms of process optimization two-dimensional electrophoresis deserves as a powerful tool to monitor these changes on protein level. For the analysis of samples derived from different states of recombinant protein production wide-range Immobiline Dry Strips pH 3-10 were used. In order to establish an efficient procedure for accelerated process optimization and to avoid costly and time-consuming analysis like mass spectrometry (MS), a database approach for the identification of significant changes of the protein pattern was evaluated. On average, 935 spots per gel were detected, whereby 50 are presumably stress-relevant. Out of these, 24 proteins could be identified by using the SWISS-2DPAGE database (www.expasy.ch/ch2d/). The identified proteins are involved in regulatory networks, energy metabolism, purine and pyrimidine nucleotide synthesis and translation. By this database approach, significant fluctuations of individual proteins in relation to recombinant protein production could be identified. Seven proteins show strong alterations (>100%) directly after induction and can therefore be stated as reliable marker proteins for the assessment of stress response. For distinctive interpretation of this highly specific information, a bioinformatic and statistic tool would be essential in order to perceive the role and contribution of individual proteins in stress response.

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

    Science.gov (United States)

    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. Restriction and sequence alterations affect DNA uptake sequence-dependent transformation in Neisseria meningitidis.

    Directory of Open Access Journals (Sweden)

    Ole Herman Ambur

    Full Text Available Transformation is a complex process that involves several interactions from the binding and uptake of naked DNA to homologous recombination. Some actions affect transformation favourably whereas others act to limit it. Here, meticulous manipulation of a single type of transforming DNA allowed for quantifying the impact of three different mediators of meningococcal transformation: NlaIV restriction, homologous recombination and the DNA Uptake Sequence (DUS. In the wildtype, an inverse relationship between the transformation frequency and the number of NlaIV restriction sites in DNA was observed when the transforming DNA harboured a heterologous region for selection (ermC but not when the transforming DNA was homologous with only a single nucleotide heterology. The influence of homologous sequence in transforming DNA was further studied using plasmids with a small interruption or larger deletions in the recombinogenic region and these alterations were found to impair transformation frequency. In contrast, a particularly potent positive driver of DNA uptake in Neisseria sp. are short DUS in the transforming DNA. However, the molecular mechanism(s responsible for DUS specificity remains unknown. Increasing the number of DUS in the transforming DNA was here shown to exert a positive effect on transformation. Furthermore, an influence of variable placement of DUS relative to the homologous region in the donor DNA was documented for the first time. No effect of altering the orientation of DUS was observed. These observations suggest that DUS is important at an early stage in the recognition of DNA, but does not exclude the existence of more than one level of DUS specificity in the sequence of events that constitute transformation. New knowledge on the positive and negative drivers of transformation may in a larger perspective illuminate both the mechanisms and the evolutionary role(s of one of the most conserved mechanisms in nature: homologous

  18. Improving recombinant protein purification yield

    Science.gov (United States)

    Production of adequate amounts of recombinant proteins is essential for antibody production, biochemical activity study, and structural determination during the post-genomic era. It’s technologically challenging and a limiting factor for tung oil research because analytical reagents such as high qua...

  19. A recombinant protein expression system

    African Journals Online (AJOL)

    Aghomotsegin

    2015-06-23

    Jun 23, 2015 ... Serum free cultivation of Leishmania is cost-effective and improves large scale production of well- defined parasite material. Moreover, the production of recombinant pharmaceutical proteins requires cultivation of the host in a culture medium free of animal materials, so several culture media for.

  20. Production and recombination of gluons

    International Nuclear Information System (INIS)

    Temiraliev, A.T.

    2006-01-01

    Full text: Nonlinear Markov process of parton production has been considered. The Kolmogorov equation is applied for the evolution equation based on the approximation of independent gluons production in every decay act. We introduced a 'crossing' parameter and used the combination relations to obtain nonlinear recombination equation for the evolution of gluon structure function. (author)

  1. Recombination in hepatitis C virus.

    Science.gov (United States)

    González-Candelas, Fernando; López-Labrador, F Xavier; Bracho, María Alma

    2011-10-01

    Hepatitis C virus (HCV) is a Flavivirus with a positive-sense, single-stranded RNA genome of about 9,600 nucleotides. It is a major cause of liver disease, infecting almost 200 million people all over the world. Similarly to most RNA viruses, HCV displays very high levels of genetic diversity which have been used to differentiate six major genotypes and about 80 subtypes. Although the different genotypes and subtypes share basic biological and pathogenic features they differ in clinical outcomes, response to treatment and epidemiology. The first HCV recombinant strain, in which different genome segments derived from parentals of different genotypes, was described in St. Petersburg (Russia) in 2002. Since then, there have been only a few more than a dozen reports including descriptions of HCV recombinants at all levels: between genotypes, between subtypes of the same genotype and even between strains of the same subtype. Here, we review the literature considering the reasons underlying the difficulties for unequivocally establishing recombination in this virus along with the analytical methods necessary to do it. Finally, we analyze the potential consequences, especially in clinical practice, of HCV recombination in light of the coming new therapeutic approaches against this virus.

  2. Long-Term Effects of Prenatal Exposure to Undernutrition on Cannabinoid Receptor-Related Behaviors: Sex and Tissue-Specific Alterations in the mRNA Expression of Cannabinoid Receptors and Lipid Metabolic Regulators

    Science.gov (United States)

    Ramírez-López, María T.; Arco, Rocío; Decara, Juan; Vázquez, Mariam; Rivera, Patricia; Blanco, Rosario Noemi; Alén, Francisco; Gómez de Heras, Raquel; Suárez, Juan; Rodríguez de Fonseca, Fernando

    2016-01-01

    Maternal malnutrition causes long-lasting alterations in feeding behavior and energy homeostasis in offspring. It is still unknown whether both, the endocannabinoid (eCB) machinery and the lipid metabolism are implicated in long-term adaptive responses to fetal reprogramming caused by maternal undernutrition. We investigated the long-term effects of maternal exposure to a 20% standard diet restriction during preconceptional and gestational periods on the metabolically-relevant tissues hypothalamus, liver, and perirenal fat (PAT) of male and female offspring at adulthood. The adult male offspring from calorie-restricted dams (RC males) exhibited a differential response to the CB1 antagonist AM251 in a chocolate preference test as well as increased body weight, perirenal adiposity, and plasma levels of triglycerides, LDL, VLDL, bilirubin, and leptin. The gene expression of the cannabinoid receptors Cnr1 and Cnr2 was increased in RC male hypothalamus, but a down-expression of most eCBs-metabolizing enzymes (Faah, Daglα, Daglβ, Mgll) and several key regulators of fatty-acid β-oxidation (Cpt1b, Acox1), mitochondrial respiration (Cox4i1), and lipid flux (Pparγ) was found in their PAT. The female offspring from calorie-restricted dams exhibited higher plasma levels of LDL and glucose as well as a reduction in chocolate and caloric intake at post-weaning periods in the feeding tests. Their liver showed a decreased gene expression of Cnr1, Pparα, Pparγ, the eCBs-degrading enzymes Faah and Mgll, the de novo lipogenic enzymes Acaca and Fasn, and the liver-specific cholesterol biosynthesis regulators Insig1 and Hmgcr. Our results suggest that the long-lasting adaptive responses to maternal caloric restriction affected cannabinoid-regulated mechanisms involved in feeding behavior, adipose β-oxidation, and hepatic lipid and cholesterol biosynthesis in a sex-dependent manner. PMID:28082878

  3. Naloxone treatment alters gene expression in the mesolimbic reward system in 'junk food' exposed offspring in a sex-specific manner but does not affect food preferences in adulthood.

    Science.gov (United States)

    Gugusheff, J R; Ong, Z Y; Muhlhausler, B S

    2014-06-22

    We have previously reported that the opioid receptor blocker, naloxone, is less effective in reducing palatable food intake in offspring exposed to a maternal cafeteria diet during the perinatal period, implicating a desensitization of the central opioid pathway in the programming of food preferences. The present study aimed to investigate the effect of a maternal cafeteria diet and naloxone treatment on the development of the mesolimbic reward pathway and food choices in adulthood. We measured mRNA expression of key components of the reward pathway (mu-opioid receptor, proenkephalin, tyrosine hydroxylase, D1 and D2 receptors and the dopamine active transporter (DAT)) in the nucleus accumbens (NAc) and ventral tegmental area (VTA) of the offspring of control and cafeteria fed (JF) dams at weaning and after a 10-day naloxone treatment post-weaning and determined food preferences in adulthood in the remaining offspring. Naloxone treatment decreased the expression of DAT by 8.2 fold in female control offspring but increased it by 4.3 fold in female offspring of JF dams relative to the saline-injected reference groups. Proenkephalin mRNA expression was higher in the NAc of female JF offspring compared to controls, independent of naloxone treatment (Pfood preferences in adulthood in either control or JF offspring. These data indicate that prenatal exposure to a cafeteria diet alters the impact of opioid signaling blockade in the early post-weaning period on gene expression in the central reward pathway in a sex specific manner, but that these changes in gene expression do not appear to have any persistent impact on food preferences in adulthood. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Recombination of hepatitis B virus DNA in patients with HIV.

    Science.gov (United States)

    Fallot, Guillaume; Halgand, Boris; Garnier, Elisabeth; Branger, Michel; Gervais, Anne; Roque-Afonso, Anne-Marie; Thiers, Valérie; Billaud, Eric; Matheron, Sophie; Samuel, Didier; Féray, Cyrille

    2012-08-01

    Hepatitis B is a major cause of death in patients with HIV who usually receive drugs active against hepatitis B virus (HBV). The variability of HBV DNA over time has been little studied. Recombination between different HBV genotypes has been described in many cross-sectional studies, but the frequency of intergenotypic and intragenotypic recombinations in individual patients is unknown. 32 HIV-positive and 11 HIV-negative patients who remained HBV viraemic despite antiviral therapy for at least 1 year were studied. Genotyping was based on line probe assays and genotype-specific PCR. The variability of HBV DNA over time was examined with restriction length and single-strand conformational polymorphism (RFLP-SSCP). HBV DNA sequences obtained by cloning a 2800 bp PCR fragment were analysed for phylogenetic parameters (diversity and selection pressure) and recombination was detected with RDP3 software. Large fragments of HBV DNA could be amplified at two different time points in 33 patients. Marked quasi-species modifications occurred in 14 patients. In seven of these patients and in one patient with no change detectable by RFLP-SSCP, the 2800 bp fragment was cloned at two time points at least. In four (57%) of these seven patients, various intergenotypic or intragenotypic recombination events were detected between subvariants present in the initial quasi-species. Recombinant fragments mostly harboured antiviral resistance determinants and reflected a large increase in diversity and in positive selection pressure on the entire HBV quasi-species. In coinfected patients, HBV DNA recombination events are frequent during antiviral therapy, corresponding to increased positive selection pressure on the HBV quasi-species and to conservation of antiviral resistance mutations. In this population and at the individual level, recombination is a significant source of HBV genetic variability.

  5. Functional, Responsive Materials Assembled from Recombinant Oleosin

    Science.gov (United States)

    Hammer, Daniel

    Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

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

    Directory of Open Access Journals (Sweden)

    Michael Mühle

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

  7. Live recombinant BHV/BRSV vaccine

    NARCIS (Netherlands)

    Keil, G.M.; Rijsewijk, F.A.M.

    1998-01-01

    The present invention refers to synthetic Bovine Respiratory Syncytium virus genes. Also the invention relates to live attenuated Bovine Herpesvirus recombinants carrying such synthetic genes. Furthermore, the invention relates to vaccines based on these live attenuated recombinants, for the

  8. Hadron production at RHIC: recombination of quarks

    Energy Technology Data Exchange (ETDEWEB)

    Fries, Rainer J [School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455 (United States)

    2005-01-01

    We discuss quark recombination applied to the hadronization of a quark gluon plasma. It has been shown that the quark recombination model can explain essential features of hadron production measured in high energy heavy ion collisions.

  9. A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc Induces Serum, Memory and Lamina Proprial Responses against OSP and Is Protective in Mice.

    Directory of Open Access Journals (Sweden)

    Md Abu Sayeed

    Full Text Available Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP component of lipopolysaccharide (LPS.Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc. We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 μg, vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1, effect of an adjuvant, and route of immunization.Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 μg. We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model.We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.

  10. Overexpression of a Mycobacterium ulcerans Ag85B-EsxH Fusion Protein in Recombinant BCG Improves Experimental Buruli Ulcer Vaccine Efficacy.

    Directory of Open Access Journals (Sweden)

    Bryan E Hart

    2016-12-01

    Full Text Available Buruli ulcer (BU vaccine design faces similar challenges to those observed during development of prophylactic tuberculosis treatments. Multiple BU vaccine candidates, based upon Mycobacterium bovis BCG, altered Mycobacterium ulcerans (MU cells, recombinant MU DNA, or MU protein prime-boosts, have shown promise by conferring transient protection to mice against the pathology of MU challenge. Recently, we have shown that a recombinant BCG vaccine expressing MU-Ag85A (BCG MU-Ag85A displayed the highest level of protection to date, by significantly extending the survival time of MU challenged mice compared to BCG vaccination alone. Here we describe the generation, immunogenicity testing, and evaluation of protection conferred by a recombinant BCG strain which overexpresses a fusion of two alternative MU antigens, Ag85B and the MU ortholog of tuberculosis TB10.4, EsxH. Vaccination with BCG MU-Ag85B-EsxH induces proliferation of Ag85 specific CD4+ T cells in greater numbers than BCG or BCG MU-Ag85A and produces IFNγ+ splenocytes responsive to whole MU and recombinant antigens. In addition, anti-Ag85A and Ag85B IgG humoral responses are significantly enhanced after administration of the fusion vaccine compared to BCG or BCG MU-Ag85A. Finally, mice challenged with MU following a single subcutaneous vaccination with BCG MU-Ag85B-EsxH display significantly less bacterial burden at 6 and 12 weeks post-infection, reduced histopathological tissue damage, and significantly longer survival times compared to vaccination with either BCG or BCG MU-Ag85A. These results further support the potential of BCG as a foundation for BU vaccine design, whereby discovery and recombinant expression of novel immunogenic antigens could lead to greater anti-MU efficacy using this highly safe and ubiquitous vaccine.

  11. Overexpression of a Mycobacterium ulcerans Ag85B-EsxH Fusion Protein in Recombinant BCG Improves Experimental Buruli Ulcer Vaccine Efficacy.

    Science.gov (United States)

    Hart, Bryan E; Lee, Sunhee

    2016-12-01

    Buruli ulcer (BU) vaccine design faces similar challenges to those observed during development of prophylactic tuberculosis treatments. Multiple BU vaccine candidates, based upon Mycobacterium bovis BCG, altered Mycobacterium ulcerans (MU) cells, recombinant MU DNA, or MU protein prime-boosts, have shown promise by conferring transient protection to mice against the pathology of MU challenge. Recently, we have shown that a recombinant BCG vaccine expressing MU-Ag85A (BCG MU-Ag85A) displayed the highest level of protection to date, by significantly extending the survival time of MU challenged mice compared to BCG vaccination alone. Here we describe the generation, immunogenicity testing, and evaluation of protection conferred by a recombinant BCG strain which overexpresses a fusion of two alternative MU antigens, Ag85B and the MU ortholog of tuberculosis TB10.4, EsxH. Vaccination with BCG MU-Ag85B-EsxH induces proliferation of Ag85 specific CD4+ T cells in greater numbers than BCG or BCG MU-Ag85A and produces IFNγ+ splenocytes responsive to whole MU and recombinant antigens. In addition, anti-Ag85A and Ag85B IgG humoral responses are significantly enhanced after administration of the fusion vaccine compared to BCG or BCG MU-Ag85A. Finally, mice challenged with MU following a single subcutaneous vaccination with BCG MU-Ag85B-EsxH display significantly less bacterial burden at 6 and 12 weeks post-infection, reduced histopathological tissue damage, and significantly longer survival times compared to vaccination with either BCG or BCG MU-Ag85A. These results further support the potential of BCG as a foundation for BU vaccine design, whereby discovery and recombinant expression of novel immunogenic antigens could lead to greater anti-MU efficacy using this highly safe and ubiquitous vaccine.

  12. Overexpression of a Mycobacterium ulcerans Ag85B-EsxH Fusion Protein in Recombinant BCG Improves Experimental Buruli Ulcer Vaccine Efficacy

    Science.gov (United States)

    Hart, Bryan E.

    2016-01-01

    Buruli ulcer (BU) vaccine design faces similar challenges to those observed during development of prophylactic tuberculosis treatments. Multiple BU vaccine candidates, based upon Mycobacterium bovis BCG, altered Mycobacterium ulcerans (MU) cells, recombinant MU DNA, or MU protein prime-boosts, have shown promise by conferring transient protection to mice against the pathology of MU challenge. Recently, we have shown that a recombinant BCG vaccine expressing MU-Ag85A (BCG MU-Ag85A) displayed the highest level of protection to date, by significantly extending the survival time of MU challenged mice compared to BCG vaccination alone. Here we describe the generation, immunogenicity testing, and evaluation of protection conferred by a recombinant BCG strain which overexpresses a fusion of two alternative MU antigens, Ag85B and the MU ortholog of tuberculosis TB10.4, EsxH. Vaccination with BCG MU-Ag85B-EsxH induces proliferation of Ag85 specific CD4+ T cells in greater numbers than BCG or BCG MU-Ag85A and produces IFNγ+ splenocytes responsive to whole MU and recombinant antigens. In addition, anti-Ag85A and Ag85B IgG humoral responses are significantly enhanced after administration of the fusion vaccine compared to BCG or BCG MU-Ag85A. Finally, mice challenged with MU following a single subcutaneous vaccination with BCG MU-Ag85B-EsxH display significantly less bacterial burden at 6 and 12 weeks post-infection, reduced histopathological tissue damage, and significantly longer survival times compared to vaccination with either BCG or BCG MU-Ag85A. These results further support the potential of BCG as a foundation for BU vaccine design, whereby discovery and recombinant expression of novel immunogenic antigens could lead to greater anti-MU efficacy using this highly safe and ubiquitous vaccine. PMID:27941982

  13. Recombinant DNA technologies for construction of precisely designed transgene constructs.

    Science.gov (United States)

    Ohtsuka, Masato; Kimura, Minoru; Tanaka, Masafumi; Inoko, Hidetoshi

    2009-02-01

    Genetically modified animals have been used as models in broad range of studies including pharmaceutical biology. Designing and construction of transgene constructs are the first indispensable task in generating model animals. In addition to the classical restriction enzyme-based method, still holds some advantages in generating precise constructs, site-specific recombinase-based and homologous recombination-based DNA engineering strategies (e.g. Gateway and Red/ET recombineering, respectively) have been developed and widely used for vector construction or BAC modification. In this review, the three construction methods are described and their applications are discussed such as tandem assemblies of multiple components and modification of large DNA molecules. Combinational use of these E. coli-based recombinant DNA technologies enables the generation of precisely designed vectors useful for desired genome modification for future analyses.

  14. High efficiency recombineering in lactic acid bacteria

    OpenAIRE

    van Pijkeren, Jan-Peter; Britton, Robert A.

    2012-01-01

    The ability to efficiently generate targeted point mutations in the chromosome without the need for antibiotics, or other means of selection, is a powerful strategy for genome engineering. Although oligonucleotide-mediated recombineering (ssDNA recombineering) has been utilized in Escherichia coli for over a decade, the successful adaptation of ssDNA recombineering to Gram-positive bacteria has not been reported. Here we describe the development and application of ssDNA recombineering in lact...

  15. Population inversion in recombining hydrogen plasma

    International Nuclear Information System (INIS)

    Furukane, Utaro; Yokota, Toshiaki; Oda, Toshiatsu.

    1978-11-01

    The collisional-radiative model is applied to a recombining hydrogen plasma in order to investigate the plasma condition in which the population inversion between the energy levels of hydrogen can be generated. The population inversion is expected in a plasma where the three body recombination has a large contribution to the recombining processes and the effective recombination rate is beyond a certain value for a given electron density and temperature. Calculated results are presented in figures and tables. (author)

  16. Cloning, purification and characterization of recombinant silkworm ...

    African Journals Online (AJOL)

    The recombinant His-tagged BmAK protein was expressed in soluble form in Escherichia coli Rosetta and purified by metal chelating affinity chromatography. The amino acid sequence of recombinant protein was confirmed by mass spectroscopic analysis and the enzyme activity assay that indicated the recombinant ...

  17. Determination of recombination in Mycoplasma hominis

    DEFF Research Database (Denmark)

    Jacobsen, Iben Søgaard; Boesen, Thomas; Mygind, Tina

    2002-01-01

    indicating the presence of recombination. In order to test for intergenic recombination, phylogenetic trees were reconstructed for each of the genes but no well-supported bifurcating phylogenetic trees could be obtained. The genes were tested for intragenic recombination using the correlation between linkage...

  18. Diagnóstico de ABPA em pacientes portadores de fibrose cística: utilidade clínica da pesquisa de IgE específica contra alérgenos recombinantes do Aspergillus fumigatus ABPA diagnosis in cystic fibrosis patients: the clinical utility of IgE specific to recombinant Aspergillus fumigatus allergens

    Directory of Open Access Journals (Sweden)

    Marina B. Almeida

    2006-06-01

    lung disease progresses. The overlap between the signs and symptoms of the two conditions makes diagnosis problematic, even if standardized criteria are used. The objective of this study was to identify, in a group of cystic fibrosis patients, cases of ABPA by assaying IgE specific to recombinant Aspergillus fumigatus antigens and to compare the method with the Cystic Fibrosis Foundation diagnostic criteria. METHODS: Fifty-four patients, aged 2 to 20 years, presenting characteristics that could occur with ABPA in isolation, were systematically assessed based on the following: clinical data, a chest CT scan, immediate hypersensitivity skin test for A. fumigatus; total serum IgE assay, RAST for A. fumigatus and serum IgE specific for the recombinant allergens Asp f1, f2, f3, f4 and f6. RESULTS: Thirty-nine patients were eligible for the study. Thirty-two of these were investigated. Sensitization to A. fumigatus was observed in 34%. Both the Cystic Fibrosis Foundation criteria and the recombinant antigen specific IgE assay defined three patients as suffering from ABPA; however, only two of these patients were diagnosed by both methods. CONCLUSIONS: The detection of A. fumigatus recombinant antigen specific IgE was a useful tool for the early detection of sensitization and diagnosis of ABPA. Nevertheless, diagnostic confirmation cannot be divorced from clinical findings, and before this method can be used for ABPA diagnosis, for detecting relapses and for defining cure criteria, longitudinal studies with larger numbers of patients are required.

  19. Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Freeman, Kathryn M. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States); Hoffmann, George R. [Department of Biology, College of the Holy Cross, One College Street, Worcester, MA 01610-2395 (United States)]. E-mail: ghoffmann@holycross.edu

    2007-03-01

    Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, {beta}-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv{sup +} revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state.

  20. Frequencies of mutagen-induced coincident mitotic recombination at unlinked loci in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Freeman, Kathryn M.; Hoffmann, George R.

    2007-01-01

    Frequencies of coincident genetic events were measured in strain D7 of Saccharomyces cerevisiae. This diploid strain permits the detection of mitotic gene conversion involving the trp5-12 and trp5-27 alleles, mitotic crossing-over and gene conversion leading to the expression of the ade2-40 and ade2-119 alleles as red and pink colonies, and reversion of the ilv1-92 allele. The three genes are on different chromosomes, and one might expect that coincident (simultaneous) genetic alterations at two loci would occur at frequencies predicted by those of the single alterations acting as independent events. Contrary to this expectation, we observed that ade2 recombinants induced by bleomycin, β-propiolactone, and ultraviolet radiation occur more frequently among trp5 convertants than among total colonies. This excess among trp5 recombinants indicates that double recombinants are more common than expected for independent events. No similar enrichment was found among Ilv + revertants. The possibility of an artifact in which haploid yeasts that mimic mitotic recombinants are generated by a low frequency of cryptic meiosis has been excluded. Several hypotheses that can explain the elevated incidence of coincident mitotic recombination have been evaluated, but the cause remains uncertain. Most evidence suggests that the excess is ascribable to a subset of the population being in a recombination-prone state

  1. Recombinant protein scaffolds for tissue engineering

    International Nuclear Information System (INIS)

    Werkmeister, Jerome A; Ramshaw, John A M

    2012-01-01

    New biological materials for tissue engineering are now being developed using common genetic engineering capabilities to clone and express a variety of genetic elements that allow cost-effective purification and scaffold fabrication from these recombinant proteins, peptides or from chimeric combinations of these. The field is limitless as long as the gene sequences are known. The utility is dependent on the ease, product yield and adaptability of these protein products to the biomedical field. The development of recombinant proteins as scaffolds, while still an emerging technology with respect to commercial products, is scientifically superior to current use of natural materials or synthetic polymer scaffolds, in terms of designing specific structures with desired degrees of biological complexities and motifs. In the field of tissue engineering, next generation scaffolds will be the key to directing appropriate tissue regeneration. The initial period of biodegradable synthetic scaffolds that provided shape and mechanical integrity, but no biological information, is phasing out. The era of protein scaffolds offers distinct advantages, particularly with the combination of powerful tools of molecular biology. These include, for example, the production of human proteins of uniform quality that are free of infectious agents and the ability to make suitable quantities of proteins that are found in low quantity or are hard to isolate from tissue. For the particular needs of tissue engineering scaffolds, fibrous proteins like collagens, elastin, silks and combinations of these offer further advantages of natural well-defined structural scaffolds as well as endless possibilities of controlling functionality by genetic manipulation. (topical review)

  2. Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate.

    Science.gov (United States)

    Smales, C M; Dinnis, D M; Stansfield, S H; Alete, D; Sage, E A; Birch, J R; Racher, A J; Marshall, C T; James, D C

    2004-11-20

    We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (c) 2004 Wiley Periodicals, Inc

  3. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    Nakai, Sayaka; Machida, Isamu; Tsuji, Satsuki

    1985-01-01

    Studies using T948 as a model system have been carried out aimed at elucidating the mechanism of sister chromatid recombination (SCR). Characterization of U.V. light- and x-ray-induced SCR, the relationiship between SCR induction and DNA repair using rad mutations, and the relationship between SCR induction and the time of cell division using cdc mutations are presented. It has been supposed that SCR is induced at the phase of S-G 2 following DNA replication, that postreplication break of DNA strands is strongly involved in the induction of SCR, and that induction type of SCR, i.e., conversion type or recombination type, is dependent upon the type of molecular damage of DNA. (Namekawa, K.)

  4. Heterogeneity in recombinant protein production

    DEFF Research Database (Denmark)

    Schalén, Martin; Johanson, Ted; Lundin, Luisa

    2012-01-01

    contribute to make a population in a fermenter heterogeneous, resulting in cell-to-cell variation in physiological parameters of the microbial culture. Our study aims at investigating how population heterogeneity and recombinant protein production is affected by environmental gradients in bioreactors....... For this purpose, a Saccharomyces cerevisiae strain, that functions as a protein production reporter, has been developed. A heterologous protein has been tagged with a fluorescent protein providing a way to measure the amount of heterologous protein produced by the cells on single cell level. Gradients...... are simulated in small bioreactors and the population heterogeneity can be visualised by analysing single cells with flow cytometry. This can give new insights to cell physiology and recombinant protein production at the industrial scale....

  5. A Case Study of a Multiobjective Elitist Recombinative Genetic Algorithm with Coevolutionary Sharing

    NARCIS (Netherlands)

    Neef, R.M.; Thierens, D.; Arciszewski, H.F.R.

    1999-01-01

    We present a multiobjective genetic algorithm that incorporates various genetic algorithm techniques that have been proven to be efficient and robust in their problem domain. More specifically, we integrate rank based selection, adaptive niching through coevolutionary sharing, elitist recombination,

  6. A case study of a multiobjective recombinative genetic algorithm with coevolutionary sharing

    NARCIS (Netherlands)

    Neef, R.M.; Thierens, D.; Arciszewski, H.F.R.

    1999-01-01

    We present a multiobjective genetic algorithm that incorporates various genetic algorithm techniques that have been proven to be efficient and robust in their problem domain. More specifically, we integrate rank based selection, adaptive niching through coevolutionary sharing, elitist recombination,

  7. Climate-Driven Reshuffling of Species and Genes: Potential Conservation Roles for Species Translocations and Recombinant Hybrid Genotypes

    Directory of Open Access Journals (Sweden)

    Jon Mark Scriber

    2013-12-01

    Full Text Available Comprising 50%–75% of the world’s fauna, insects are a prominent part of biodiversity in communities and ecosystems globally. Biodiversity across all levels of biological classifications is fundamentally based on genetic diversity. However, the integration of genomics and phylogenetics into conservation management may not be as rapid as climate change. The genetics of hybrid introgression as a source of novel variation for ecological divergence and evolutionary speciation (and resilience may generate adaptive potential and diversity fast enough to respond to locally-altered environmental conditions. Major plant and herbivore hybrid zones with associated communities deserve conservation consideration. This review addresses functional genetics across multi-trophic-level interactions including “invasive species” in various ecosystems as they may become disrupted in different ways by rapid climate change. “Invasive genes” (into new species and populations need to be recognized for their positive creative potential and addressed in conservation programs. “Genetic rescue” via hybrid translocations may provide needed adaptive flexibility for rapid adaptation to environmental change. While concerns persist for some conservationists, this review emphasizes the positive aspects of hybrids and hybridization. Specific implications of natural genetic introgression are addressed with a few examples from butterflies, including transgressive phenotypes and climate-driven homoploid recombinant hybrid speciation. Some specific examples illustrate these points using the swallowtail butterflies (Papilionidae with their long-term historical data base (phylogeographical diversity changes and recent (3-decade climate-driven temporal and genetic divergence in recombinant homoploid hybrids and relatively recent hybrid speciation of Papilio appalachiensis in North America. Climate-induced “reshuffling” (recombinations of species composition, genotypes

  8. Workshop on Radio Recombination Lines

    CERN Document Server

    1980-01-01

    Since their first detection 15 years ago, radio recombination lines from several elements have been observed in a wide variety of objects including HII regions, planetary nebulae, molecular clouds, the diffuse interstellar medium, and recently, other galaxies. The observations span almost the entire range from 0.1 to 100 GHz, and employ both single­ djsh and aperture synthesis techniques. The theory of radio recombination lines has also advanced strongly, to the point where it is perhaps one of the best-understood in astro­ physics. In a parallel development, it has become possible over the last decade to study these same highly-excited atoms in the laboratory; this work provides further confirmation of the theoretical framework. However there has been continuing controversy over the astrophysical interpre­ tation of radio recombination line observations, especially regarding the role of stimulated emission. A workshop was held in Ottawa on 24-25 August, 1979, bringing together many of the active scientist...

  9. Consequences of recombination on traditional phylogenetic analysis

    DEFF Research Database (Denmark)

    Schierup, M H; Hein, J

    2000-01-01

    We investigate the shape of a phylogenetic tree reconstructed from sequences evolving under the coalescent with recombination. The motivation is that evolutionary inferences are often made from phylogenetic trees reconstructed from population data even though recombination may well occur (mt......DNA or viral sequences) or does occur (nuclear sequences). We investigate the size and direction of biases when a single tree is reconstructed ignoring recombination. Standard software (PHYLIP) was used to construct the best phylogenetic tree from sequences simulated under the coalescent with recombination....... With recombination present, the length of terminal branches and the total branch length are larger, and the time to the most recent common ancestor smaller, than for a tree reconstructed from sequences evolving with no recombination. The effects are pronounced even for small levels of recombination that may...

  10. Quantitative metabolomics analysis of amino acid metabolism in recombinant Pichia pastoris under different oxygen availability conditions

    NARCIS (Netherlands)

    Carnicer, M.; Ten Pierick, A.; Van Dam, J.; Heijnen, J.J.; Albiol, J.; Van Gulik, W.; Ferrer, P.

    2012-01-01

    Background: Environmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies. Hypoxia has been shown to have a beneficial effect on the expression of recombinant proteins in Pichia pastoris growing on glucose.

  11. Classification of Recombinant Biologics in the EU

    DEFF Research Database (Denmark)

    Klein, Kevin; De Bruin, Marie L; Broekmans, Andre W

    2015-01-01

    BACKGROUND AND OBJECTIVE: Biological medicinal products (biologics) are subject to specific pharmacovigilance requirements to ensure that biologics are identifiable by brand name and batch number in adverse d