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Sample records for altered recombination specificity

  1. Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans

    OpenAIRE

    Romano, Patrick R.; Mackay, Andrew; Vong, Minh; deSa, Johann; Lamontagne, Anne; Comunale, Mary Ann; Hafner, Julie; Block, Timothy; Lec, Ryszard; Mehta, Anand

    2011-01-01

    Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and soluti...

  2. Site-Specific Recombination Strategies for Engineering Actinomycete Genomes

    OpenAIRE

    Herrmann, Simone; Siegl, Theresa; Luzhetska, Marta; Petzke, Lutz; Jilg, Caroline; Welle, Elisabeth; Erb, Annette; Leadlay, Peter F; Bechthold, Andreas; Luzhetskyy, Andriy

    2012-01-01

    The feasibility of using technologies based on site-specific recombination in actinomycetes was shown several years ago. Despite their huge potential, these technologies mostly have been used for simple marker removal from a chromosome. In this paper, we present different site-specific recombination strategies for genome engineering in several actinomycetes belonging to the genera Streptomyces, Micromonospora, and Saccharothrix. Two different systems based on Cre/loxP and Dre/rox have been ut...

  3. DNA Cloning Using In Vitro Site-Specific Recombination

    OpenAIRE

    Hartley, James L.; Temple, Gary F.; Brasch, Michael A.

    2000-01-01

    As a result of numerous genome sequencing projects, large numbers of candidate open reading frames are being identified, many of which have no known function. Analysis of these genes typically involves the transfer of DNA segments into a variety of vector backgrounds for protein expression and functional analysis. We describe a method called recombinational cloning that uses in vitro site-specific recombination to accomplish the directional cloning of PCR products and the subsequent automatic...

  4. 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.

  5. Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens.

    Science.gov (United States)

    Valenta, Rudolf; Linhart, B; Swoboda, I; Niederberger, V

    2011-06-01

    The broad applicability of allergen-specific immunotherapy for the treatment and eventually prevention of IgE-mediated allergy is limited by the poor quality and allergenic activity of natural allergen extracts that are used for the production of current allergy vaccines. Today, the genetic code of the most important allergens has been deciphered; recombinant allergens equalling their natural counterparts have been produced for diagnosis and immunotherapy, and a large panel of genetically modified allergens with reduced allergenic activity has been characterized to improve safety of immunotherapy and explore allergen-specific prevention strategies. Successful immunotherapy studies have been performed with recombinant allergens and hypoallergenic allergen derivatives and will lead to the registration of the first recombinant allergen-based vaccines in the near future. There is no doubt that recombinant allergen-based vaccination strategies will be generally applicable to most allergen sources, including respiratory, food and venom allergens and allow to produce safe allergy vaccines for the treatment of the most common forms of IgE-mediated allergies.

  6. Development of an EGFRvIII specific recombinant antibody

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    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC and immunofluorescence (IF and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as

  7. Alteration of the Specificity of PstⅠRestriction Endonuclease

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The influence of factors on the substrate-specificity of Pst Ⅰ restriction endonuclease has been studied with the method of electrophoresis. The results show that, the specificity of Pst Ⅰ almost can not be influenced by the single alteration of the concentration of Tris*HCl, Mg2+ or Na+ in the reaction system, but it can be altered by the reduction of any two of them. The specificity can not be altered by the single alteration of pH or the replacement of Mg2+ with Mn2+. The addition of glycerol or dimethylsulphoxide (DMSO) to the reaction system results in the relaxation of the substrate-specificity of Pst Ⅰ , but dimethylmethylformide, glycol and ethyl alcohol can not bring about the alteration of Pst Ⅰ specificity. Through the method of cloning and sequencing, the nucleotides of No.1 and 6 in the recognition sequence of Pst Ⅰ have changed (1C→A or 6G→T). Used with the enzyme analysis of an artificially synthetic DNA segment containing a special sequence, the nucleotides of No.1 and 6 have both changed (1C→A and 6G→T). The recognition sequence of Pst Ⅰ is speculated to be changed from CTGCA↓G to TGCA↓.

  8. An approach to mapping haplotype-specific recombination sites in human MHC class III

    Energy Technology Data Exchange (ETDEWEB)

    Levo, A.; Westman, P.; Partanen, J. [Finnish Red Cross Blood Transfusion Service, Helsinki (Finland)

    1996-12-31

    Studies of the major histocompatibility complex (MHC) in mouse indicate that the recombination sites are not randomly distributed and their occurrence is haplotype-dependent. No data concerning haplotype-specific recombination sites in human are available due to the low number of informative families. To investigate haplotype-specific recombination sites in human MHC, we describe an approach based on identification of recombinant haplotypes derived from one conserved haplotype at the population level. The recombination sites were mapped by comparing polymorphic markers between the recombinant and assumed original haplotypes. We tested this approach on the extended haplotype HLA A3; B47; Bf{sup *}F; C4A{sup *}1; C4B{sup *}Q0; DR7, which is most suitable for this analysis. First, it carries a number of rare markers, and second, the haplotype, albeit rare in the general population, is frequent in patients with 21-hydroxylase (21OH) defect. We observed recombinants derived from this haplotype in patients with 21OH defect. All these haplotypes had the centromeric part (from Bf to DR) identical to the original haplotype, but they differed in HLA A and B. We therefore assumed that they underwent recombinations in the segment that separates the Bf and HLA B genes. Polymorphic markers indicated that all break points mapped to two segments near the TNF locus. This approach makes possible the mapping of preferential recombination sites in different haplotypes. 20 refs., 1 fig., 1 tab.

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

    Science.gov (United States)

    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.

  10. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    Science.gov (United States)

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

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. 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 analysis7. In addition, we identified and characterized 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 found 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. PMID:26098369

  11. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    OpenAIRE

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

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. 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 commo...

  12. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    OpenAIRE

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

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing 1, 2 , the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM) 3–6 . 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 c...

  13. 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

  14. An Overview of Tyrosine Site-specific Recombination: From an Flp Perspective.

    Science.gov (United States)

    Jayaram, Makkuni; Ma, Chien-Hui; Kachroo, Aashiq H; Rowley, Paul A; Guga, Piotr; Fan, Hsui-Fang; Voziyanov, Yuri

    2015-08-01

    Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been employed to trace the topological features of DNA within high-order DNA interactions established by protein machines. The directed evolution of altered specificity YRs, combined with their spatially and temporally regulated expression, heralds their emergence as vital tools in genome engineering projects with wide-ranging biotechnological and medical applications. PMID:26350308

  15. Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells.

    Science.gov (United States)

    Onouchi, H; Yokoi, K; Machida, C; Matsuzaki, H; Oshima, Y; Matsuoka, K; Nakamura, K; Machida, Y

    1991-12-11

    Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.

  16. Targeted in vivo inhibition of specific protein-protein interactions using recombinant antibodies.

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    Matej Zábrady

    Full Text Available With the growing availability of genomic sequence information, there is an increasing need for gene function analysis. Antibody-mediated "silencing" represents an intriguing alternative for the precise inhibition of a particular function of biomolecules. Here, we describe a method for selecting recombinant antibodies with a specific purpose in mind, which is to inhibit intrinsic protein-protein interactions in the cytosol of plant cells. Experimental procedures were designed for conveniently evaluating desired properties of recombinant antibodies in consecutive steps. Our selection method was successfully used to develop a recombinant antibody inhibiting the interaction of ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 3 with such of its upstream interaction partners as the receiver domain of CYTOKININ INDEPENDENT HISTIDINE KINASE 1. The specific down-regulation of the cytokinin signaling pathway in vivo demonstrates the validity of our approach. This selection method can serve as a prototype for developing unique recombinant antibodies able to interfere with virtually any biomolecule in the living cell.

  17. Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    For the temporally and spatially regulated expression of the barnase gene in plant,two kinds of plasmids with cre gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure.The transgenic plants were conformed by PCR analysis.The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome.Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.

  18. Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana.

    Science.gov (United States)

    Onouchi, H; Nishihama, R; Kudo, M; Machida, Y; Machida, C

    1995-06-25

    Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

  19. Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

    Institute of Scientific and Technical Information of China (English)

    LIU Zuqiang; WANG Zuguang; CHEN Yinghua

    2005-01-01

    Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.

  20. 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.

  1. Trauma-specific Grey Matter Alterations in PTSD.

    Science.gov (United States)

    Meng, Linghui; Jiang, Jing; Jin, Changfeng; Liu, Jia; Zhao, Youjin; Wang, Weina; Li, Kaiming; Gong, Qiyong

    2016-01-01

    Previous studies have demonstrated that patients with posttraumatic stress disorder (PTSD) caused by different types of trauma may show divergence in epidemiology, clinical manifestation and treatment outcome. However, it is still unclear whether this divergence has neuroanatomic correlates in PTSD brains. To elucidate the general and trauma-specific cortical morphometric alterations, we performed a meta-analysis of grey matter (GM) changes in PTSD (N = 246) with different traumas and trauma-exposed controls (TECs, N = 347) using anisotropic effect-size signed differential mapping and its subgroup analysis. Our results revealed general GM reduction (GMR) foci in the prefrontal-limbic-striatal system of PTSD brains when compared with those of TECs. Notably, the GMR patterns were trauma-specific. For PTSD by single-incident traumas, GMR foci were found in bilateral medial prefrontal cortex (mPFC), anterior cingulate cortex (ACC), insula, striatum, left hippocampus and amygdala; and for PTSD by prolonged traumas in the left insula, striatum, amygdala and middle temporal gyrus. Moreover, Clinician-Administered PTSD Scale scores were found to be negatively associated with the GM changes in bilateral ACC and mPFC. Our study indicates that the GMR patterns of PTSD are associated with specific traumas, suggesting a stratified diagnosis and treatment for PTSD patients. PMID:27651030

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

    Science.gov (United States)

    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

  3. Gated rotation mechanism of site-specific recombination by ϕC31 integrase.

    Science.gov (United States)

    Olorunniji, Femi J; Buck, Dorothy E; Colloms, Sean D; McEwan, Andrew R; Smith, Margaret C M; Stark, W Marshall; Rosser, Susan J

    2012-11-27

    Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a "subunit rotation" mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid "phes" recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive "360° rotation" rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory "gating" mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round.

  4. 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.

  5. Construction of Marker-Free GFP Transgenic Tobacco by Cre/Iox Site-Specific Recombination System

    Institute of Scientific and Technical Information of China (English)

    SONG Hong-yuan; REN Xue-song; SI Jun; LI Cheng-qiong; SONG Ming

    2008-01-01

    Marker-free GFP transgenic tobacco plants were constructed based on Cre/lox site-specific recombination system.A GFP gene was introduced into the tobacco genome using the Bar gene as a linked selectable marker flanked by recombination sites in a directed orientation.The Bar gene expression box was subsequently excised from the plant genome by a strategy of Cre gene retransformation.After removal of the Cre-NPT H locus by genetic segregation through self-cross,plants that incorporated only the GFP transgene were obtained.Transgenic tobacco plants mediated by Agrobacterium tumefaciens were obtained,which resisted herbicide Basta and GFP expressed well,then the Cre gene was subsequently introduced into 5 plants of them,respectively,by retransformation.The leaf disks from Cre transgenic plants were used to test the resistance to Basta on the medium with 8 mg L-1 of PPT.The results showed that few discs were able to regenerate normally,and the excision at 76-100% efficiency depended on individual retransformation events.Evidence for a precise recombination event was confirmed by cloning the nucleotides sequence surrounding the lox sites of the Basts sensitive plants.The result indicated that the excision event in the recombination sites was precise and conservative,without loss or alteration of any submarginal nucleotides of the recombination sites.Bar gene excised plants were self-pollinated to allow segregation of the GFP gene from the Cre-NPT II locus.The progenies from self-pollinated plants were scored for Kan senstivity,then the segregation of GFP gene from Cre-NPT II locus in the Kan senstive plants were confirmed by PCR analysis subsequently.Hence,constructing marker-free transgenic tobacco plants by Cre/lox site-specific recombination system was reliable,and the strategy presented here should be applicable to other plants for the construction of marker-free transgenic plants as well.

  6. Recombinant methods and materials

    Energy Technology Data Exchange (ETDEWEB)

    Roizman, B.; Post, L.E.

    1988-09-06

    This patent describes a method for stably effecting the insertion or deletion of a selected DNA sequence at a specific site in a viral genome. The method consists of: (1) isolating from the genome a linear DNA fragment comprising both (a) the specific site determined for insertion or deletion of selected DNA sequence and (b) flanking DNA sequences normally preceding and following the site; (2) preparing first and second altered genome fragments from the fragment isolated in step (1). (a) the first altered fragment comprising the fragment comprising a thymidine kinase gene in a position intermediate the ends of the fragment, and (b) the second altered fragment comprising the fragment having the selected DNA sequence inserted therein or deleted therefrom; (3) contacting the genome with the first altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome comprising the thymidine kinase gene, and isolating the recombinant genome; and (4) contacting the recombinant genome isolated in step (3) with the second altered fragment under conditions permitting recombination at sites of DNA sequence homology, selecting for a recombinant genome lacking the thymidine kinase gene, and isolating the recombinant genome product.

  7. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III

    Science.gov (United States)

    Richard-Fogal, Cynthia L.; Francisco, Brian San; Frawley, Elaine R.; Kranz, Robert G.

    2011-01-01

    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E.coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E.coli periplasmic DsbC/DsbD thiol-reduction pathway. PMID:21945855

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

    Science.gov (United States)

    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.

  9. Producing Recombinant mTEX101; a Murine Testis Specific Protein

    Science.gov (United States)

    Barzegar Yarmohammadi, Leila; Modarresi, Mohammad Hossein; Talebi, Saeed; Hadavi, Reza; Ostad Karampour, Mahyar; Mahmoudi, Ahmad Reza; Akhondi, Mohammad Mehdi; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2009-01-01

    Introduction Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous. Materials and Methods RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a (+). The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 (DE3) E. coli strain. Results A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies. Conclusion We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies. PMID:23926468

  10. Producing Recombinant mTEX101; a Murine Testis Specific Protein

    OpenAIRE

    Barzegar Yarmohammadi, Leila; Modarresi, Mohammad Hossein; Talebi, Saeed; Hadavi, Reza; Ostad Karampour, Mahyar; Mahmoudi, Ahmad Reza; Akhondi, Mohammad mehdi; Rabbani, Hodjattallah; Jeddi-Tehrani, Mahmood

    2009-01-01

    Introduction Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 (mTEX101) is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and fun...

  11. [Recombinant allergens for diagnosis and specific immunotherapy--value in pediatric patients].

    Science.gov (United States)

    Couderc, Rémy; Just, Jocelyne

    2013-03-01

    Identification of culprit allergens is important for prophylactic measures and specific allergen immunotherapy (SIT). Since the late 1980s, the use of molecular cloning technology has led to a major improvement in our knowledge of epitopes involved in IgE-mediated allergy, and has also allowed in vitro production of recombinant allergens of interest for the diagnosis of allergenic sensitization. It has also improved our understanding of allergen cross-reactivity, which can be responsible for severe clinical manifestations, particularly in children with food allergy and allergic asthma. Better knowledge of molecular and cellular mechanisms of allergenic sensitization, based on the use of natural or modified recombinant allergens, has led to the development of effective SIT strategies which, in the foreseeable future, could provide genuine cure, therefore avoiding use of symptomatic therapeutics, starting very early in childhood. PMID:25163347

  12. Transgene expression in Penaeus monodon cells: evaluation of recombinant baculoviral vectors with shrimp specific hybrid promoters.

    Science.gov (United States)

    Puthumana, Jayesh; Philip, Rosamma; Bright Singh, I S

    2016-08-01

    It has been realized that shrimp cell immortalization may not be accomplished without in vitro transformation by expressing immortalizing gene in cells. In this process, efficiency of transgene expression is confined to the ability of vectors to transmit gene of interests to the genome. Over the years, unavailability of such vectors has been hampering application of such a strategy in shrimp cells. We report the use of recombinant baculovirus mediated transduction using hybrid promoter system for transgene expression in lymphoid cells of Penaeus monodon. Two recombinant baculovirus vectors with shrimp viral promoters (WSSV-Ie1 and IHHNV-P2) were constructed (BacIe1-GFP and BacP2-GFP) and green fluorescent protein (GFP) used as the transgene. The GFP expression in cells under the control of hybrid promoters, PH-Ie1 or PH-P2, were analyzed and confirmed in shrimp cells. The results indicate that the recombinant baculovirus with shrimp specific viral promoters (hybrid) can be employed for delivery of foreign genes to shrimp cells for in vitro transformation.

  13. Preparation of a recombinant adenoviral encoding human NIS gene and its specific expression in cardiomyocytes

    International Nuclear Information System (INIS)

    Objective: To construct a recombinant adenovirus vector containing the human NIS gene with the myosin light chain-2(MLC-2v) gene as the promoter and evaluate its specific expression and feasibility as a reporter gene in cardiomyocytes. Methods: MLC-2v promoter and NIS were subcloned into an adenovirus shuttle vector, and forwarded by homologous recombination in the bacteria BJ5183 containing AdEasy-1 plasmid. Positive recombinant adenovirus vector was selected, packaged and amplified in the HEK293 cells to obtain recombinant adenovirus Ad-MLC-NIS. Ad-cytomegalovirus (CMV)-NIS with cytomegalovirus as the promoter, Ad-MLC without NIS and Ad-NIS without promoter were constructed as the controls. Cardiomyocytes and non-cardiomyocytes were then infected by the adenovirus. The protein expression was tested by Western blot analysis. The function and features of NIS protein were evaluated by dynamic iodide uptake and NaClO4 iodine uptake inhibition test in vitro. The viability and proliferation of cardiomyocytes after adenovirus transfection and radioiodine incubation were checked by trypan blue staining. Results: Recombinant NIS adenovirus was successfully constructed. Western blot analysis showed that the NIS protein was highly expressed in cardiomyocytes transfected with Ad-MLC-NIS, and all cells transfected with Ad-CMV-NIS. However, in non-cardiomyocytes transfected with Ad-MLC-NIS, little NIS protein was detected. Dynamic iodine uptake tests showed that the peaks of iodide uptake of the three different cell lines (H9C2, A549, U87 cell) transfected with Ad-MLC-NIS were 5844.0, 833.6 and 846.0 counts · min-1, respectively. The iodide uptake function of H9C2 was inhibited by NaClO4. There was almost no change in cell viability and proliferation when the MOI was 100. Conclusions: Ad-MLC-NIS allows myocardial specific expression of an external gene, and the cardiomyocytes with NIS expression are capable of iodine uptake. Further research of NIS as a reporter gene in

  14. 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

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perfor...

  15. Protein-protein interactions in a higher-order structure direct lambda site-specific recombination.

    Science.gov (United States)

    Thompson, J F; de Vargas, L M; Skinner, S E; Landy, A

    1987-06-01

    The highly directional site-specific recombination of bacteriophage lambda is tightly regulated by the binding of three different proteins to a complex array of sites. The manner in which these reactions are both stimulated and inhibited by co-operative binding of proteins to specific sites on the P arm of attP and AttR has been elucidated by correlation of nuclease protection with recombination studies of both wild-type and mutant DNAs. In addition to co-operative forces, there is a specific competitive interaction that allows the protein-DNA complex to serve as a "biological switch". This switch does not depend upon the simple occlusion of DNA binding sites by neighboring proteins; but, rather, the outcome of this competition is dependent on long-range interactions that vary between the higher-order structures of attP and attR. These higher-order structures are dependent on cooperative interactions involving three proteins binding to five or more sites. PMID:2958633

  16. 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

  17. ALTERATION OF GENE EXPRESSION IN LEUKOCYTES FROM RECOMBINANT SOMATOTROPIN TREATED ANIMALS: SEARCHING FOR INSPECTION INDICATORS

    Directory of Open Access Journals (Sweden)

    NR Brizioli

    2008-12-01

    Full Text Available Besides immunochemical approaches, biomolecular studies can be carried out in order to discover a greater number of biological indicators to be exploited for the identification of bovines treated with recombinant somatotropin (rbST. With this aim, we analysed the expression of a number of genes related to the somatotropic axis in leucocytes from rbST treated cows and non-treated animals. Significant differences were observed in the genes IGF-1,IGFBP-1, IGFBP-4 and the I- 5’UTR variant of the GHR gene.

  18. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    Energy Technology Data Exchange (ETDEWEB)

    X. Li; A.M. Kriegel; T.C. Bishop; R.C. Blake; E. Figueiredo; H. Yu; D.A. Blake

    2005-04-18

    The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO{sub 2}{sup 2+} was used as a source of RNA for the development of a recombinant (Fab){sub 2} fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire {kappa} light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab){sub 2} protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab){sub 2} fragments that bound to the UO{sub 2}{sup 2+}-DCP complex with an affinity indistinguishable from that of a (Fab){sub 2} fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the

  19. Development of a Recombinant Antibody with Specificity for Chelated Uranyl Ions

    International Nuclear Information System (INIS)

    The goal of our project is to continue the development of new techniques for rapid, automated identification of radionuclides, metals, and chelators that may contaminant sur face and groundwater at DOE sites. One of the four specific aims of the present project is to develop new technologies in antibody engineering that will enhance our immunosensor program. Recombinant antibodies have potential advantages over monoclonal antibodies produced by standard hybridoma technology. The cloned genes represent a stable, recoverable source for antibody production. In addition, the recombinant format offers opportunities for protein engineering that enhances antibody performance and for studies that relate antibody sequence to binding activity. In this study, a hybridoma that synthesized an antibody (12F6) that recognized a 1:1 complex between 2,9-dicarboxyl-1,10- phenanthroline (DCP) and UO22+ was used as a source of RNA for the development of a recombinant (Fab)2 fragment. RNA was isolated from the 12F6 hybridoma and the cDNA encoding the entire κ light chain and the linked VH and C1 portions of the heavy chain were amplified from total RNA. cDNA sequences were verified by comparison with the N-terminal amino acid sequences of the light and heavy chains of the native 12F6 monoclonal antibody. A leader sequence and appropriate restriction sites were added to each chain, and the fragments were ligated into a commercial dicistronic vector (pBudCE4.1, Invitrogen, Inc.). COS-1 cells were transfected with this vector and the culture supernatant was assayed for activity and the (Fab)2 protein. Cells transfected with vector containing 12F6 cDNA synthesized and secreted recombinant (Fab)2 fragments that bound to the UO22+-DCP complex with an affinity indistinguishable from that of a (Fab)2 fragment prepared from the native antibody. Molecular models of the heavy and light chain variable domains were constructed according to the canonical structures method detailed by Morea et al

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

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available 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.

  1. 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

    in vitro T cell stimulation was investigated. By use of an antigenic peptide derived from the cytomegalovirus (CMVp) we tested the stimulatory efficacy of recombinant plate bound MHC molecules (PB-MHC), being immobilized in culture plates. A single stimulation of non-adherent peripheral blood...... effect of new stimulatory cocktails, e.g. cytokines and co-stimulatory molecules, by use of the present rapid and easy-to-use method of expanding peptide specific T cells.......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 to...

  2. Fanca deficiency reduces A/T transitions in somatic hypermutation and alters class switch recombination junctions in mouse B cells.

    Science.gov (United States)

    Nguyen, Thuy Vy; Riou, Lydia; Aoufouchi, Saïd; Rosselli, Filippo

    2014-06-01

    Fanconi anemia is a rare genetic disorder that can lead to bone marrow failure, congenital abnormalities, and increased risk for leukemia and cancer. Cells with loss-of-function mutations in the FANC pathway are characterized by chromosome fragility, altered mutability, and abnormal regulation of the nonhomologous end-joining (NHEJ) pathway. Somatic hypermutation (SHM) and immunoglobulin (Ig) class switch recombination (CSR) enable B cells to produce high-affinity antibodies of various isotypes. Both processes are initiated after the generation of dG:dU mismatches by activation-induced cytidine deaminase. Whereas SHM involves an error-prone repair process that introduces novel point mutations into the Ig gene, the mismatches generated during CSR are processed to create double-stranded breaks (DSBs) in DNA, which are then repaired by the NHEJ pathway. As several lines of evidence suggest a possible role for the FANC pathway in SHM and CSR, we analyzed both processes in B cells derived from Fanca(-/-) mice. Here we show that Fanca is required for the induction of transition mutations at A/T residues during SHM and that despite globally normal CSR function in splenic B cells, Fanca is required during CSR to stabilize duplexes between pairs of short microhomology regions, thereby impeding short-range recombination downstream of DSB formation. PMID:24799500

  3. Detection of Q Fever Specific Antibodies Using Recombinant Antigen in ELISA with Peroxidase Based Signal Amplification

    Directory of Open Access Journals (Sweden)

    Hua-Wei Chen

    2014-01-01

    Full Text Available Currently, the accepted method for Q fever serodiagnosis is indirect immunofluorescent antibody assay (IFA using the whole cell antigen. In this study, we prepared the recombinant antigen of the 27-kDa outer membrane protein (Com1 which has been shown to be recognized by Q fever patient sera. The performance of recombinant Com1 was evaluated in ELISA by IFA confirmed serum samples. Due to the low titers of IgG and IgM in Q fever patients, the standard ELISA signals were further amplified by using biotinylated anti-human IgG or IgM plus streptavidin-HRP polymer. The modified ELISA can detect 88% (29 out of 33 of Q fever patient sera collected from Marines deployed to Iraq. Less than 5% (5 out of 156 of the sera from patients with other febrile diseases reacted with the Com1. These results suggest that the modified ELISA using Com1 may have the potential to improve the detection of Q fever specific antibodies.

  4. Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LCs0 value of 18.4 uM and 0.70 μM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 μM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3μM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

  5. Different thermostabilities of FLP and Cre recombinases: implications for applied site-specific recombination.

    Science.gov (United States)

    Buchholz, F; Ringrose, L; Angrand, P O; Rossi, F; Stewart, A F

    1996-11-01

    Genomic manipulations using site-specific recombinases rely on their applied characteristics in living systems. To understand their applied properties so that they can be optimally deployed, we compared the recombinases FLP and Cre in two assays. In both Escherichia coli and in vitro, FLP shows a different temperature optimum than Cre. FLP is more thermolabile, having an optimum near 30 degrees C and little detectable activity above 39 degrees C. Cre is optimally efficient at 37 degrees C and above. Consistent with FLP thermolability, recombination in a mammalian cell line mediated by a ligand- regulated FLP-androgen receptor fusion protein is more efficient at 35 degrees C than at higher temperatures. We also document a mutation in a commercially available FLP plasmid (FLP-F70L) which renders this recombinase even more thermolabile. The different temperature optima of FLP, FLP-F70L and Cre influence their strategies of usage. Our results recommend the use of Cre for applications in mice that require efficient recombination. The thermolabilities of FLP and FLP-F70L can be usefully exploited for gain of function and cell culture applications.

  6. Recombinant Human Leptin Does Not Alter Gut Hormone Levels after Gastric Bypass but May Attenuate Sweet Cravings

    Directory of Open Access Journals (Sweden)

    Rushika Conroy

    2014-01-01

    Full Text Available Bariatric surgery improves glucose homeostasis and alters gut hormones partly independent of weight loss. Leptin plays a role in these processes; levels are decreased following bariatric surgery, creating a relative leptin insufficiency. We previously showed that leptin administration in a weight-reduced state after Roux-en-Y gastric bypass (RYGB caused no further weight loss. Here, we discuss the impact of leptin administration on gut hormones, glucostasis, and appetite. Weight stable women after RYGB were randomized to receive placebo or recombinant human metreleptin (0.05 mg/kg twice daily. At weeks 0 and 16, a liquid meal challenge was performed. Glucose, insulin, C-peptide, GLP-1, PYY, glucagon, and ghrelin (total, acyl, and desacyl were measured fasting and postprandially. Appetite was assessed using a visual analog scale. Mean post-op period was 53±2.3 months; mean BMI was 34.6±0.2 kg/m2. At 16 weeks, there was no significant change in weight within or between groups. Fasting PYY was significantly different between groups and the leptin group had lower sweets craving at week 16 than the placebo group (P<0.05. No other differences were observed. Leptin replacement does not alter gut hormones or glucostasis but may diminish sweet cravings compared to placebo in this population of post-RYGB women.

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

    Directory of Open Access Journals (Sweden)

    Thuc-vy L Le

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS: 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.

  8. 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...

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Enhanced cell-permeant Cre protein for site-specific recombination in cultured cells

    Directory of Open Access Journals (Sweden)

    Ruley H Earl

    2004-10-01

    Full Text Available Abstract Background Cell-permeant Cre DNA site-specific recombinases provide an easily controlled means to regulate gene structure and function in living cells. Since recombination provides a stable and unambiguous record of protein uptake, the enzyme may also be used for quantitative studies of cis- and trans-acting factors that influence the delivery of proteins into cells. Results In the present study, 11 recombinant fusion proteins were analyzed to characterize sequences and conditions that affect protein uptake and/or activity and to develop more active cell-permeant enzymes. We report that the native enzyme has a low, but intrinsic ability to enter cells. The most active Cre proteins tested contained either an N-terminal 6xHis tag and a nuclear localization sequence from SV40 large T antigen (HNC or the HIV Tat transduction sequence and a C-terminal 6xHis tag (TCH6. The NLS and 6xHis elements separately enhanced the delivery of the HNC protein into cells; moreover, transduction sequences from fibroblast growth factor 4, HIV Tat or consisting of the (KFF3K sequence were not required for efficient protein transduction and adversely affected enzyme solubility. Transduction of the HNC protein required 10 to 15 min for half-maximum uptake, was greatly decreased at 4°C and was inhibited by serum. Efficient recombination was observed in all cell types tested (a T-cell line, NIH3T3, Cos7, murine ES cells, and primary splenocytes, and did not require localization of the enzyme to the nucleus. Conclusions The effects of different sequences on the delivery and/or activity of Cre in cultured cells could not be predicted in advance. Consequently, the process of developing more active cell-permeant recombinases was largely empirical. The HNC protein, with an excellent combination of activity, solubility and yield, will enhance the use of cell-permeant Cre proteins to regulate gene structure and function in living cells.

  12. Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

    Science.gov (United States)

    Modi, Souvik; Halder, Saheli; Nizak, Clément; Krishnan, Yamuna

    2013-12-01

    DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing

  13. A pathogen-specific epitope inserted into recombinant secretory immunoglobulin A is immunogenic by the oral route.

    Science.gov (United States)

    Corthésy, B; Kaufmann, M; Phalipon, A; Peitsch, M; Neutra, M R; Kraehenbuhl, J P

    1996-12-27

    Oral administration of rabbit secretory IgA (sIgA) to adult BALB/c mice induced IgA+, IgM+, and IgG+ lymphoblasts in the Peyer's patches, whose fusion with myeloma cells resulted in hybridomas producing IgA, IgM, and IgG1 antibodies to the secretory component (SC). This suggests that SC could serve as a vector to target protective epitopes into mucosal lymphoid tissue and elicit an immune response. We tested this concept by inserting a Shigella flexneri invasin B epitope into SC, which, following reassociation with IgA, was delivered orally to mice. To identify potential insertion sites at the surface of SC, we constructed a molecular model of the first and second Ig-like domains of rabbit SC. A surface epitope recognized by an SC-specific antibody was mapped to the loop connecting the E and F beta strands of domain I. This 8-amino acid sequence was replaced by a 9-amino acid linear epitope from S. flexneri invasin B. We found that cellular trafficking of recombinant SC produced in mammalian CV-1 cells was drastically altered and resulted in a 50-fold lower rate of secretion. However, purification of chimeric SC could be achieved by Ni2+-chelate affinity chromatoraphy. Both wild-type and chimeric SC bound to dimeric IgA, but not to monomeric IgA. Reconstituted sIgA carrying the invasin B epitope within the SC moiety triggers the appearance of seric and salivary invasin B-specific antibodies. Thus, neo-antigenized sIgA can serve as a mucosal vaccine delivery system inducing systemic and mucosal immune responses. PMID:8969237

  14. 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.

  15. 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. PMID:27378621

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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

  18. Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.

    OpenAIRE

    Yu, A.; Haggård-Ljungquist, E

    1993-01-01

    Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both t...

  19. Species-specific antibody responses to the recombinant 53-kilodalton excretory and secretory proteins in mice infected with Trichinella spp.

    Science.gov (United States)

    Nagano, Isao; Wu, Zhiliang; Takahashi, Yuzo

    2008-03-01

    The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp. PMID:18184826

  20. Specific IgE response to purified and recombinant allergens in latex allergy

    Directory of Open Access Journals (Sweden)

    Arif Siti AM

    2005-08-01

    Full Text Available Abstract Background In recent years, allergy to natural rubber latex has emerged as a major allergy among certain occupational groups and patients with underlying diseases. The sensitization and development of latex allergy has been attributed to exposure to products containing residual latex proteins. Although improved manufacturing procedures resulted in a considerable reduction of new cases, the potential risk for some patient groups is still great. In addition the prevalent cross-reactivity of latex proteins with other food allergens poses a major concern. A number of purified allergens and a few commercial kits are currently available, but no concerted effort was undertaken to evaluate them. Methods We studied 11 purified latex allergens, Hev b 1 to Hev b 10, and Hev b 13 along with several crude allergen extracts and two commercial ImmunoCAP assays to evaluate specific IgE antibody in the sera from latex allergic patients and controls. Health care workers and spina bifida patients with clinical symptoms of latex allergy, spina bifida patients without latex allergy, and non-atopic health care workers have been studied. Results The results suggest that Hev b 2, 5, 6, and 13 together identified over 80 percent health care workers with latex allergy, while Hev b 6 along with Hev b 1 or 3 detected specific IgE antibody in all sera studied from patients with spina bifida and latex allergy. The ImmunoCAP results using both Hev b 5 amplified and non-amplified closely agreed with the clinical diagnosis of latex allergy in health care workers and in spina bifida. Conclusion Although the purified allergens and crude extracts reacted diversely with IgE from different patient groups, the results indicated that use of certain combinations of purified recombinant antigens will be useful in commercial kits or in in-house assays for detecting specific IgE antibody in the sera. The results suggest that a combination of Hev b 2, 3, 5, 6, and 13 together

  1. Generation of recombinant porcine parvovirus virus-like particles in Saccharomyces cerevisiae and development of virus-specific monoclonal antibodies.

    Science.gov (United States)

    Tamošiūnas, Paulius Lukas; Petraitytė-Burneikienė, Rasa; Lasickienė, Rita; Akatov, Artiomas; Kundrotas, Gabrielis; Sereika, Vilimas; Lelešius, Raimundas; Žvirblienė, Aurelija; Sasnauskas, Kęstutis

    2014-01-01

    Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance.

  2. Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III

    OpenAIRE

    Richard-Fogal, Cynthia L; Francisco, Brian San; Frawley, Elaine R.; Kranz, Robert G.

    2011-01-01

    The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA...

  3. Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules.

    Science.gov (United States)

    Ahlgren, Sara; Orlova, Anna; Rosik, Daniel; Sandström, Mattias; Sjöberg, Anna; Baastrup, Barbro; Widmark, Olof; Fant, Gunilla; Feldwisch, Joachim; Tolmachev, Vladimir

    2008-01-01

    Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for 111 In-labeling of anti-HER2 Affibody molecules His 6-Z HER2:342-Cys and Z HER2:2395-Cys has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for His 6-Z HER2:342-Cys and 27 pM for Z HER2:2395-Cys, comparable with 22 pM for the parental Z HER2:342. MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with 111 In to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non-His-tagged variant 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys demonstrated appreciably lower liver uptake than its His-tag-containing counterpart. In mice bearing HER2-expressing LS174T xenografts, 111 In-[MMA-DOTA-Cys61]-Z HER2:2395-Cys showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i. PMID:18163536

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    Science.gov (United States)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-07-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  6. Specific targeting of infectious foci with radioiodinated human recombinant interleukin-1 in an experimental model

    International Nuclear Information System (INIS)

    In the present study, radioiodinated human recombinant interleukin-1 (IL-1) was investigated for its potential to image infectious foci in vivo in an animal model of infection. Twenty-four hours after induction of a Staphylococcus aureus abscess in the left calf muscle, mice were i.v. injected with both iodine-125 labelled IL-1 and iodine-131 labelled myoglobin, a size-matched control agent. The animals were killed for tissue biodistribution studies at 2, 6, 12, 24 and 48 h p.i. Gamma camera images were obtained at 6, 24 and 48 h after injecting mice with 123I-IL-1. Radioiodinated IL-1 rapidly cleared from the body; after 12 h the abscess was the organ with the highest activity. The absolute abscess uptake of 125I-IL-1 remained high compared to 131I-myoglobin, resulting in significantly higher abscess-to-muscle ratios of 125I-IL-1 compared to 131I-myoglobin. The ratios of 125I-IL-1 reached the ultimate value of 44.4 ± 10.8 at 48 h p.i., whereas the ratios of 131I-myoglobin did not exceed 5.9 ± 0.7. Gamma camera imaging revealed clearly visible abscesses. In conclusion, our results demonstrate specific retention of radioiodinated IL-1 in the abscess, presumably by interaction of IL-1 with its receptor on the inflammatory cells. The high target-to-background ratios that were obtained over the course of time indicate that the IL-1 receptor may be a valuable target for the imaging of infectious foci. (orig.)

  7. 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 heteroal...

  8. Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.

    Science.gov (United States)

    Vega, Juan M; Yu, Weichang; Han, Fangpu; Kato, Akio; Peters, Eric M; Zhang, Zhanyuan J; Birchler, James A

    2008-04-01

    The Cre/loxP site-specific recombination system has been applied in various plant species including maize (Zea mays) for marker gene removal, gene targeting, and functional genomics. A BIBAC vector system was adapted for maize transformation with a large fragment of genetic material including a herbicide resistance marker gene, a 30 kb yeast genomic fragment as a marker for fluorescence in situ hybridization (FISH), and a 35S-lox-cre recombination cassette. Seventy-five transgenic lines were generated from Agrobacterium-mediated transformation of a maize Hi II line with multiple B chromosomes. Eighty-four inserts have been localized among all 10 A chromosome pairs by FISH using the yeast DNA probe together with a karyotyping cocktail. No inserts were found on the B chromosomes; thus a bias against the B chromosomes by the Agrobacterium-mediated transformation was revealed. The expression of a cre gene was confirmed in 68 of the 75 transgenic lines by a reporter construct for cre/lox mediated recombination. The placement of the cre/lox site-specific recombination system in many locations in the maize genome will be valuable materials for gene targeting and chromosome engineering.

  9. 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. PMID:27393468

  10. Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

    Institute of Scientific and Technical Information of China (English)

    Hyun Jung Park; Won Young Lee; Jin Hoi Kim; Jae Hwan Kim; Hun Jong Jung; Nam Hyung Kim; Bo Kyung Kim; Hyuk Song

    2009-01-01

    The purpose of this study is to establish a gene delivery system for interstitial tissue-specific protein expression in mice testes using modified recombinant baculovirus. Green fluorescent protein (GFP)-expressing recombinant bacuiovirus (GFP-baculovirus), in which the insect cell-specific polyhedron promoter was replaced by the cytomegalovirus (CMV)-IE promoter, was used to transfect testicular cells in vitro, and for intra-tunica albuguineai injection of the interstitial tissue of the testis. GFP expression was monitored in frozen testes sections by fluorescence microscopy. Expression of GFP in testicular tissues was also assessed by reverse transcription polymerase chain reaction (RT-PCR), and protein expression was assessed by Western blot. Testicular cells in vitro were infected efficiently by modified recombinant GFP-baculovirus. Intra-tunica albuguineal injection of GFP-baculovirus into the mouse testis resulted in a high level of GFP expression in the interstitial tissues. RT-PCR analysis clearly showed GFP gene expression in the testis, particularly interstitial tissues. Intra-tunica albuguineal injection of a modified baculovirus that encoded recombinant rat insulin-like growth factor binding protein (IGFBP)-5 resulted in an increase in IGFBP-5 in testis and semen. In conclusion, we have developed an efficient delivery system for gene expression in vivo in testicular cells, particularly cells of the interstitial tissue using intra-tunica albuguineal injection of a modified recombinant baculovirus. This method will be particularly relevant for application that requires gene delivery and protein expression in the testicular cells of the outer seminiferous tubule of the testis.

  11. Cattle sex-specific recombination and genetic control from a very large pedigree

    Science.gov (United States)

    Meiotic recombination is an essential biological process that generates novel genetic variants and ensures proper segregation of chromosomes during meiosis. From a large USDA dairy cattle pedigree with over half million genotyped animals, we extracted 186,927 three-generation families, identified ov...

  12. Specific features of radiative recombination spectra of ZnIn2S4 single crystals

    International Nuclear Information System (INIS)

    Radiative recombination spectra of Mn-doped zinc tioindat single crystals have been analyzed in the work. The emission spectra interval close to its maximum (1.91 ± 0.2 eV) contains a number of special features which were identified by us as intra-center transitions. We attribute the special features observed on the complex emission spectra to this type of transition by their decomposition into simple lines using the Alentsev-Foch method. (authors)

  13. Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

    OpenAIRE

    Larionov, V.; Kouprina, N; Graves, J.; Chen, X N; Korenberg, J R; Resnick, M A

    1996-01-01

    DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized v...

  14. 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

    The genetic information of retroviruses is retained within a dimeric RNA genome held together by intermolecular RNA-RNA interactions near the 5' ends. Coencapsidation of retrovirus-derived RNA molecules allows frequent template switching of the virus-encoded reverse transcriptase during DNA...... 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...... 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...

  15. Role of the beta subunit of casein kinase-2 on the stability and specificity of the recombinant reconstituted holoenzyme

    DEFF Research Database (Denmark)

    Meggio, F; Boldyreff, B; Marin, O;

    1992-01-01

    Recombinant human alpha subunit from casein kinase-2 (CK-2) was subjected, either alone or in combination with recombinant human beta subunit, to high temperature, tryptic digestion and urea treatment. In all three cases, it was shown that the presence of the beta subunit could drastically reduce...... alpha subunit. Therefore, a dual function for the beta subunit is proposed which confers both specificity and stability to the catalytic alpha subunit within the CK-2 holoenzyme complex. The peptide DLEPDEELEDNPNQSDL, reproducing the highly acidic amino acid 55-71 segment of the human beta subunit......, counteracts the stimulatory effect of the beta subunit on the alpha subunit activity and partially substitutes the beta subunit in conferring thermal stability to the alpha subunit. No such effect is induced by the peptide MSSSEEVSW, reproducing the N-terminal segment of the beta subunit including...

  16. Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

    Directory of Open Access Journals (Sweden)

    Greta A Van Slyke

    2015-06-01

    Full Text Available High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i demonstrate the importance of allosteric interactions in regulating mutation rates, (ii establish that mutational spectra can be both sequence and strain-dependent, and (iii display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

  17. Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subse...

  18. Overlapping yet response-specific transcriptome alterations characterize the nature of tobacco-Pseudomonas syringae interactions

    Directory of Open Access Journals (Sweden)

    Zoltán eBozsó

    2016-03-01

    Full Text Available In this study transcriptomic alterations of bacterially induced pattern triggered immunity (PTI were compared with other types of tobacco-Pseudomonas interactions. In addition, using pharmacological agents we blocked some signal transduction pathways (Ca2+ influx, kinases, phospholipases, proteasomic protein degradation to find out how they contribute to gene expression during PTI. PTI is the first defense response of plant cells to microbes, elicited by their widely conserved molecular patterns. Tobacco is an important model of Solanaceae to study resistance responses, including defense mechanisms against bacteria. In spite of these facts the transcription regulation of tobacco genes during different types of plant bacterial interactions is not well described. In this paper we compared the tobacco transcriptomic alterations in microarray experiments induced by (i PTI inducer Pseudomonas syringae pv. syringae type III secretion mutant (hrcC at earlier (6 hours post inoculation and later (48 hpi stages of defense, (ii wild type P. syringae (6 hpi that causes effector triggered immunity (ETI and cell death (HR and (iii disease-causing Pseudomonas syringae pv. tabaci (6 hpi. Among the different treatments the highest overlap was between the PTI and ETI at 6 hpi, however, there were groups of genes with specifically altered activity for either type of defenses. Instead of quantitative effects of the virulent P. tabaci on PTI-related genes it influenced transcription qualitatively and blocked the expression changes of a special set of genes including ones involved in signal transduction and transcription regulation. P. tabaci specifically activated or repressed other groups of genes seemingly not related to either PTI or ETI.Kinase and phospholipase A inhibitors had highest impacts on the PTI response and effects of signal inhibitors on transcription greatly overlapped. Remarkable interactions of phospholipase C-related pathways with the proteasomal

  19. Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

    OpenAIRE

    Dahmen, Volker; Kriehuber, Ralf

    2012-01-01

    Purpose: Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of ...

  20. Altered Specificity of DNA-Binding Proteins with Transition Metal Dimerization Domains

    Science.gov (United States)

    Cuenoud, Bernard; Schepartz, Alanna

    1993-01-01

    The bZIP motif is characterized by a leucine zipper domain that mediates dimerization and a basic domain that contacts DNA. A series of transition metal dimerization domains were used to alter systematically the relative orientation of basic domain peptides. Both the affinity and the specificity of the peptide-DNA interaction depend on domain orientation. These results indicate that the precise configuration linking the domains is important; dimerization is not always sufficient for DNA binding. This approach to studying the effect of orientation on protein function complements mutagenesis and could be used in many systems.

  1. Design of Deinococcus radiodurans thioredoxin reductase with altered thioredoxin specificity using computational alanine mutagenesis

    OpenAIRE

    Obiero, Josiah; Sanders, David AR

    2011-01-01

    In this study, the X-ray crystal structure of the complex between Escherichia coli thioredoxin reductase (EC TrxR) and its substrate thioredoxin (Trx) was used as a guide to design a Deinococcus radiodurans TrxR (DR TrxR) mutant with altered Trx specificity. Previous studies have shown that TrxRs have higher affinity for cognate Trxs (same species) than that for Trxs from different species. Computational alanine scanning mutagenesis and visual inspection of the EC TrxR–Trx interface suggested...

  2. An African horse sickness virus serotype 4 recombinant canarypox virus vaccine elicits specific cell-mediated immune responses in horses.

    Science.gov (United States)

    El Garch, H; Crafford, J E; Amouyal, P; Durand, P Y; Edlund Toulemonde, C; Lemaitre, L; Cozette, V; Guthrie, A; Minke, J M

    2012-09-15

    A recombinant canarypox virus vectored vaccine co-expressing synthetic genes encoding outer capsid proteins, VP2 and VP5, of African horse sickness virus (AHSV) serotype 4 (ALVAC(®)-AHSV4) has been demonstrated to fully protect horses against homologous challenge with virulent field virus. Guthrie et al. (2009) detected weak and variable titres of neutralizing antibody (ranging from horses received two vaccinations twenty-eight days apart and three horses remained unvaccinated. The detection of VP2/VP5 specific IFN-γ responses was assessed by enzyme linked immune spot (ELISpot) assay and clearly demonstrated that all ALVAC(®)-AHSV4 vaccinated horses developed significant IFN-γ production compared to unvaccinated horses. More detailed immune responses obtained by flow cytometry demonstrated that ALVAC(®)-AHSV4 vaccinations induced immune cells, mainly CD8(+) T cells, able to recognize multiple T-epitopes through all VP2 and only the N-terminus sequence of VP5. Neither VP2 nor VP5 specific IFN-γ responses were detected in unvaccinated horses. Overall, our data demonstrated that an experimental recombinant canarypox based vaccine induced significant CMI specific for both VP2 and VP5 proteins of AHSV4.

  3. Renal and hepatotoxic alterations in adult mice on inhalation of specific mixture of organic solvents.

    Science.gov (United States)

    Ketan, Vaghasia K; Bhavyata, Kalariya; Linzbuoy, George; Hyacinth, Highland N

    2015-12-01

    This study was aimed at investigating alterations in renal and hepatic toxicity induced by exposing to a combination of three solvents, namely, benzene, toluene and xylene in adult mice. The mice were divided into three groups (control, low-dose-treated (450 ppm) and high-dose (675 ppm) groups) using randomization methods. The treated groups were exposed to vapours of a mixture of benzene, toluene and xylene at doses of 450 and 675 ppm, for 6 h day(-1) for a short-term of 7-day exposure period. The study revealed that the solvent exposure resulted in an increase in the weight of liver and kidney as compared to the control. Biochemical analyses indicated a significant decline in the activities of superoxide dismutase and catalase in both the treated groups, with concomitant increase in lipid peroxidation. Liver aminotransferases (alanine aminotransferase and aspartate aminotransferase) were elevated with significant alterations in the levels of protein, creatinine and cholesterol in these tissues upon solvent exposure. Correlated with these changes, serum thyroid hormones T3 and T4 were also significantly altered. This study, therefore, demonstrates that inhalation of vapours from the solvent mixture resulted in significant dose-dependent biochemical and functional changes in the vital tissues (liver and kidney) studied. The study has specific relevance since humans are increasingly being exposed to such solvents due to increased industrial use in such combinations.

  4. Pseudomonas putida KT2440 markerless gene deletion using a combination of λ Red recombineering and Cre/loxP site-specific recombination.

    Science.gov (United States)

    Luo, Xi; Yang, Yunwen; Ling, Wen; Zhuang, Hao; Li, Qin; Shang, Guangdong

    2016-02-01

    Pseudomonas putida KT2440 is a saprophytic, environmental microorganism that plays important roles in the biodegradation of environmental toxic compounds and production of polymers, chemicals and secondary metabolites. Gene deletion of KT2440 usually involves cloning of the flanking homologous fragments of the gene of interest into a suicide vector followed by transferring into KT2440 via triparental conjugation. Selection and counterselection steps are then employed to generate gene deletion mutant. However, these methods are tedious and are not suitable for the manipulation of multiple genes simultaneously. Herein, a two-step, markerless gene deletion method is presented. First, homologous armsflanked loxP-neo-loxP was knocked-in to replace the gene of interest, then the kanamycin resistance marker is removed by Cre recombinase catalyzed site-specific recombination. Both two-plasmid and one-plasmid gene systems were established. MekR/PmekA regulated gene expression system was found to be suitable for tight Cre expression in one-plasmid deletion system. The straightforward, time saving and highly efficient markerless gene deletion strategy has the potential to facilitate the genetics and functional genomics study of P. putida KT2440. PMID:26802072

  5. Cytological studies of human meiosis: sex-specific differences in recombination originate at, or prior to, establishment of double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Jennifer R Gruhn

    Full Text Available Meiotic recombination is sexually dimorphic in most mammalian species, including humans, but the basis for the male:female differences remains unclear. In the present study, we used cytological methodology to directly compare recombination levels between human males and females, and to examine possible sex-specific differences in upstream events of double-strand break (DSB formation and synaptic initiation. Specifically, we utilized the DNA mismatch repair protein MLH1 as a marker of recombination events, the RecA homologue RAD51 as a surrogate for DSBs, and the synaptonemal complex proteins SYCP3 and/or SYCP1 to examine synapsis between homologs. Consistent with linkage studies, genome-wide recombination levels were higher in females than in males, and the placement of exchanges varied between the sexes. Subsequent analyses of DSBs and synaptic initiation sites indicated similar male:female differences, providing strong evidence that sex-specific differences in recombination rates are established at or before the formation of meiotic DSBs. We then asked whether these differences might be linked to variation in the organization of the meiotic axis and/or axis-associated DNA and, indeed, we observed striking male:female differences in synaptonemal complex (SC length and DNA loop size. Taken together, our observations suggest that sex specific differences in recombination in humans may derive from chromatin differences established prior to the onset of the recombination pathway.

  6. Concerted Assembly and Cloning of Multiple DNA Segments Using In Vitro Site-Specific Recombination: Functional Analysis of Multi-Segment Expression Clones

    OpenAIRE

    Cheo, David L.; Titus, Steven A.; Byrd, Devon R.N.; Hartley, James L.; Temple, Gary F.; Brasch, Michael A.

    2004-01-01

    The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have e...

  7. 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.

  8. 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.

  9. Tissue-specific alterations in expression and function of P-glycoprotein in streptozotocininduced diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Lu-lu ZHANG; Guang-ji WANG; Lin XIE; Liang LU; Shi JIN; Xin-yue JING; Dan YAO; Nan HU; Li LIU; Ru DUAN; Xiao-dong LIU

    2011-01-01

    Aim: To investigate the changes of expression and function of P-glycoprotein (P-GP) in cerebral cortex, hippocampus, liver, intestinal mucosa and kidney of streptozocin-induced diabetic rats.Methods: Diabetic rats were prepared via a single dose of streptozocin (65 mg/kg, ip). Abcb1/P-GP mRNA and protein expression levels in tissues were evaluated using quantitative real time polymerase chain reaction (QRT-PCR) analysis and Western blot, respectively.P-GP function was investigated via measuring tissue-to-plasma concentration ratios and body fluid excretion percentages of rhodamine 123.Results: In 5- and 8-week diabetic rats, Abcb1a mRNA levels were significantly decreased in cerebral cortices and intestinal mucosa,but dramatically increased in hippocampus and kidney. In liver, the level was increased in 5-week diabetic rats, and decreased in 8-week diabetic rats. Abcb1b mRNA levels were increased in cerebral cortex, hippocampus and kidney, but reduced in liver and intestinal mucosa in the diabetic rats. Western blot results were in accordance with the alterations of Abcb1a mRNA levels in most tissues examined. P-GP activity was markedly decreased in most tissues of diabetic rats, except kidney tissues.Conclusion: Alterations in the expression and function of Abcb1/P-GP under diabetic conditions are tissue specific, Abcb1 specific and diabetic duration-dependent.

  10. Serodiagnosis of tuberculosis: specific detection of free and complex-dissociated antibodies anti-mycobacterium tuberculosis recombinant antigens

    Directory of Open Access Journals (Sweden)

    María Susana Imaz

    2008-06-01

    Full Text Available The diagnostic test characteristics of detecting free and complex-dissociated IgG to three recombinant antigens of Mycobacterium tuberculosis (38-kDa, Ag16 and Ag85B, singly and in combination, were evaluated in sera from 161 tuberculous patients [smear-positive pulmonary TB (50, smear-negative pulmonary TB (pTBsm- (60 and extrapulmonary TB (51 and 214 control patients (mycobacteriosis (14, mycoses(14, leprosy(4, other underlying diseases (82 and healthy people (100]. The individual antigens ranged from 25% to 42% in sensitivity and from 93% to 96% in specificity, while considering free IgG response. Addition of complex-dissociated antibodies against each individual antigen improved the sensitivity up to 55%. The number and levels of specific antibodies varied greatly from individual to individual. Combination of individual results for free and complex-dissociated IgG to 38-kDa, Ag16 and Ag85B offered 76% sensitivity and 83% specificity. When the three antigens were placed in the same well, the sensitivity was lower than that expected on the basis of single antigen (63% but with a good specificity (95%, even in the group of mycobacteriosis or mycoses. The highest contribution of complex-dissociated IgG results to free IgG results was seen for the diagnosis of pTBsm- patients. In conclusion, although neither single recombinant antigen was reactive with most sera from TB patients even after the measurement of both free and complex-dissociated antibodies, the use of multi-antigen cocktails improved the diagnostic utility of the ELISA assay, allowing the identification of almost 70% of pTBsm-, with a high level of specificity; the use of additional, well selected antigens should lead to the detection of almost all patients with TB.

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

    OpenAIRE

    Karimova, M.; Abi-Ghanem, J.; N. Berger; Surendranath, V.; Pisabarro, M. T.; Buchholz, F

    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 di...

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  14. Region-Specific Genetic Alterations in the Aging Hippocampus: Implications for Cognitive Aging

    Directory of Open Access Journals (Sweden)

    Corinna eBurger

    2010-10-01

    Full Text Available Aging is associated with cognitive decline in both humans and animals and of all brain regions, the hippocampus appears to be particularly vulnerable to senescence. Age-related spatial learning deficits result from alterations in hippocampal connectivity and plasticity. These changes are differentially expressed in each of the hippocampal fields known as cornu ammonis 1 (CA1, cornu ammonis 3 (CA3, and the dentate gyrus. Each sub-region displays varying degrees of susceptibility to aging. For example, the CA1 region is particularly susceptible in Alzheimer’s disease while the CA3 region shows vulnerability to stress and glucocorticoids. Further, in animals, aging is the main factor associated with the decline in adult neurogenesis in the dentate gyrus. This review discusses the relationship between region-specific hippocampal connectivity, morphology, and gene expression alterations and the cognitive deficits associated with senescence. In particular, data are reviewed that illustrate how the molecular changes observed in the CA1, CA3, and dentate regions are associated with age-related learning deficits. This topic is of importance because increased understanding of how gene expression patterns reflect individual differences in cognitive performance is critical to the process of identifying new and clinically-useful biomarkers for cognitive aging.

  15. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

    Science.gov (United States)

    Canals, Isaac; Soriano, Jordi; Orlandi, Javier G.; Torrent, Roger; Richaud-Patin, Yvonne; Jiménez-Delgado, Senda; Merlin, Simone; Follenzi, Antonia; Consiglio, Antonella; Vilageliu, Lluïsa; Grinberg, Daniel; Raya, Angel

    2015-01-01

    Summary Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration. PMID:26411903

  16. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

    Directory of Open Access Journals (Sweden)

    Isaac Canals

    2015-10-01

    Full Text Available Induced pluripotent stem cell (iPSC technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

  17. Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks.

    Science.gov (United States)

    Canals, Isaac; Soriano, Jordi; Orlandi, Javier G; Torrent, Roger; Richaud-Patin, Yvonne; Jiménez-Delgado, Senda; Merlin, Simone; Follenzi, Antonia; Consiglio, Antonella; Vilageliu, Lluïsa; Grinberg, Daniel; Raya, Angel

    2015-10-13

    Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

  18. Induction of Epstein-Barr Virus Lytic Replication by Recombinant Adenoviruses Expressing the Zebra Gene with EBV Specific Promoters

    Institute of Scientific and Technical Information of China (English)

    Lu CHEN; Juan YIN; Yi CHEN; Jiang ZHONG

    2005-01-01

    The latent Epstein-Barr virus (EBV) is found in the cells of many tumors. For example, EBV is detectable in almost all cases, and in almost all tumor cells, of non-keratinizing nasopharyngeal carcinoma.Activating the latent virus, which will result in its lytic replication and the death of tumor cells, is a potential approach for the treatment of EBV-associated cancers. In this study, three recombinant adenoviruses were constructed to express the Zebra gene, an EBV gene responsible for switching from the latent state to lytic replication. EBV-specific promoters were used in order to limit Zebra expression in EBV-positive cells, and reduce the potential side effects. The EBV promoters used were Cp, Zp and a dual promoter combining both promoters, CpZp. The Zebra protein was detected in HEK293 cells as well as the EBV-positive D98-HR1 cells infected with recombinant viruses. An EBV lytic replication early antigen, EA-D, was also detected in infected D98-HR1, implying the initiation of lytic replication. In the cell viability assay, Zebra-expressing adenoviruses had little effect on EBV-negative HeLa cells, while significantly reducing the cell viability and proliferation of D98-HR1 cells. The results indicate that EBV virus promoters can be used in adenovirus vectors to express the Zebra gene and induce EBV lytic replication in D98-HR1 cells.

  19. 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.

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

    Science.gov (United States)

    Rockstroh, Alexandra; Barzon, Luisa; Pacenti, Monia; Palù, Giorgio; Niedrig, Matthias; Ulbert, Sebastian

    2015-11-01

    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. PMID:26565964

  1. Continuous Monitoring of Specific mRNA Expression Responses with a Fluorescence Resonance Energy Transfer-Based DNA Nano-tweezer Technique That Does Not Require Gene Recombination.

    Science.gov (United States)

    Shigeto, Hajime; Nakatsuka, Keisuke; Ikeda, Takeshi; Hirota, Ryuichi; Kuroda, Akio; Funabashi, Hisakage

    2016-08-16

    This letter discusses the feasibility of continuously monitoring specific mRNA expression responses in a living cell with a probe structured as a fluorescence resonance energy transfer (FRET)-based DNA nano-tweezer (DNA-NT). The FRET-based DNA-NT, self-assembled from three single-stranded DNAs, alters its structure from an open state to a closed state in recognition of a target mRNA, resulting in the closing of the distal relation of previously modified FRET-paired fluorescent dyes and generating a FRET signal. The expressions of glucose transporters (GLUT) 1 and 4 in a mouse hepato-carcinoma (Hepa 1-6 cells) were selected as the target model. Live-cell imaging analysis of Hepa 1-6 cells with both FRET-based DNA-NTs indicated that the behaviors of the FRET signals integrated in each individual cell were similar to those measured with the conventional mass analysis technique of semiquantitative real-time (RT) polymerase chain reaction (PCR). From these results, it is concluded that continuous monitoring of gene expression response without gene recombination is feasible with a FRET-based DNA-NT, even in a single cell manner. PMID:27458920

  2. Development of specific enzyme-linked immunosorbent assay for determining LH and FSH levels in tilapia, using recombinant gonadotropins.

    Science.gov (United States)

    Aizen, Joseph; Kasuto, Harel; Levavi-Sivan, Berta

    2007-01-01

    We recently produced Oreochromis niloticus recombinant LH and FSH as single-chain polypeptides in the methylotrophic yeast Pichia pastoris. Glycoprotein subunit alpha was joined with tilapia (t) LHbeta or tFSHbeta mature protein-coding sequences to form a fusion gene that encodes a ;;tethered" polypeptide, in which the gonadotropin beta-subunit forms the N-terminal part and the alpha-subunit forms the C-terminal part. Recombinant (r) gonadotropins were used to develop specific and homologous competitive ELISAs for measurements of FSH and LH in the plasma and pituitary of tilapia, using primary antibodies against rtLHbeta or rtFSHbeta, respectively, and rtLHbetaalpha or rtFSHbetaalpha for the standard curves. The wells were coated with either rtLHbeta (2ng/ml) or rtFSHbeta (0.5ng/well), and the final concentrations of the antisera were 1:5000 (for tLH) or 1:50,000 (for tFSH). The sensitivity of the assay was 15.84pg/ml for tLH and 0.24pg/ml for tFSH measurements in the plasma, whereas for the measurements in the pituitary, the sensitivity was 2.43ng/ml and 1.52ng/ml for tLH and tFSH, respectively. The standard curves for tFSH and tLH paralleled those of serially diluted pituitary extracts of other cichlids, as well as of serially diluted pituitary extract of seabream, European seabass and hybrid bass. We examined plasma tFSH and tLH levels in the course of one reproductive cycle, between two successive spawnings, in three individual tilapia females. Plasma levels of both FSH and LH increased during the second day after the eggs had been removed, probably related to the vitellogenic phase. LH levels increased toward spawning, which occurred on the 11th day. FSH levels also increased on day of cycle, probably due to recruitment of a new generation of follicles for the successive spawning. The development of specific ELISAs using recombinant gonadotropins is expected to advance the study of the distinct functions of each of these important hormones. PMID:17507016

  3. A New Recombinant BCG Vaccine Induces Specific Th17 and Th1 Effector Cells with Higher Protective Efficacy against Tuberculosis

    Science.gov (United States)

    da Costa, Adeliane Castro; Costa-Júnior, Abadio de Oliveira; de Oliveira, Fábio Muniz; Nogueira, Sarah Veloso; Rosa, Joseane Damaceno; Resende, Danilo Pires; Kipnis, André; Junqueira-Kipnis, Ana Paula

    2014-01-01

    Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb) that is a major public health problem. The vaccine used for TB prevention is Mycobacterium bovis bacillus Calmette-Guérin (BCG), which provides variable efficacy in protecting against pulmonary TB among adults. Consequently, several groups have pursued the development of a new vaccine with a superior protective capacity to that of BCG. Here we constructed a new recombinant BCG (rBCG) vaccine expressing a fusion protein (CMX) composed of immune dominant epitopes from Ag85C, MPT51, and HspX and evaluated its immunogenicity and protection in a murine model of infection. The stability of the vaccine in vivo was maintained for up to 20 days post-vaccination. rBCG-CMX was efficiently phagocytized by peritoneal macrophages and induced nitric oxide (NO) production. Following mouse immunization, this vaccine induced a specific immune response in cells from lungs and spleen to the fusion protein and to each of the component recombinant proteins by themselves. Vaccinated mice presented higher amounts of Th1, Th17, and polyfunctional specific T cells. rBCG-CMX vaccination reduced the extension of lung lesions caused by challenge with Mtb as well as the lung bacterial load. In addition, when this vaccine was used in a prime-boost strategy together with rCMX, the lung bacterial load was lower than the result observed by BCG vaccination. This study describes the creation of a new promising vaccine for TB that we hope will be used in further studies to address its safety before proceeding to clinical trials. PMID:25398087

  4. A recombination based method to rapidly assess specificity of two-hybrid clones in yeast.

    OpenAIRE

    Petermann, R; Mossier, B M; Aryee, D N; Kovar, H.

    1998-01-01

    The yeast two-hybrid system is frequently used to identify protein-protein interactions. Confirming the specificity of candidate clones requires separation and isolation of yeast plasmids, propagation in bacteria and testing combinations of DNA-binding and activation domain hybrids in yeast. In order to simplify this procedure, we developed a rapid method based on PCR amplification of library insert DNAs and in vivo cloning into the activation domain hybrid vector. Reporter gene activity is a...

  5. Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

    OpenAIRE

    Butcher, S A; Gould, E. A.

    1997-01-01

    Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no conve...

  6. The alteration of pain sensitivity at disease-specific acupuncture points in premenstrual syndrome.

    Science.gov (United States)

    Chae, Younbyoung; Kim, Hee-Young; Lee, Hwa-Jin; Park, Hi-Joon; Hahm, Dae-Hyun; An, Kyungeh; Lee, Hyejung

    2007-04-01

    Acupuncture points (APs) are well known to be small regions of local or referred pain that are more sensitive than surrounding tissue. Based on bibliographical and clinical data, specific conditions are commonly believed to change the pain sensitivity at corresponding APs. The aim of the present study was to investigate whether the pressure pain threshold (PPT) of specific APs is associated with the severity of premenstrual syndrome. The 46 participants were female students attending a middle school. Premenstrual syndrome (PMS) was measured using a structured questionnaire, the menstruation distress questionnaire (MDQ). High PMS (HP) and low PMS (LP) groups were divided based on their MDQ scores. The PPTs at sites in the leg (the APs SP6, GB39, and LR3 and a non-AP 2-cm anterior to SP6) and in the arm (the APs PC6, TE5, and LI4 and a non-AP 2-cm proximal to PC6) were measured using an algometer. The PPT of the HP group at SP6 was significantly lower than that of the LP group (13.50 +/- 0.73 vs. 16.30 +/- 0.66 kilopascals, P < 0.05), but not at other APs or at non-APs. The findings of our study support the hypothesis that the alteration of pain threshold at specific APs is associated with the severity of corresponding diseases. Further studies are needed to determine whether an observation of pain sensitivity at the APs could be used as an adjunctive tool for the diagnosis of a clinical problem. PMID:17378970

  7. Evolutionary alteration of ALOX15 specificity optimizes the biosynthesis of antiinflammatory and proresolving lipoxins.

    Science.gov (United States)

    Adel, Susan; Karst, Felix; González-Lafont, Àngels; Pekárová, Mária; Saura, Patricia; Masgrau, Laura; Lluch, José M; Stehling, Sabine; Horn, Thomas; Kuhn, Hartmut; Heydeck, Dagmar

    2016-07-26

    ALOX15 (12/15-lipoxygenase) orthologs have been implicated in maturational degradation of intracellular organelles and in the biosynthesis of antiinflammatory and proresolving eicosanoids. Here we hypothesized that lower mammals (mice, rats, pigs) express 12-lipoxygenating ALOX15 orthologs. In contrast, 15-lipoxygenating isoforms are found in higher primates (orangutans, men), and these results suggest an evolution of ALOX15 specificity. To test this hypothesis we first cloned and characterized ALOX15 orthologs of selected Catarrhini representing different stages of late primate evolution and found that higher primates (men, chimpanzees) express 15-lipoxygenating orthologs. In contrast, lower primates (baboons, rhesus monkeys) express 12-lipoxygenating enzymes. Gibbons, which are flanked in evolution by rhesus monkeys (12-lipoxygenating ALOX15) and orangutans (15-lipoxygenating ALOX15), express an ALOX15 ortholog with pronounced dual specificity. To explore the driving force for this evolutionary alterations, we quantified the lipoxin synthase activity of 12-lipoxygenating (rhesus monkey, mouse, rat, pig, humIle418Ala) and 15-lipoxygenating (man, chimpanzee, orangutan, rabbit, ratPhe353Ala) ALOX15 variants and found that, when normalized to their arachidonic acid oxygenase activities, the lipoxin synthase activities of 15-lipoxygenating ALOX15 variants were more than fivefold higher (P < 0.01). Comparative molecular dynamics simulations and quantum mechanics/molecular mechanics calculations indicated that, for the 15-lipoxygenating rabbit ALOX15, the energy barrier for C13-hydrogen abstraction (15-lipoxygenation) was 17 kJ/mol lower than for arachidonic acid 12-lipoxygenation. In contrast, for the 12-lipoxygenating Ile418Ala mutant, the energy barrier for 15-lipoxygenation was 10 kJ/mol higher than for 12-lipoxygenation. Taken together, our data suggest an evolution of ALOX15 specificity, which is aimed at optimizing the biosynthetic capacity for antiinflammatory

  8. Directed evolution of estrogen receptor proteins with altered ligand-binding specificities.

    Science.gov (United States)

    Islam, Kazi Mohammed Didarul; Dilcher, Meik; Thurow, Corinna; Vock, Carsten; Krimmelbein, Ilga Kristine; Tietze, Lutz Friedjan; Gonzalez, Victor; Zhao, Huimin; Gatz, Christiane

    2009-01-01

    Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor alpha (hER alpha) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER alpha and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 microM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 microM) and a 10-fold decreased activity to 17beta-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Kibat, Janek; Schirrmann, Thomas; Knape, Matthias J; Helmsing, Saskia; Meier, Doris; Hust, Michael; Schröder, Christoph; Bertinetti, Daniela; Winter, Gerhard; Pardes, Khalid; Funk, Mia; Vala, Andrea; Giese, Nathalia; Herberg, Friedrich W; Dübel, Stefan; Hoheisel, Jörg D

    2016-09-25

    Many diagnostic and therapeutic concepts require antibodies of high specificity. Recombinant binder libraries and related selection approaches allow the efficient isolation of antibodies against almost every target of interest. Nevertheless, it cannot be guaranteed that selected antibodies perform well and interact specifically enough with analytes unless an elaborate characterisation is performed. Here, we present an approach to shorten this process by combining the selection of suitable antibodies with the identification of informative target molecules by means of antibody microarrays, thereby 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 a microarray result by immunohistochemistry analysis. PMID:26709003

  11. Recombinant antibodies for specific detection of clostridial [Fe-Fe] hydrogenases

    Science.gov (United States)

    Mangayil, Rahul; Karp, Matti; Lamminmäki, Urpo; Santala, Ville

    2016-01-01

    Biological hydrogen production is based on activity of specific enzymes called hydrogenases. Hydrogenases are oxygen sensitive metalloenzymes containing Ni and/or Fe atoms at the active site, catalyzing reversible reduction of protons. Generally, [Fe-Fe] hydrogenases prefer proton reduction to molecular hydrogen, a potential energy carrier molecule that can be produced by bioprocesses in sustainable manner. Thus, monitoring tools have been developed to study the relationship between [Fe-Fe] hydrogenases and biohydrogen production in bioreactors at DNA and RNA levels. In the present study, novel molecular tools are introduced for quantitative monitoring of clostridial [Fe-Fe] hydrogenases at the protein level. Aerobic and anaerobic biopanning (for inactive and active [Fe-Fe] hydrogenase, respectively) of phage displayed single-chain variable fragment (scFv) antibody libraries aided in isolating nine potential scFvs. The enriched antibodies demonstrated high specificity towards Clostridium spp. [Fe-Fe] hydrogenases allowing detection from pure and mixed cultures. Additionally, the antibodies showed different binding characteristics towards hydrogenase catalytic states, providing a possible means for functional detection of clostridial [Fe-Fe] hydrogenases. From hydrogenase-antibody interaction studies we observed that though antibody binding reduced the enzyme catalytic activity, it facilitated to retain hydrogen evolution from oxygen exposed hydrogenases. PMID:27786270

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

    International Nuclear Information System (INIS)

    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

  13. Site-specific DOTA/europium-labeling of recombinant human relaxin-3 for receptor-ligand interaction studies.

    Science.gov (United States)

    Zhang, Wei-Jie; Luo, Xiao; Liu, Ya-Li; Shao, Xiao-Xia; Wade, John D; Bathgate, Ross A D; Guo, Zhan-Yun

    2012-08-01

    Relaxin-3 (also known as INSL7) is a recently identified neuropeptide belonging to the insulin/relaxin superfamily. It has putative roles in the regulation of stress responses, food intake, and reproduction by activation of its cognate G-protein-coupled receptor RXFP3. It also binds and activates the relaxin family peptide receptors RXFP1 and RXFP4 in vitro. To obtain a europium-labeled relaxin-3 as tracer for studying the interaction of these receptors with various ligands, in the present work we propose a novel site-specific labeling strategy for the recombinant human relaxin-3 that has been previously prepared in our laboratory. First, the N-terminal 6 × His-tag of the single-chain relaxin-3 precursor was removed by Aeromonas aminopeptidase and all of the primary amines of the resultant peptide were reversibly blocked by citroconic anhydride. Second, the A-chain N-terminus of the blocked peptide was released by endoproteinase Asp-N cleavage that removed the linker peptide between the B- and A-chains. Third, an alkyne moiety was introduced to the newly released A-chain N-terminus by reaction with the highly active primary amine-specific N-hydroxysuccinimide ester. Fourth, after removal of the reversible blockage under mild acidic condition, europium-loaded DOTA with an azide moiety was introduced to the two-chain relaxin-3 carrying the alkyne moiety through click chemistry. Using this site-specific labeling strategy, homogeneous monoeuropium-labeled human relaxin-3 could be obtained with good overall yield. In contrast, conventional random labeling resulted in a complex mixture that was poorly resolved because human relaxin-3 has four primary amine moieties that all react with the modification reagent. Both saturation and competition binding assays demonstrated that the DOTA/Eu(3+)-labeled relaxin-3 retained high binding affinity for human RXFP3, RXFP4, and RXFP1 and was therefore a suitable non-radioactive and stable tracer to study the interaction of various

  14. Secreted lipases from Malassezia globosa: recombinant expression and determination of their substrate specificities.

    Science.gov (United States)

    Sommer, Bettina; Overy, David P; Haltli, Bradley; Kerr, Russell G

    2016-07-01

    Malassezia globosa, which is associated with skin conditions such as dandruff and seborrhoeic dermatitis, possesses 13 secreted lipases, but only MgLip1, MgMDL2 and MgLip2 have been characterized. To understand the substrate preferences of these lipases and by extension their potential role in colonizing human skin, we expressed all 13 predicted secreted lipases in Pichia pastoris and evaluated their ability to utilize mono-, di- and triolein substrates. The M. globosa family class 3 lipases were shown to be specific for mono- and diacylglycerols, but exhibited no regio-selective production of diacylglycerols, which are of special interest for industrial applications. Lipases belonging to the Lip family utilized all substrates. In a further step, five lipases previously demonstrated to be expressed on human skin were tested against the eight most common di- and triacylglycerols in human sebum. All lipases liberated free fatty acids from three to eight of these substrates, proving their ability to hydrolyse key components of human sebum. Again, only Lip family lipases showed activity on triacylglycerides. Based on the demonstrated activity and expression levels of MgLip2 in M. globosa, the Lip lipase family appears to have the highest impact for the pathogenicity of M. globosa. PMID:27130210

  15. Frequency-specific Alterations of Large-scale Functional Brain Networks in Patients with Alzheimer's Disease

    Institute of Scientific and Technical Information of China (English)

    Yuan-Yuan Qin; Ya-Peng Li; Shun Zhang; Ying Xiong; Lin-Ying Guo; Shi-Qi Yang; Yi-Hao Yao

    2015-01-01

    Background:Previous studies have indicated that the cognitive deficits in patients with Alzheimer's disease (AD) may be due to topological deteriorations of the brain network.However,whether the selection of a specific frequency band could impact the topological properties is still not clear.Our hypothesis is that the topological properties of AD patients are also frequency-specific.Methods:Resting state functional magnetic resonance imaging data from l0 right-handed moderate AD patients (mean age:64.3 years; mean mini mental state examination [MMSE]:18.0) and 10 age and gender-matched healthy controls (mean age:63.6 years; mean MMSE:28.2) were enrolled in this study.The global efficiency,the clustering coefficient (CC),the characteristic path length (CpL),and "small-world" property were calculated in a wide range of thresholds and averaged within each group,at three different frequency bands (0.01-0.06 Hz,0.06-0.11 Hz,and 0.11-0.25 Hz).Results:At lower-frequency bands (0.01-0.06 Hz,0.06-0.11 Hz),the global efficiency,the CC and the "small-world" properties of AD patients decreased compared to controls.While at higher-frequency bands (0.11-0.25 Hz),the CpL was much longer,and the "small-world" property was disrupted in AD,particularly at a higher threshold.The topological properties changed with different frequency bands,suggesting the existence of disrupted global and local functional organization associated with AD.Conclusions:This study demonstrates that the topological alterations of large-scale functional brain networks inAD patients are frequency dependent,thus providing fundamental support for optimal frequency selection in future related research.

  16. Structural conservation of prion strain specificities in recombinant prion protein fibrils in real-time quaking-induced conversion.

    Science.gov (United States)

    Sano, Kazunori; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    A major unsolved issue of prion biology is the existence of multiple strains with distinct phenotypes and this strain phenomenon is postulated to be associated with the conformational diversity of the abnormal prion protein (PrP(Sc)). Real-time quaking-induced conversion (RT-QUIC) assay that uses Escherichia coli-derived recombinant prion protein (rPrP) for the sensitive detection of PrP(Sc) results in the formation of rPrP-fibrils seeded with various strains. We demonstrated that there are differences in the secondary structures, especially in the β-sheets, and conformational stability between 2 rPrP-fibrils seeded with either Chandler or 22L strains in the first round of RT-QUIC. In particular, the differences in conformational properties of these 2 rPrP-fibrils were common to those of the original PrP(Sc). However, the strain specificities of rPrP-fibrils seen in the first round were lost in subsequent rounds. Instead, our findings suggest that nonspecific fibrils became the major species, probable owing to their selective growth advantage in the RT-QUIC. This study shows that at least some strain-specific conformational properties of the original PrP(Sc) can be transmitted to rPrP-fibrils in vitro, but further conservation appears to require unknown cofactors or environmental conditions or both.

  17. A recombinant horseshoe crab plasma lectin recognizes specific pathogen-associated molecular patterns of bacteria through rhamnose.

    Directory of Open Access Journals (Sweden)

    Sim-Kun Ng

    Full Text Available Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs, yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.

  18. Altered Nutrition State in the Severe Multiple Trauma Patients Undergoing Adjuvant Recombinant Human Growth Hormone Nutritional Support Therapy

    Institute of Scientific and Technical Information of China (English)

    GUO Yanqing; BAI Xiangjun; LIN Guanyu; TANG Zhaohui

    2007-01-01

    In order to observe the nutrition state in the severe multiple trauma patients undergoing adjuvant recombinant human growth hormone (rhGH) nutritional support therapy, 45 patients with severe multiple traumas (ISS>25) were randomly divided into 3 groups. All the 3 groups had been supplied with nitrogen and caloricity according to the need of patients for 16 days. The rhGH therapy started 48 h after surgery and lasted for 14 days in two rhGH-treated groups in which rhGH was 0.2 and 0.4 U/(kg·d) respectively, and the resting group served as control one. The levels of nitrogen balance, prealbumin and safety variables (blood sugar, Na+, TT3 and TT4) were observed and compared among the three groups. The levels of nitrogen balance on the postoperative day (POD) 3 and 5 in the rhGH-treated groups were - 1.28±3.19, 5.45±2.00 and -0.18±2.55, 6.11±1.60, respectively,which were significantly higher than those in the control group (-5.17±1.68 and -1.08±3.31, P<0.01). The values of prealbumin on the POD 3 and 5 in the rhGH-treated groups were 180.19±27.15, 194.44±50.82 and 194.94±29.65, 194.11±16.17, respectively, which were significantly higher than those in the control group (117.42±19.10 and 135.63±28.31, P<0.01). There was no significant difference between the rhGH 0.2 U/(kg·d) group and rhGH 0.4 U/(kg·d) group in both of the levels of nitrogen balance and prealbumin. It is concluded that the nutritional support therapy with adjuvant rhGH which starts 48 h after surgery improves the nutrition state of the patients with severe multiple trauma. It is safe for severe multiple trauma patients who accept rhGH at the dose of 0.2 and 0.4 U/(kg·d).

  19. 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.

  20. Species-specific fine root biomass distribution alters competition in mixed forests under climate change

    Science.gov (United States)

    Reyer, Christopher; Gutsch, Martin; Lasch, Petra; Suckow, Felicitas; Sterck, Frank; Mohren, Frits

    2010-05-01

    The importance of mixed forests in European silviculture has increased due to forest conversion policies and multifunctional forest management. Concurrently, evidences for substantial impacts of climate change on forest ecosystems accumulate. Projected drier and warmer conditions alter the water relations of tree species, their growth and ultimately their inter-specific competition in mixed stands. Process-based models are scientific tools to study the impact of climate change on and to deepen the understanding of the functioning of these systems based on ecological mechanisms. They allow for long-term, stand-level studies of forest dynamics which could only be addressed with great difficulty in an experimental or empirical setup. We used the process-based forest model 4C to simulate inter-specific competition in mixed stands of Douglas-fir (Pseudotsuga menziesii) and Common beech (Fagus sylvatica) as well as Scots pine (Pinus sylvestris) and Sessile / Pedunculate oak (Quercus petraea and Quercus robur) under a) historical climate for model verification and b) under climate change scenario realizations of the climate model STAR 2.0 in Brandenburg, Germany. Some of the climate change scenario realizations feature a substantially drier and warmer summer climate which decreases the climatic water balance during the growing season. We assumed species-specific fine root biomass distributions which feature broadleaved fine roots in deeper soil layers and coniferous fine roots in upper soil layers according to several root excavation studies from mixed stands. The stands themselves were constructed from yield tables of the contributing species. The model verification provided good results for the basal area predictions under the historical climate. Under climate change, the number of days when the tree water demand exceeded the soil water supply was higher for the coniferous species than for broadleaved species. Furthermore, after 45 simulation years the basal area

  1. Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage phiC31 site-specific recombination system.

    Science.gov (United States)

    Thomason, L C; Calendar, R; Ow, D W

    2001-08-01

    The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.

  2. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    Science.gov (United States)

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc.

  3. Chemical inhibition of autophagy: Examining its potential to increase the specific productivity of recombinant CHO cell lines.

    Science.gov (United States)

    Baek, Eric; Kim, Che Lin; Kim, Mi Gyeom; Lee, Jae Seong; Lee, Gyun Min

    2016-09-01

    Chinese hamster ovary (CHO) cells activate and undergo apoptosis and autophagy for various environmental stresses. Unlike apoptosis, studies on increasing the production of therapeutic proteins in CHO cells by targeting the autophagy pathway are limited. In order to identify the effects of chemical autophagy inhibitors on the specific productivity (qp ), nine chemical inhibitors that had been reported to target three different phases of autophagy (metformin, dorsomorphin, resveratrol, and SP600125 against initiation and nucleation; 3-MA, wortmannin, and LY294002 against elongation, and chloroquine and bafilomycin A1 against autophagosome fusion) were used to treat three recombinant CHO (rCHO) cell lines: the Fc-fusion protein-producing DG44 (DG44-Fc) and DUKX-B11 (DUKX-Fc) and antibody-producing DG44 (DG44-Ab) cell lines. Among the nine chemical inhibitors tested, 3-MA, dorsomorphin, and SP600125 significantly increased the qp of DG44-Fc and DUKX-Fc. In contrast, for DG44-Ab, only 3-MA significantly increased the qp . The autophagy-inhibiting activity of the nine chemical inhibitors on the rCHO cell lines was evaluated through Western blot analysis and flow cytometry. Unexpectedly, some chemical inhibitors did not exhibit any apparent inhibition activity on autophagy. The chemical inhibitors that enhanced the qp , 3-MA, dorsomorphin, and SP600125, exhibited instead an increased autophagic flux. Taken all together, the chemical inhibition of autophagy was not effective in increasing the qp in rCHO cell lines and the positive effect of 3-MA, dorsomorphin, and SP600125 on the qp was not due to the inhibition of autophagy. Biotechnol. Bioeng. 2016;113: 1953-1961. © 2016 Wiley Periodicals, Inc. PMID:26914152

  4. Specific inhibition of T-cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin-1.

    Science.gov (United States)

    Rabinovich, G A; Ariel, A; Hershkoviz, R; Hirabayashi, J; Kasai, K I; Lider, O

    1999-05-01

    The migration of immune cells through the extracellular matrix (ECM) towards inflammatory sites is co-ordinated by receptors recognizing ECM glycoproteins, chemokines and proinflammatory cytokines. In this context, galectins are secreted to the extracellular milieu, where they recognize poly-N-acetyllactosamine chains on major ECM glycoproteins, such as fibronectin and laminin. We investigated the possibility that galectin-1 could modulate the adhesion of human T cells to ECM and ECM components. T cells were purified from human blood, activated with interleukin-2 (IL-2), labelled, and incubated further with intact immobilized ECM and ECM glycoproteins in the presence of increasing concentrations of human recombinant galectin-1, or its more stable, related, C2-S molecule obtained by site-directed mutagenesis. The presence of galectin-1 was shown to inhibit T-cell adhesion to intact ECM, laminin and fibronectin, and to a lesser extent to collagen type IV, in a dose-dependent manner. This effect was specifically blocked by anti-galectin-1 antibody and was dependent on the lectin's carbohydrate-binding properties. The inhibition of T-cell adhesion by galectin-1 correlates with the ability of this molecule to block the re-organization of the activated cell's actin cytoskeleton. Furthermore, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production was markedly reduced when IL-2-activated T cells were incubated with galectin-1 or its mutant. This effect was prevented by beta-galactoside-related sugars. The present study reveals an alternative inhibitory mechanism for explaining the suppressive properties of the galectin-1 subfamily on inflammatory and autoimmune processes. PMID:10447720

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Cellular responses to HSV-1 infection are linked to specific types of alterations in the host transcriptome.

    Science.gov (United States)

    Hu, Benxia; Li, Xin; Huo, Yongxia; Yu, Yafen; Zhang, Qiuping; Chen, Guijun; Zhang, Yaping; Fraser, Nigel W; Wu, Dongdong; Zhou, Jumin

    2016-01-01

    Pathogen invasion triggers a number of cellular responses and alters the host transcriptome. Here we report that the type of changes to cellular transcriptome is related to the type of cellular functions affected by lytic infection of Herpes Simplex Virus type I in Human primary fibroblasts. Specifically, genes involved in stress responses and nuclear transport exhibited mostly changes in alternative polyadenylation (APA), cell cycle genes showed mostly alternative splicing (AS) changes, while genes in neurogenesis, rarely underwent these changes. Transcriptome wide, the infection resulted in 1,032 cases of AS, 161 incidences of APA, 1,827 events of isoform changes, and up regulation of 596 genes and down regulations of 61 genes compared to uninfected cells. Thus, these findings provided important and specific links between cellular responses to HSV-1 infection and the type of alterations to the host transcriptome, highlighting important roles of RNA processing in virus-host interactions. PMID:27354008

  7. 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

  8. 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

    OpenAIRE

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

    2016-01-01

    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 char...

  9. Adaptive mass alteration to model ion-ion recombination in a Particle-in-Cell simulation of silane radio-frequency discharges

    NARCIS (Netherlands)

    Snytnikov, A. V.; Vshivkov, V. A.; W. J. Goedheer,

    2010-01-01

    In discharges in electronegative gases, negative ions are generated by (dissociative) attachment and lost by recombination with positive ions. Straightforward modeling of the recombination in a Particle-in-Cell simulation by removing a positive and negative super-particle gives rise to large numeric

  10. 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.

  11. Mcp7, a meiosis-specific coiled-coil protein of fission yeast, associates with Meu13 and is required for meiotic recombination.

    Science.gov (United States)

    Saito, Takamune T; Tougan, Takahiro; Kasama, Takashi; Okuzaki, Daisuke; Nojima, Hiroshi

    2004-01-01

    We previously showed that Meu13 of Schizosaccharomyces pombe functions in homologous pairing and recombination at meiosis I. Here we show that a meiosis-specific gene encodes a coiled-coil protein that complexes with Meu13 during meiosis in vivo. This gene denoted as mcp7+ (after meiotic coiled-coil protein) is an ortholog of Mnd1 of Saccharomyces cerevisiae. Mcp7 proteins are detected on meiotic chromatin. The phenotypes of mcp7Delta cells are similar to those of meu13Delta cells as they show reduced recombination rates and spore viability and produce spores with abnormal morphology. However, a delay in initiation of meiosis I chromosome segregation of mcp7Delta cells is not so conspicuous as meu13Delta cells, and no meiotic delay is observed in mcp7Deltameu13Delta cells. Mcp7 and Meu13 proteins depend on each other differently; Mcp7 becomes more stable in meu13Delta cells, whereas Meu13 becomes less stable in mcp7Delta cells. Genetic analysis shows that Mcp7 acts in the downstream of Dmc1, homologs of Escherichia coli RecA protein, for both recombination and subsequent sporulation. Taken together, we conclude that Mcp7 associates with Meu13 and together they play a key role in meiotic recombination. PMID:15210864

  12. 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)

    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

  13. Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2.

    Science.gov (United States)

    Fogel, Brent L; Cho, Ellen; Wahnich, Amanda; Gao, Fuying; Becherel, Olivier J; Wang, Xizhe; Fike, Francesca; Chen, Leslie; Criscuolo, Chiara; De Michele, Giuseppe; Filla, Alessandro; Collins, Abigail; Hahn, Angelika F; Gatti, Richard A; Konopka, Genevieve; Perlman, Susan; Lavin, Martin F; Geschwind, Daniel H; Coppola, Giovanni

    2014-09-15

    Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and RNA-sequencing. To determine whether AOA2 and ALS4 mutations differentially affect gene expression, we overexpressed disease-specific SETX mutations in senataxin-haploinsufficient fibroblasts and observed changes in distinct sets of genes. This implicates mutation-specific alterations of senataxin function in disease pathogenesis and provides a novel example of allelic neurogenetic disorders with differing gene expression profiles. Weighted gene co-expression network analysis (WGCNA) demonstrated these senataxin-associated genes to be involved in both mutation-specific and shared functional gene networks. To assess this in vivo, we performed gene expression analysis on peripheral blood from members of 12 different AOA2 families and identified an AOA2-specific transcriptional signature. WGCNA identified two gene modules highly enriched for this transcriptional signature in the peripheral blood of all AOA2 patients studied. These modules were disease-specific and preserved in patient fibroblasts and in the cerebellum of Setx knockout mice demonstrating conservation across species and cell types, including neurons. These results identify novel genes and cellular pathways related to senataxin function in normal and disease states, and implicate alterations in gene expression as underlying the phenotypic differences between AOA2 and ALS4. PMID:24760770

  14. Sex-specific responses to climate change in plants alter population sex ratio and performance.

    Science.gov (United States)

    Petry, William K; Soule, Judith D; Iler, Amy M; Chicas-Mosier, Ana; Inouye, David W; Miller, Tom E X; Mooney, Kailen A

    2016-07-01

    Males and females are ecologically distinct in many species, but whether responses to climate change are sex-specific is unknown. We document sex-specific responses to climate change in the plant Valeriana edulis (valerian) over four decades and across its 1800-meter elevation range. Increased elevation was associated with increased water availability and female frequency, likely owing to sex-specific water use efficiency and survival. Recent aridification caused male frequency to move upslope at 175 meters per decade, a rate of trait shift outpacing reported species' range shifts by an order of magnitude. This increase in male frequency reduced pollen limitation and increased seedset. Coupled with previous studies reporting sex-specific arthropod communities, these results underscore the importance of ecological differences between the sexes in mediating biological responses to climate change. PMID:27365446

  15. A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σK and the Fidelity of Spore Development in Clostridium difficile

    Science.gov (United States)

    Serrano, Mónica; Kint, Nicolas; Pereira, Fátima C.; Saujet, Laure; Boudry, Pierre; Dupuy, Bruno; Martin-Verstraete, Isabelle

    2016-01-01

    The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σK. In Bacillus subtilis, the onset of σK activity requires both excision of a prophage-like element (skinBs) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skinBs recombinase is produced specifically in this cell. In C. difficile, σK lacks a pro-sequence but a skinCd element is present. The product of the skinCd gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skinCd excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skinCd excision is under the control of mother cell regulatory proteins σE and SpoIIID. We then demonstrate that σE and SpoIIID control the expression of the skinCd gene CD1234, and that this gene is required for sporulation and skinCd excision. CD1231 and CD1234 appear to interact and both proteins are required for skinCd excision while only CD1231 is necessary for skinCd integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skinCd excision to the terminal mother cell. Finally, while the skinCd element is not essential for sporulation, deletion of skinCd results in premature activity of σK and in spores with altered surface layers. Thus, skinCd excision is a key element controlling the onset of σK activity and the fidelity of spore development. PMID:27631621

  16. In Vitro Neutralisation of Rotavirus Infection by Two Broadly Specific Recombinant Monovalent Llama-Derived Antibody Fragments

    OpenAIRE

    Farah Aladin; Einerhand, Alexandra W. C.; Janneke Bouma; Sandra Bezemer; Pim Hermans; Danielle Wolvers; Kate Bellamy; Frenken, Leon G J; Jim Gray; Miren Iturriza-Gómara

    2012-01-01

    textabstractRotavirus is the main cause of viral gastroenteritis in young children. Therefore, the development of inexpensive antiviral products for the prevention and/or treatment of rotavirus disease remains a priority. Previously we have shown that a recombinant monovalent antibody fragment (referred to as Anti-Rotavirus Proteins or ARP1) derived from a heavy chain antibody of a llama immunised with rotavirus was able to neutralise rotavirus infection in a mouse model system. In the presen...

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

    NARCIS (Netherlands)

    J. Müller (Julia); S. Mayerl (Steffen); T.J. Visser (Ton); 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

  18. TFIIH subunit alterations causing xeroderma pigmentosum and trichothiodystrophy specifically disturb several steps during transcription.

    Science.gov (United States)

    Singh, Amita; Compe, Emanuel; Le May, Nicolas; Egly, Jean-Marc

    2015-02-01

    Mutations in genes encoding the ERCC3 (XPB), ERCC2 (XPD), and GTF2H5 (p8 or TTD-A) subunits of the transcription and DNA-repair factor TFIIH lead to three autosomal-recessive disorders: xeroderma pigmentosum (XP), XP associated with Cockayne syndrome (XP/CS), and trichothiodystrophy (TTD). Although these diseases were originally associated with defects in DNA repair, transcription deficiencies might be also implicated. By using retinoic acid receptor beta isoform 2 (RARB2) as a model in several cells bearing mutations in genes encoding TFIIH subunits, we observed that (1) the recruitment of the TFIIH complex was altered at the activated RARB2 promoter, (2) TFIIH participated in the recruitment of nucleotide excision repair (NER) factors during transcription in a manner different from that observed during NER, and (3) the different TFIIH variants disturbed transcription by having distinct consequences on post-translational modifications of histones, DNA-break induction, DNA demethylation, and gene-loop formation. The transition from heterochromatin to euchromatin was disrupted depending on the variant, illustrating the fact that TFIIH, by contributing to NER factor recruitment, orchestrates chromatin remodeling. The subtle transcriptional differences found between various TFIIH variants thus participate in the phenotypic variability observed among XP, XP/CS, and TTD individuals.

  19. Characterization of recombinant CEL-I, a GalNAc-specific C-type lectin, expressed in Escherichia coli using an artificial synthetic gene.

    Science.gov (United States)

    Hatakeyama, Tomomitsu; Shiba, Kouhei; Matsuo, Noriaki; Fujimoto, Tokiko; Oda, Tatsuya; Sugawara, Hajime; Aoyagi, Haruhiko

    2004-01-01

    CEL-I is a C-type lectin isolated from the Holothuroidea Cucumaria echinata. This lectin shows very high N-acetylgalactosamine-binding specificity. We constructed an artificial gene encoding recombinant CEL-I (rCEL-I) using a combination of synthetic oligonucleotides, and expressed it in Escherichia coli cells. Since the recombinant protein was obtained as inclusion bodies, the latter were solubilized using urea and 2-mercaptoethanol, and the protein was refolded during the purification and dialysis steps. The purified rCEL-I showed comparable hemagglutinating activity to that of native CEL-I at relatively high Ca(2+)-concentrations, whereas it was weaker at lower Ca(2+)-concentrations due to decreased Ca(2+)-binding affinity. rCEL-I exhibited similar carbohydrate-binding specificity to native CEL-I, including strong GalNAc-binding specificity, as examined by hemagglutination inhibition assay. Comparison of the far UV-CD spectra of recombinant and native CEL-I revealed that the two proteins undergo a similar conformational change upon binding of Ca(2+). Single crystals of rCEL-I were also obtained under the same conditions as those used for the native protein, suggesting that they have similar tertiary structures. Although native CEL-I exhibited strong cytotoxicity toward cultured cells, rCEL-I showed low cytotoxicity. These results indicate that rCEL-I has a tertiary structure and carbohydrate-binding specificity similar to those of native CEL-I. Howeger, there is a subtle difference in the properties between the two proteins probably due to the additional methionine residue at the N-terminus of rCEL-I. PMID:14999015

  20. Production, characterization, and antigen specificity of recombinant 62-71-3, a candidate monoclonal antibody for rabies prophylaxis in humans

    OpenAIRE

    Both, L.; van Dolleweerd, C.; Wright, E.; Banyard, A. C.; Bulmer-Thomas, B.; Selden, D.; Altmann, F.; Fooks, A.R.; Ma, J. K.- C.

    2013-01-01

    Rabies kills many people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies postexposure prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with rabies virus glycoprotein. Chimeric 62-71-3 was successfully expressed ...

  1. 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 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 < 0.05) compared with RES ewes. Even though ewes were transitioned to a common diet after parturition, endocrine status continued to be affected into lactation. Moreover, it appears that gestational diet may partially affect lactational performance through altered endocrine status.

  2. Genomic alterations in advanced esophageal cancer may lead to subtype-specific therapies.

    Science.gov (United States)

    Forde, Patrick M; Kelly, Ronan J

    2013-01-01

    The development of targeted agents for metastatic esophageal or gastroesophageal junction (GEJ) tumors has been limited when compared with that for other common tumors. To date, the anti-human epidermal growth factor receptor-2 (HER-2) antibody, trastuzumab, in combination with chemotherapy, is the only approved novel agent for these cancers, and its use is limited to the small population of patients whose tumors overexpress HER-2. Despite recent progress in the field, median overall survival remains only 8-12 months for patients with stage IV esophageal or GEJ cancer. In this article, we examine the molecular aberrations thought to drive the development and spread of esophageal cancer and identify promising targets for specific tumor inhibition. Data from clinical studies of targeted agents are reviewed, including epidermal growth factor receptor antibodies, tyrosine kinase inhibitors, HER-2, and vascular endothelial growth factor-directed therapy. Current and future targets include MET, fibroblast growth factor receptor, and immune-based therapies. Evidence from trials to date suggests that molecularly unselected patient cohorts derive minimal benefit from most target-specific agents, suggesting that future collaborative investigation should focus on preselected molecular subgroups of patients with this challenging heterogeneous disease.

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

    Science.gov (United States)

    Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

    2015-02-15

    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 for the ZZ protein.

  4. Structural alterations of the language connectome in children with specific language impairment.

    Science.gov (United States)

    Vydrova, Rosa; Komarek, Vladimir; Sanda, Jan; Sterbova, Katalin; Jahodova, Alena; Maulisova, Alice; Zackova, Jitka; Reissigova, Jindra; Krsek, Pavel; Kyncl, Martin

    2015-12-01

    We evaluated brain white matter pathways associated with language processing in 37 children with specific language impairment aged 6-12 years and 34 controls, matched for age, sex and handedness. Arcuate fascicle (AF), inferior fronto-occipital fascicle (IFOF), inferior longitudinal fascicle (ILF) and uncinate fascicle (UF) were identified using magnetic resonance diffusion tensor imaging (DTI). Diffusivity parameters and volume of the tracts were compared between the SLI and control group. Children with SLI showed decreased fractional anisotropy in all investigated tracts, increased mean diffusivity and radial diffusivity component in arcuate fascicle bilaterally, left IFOF and left ILF. Further, bilaterally increased volume of the ILF in children with SLI was found. We confirmed previous findings indicating deficient connectivity of the arcuate fascicle and as a novel finding, demonstrate abnormal development of the ventral language stream in patients with SLI.

  5. DNA cleavage is independent of synapsis during Streptomyces phage phiBT1 integrase-mediated site-specific recombination.

    Science.gov (United States)

    Zhang, Lin; Wang, Lu; Wang, Jin; Ou, Xijun; Zhao, Guoping; Ding, Xiaoming

    2010-10-01

    Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.

  6. 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. PMID:27506654

  7. 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

  8. A pilot study comparing the development of EIAV Env-specific antibodies induced by DNA/recombinant vaccinia-vectored vaccines and an attenuated Chinese EIAV vaccine.

    Science.gov (United States)

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Yang, Kai; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2012-12-01

    Data from successful attenuated lentiviral vaccine studies indicate that fully mature Env-specific antibodies characterized by high titer, high avidity, and the predominant recognition of conformational epitopes are associated with protective efficacy. Although vaccination with a DNA prime/recombinant vaccinia-vectored vaccine boost strategy has been found to be effective in some trials with non-human primate/simian/human immunodeficiency virus (SHIV) models, it remains unclear whether this vaccination strategy could elicit mature equine infectious anemia virus (EIAV) Env-specific antibodies, thus protecting vaccinated horses against EIAV infection. Therefore, in this pilot study we vaccinated horses using a strategy based on DNA prime/recombinant Tiantan vaccinia (rTTV)-vectored vaccines encoding EIAV env and gag genes, and observed the development of Env-specific antibodies, neutralizing antibodies, and p26-specific antibodies. Vaccination with DNA induced low titer, low avidity, and the predominant recognition of linear epitopes by Env-specific antibodies, which was enhanced by boosting vaccinations with rTTV vaccines. However, the maturation levels of Env-specific antibodies induced by the DNA/rTTV vaccines were significantly lower than those induced by the attenuated vaccine EIAV(FDDV). Additionally, DNA/rTTV vaccines did not elicit broadly neutralizing antibodies. After challenge with a virulent EIAV strain, all of the vaccinees and control horses died from EIAV disease. These data indicate that the regimen of DNA prime/rTTV vaccine boost did not induce mature Env-specific antibodies, which might have contributed to immune protection failure. PMID:23171359

  9. Photoionization and Recombination

    Science.gov (United States)

    Nahar, Sultana N.

    2000-01-01

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

  10. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Directory of Open Access Journals (Sweden)

    Tatsuaki Tsuruyama

    Full Text Available It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1 recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  11. Dialysis purification of integrase-DNA complexes provides high-resolution atomic force microscopy images: dimeric recombinant HIV-1 integrase binding and specific looping on DNA.

    Science.gov (United States)

    Tsuruyama, Tatsuaki; Nakai, Tonau; Ohmori, Rei; Ozeki, Munetaka; Tamaki, Keiji; Yoshikawa, Kenichi

    2013-01-01

    It remains difficult to obtain high-resolution atomic force microscopy images of HIV-1 integrase bound to DNA in a dimeric or tetrameric fashion. We therefore constructed specific target DNAs to assess HIV-1 integrase binding and purified the complex by dialysis prior to analysis. Our resulting atomic force microscopy analyses indicated precise size of binding human immunodeficiency virus type 1 (HIV-1) recombinant integrase in a tetrameric manner, inducing formation of a loop-like or figure-eight-like secondary structure in the target DNA. Our findings regarding the target DNA secondary structure provide new insights into the intermediate states of retroviral integration.

  12. Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

    OpenAIRE

    Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

    2000-01-01

    We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T ...

  13. Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues.

    Directory of Open Access Journals (Sweden)

    J Muse Davis

    Full Text Available Leptospirosis is an extremely widespread zoonotic infection with outcomes ranging from subclinical infection to fatal Weil's syndrome. Despite the global impact of the disease, key aspects of its pathogenesis remain unclear. To examine in detail the earliest steps in the host response to leptospires, we used fluorescently labelled Leptospira interrogans serovar Copenhageni to infect 30 hour post fertilization zebrafish embryos by either the caudal vein or hindbrain ventricle. These embryos have functional innate immunity but have not yet developed an adaptive immune system. Furthermore, they are optically transparent, allowing direct visualization of host-pathogen interactions from the moment of infection. We observed rapid uptake of leptospires by phagocytes, followed by persistent, intracellular infection over the first 48 hours. Phagocytosis of leptospires occasionally resulted in formation of large cellular vesicles consistent with apoptotic bodies. By 24 hours, clusters of infected phagocytes were accumulating lateral to the dorsal artery, presumably in early hematopoietic tissue. Our observations suggest that phagocytosis may be a key defense mechanism in the early stages of leptospirosis, and that phagocytic cells play roles in immunopathogenesis and likely in the dissemination of leptospires to specific target tissues.

  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. Chromosome Specific Substitution Lines of Aegilops geniculata Alter Parameters of Bread Making Quality of Wheat

    Science.gov (United States)

    Tsujimoto, Hisashi; Gupta, Raj Kumar; Kumar, Aman; Kaur, Navneet; Kumar, Rohit; Chunduri, Venkatesh; Sharma, Nand Kishor; Chawla, Meenakshi; Sharma, Saloni; Mundey, Jaspreet Kaur

    2016-01-01

    Wheat cultivars with wide introgression have strongly impacted global wheat production. Aegilops geniculata (MgUg) is an important wild relative with several useful traits that can be exploited for wheat improvement. Screening of Ae. geniculata addition lines indicated a negative effect of 1Ug and the positive effect of 1Mg chromosome on wheat dough strength. Negative effect of 1Ug is probably associated with variation in number and position of the tripeptide repeat motif in the high molecular weight glutenin (HMW-G) gene. To utilize the positive potential of 1Mg chromosome, three disomic substitution lines (DSLs) 1Mg(1A), 1Mg(1B) and 1Mg(1D) were created. These lines were characterized for morphological, cytogenetic properties and biochemical signatures using FISH, 1D-, 2D-PAGE and RP-HPLC. Contribution of wheat 1A, 1B and 1D chromosomes towards dough mixing and baking parameters, chapatti quality, Fe/Zn content and glume color were identified. Observed order of variation in the dough mixing and baking parameters {1Mg(1D) ≤wheat ≤1Mg(1B) ≤1Mg(1A)} indicated that chromosome specific introgression is desirable for best utilization of wild species’ potential. PMID:27755540

  16. 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. PMID:27000396

  17. Altered myocardial metabolic adaptation to increased fatty acid availability in cardiomyocyte-specific CLOCK mutant mice.

    Science.gov (United States)

    Peliciari-Garcia, Rodrigo A; Goel, Mehak; Aristorenas, Jonathan A; Shah, Krishna; He, Lan; Yang, Qinglin; Shalev, Anath; Bailey, Shannon M; Prabhu, Sumanth D; Chatham, John C; Gamble, Karen L; Young, Martin E

    2016-10-01

    A mismatch between fatty acid availability and utilization leads to cellular/organ dysfunction during cardiometabolic disease states (e.g., obesity, diabetes mellitus). This can precipitate cardiac dysfunction. The heart adapts to increased fatty acid availability at transcriptional, translational, post-translational and metabolic levels, thereby attenuating cardiomyopathy development. We have previously reported that the cardiomyocyte circadian clock regulates transcriptional responsiveness of the heart to acute increases in fatty acid availability (e.g., short-term fasting). The purpose of the present study was to investigate whether the cardiomyocyte circadian clock plays a role in adaptation of the heart to chronic elevations in fatty acid availability. Fatty acid availability was increased in cardiomyocyte-specific CLOCK mutant (CCM) and wild-type (WT) littermate mice for 9weeks in time-of-day-independent (streptozotocin (STZ) induced diabetes) and dependent (high fat diet meal feeding) manners. Indices of myocardial metabolic adaptation (e.g., substrate reliance perturbations) to STZ-induced diabetes and high fat meal feeding were found to be dependent on genotype. Various transcriptional and post-translational mechanisms were investigated, revealing that Cte1 mRNA induction in the heart during STZ-induced diabetes is attenuated in CCM hearts. At the functional level, time-of-day-dependent high fat meal feeding tended to influence cardiac function to a greater extent in WT versus CCM mice. Collectively, these data suggest that CLOCK (a circadian clock component) is important for metabolic adaption of the heart to prolonged elevations in fatty acid availability. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk. PMID:26721420

  18. Site-specific recombination of the circular 2 microns-like plasmid pKD1 requires integrity of the recombinase gene A and of the partitioning genes B and C.

    OpenAIRE

    Bianchi, M. M.

    1992-01-01

    In the circular plasmid pKD1, which stably replicates in Kluyveromyces lactis, the three open reading frames encode a site-specific recombinase (gene A) and two proteins involved in mitotic stability (genes B and C). A recombination analysis of plasmids in which gene B or C is inactivated reveals that unlike the 2 microns plasmid of Saccharomyces cerevisiae, these genes are also required for the site specificity of plasmid recombination.

  19. 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.

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

    International Nuclear Information System (INIS)

    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)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 for the

  1. 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

  2. Discovery of specific metastasis-related N-glycan alterations in epithelial ovarian cancer based on quantitative glycomics.

    Directory of Open Access Journals (Sweden)

    Xingwang Zhang

    Full Text Available Generally, most of ovarian cancer cannot be detected until large scale and remote metastasis occurs, which is the major cause of high mortality in ovarian cancer. Therefore, it is urgent to discover metastasis-related biomarkers for the detection of ovarian cancer in its occult metastasis stage. Altered glycosylation is a universal feature of malignancy and certain types of glycan structures are well-known markers for tumor progressions. Thus, this study aimed to reveal specific changes of N-glycans in the secretome of the metastatic ovarian cancer. We employed a quantitative glycomics approach based on metabolic stable isotope labeling to compare the differential N-glycosylation of secretome between an ovarian cancer cell line SKOV3 and its high metastatic derivative SKOV3-ip. Intriguingly, among total 17 N-glycans identified, the N-glycans with bisecting GlcNAc were all significantly decreased in SKOV3-ip in comparison to SKOV3. This alteration in bisecting GlcNAc glycoforms as well as its corresponding association with ovarian cancer metastatic behavior was further validated at the glycotransferase level with multiple techniques including real-time PCR, western blotting, transwell assay, lectin blotting and immunohistochemistry analysis. This study illustrated metastasis-related N-glycan alterations in ovarian cancer secretome in vitro for the first time, which is a valuable source for biomarker discovery as well. Moreover, N-glycans with bisecting GlcNAc shed light on the detection of ovarian cancer in early peritoneal metastasis stage which may accordingly improve the prognosis of ovarian cancer patients.

  3. Protection conferred by a recombinant Marek’s disease virus that expresses the spike protein from infectious bronchitis virus in specific pathogen-free chicken

    Directory of Open Access Journals (Sweden)

    Zhang Xiaorong

    2012-05-01

    Full Text Available Abstract Background In many countries, the predominant field isolates of infectious bronchitis virus (IBV have been classified as QX-like strains since 1996. However, no commercial vaccines that are specific for this type of IBV are currently available. Therefore, there is an urgent need to develop novel vaccines that prevent QX-like IBV infection. Results A recombinant Marek’s disease virus (MDV, rMDV-S1, that expresses the S1 subunit of the spike (S protein from the QX-like infectious bronchitis virus (IBV was constructed by inserting the IBV S1 gene into the genome of the CVI988/Rispens strain of MDV. Specific pathogen-free (SPF chickens that were vaccinated with rMDV-S1 were protected when challenged with the QX-like IBV. They were observed to have mild clinical signs of disease, a short virus-shedding period and low mortality. Additionally, the rMDV-S1 conferred full protection to chickens against virulent MDV, as did the CVI988/Rispens strain. Conclusions Our results demonstrate that rMDV-S1 is an effective and promising recombinant vaccine for the prevention of QX-like IBV infection.

  4. Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

    Science.gov (United States)

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-04-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. PMID:26848818

  5. Fmr1 knockout mice show reduced anxiety and alterations in neurogenesis that are specific to the ventral dentate gyrus.

    Science.gov (United States)

    Eadie, B D; Zhang, W N; Boehme, F; Gil-Mohapel, J; Kainer, L; Simpson, J M; Christie, B R

    2009-11-01

    Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by the selective loss of the expression of the Fmr1 gene. Key symptoms in FXS include intellectual impairment and abnormal anxiety-related behaviors. Fmr1 knockout (KO) mice exhibited reduced anxiety on two behavioral tests as well as a blunted corticosterone response to acute stress. Spatial learning and memory was not impaired when tested with both the classic Morris water and Plus-shaped mazes. Adult hippocampal neurogenesis has been associated with spatial learning and memory and emotions such as anxiety and depression. The process of neurogenesis appears abnormal in young adult Fmr1 KO mice, with significantly fewer bromodeoxyuridine-positive cells surviving for at least 4 weeks in the ventral subregion of the dentate gyrus (DG), a hippocampal subregion more closely associated with emotion than the dorsal DG. Within this smaller pool of surviving cells, we observed a concomitant increase in the proportion of surviving cells that acquire a neuronal phenotype. We did not observe a clear difference in cell proliferation using both endogenous and exogenous markers. This work indicates that loss of Fmr1 expression can alter anxiety-related behaviors in mice as well as produce region-specific alterations in hippocampal adult neurogenesis.

  6. Alterations in catecholamine turnover in specific regions of the rat brain following acute exposure to nitrous oxide.

    Science.gov (United States)

    Karuri, A R; Kugel, G; Engelking, L R; Kumar, M S

    1998-04-01

    The effects of nitrous oxide (N2O) on steady-state concentrations and turnover rates of catecholamines in the olfactory bulb, hypothalamus, brain stem, hippocampus, striatum, thalamus, cerebral cortex, and spinal cord were determined in rats. Animals were exposed for 2 h to either 60% N2O or air. Immediately following exposure, all animals were injected intraperitoneally with alpha-methylparatyrosine (alphaMPT), a competitive inhibitor of tyrosine hydroxylase, and sacrificed at 0, 30, or 90 min postinjection. Brain catecholamine concentrations were determined using high-performance liquid chromatography coupled with electrochemical detection (HPLC-EC). Results indicate that N2O exposure significantly elevates steady-state concentrations of norepinephrine (NE) in the hypothalamus and striatum yet decreases amine levels in the brain stem region. Steady-state levels of dopamine (DA) were not significantly altered in any region of the CNS by N2O exposure. Acute exposure to N2O also resulted in significant decreases in the turnover rate of NE in the brain stem, yet it increased turnover of this amine in the olfactory bulb, hypothalamus, and striatum. Acute exposure to N2O resulted in a decreased turnover rate of DA in the hippocampus and striatum. In contrast, N2O appears to increase DA turnover in the olfactory bulb. These results indicate that acute exposure to N2O in rats causes region-specific alterations in steady-state levels and turnover rates of DA and NE within the central nervous system.

  7. 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...

  8. 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

  9. 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

    2014-04-01

    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

  10. Immune Response to Recombinant Adenovirus in Humans: Capsid Components from Viral Input Are Targets for Vector-Specific Cytotoxic T Lymphocytes

    Science.gov (United States)

    Molinier-Frenkel, Valérie; Gahery-Segard, Hanne; Mehtali, Majid; Le Boulaire, Christophe; Ribault, Sébastien; Boulanger, Pierre; Tursz, Thomas; Guillet, Jean-Gérard; Farace, Françoise

    2000-01-01

    We previously demonstrated that a single injection of 109 PFU of recombinant adenovirus into patients induces strong vector-specific immune responses (H. Gahéry-Ségard, V. Molinier-Frenkel, C. Le Boulaire, P. Saulnier, P. Opolon, R. Lengagne, E. Gautier, A. Le Cesne, L. Zitvogel, A. Venet, C. Schatz, M. Courtney, T. Le Chevalier, T. Tursz, J.-G. Guillet, and F. Farace, J. Clin. Investig. 100:2218–2226, 1997). In the present study we analyzed the mechanism of vector recognition by cytotoxic T lymphocytes (CTL). CD8+ CTL lines were derived from two patients and maintained in long-term cultures. Target cell infections with E1-deleted and E1-plus E2-deleted adenoviruses, as well as transcription-blocking experiments with actinomycin D, revealed that host T-cell recognition did not require viral gene transcription. Target cells treated with brefeldin A were not lysed, indicating that viral input protein-derived peptides are associated with HLA class I molecules. Using recombinant capsid component-loaded targets, we observed that the three major proteins could be recognized. These results raise the question of the use of multideleted adenoviruses for gene therapy in the quest to diminish antivector CTL responses. PMID:10906225

  11. Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification.

    Science.gov (United States)

    Gandillet, Arnaud; Serrano, Alicia G; Pearson, Stella; Lie-A-Ling, Michael; Lacaud, Georges; Kouskoff, Valerie

    2009-11-26

    The molecular mechanisms that regulate the balance between proliferation and differentiation of precursors at the onset of hematopoiesis specification are poorly understood. By using a global gene expression profiling approach during the course of embryonic stem cell differentiation, we identified Sox7 as a potential candidate gene involved in the regulation of blood lineage formation from the mesoderm germ layer. In the present study, we show that Sox7 is transiently expressed in mesodermal precursors as they undergo specification to the hematopoietic program. Sox7 knockdown in vitro significantly decreases the formation of both primitive erythroid and definitive hematopoietic progenitors as well as endothelial progenitors. In contrast, Sox7-sustained expression in the earliest committed hematopoietic precursors promotes the maintenance of their multipotent and self-renewing status. Removal of this differentiation block driven by Sox7-enforced expression leads to the efficient differentiation of hematopoietic progenitors to all erythroid and myeloid lineages. This study identifies Sox7 as a novel and important player in the molecular regulation of the first committed blood precursors. Furthermore, our data demonstrate that the mere sustained expression of Sox7 is sufficient to completely alter the balance between proliferation and differentiation at the onset of hematopoiesis.

  12. Sex-specific alterations in behavioral and cognitive functions in a "three hit" animal model of schizophrenia.

    Science.gov (United States)

    Kekesi, G; Petrovszki, Z; Benedek, G; Horvath, G

    2015-05-01

    Whereas schizophrenia affects both human sexes, there are known sex-dependent disparities. We developed a chronic animal model that shows some schizophrenia-related deficits in rats by applying selective breeding after subchronic ketamine administration connected with postweaning social isolation (complex treatment). Our aim was to determine the sex-specific effects of these interventions on several processes. Sensory gating to acoustic stimulation, pain sensitivity, motor behavior, spatial learning and memory deficits on the hole-board test were assessed in the 17th generation of selectively bred Wistar rats compared to their naive counterparts with or without complex treatment. We found differences between the sexes: selectively bred males with complex treatment showed the lowest pain sensitivity; however, the results of the prepulse inhibition test indicated that female rats showed enhanced impairment of sensory gating and increased acoustic startle reaction. The cognitive performance, working and reference memory ratios were significantly decreased by selective breeding and showed sex-specific alterations, with the smallest value in male rats of the new substrain. Based on these results, the animals of the new substrain could be classified into the high-risk for schizophreniform phenotype with the highest sensitivity of males with complex treatment. Decreased cognitive performance highlighted spatial learning deficits in the selectively bred and treated rats that escalate the validity of our new and complex rat model of schizophrenia. The results indicate the same sex selectivity as observed in humans, with increased incidence of risk ratios for men to develop schizophrenia relative to women.

  13. Alteration of substrate specificities of thermophilic α/β hydrolases through domain swapping and domain interface optimization

    Institute of Scientific and Technical Information of China (English)

    Xiaoli Zhou; Honglei Wang; Yuhang Zhang; Le Gao; Yan Feng

    2012-01-01

    Protein domain swapping is an efficient way in protein functional evolution in vivo and also has been proved to be an effective strategy to modify the function of the multidomain proteins in vitro.To explore the potentials of domain swapping for alteration of the enzyme substrate specificities and the structure-function relationship of the homologous proteins,here we constructed two chimeras from a pair of thermophilic members of the α/β hydrolase superfamily by grafting their functional domains to the conserved α/β hydrolase fold domain:a carboxylesterase from Archaeoglobus fulgidus (AFEST) and an acylpeptide hydrolase from Aeropyrum pernix K1 (apAPH) and explored their activities on hydrolyze p-nitrophenyl esters (pNP) with different acyl chain lengths.We took two approaches to reduce the crossover disruptions when creating the chimeras:chose the residue which involved in the least contacts as the splicing site and optimized the newly formed domain interfaces of the chimeras by sitedirected mutations.Characterizations of AAM7 and PAR showed that these chimeras inherited the thermophilic property of both parents.In the aspect of substrate specificity,AAM7 and PAR showed highest activity towards short chain length substrate pNPC4 and middle chain length substrate pNPC8,similar to parent AFEST and apAPH,respectively.These results suggested that the substrate-binding domain is the dominant factor on enzyme substrate specificity,and the optimization of the newly formed domain interface is an important guarantee for successful domain swapping of proteins with low-sequence homology.

  14. Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination.

    Directory of Open Access Journals (Sweden)

    Corinne Grey

    2011-10-01

    Full Text Available Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3 levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.

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

    Directory of Open Access Journals (Sweden)

    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

  16. Mcp6, a meiosis-specific coiled-coil protein of Schizosaccharomyces pombe, localizes to the spindle pole body and is required for horsetail movement and recombination.

    Science.gov (United States)

    Saito, Takamune T; Tougan, Takahiro; Okuzaki, Daisuke; Kasama, Takashi; Nojima, Hiroshi

    2005-01-15

    We report here that a meiosis-specific gene of Schizosaccharomyces pombe denoted mcp6+ (meiotic coiled-coil protein) encodes a protein that is required for the horsetail movement of chromosomes at meiosis I. The mcp6+ gene is specifically transcribed during the horsetail phase. Green fluorescent protein (GFP)-tagged Mcp6 appears at the start of karyogamy, localizes to the spindle-pole body (SPB) and then disappears before chromosome segregation at meiosis I. In the mcp6Delta strain, the horsetail movement was either hampered (zygotic meiosis) or abolished (azygotic meiosis) and the pairing of homologous chromosomes was impaired. Accordingly, the allelic recombination rates of the mcp6Delta strain were only 10-40% of the wild-type rates. By contrast, the ectopic recombination rate of the mcp6Delta strain was twice the wild-type rate. This is probably caused by abnormal homologous pairing in mcp6Delta cells because of aberrant horsetail movement. Fluorescent microscopy indicates that SPB components such as Sad1, Kms1 and Spo15 localize normally in mcp6Delta cells. Because Taz1 and Swi6 also localized with Sad1 in mcp6Delta cells, Mcp6 is not required for telomere clustering. In a taz1Delta strain, which does not display telomere clustering, and the dhc1-d3 mutant, which lacks horsetail movement, Mcp6 localized with Sad1 normally. However, we observed abnormal astral microtubule organization in mcp6Delta cells. From these results, we conclude that Mcp6 is necessary for neither SPB organization nor telomere clustering, but is required for proper astral microtubule positioning to maintain horsetail movement. PMID:15654021

  17. Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system

    Directory of Open Access Journals (Sweden)

    Bihao Cao

    2010-01-01

    Full Text Available This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line ('E-38' was transformed with Cre gene and the inbred/pure line ('E-8' was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T0-plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T0-plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.

  18. A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition

    OpenAIRE

    Kvajo, Mirna; McKellar, Heather; Arguello, P. Alexander; Liam J Drew; Moore, Holly; Macdermott, Amy B; Karayiorgou, Maria; Gogos, Joseph A.

    2008-01-01

    DISC1 is a strong candidate susceptibility gene for schizophrenia, bipolar disorder, and depression. Using a mouse strain carrying an endogenous Disc1 orthologue engineered to model the putative effects of the disease-associated chromosomal translocation we demonstrate that impaired Disc1 function results in region-specific morphological alterations, including alterations in the organization of newly born and mature neurons of the dentate gyrus. Field recordings at CA3/CA1 synapses revealed a...

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

    Science.gov (United States)

    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. PMID:26255274

  20. The adrenal specific toxicant mitotane directly interacts with lipid membranes and alters membrane properties depending on lipid composition.

    Science.gov (United States)

    Scheidt, Holger A; Haralampiev, Ivan; Theisgen, Stephan; Schirbel, Andreas; Sbiera, Silviu; Huster, Daniel; Kroiss, Matthias; Müller, Peter

    2016-06-15

    Mitotane (o,p'.-DDD) is an orphan drug approved for the treatment of adrenocortical carcinoma. The mechanisms, which are responsible for this activity of the drug, are not completely understood. It can be hypothesized that an impact of mitotane is mediated by the interaction with cellular membranes. However, an interaction of mitotane with (lipid) membranes has not yet been investigated in detail. Here, we characterized the interaction of mitotane and its main metabolite o,p'-dichlorodiphenyldichloroacetic acid (o,p'-DDA) with lipid membranes by applying a variety of biophysical approaches of nuclear magnetic resonance, electron spin resonance, and fluorescence spectroscopy. We found that mitotane and o,p'-DDA bind to lipid membranes by inserting into the lipid-water interface of the bilayer. Mitotane but not o,p'-DDA directly causes a disturbance of bilayer structure leading to an increased permeability of the membrane for polar molecules. Mitotane induced alterations of the membrane integrity required the presence of phosphatidylethanolamine and/or cholesterol. Collectively, our data for the first time characterize the impact of mitotane on the lipid membrane structure and dynamics, which may contribute to a better understanding of specific mitotane effects and side effects. PMID:27002491

  1. The mutated S1-haplotype in sour cherry has an altered S-haplotype-specific F-box protein gene.

    Science.gov (United States)

    Hauck, Nathanael R; Ikeda, Kazuo; Tao, Ryutaro; Iezzoni, Amy F

    2006-01-01

    Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S(1)-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Ujfehértói Fürtös carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI. PMID:16985081

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

    Science.gov (United States)

    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.

  3. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    Science.gov (United States)

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  4. Altered immune response of immature dendritic cells following dengue virus infection in the presence of specific antibodies.

    Science.gov (United States)

    Torres, Silvia; Flipse, Jacky; Upasani, Vinit C; van der Ende-Metselaar, Heidi; Urcuqui-Inchima, Silvio; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2016-07-01

    Dengue virus (DENV) replication is known to prevent maturation of infected dendritic cells (DCs) thereby impeding the development of adequate immunity. During secondary DENV infection, dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), however how dengue-antibody complexes (DENV-IC) influence the phenotype of DCs remains elusive. Here, we evaluated the maturation state and cytokine profile of immDCs exposed to DENV-ICs. Indeed, DENV infection of immDCs in the absence of antibodies was hallmarked by blunted upregulation of CD83, CD86 and the major histocompatibility complex molecule HLA-DR. In contrast, DENV infection in the presence of neutralizing antibodies triggered full DC maturation and induced a balanced inflammatory cytokine response. Moreover, DENV infection under non-neutralizing conditions prompted upregulation of CD83 and CD86 but not HLA-DR, and triggered production of pro-inflammatory cytokines. The effect of DENV-IC was found to be dependent on the engagement of FcγRIIa. Altogether, our data show that the presence of DENV-IC alters the phenotype and cytokine profile of DCs. PMID:27121645

  5. 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. PMID:27416794

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

    International Nuclear Information System (INIS)

    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. 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. 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 heparanase 2, which displays anti-metastatic features

  7. Site-specific DNA double-strand break generated by I-SceI endonuclease enhances ectopic homologous recombination in Pyricularia oryzae.

    Science.gov (United States)

    Arazoe, Takayuki; Younomaru, Tetsuya; Ohsato, Shuichi; Kimura, Makoto; Arie, Tsutomu; Kuwata, Shigeru

    2014-03-01

    To evaluate the contribution of DNA double-strand breaks (DSBs) to somatic homologous recombination (HR) in Pyricularia oryzae, we established a novel detection/selection system of DSBs-mediated ectopic HR. This system consists of donor and recipient nonfunctional yellow fluorescent protein (YFP)/blasticidin S deaminase (BSD) fusion genes and the yeast endonuclease I-SceI gene as a recipient-specific DSB inducer. The system enables to detect and select ectopic HR events by the restoration of YFP fluorescence and blasticidin S resistance. The transformed lines with donor and recipient showed low frequencies of endogenous ectopic HR (> 2.1%). Compared with spontaneous HR, c. 20-fold increases in HR and absolute frequency of HR as high as 40% were obtained by integration of I-SceI gene, indicating that I-SceI-mediated DSB was efficiently repaired via ectopic HR. Furthermore, to validate the impact of DSB on targeted gene replacement (TGR), the transformed lines with a recipient gene were transfected with an exogenous donor plasmid in combination with the DSB inducer. TGR events were not observed without the DSB inducer, whereas hundreds of colonies resulting from TGR events were obtained with the DSB inducer. These results clearly demonstrated that the introduction of site-specific DSB promotes ectopic HR repair in P. oryzae. PMID:24517488

  8. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

    Recombinant antibodies are highly specific detection probes in research, diagnostics, and have emerged over the last two decades as the fastest growing class of therapeutic proteins. Antibody generation has been dramatically accelerated by in vitro selection systems, particularly phage display. An increasing variety of recombinant production systems have been developed, ranging from Gram-negative and positive bacteria, yeasts and filamentous fungi, insect cell lines, mammalian cells to transg...

  9. A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation, reduced recombination of nuclear genes, and altered transmission of chloroplast genes.

    Science.gov (United States)

    Rosen, H; Newman, S M; Boynton, J E; Gillham, N W

    1991-01-01

    Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt+ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt- parent, arise at a low frequency. Mutations at the mt(+)-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt- parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically with the mat-3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt+ parent.

  10. 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

  11. 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

  12. A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Deng, Guangcun; Li, Min; Zeng, Jin; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2014-11-01

    Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.

  13. Recombination instability

    DEFF Research Database (Denmark)

    D'Angelo, N.

    1967-01-01

    A recombination instability is considered which may arise in a plasma if the temperature dependence of the volume recombination coefficient, alpha, is sufficiently strong. Two cases are analyzed: (a) a steady-state plasma produced in a neutral gas by X-rays or high energy electrons; and (b...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

    Science.gov (United States)

    Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

    2010-01-01

    Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb. PMID:19597742

  16. Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice.

    Science.gov (United States)

    Sabio y García, Julia V; Bigi, Fabiana; Rossetti, Osvaldo; Campos, Eleonora

    2010-12-01

    Immunodominant MPB83 antigen from Mycobacterium bovis was expressed as a chimeric protein fused to either β-galactosidase, outer membrane lipoprotein OMP19 or periplasmic protein BP26 in gram-negative Brucella abortus S19, in all cases driven by each gene's own promoter. All fusion proteins were successfully expressed and localized in the expected subcellular fraction. Moreover, OMP19-MPB83 was processed as a lipoprotein when expressed in B. abortus. Splenocytes from BALB/c mice immunized with the recombinant S19 strains carrying the genes coding for the heterologous antigens in replicative plasmids, showed equally specific INF-γ production in response to MPB83 stimulation. Association to the lipid moiety of OMP19 presented no advantage in terms of immunogenicity for MPB83. In contrast, fusion to BP26, which was encoded by an integrative plasmid, resulted in a weaker immune response. None of the constructions affected the survival rate or the infection pattern of Brucella. We concluded that B. abortus S19 is an appropriate candidate for the expression of M. bovis antigens both associated to the membrane or cytosolic fraction and may provide the basis for a future combined vaccine for bovine brucellosis and tuberculosis. PMID:20888425

  17. Comparison of immunoglobulin E measurements on IMMULITE and ImmunoCAP in samples consisting of allergen-specific mouse-human chimeric monoclonal antibodies towards allergen extracts and four recombinant allergens

    DEFF Research Database (Denmark)

    Szecsi, Pal B; Stender, Steen

    2013-01-01

    Specific immunoglobulin E (IgE) antibody in vitro tests are performed on enzyme immunoassay systems. Poor agreement among systems has been reported and comparisons have been made exclusively with allergen extracts - not with recombinant allergens. Here we compare the ImmunoCAP and the IMMULITE...

  18. Enhancement of human immunodeficiency virus (HIV)-specific CD4+ and CD8+ cytotoxic T-lymphocyte activities in HIV-infected asymptomatic patients given recombinant gp160 vaccine.

    OpenAIRE

    Kundu, S K; Katzenstein, D; Moses, L E; Merigan, T C

    1992-01-01

    Twenty-six human immunodeficiency virus (HIV)-infected asymptomatic patients with CD4+ lymphocytes > 400 per mm3 were randomly allocated to a range of doses of recombinant gp160 or a control (recombinant hepatitis B vaccine) on a double-blind basis. Each patient received an injection at 0, 4, 12, 24, 36, and 48 weeks. Treatment assignments were decoded when all patients reached 28 weeks of the study period. HIV-1-specific CD4+ and CD8+ cytotoxic T lymphocyte (CTL) activities were assessed in ...

  19. Location, degree, and direction of DNA bending associated with the Hin recombinational enhancer sequence and Fis-enhancer complex.

    OpenAIRE

    Perkins-Balding, D; D. P Dias; Glasgow, A. C.

    1997-01-01

    The Fis protein of Escherichia coli and Salmonella typhimurium stimulates several site-specific DNA recombination reactions, as well as transcription of a number of genes. Fis binds to a 15-bp core recognition sequence and induces DNA bending. Mutations in Fis which alter its ability to bend DNA have been shown to reduce the stimulatory activity of Fis in both site-specific recombination and transcription systems. To examine the role of DNA bending in the activity of the Fis-recombinational e...

  20. Zebrafish fed on recombinant Artemia expressing epinecidin-1 exhibit increased survival and altered expression of immunomodulatory genes upon Vibrio vulnificus infection.

    Science.gov (United States)

    Jheng, Yu-Hsuan; Lee, Lin-Han; Ting, Chen-Hung; Pan, Chieh-Yu; Hui, Cho-Fat; Chen, Jyh-Yih

    2015-01-01

    Artemia has been used extensively in aquaculture as fodder for larval fish, shrimp, and shellfish. Epinecidin-1, an antimicrobial peptide, was isolated from grouper (Epinephelus coioides) in 2005. Epinecidin-1 has been previously reported to possess antimicrobial activity against several Gram-positive and Gram-negative bacterial species, including Staphylococcus coagulase, Pseudomonas aeruginosa, Streptococcus pyogenes, and Vibrio vulnificus. In this study, we used electroporation to introduce plasmid DNA encoding a green fluorescent protein (EGFP)-epinecidin-1 fusion protein under the control of the cytomegalovirus (CMV) promoter into decapsulated Artemia cysts. Optimization of various properties (including cyst weight (0.2 g), plasmid concentration (50 μg/100 μl), and pulse voltage (150 V), length (10 ms), and number (2)) resulted in a hatching rate of 41.15%, a transfection efficiency of 49.81%, and a fluorescence intensity (A.U.) of 47.46. The expression of EGFP-epinecidin-1 was first detected by quantitative RT-PCR at 120 h post-electroporation, and protein was identified by Western blot at the same time. Furthermore, the EGFP-epinecidin-1 protein inhibited V. vulnificus (204) growth, as demonstrated by zone of inhibition studies. Zebrafish fed on transgenic Artemia expressing CMV-gfp-epi combined with commercial fodder were more resistant to infection by V. vulnificus (204): survival rate was enhanced by over 70% at 7, 14, and 21 days post-infection, and bacterial numbers in the liver and intestine were reduced. In addition, feeding of transgenic Artemia to zebrafish affected the immunomodulatory response to V. vulnificus (204) infection; expression of immune-responsive genes, including hepcidin and defbl2, was altered, as shown by qPCR. These findings suggest that feeding transgenic Artemia expressing CMV-gfp-epi to larval fish has antimicrobial effects, without the drawbacks of introducing drug residues or inducing bacterial drug resistance.

  1. 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.

  2. A common SNP in ER aminopeptidase 2 induces a specificity switch that leads to altered antigen processing

    OpenAIRE

    Evnouchidou, Irini; Birtley, James; Seregin, Sergey; Papakyriakou, Athanasios; Zervoudi, Efthalia; Samiotaki, Martina; Panayotou, George; Giastas, Petros; Petrakis, Olivia; Georgiadis, Dimitris; Amalfitano, Andrea; Saridakis, Emmanuel; Mavridis, Irene M.; Stratikos, Efstratios

    2012-01-01

    ER aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms (SNPs) in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered antigen processing by these enzymes is a causal link to disease etiology but the ...

  3. Mutation of senataxin alters disease-specific transcriptional networks in patients with ataxia with oculomotor apraxia type 2

    OpenAIRE

    Fogel, Brent L.; Cho, Ellen; Wahnich, Amanda; Gao, Fuying; Becherel, Olivier J.; Wang, Xizhe; Fike, Francesca; Chen, Leslie; Criscuolo, Chiara; De Michele, Giuseppe; Filla, Alessandro; Collins, Abigail; Hahn, Angelika F.; Gatti, Richard A.; Konopka, Genevieve

    2014-01-01

    Senataxin, encoded by the SETX gene, contributes to multiple aspects of gene expression, including transcription and RNA processing. Mutations in SETX cause the recessive disorder ataxia with oculomotor apraxia type 2 (AOA2) and a dominant juvenile form of amyotrophic lateral sclerosis (ALS4). To assess the functional role of senataxin in disease, we examined differential gene expression in AOA2 patient fibroblasts, identifying a core set of genes showing altered expression by microarray and ...

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

    Science.gov (United States)

    Gach, Johannes S; Gorlani, Andrea; Dotsey, Emmanuel Y; Becerra, Juan C; Anderson, Chase T M; Berzins, Baiba; Felgner, Philip L; Forthal, Donald N; Deeks, Steven G; Wilkin, Timothy J; Casazza, Joseph P; Koup, Richard A; Katlama, Christine; Autran, Brigitte; Murphy, Robert L; Achenbach, Chad J

    2016-01-01

    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. PMID:27500639

  5. Alteration of the carbohydrate-binding specificity of a C-type lectin CEL-I mutant with an EPN carbohydrate-binding motif.

    Science.gov (United States)

    Hatakeyama, Tomomitsu; Ishimine, Tomohiro; Baba, Tomohiro; Kimura, Masanari; Unno, Hideaki; Goda, Shuichiro

    2013-07-01

    CEL-I is a Gal/GalNAc-specific C-type lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-recognition domains (CRDs) with the carbohydrate-recognition motif QPD (Gln-Pro- Asp), which is generally known to exist in galactose-specific C-type CRDs. In the present study, a mutant CEL-I with EPN (Glu-Pro-Asn) motif, which is thought to be responsible for the carbohydrate-recognition of mannose-specific Ctype CRDs, was produced in Escherichia coli, and its effects on the carbohydrate-binding specificity were examined using polyamidoamine dendrimer (PD) conjugated with carbohydrates. Although wild-type CEL-I effectively formed complexes with N-acetylgalactosamine (GalNAc)-PD but not with mannose-PD, the mutant CEL-I showed relatively weak but definite affinity for mannose-PD. These results indicated that the QPD and EPN motifs play a significant role in the carbohydrate-recognition mechanism of CEL-I, especially in the discrimination of galactose and mannose. Additional mutations in the recombinant CEL-I binding site may further increase its specificity for mannose, and should provide insights into designing novel carbohydrate-recognition proteins. PMID:23157284

  6. Specifications

    International Nuclear Information System (INIS)

    As part of the Danish RERTR Program, three fuel elements with LEU U3O8-Al fuel and three fuel elements with LEU U3Si2-Al fuel were manufactured by NUKEM for irradiation testing in the DR-3 reactor at the Risoe National Laboratory in Denmark. The specifications for the elements with U3O8-Al fuel are presented here as an illustration only. Specifications for the elements with U3Si2-Al fuel were very similar. In this example, materials, material numbers, documents numbers, and drawing numbers specific to a single fabricator have been deleted. (author)

  7. Fms-like tyrosine kinase-3 ligand alters antigen-specific responses to infections after severe burn injury

    OpenAIRE

    Bohannon, Julia; Fang, Geping; Cui, Weihua; Sherwood, Edward; Toliver-Kinsky, Tracy

    2009-01-01

    Burn patients are susceptible to opportunistic infections due partly to decreased immune functions, especially Th1-driven antigen-specific responses, which are regulated by dendritic cells. The dendritic cell growth factor, fms-like tyrosine kinase-3 ligand (FL), has been shown to increase resistance to P. aeruginosa, in a dendritic cell-dependent manner, in a mouse model of burn wound infection. The specific mechanisms of protection are not known. This study tested the hypothesis that FL can...

  8. Framing alters risk-taking behavior on a modified Balloon Analogue Risk Task (BART) in a sex-specific manner.

    Science.gov (United States)

    Gabriel, Kara I; Williamson, Ashley

    2010-12-01

    Framing uncertain scenarios to emphasize potential positive or negative elements influences decision making and behavior. The current experiment investigated sex differences in framing effects on risk-taking propensity in a modified version of the Balloon Analogue Risk Task (BART). Male and female undergraduates completed questionnaires on sensation seeking, impulsiveness, and risk and benefit perception prior to viewing one of three framing conditions for the BART: (1) positively-framed instructions emphasizing the ability to earn money if balloons were inflated to large size; (2) negatively framed instructions emphasizing the possibility that money could be lost if balloons were inflated to bursting; and (3) completely framed instructions noting both possible outcomes. Results revealed correlations between BART performance and impulsiveness for both sexes. Compared to positive and complete framing, negatively framed instructions decreased balloon inflation time in women but not men, indicating sex differences in response to treatments designed to alter risk-taking behavior. PMID:21323127

  9. Chronic stress induces sex-specific alterations in methylation and expression of corticotropin-releasing factor gene in the rat.

    Directory of Open Access Journals (Sweden)

    Linda Sterrenburg

    Full Text Available BACKGROUND: Although the higher prevalence of depression in women than in men is well known, the neuronal basis of this sex difference is largely elusive. METHODS: Male and female rats were exposed to chronic variable mild stress (CVMS after which immediate early gene products, corticotropin-releasing factor (CRF mRNA and peptide, various epigenetic-associated enzymes and DNA methylation of the Crf gene were determined in the hypothalamic paraventricular nucleus (PVN, oval (BSTov and fusiform (BSTfu parts of the bed nucleus of the stria terminalis, and central amygdala (CeA. RESULTS: CVMS induced site-specific changes in Crf gene methylation in all brain centers studied in female rats and in the male BST and CeA, whereas the histone acetyltransferase, CREB-binding protein was increased in the female BST and the histone-deacetylase-5 decreased in the male CeA. These changes were accompanied by an increased amount of c-Fos in the PVN, BSTfu and CeA in males, and of FosB in the PVN of both sexes and in the male BSTov and BSTfu. In the PVN, CVMS increased CRF mRNA in males and CRF peptide decreased in females. CONCLUSIONS: The data confirm our hypothesis that chronic stress affects gene expression and CRF transcriptional, translational and secretory activities in the PVN, BSTov, BSTfu and CeA, in a brain center-specific and sex-specific manner. Brain region-specific and sex-specific changes in epigenetic activity and neuronal activation may play, too, an important role in the sex specificity of the stress response and the susceptibility to depression.

  10. Altered immune response of immature dendritic cells upon dengue virus infection in the presence of specific antibodies

    NARCIS (Netherlands)

    Torres, Silvia; Flipse, Jacky; Upasani, Vinit C; van der Ende-Metselaar, Heidi; Urcuqui-Inchima, Silvio; Smit, Jolanda M; Rodenhuis-Zybert, Izabela A

    2016-01-01

    Dengue virus (DENV) replication is known to prevent maturation of infected DCs thereby impeding the development of adequate immunity. During secondary DENV infection, dengue-specific antibodies can suppress DENV replication in immature DCs (immDCs), however how dengue-antibody complexes (DENV-IC) in

  11. Cold-Induced Browning Dynamically Alters the Expression Profiles of Inflammatory Adipokines with Tissue Specificity in Mice

    Directory of Open Access Journals (Sweden)

    Xiao Luo

    2016-05-01

    Full Text Available Cold exposure or β3-adrenoceptor agonist treatment induces the adipose tissues remodeling, relevant for beige adipogenesis within white adipose tissue (WAT. It remains unclear whether this process influences inflammatory adipokines expression in adipose tissues. We determine the temporal profile of cold or β3-adrenoceptor agonist (CL316,243-induced changes in the expression of inflammatory adipokines in adipose tissues in mice or primary mice adipocytes. Male C57BL/6J mice at eight weeks old were exposed to 4 °C for 1–5 days. Interscapular brown adipose tissue (iBAT, inguinal subcutaneous WAT (sWAT and epididymal WAT (eWAT were harvested for gene and protein expression analysis. In addition, cultured primary mice brown adipocyte (BA and white adipocyte (WA treated with or without CL316,243 were harvested for gene expression analysis. The inflammatory adipokines expressed significantly higher in WAT than BAT at baseline. They were rapidly changed in iBAT, while down-regulated in sWAT and up-regulated in eWAT during the cold acclimation. Upon CL316,243 treatment, detected inflammatory adipokines except Leptin were transiently increased in both BA and WA. Our in vivo and in vitro data demonstrate that the browning process alters the inflammatory adipokines expression in adipose tissues, which is acutely responded to in iBAT, dynamically decreased in sWAT whilst increased in eWAT for compensation.

  12. The Contribution of the C-Terminal Tails of Microtubules in Altering the Force Production Specifications of Multiple Kinesin-1.

    Science.gov (United States)

    Feizabadi, Mitra Shojania

    2016-09-01

    The extent to which beta tubulin isotypes contribute to the function of microtubules and the microtubule-driven transport of molecular motors is poorly understood. The major differences in these isotypes are associated with the structure of their C-terminal tails. Recent studies have revealed a few aspects of the C-terminal tails' regulatory role on the activities of some of the motor proteins on a single-molecule level. However, little attention is given to the degree to which the function of a team of motor proteins can be altered by the microtubule's tail. In a set of parallel experiments, we investigated this open question by studying the force production of several kinesin-1 (kinesin) molecular motors along two groups of microtubules: regular ones and those microtubules whose C-terminals are cleaved by subtilisin digestion. The results indicate that the difference between the average of the force production of motors along two types of microtubules is statistically significant. The underlying mechanism of such production is substantially different as well. As compared to untreated microtubules, the magnitude of the binding time of several kinesin-1 is almost three times greater along subtilisin-treated microtubules. Also, the velocity of the group of kinesin molecules shows a higher sensitivity to external loads and reduces significantly under higher loads along subtilisin-treated microtubules. Together, this work shows the capacity of the tails in fine-tuning the force production characteristics of several kinesin molecules. PMID:27503105

  13. Maternal deprivation of rat pups reduces body weight and alters behavior in adulthood in a gender-specific manner

    Directory of Open Access Journals (Sweden)

    Đorđević Jelena

    2015-01-01

    Full Text Available The early postnatal environment is critical for its capacity to influence adult behavior, and is associated with traits of altered physiological and neurobiological function and long-term predisposition to depression. Here we describe the delayed effects of maternal deprivation (MD in male and female Wistar pups on their physical development and behavior in adulthood in tasks designed to explore depressive-like (forced swimming test, FST, and anxiety-like behaviors (elevated plus maze, EPM. We observed that MD led to reduced body weight in adulthood, anxiety-like traits in the EPM test and increased activity in the phases of the FST. Particularly, a consistent sexual dimorphism was observed in the responses to MD. A lower increase in body weight during maturation of MD rats was more pronounced in males than in females. MD anxiogenic effects were more pronounced in females, while in FST only MD males showed a marked increase in swimming activity followed by decreased immobility. [Projekat Ministarstva nauke Republike Srbije, br. III41029

  14. Specific siRNA Downregulated TLR9 and Altered Cytokine Expression Pattern in Macrophage after CpG DNA Stimulation

    Institute of Scientific and Technical Information of China (English)

    Bin Qiao; Baohua Li; Xiuli Yang; Hongyong Zhang; Yiwei Chu; Ying Wang; Sidong Xiong

    2005-01-01

    Bacterial CpG DNA or synthetic oligonucleotides (ODNs) that contain unmethylated CpG motifs (CpG ODN) can directly activate antigen-presenting cells (APCs) to secrete various cytokines through the intracellular receptor TLR9. Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses. To date, cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated. In the present study, vector-based siRNA was used to downregulate TLR9 expression. Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid upon CpG ODN stimulation. We found that not all the cytokine expressions by the macrophage were decreased while TLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased, but IL-6,IFN-β and IL-10 expressions were not affected. Interestingly, the level of IFN-α was even increased. This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases. Cellular & Molecular Immunology.2005;2(2):130-135.

  15. Specific siRNA Downregulated TLR9 and Altered Cytokine Expression Pattern in Macrophage after CpG DNA Stimulation

    Institute of Scientific and Technical Information of China (English)

    BinQiao; BaohuaLi; XiuliYang; HongyongZhang; YiweiChu; YingWang; SidongXiong

    2005-01-01

    Bacterial CpG DNA or synthetic oligonucleotides (ODNs) that contain unmethylated CpG motifs (CpG ODN) can directly activate antigen-presenting cells (APCs) to secrete various cytokines through the intraceilular receptor TL R9. Cytokine profiles elicited by the actions of stimulatory CpG DNA on TLR9 expressed APCs are crucial to the subsequent immune responses. To date, cytokine profiles in APCs upon CpG ODN stimulation in vitro are not fully investigated. In the present study, vector-based siRNA was used to downregulate TLR9 expression. Cytokine profiles were observed in murine macrophage cell line RAW264.7 transfected with TLR9-siRNA plasmid uponCpG ODN stimulation. We found that not all the cytokine expressions by the macrophage were decreased whileTLR9 was downregulated. IL-12, TNF-α, IFN-γ and IL-1β expressions were significantly decreased, but IL-6, IFN-β and IL-10 expressions were not affected. Interestingly, the level of IFN-α was even increased. This alteration of cytokines produced by TLR9-downregulated APCs upon CpG ODN stimulation might indicate that the role of CpG DNA is more complicated in the pathogenesis and prevention of diseases. Cellular & Molecular Immunology. 2005;2(2):130-135.

  16. 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

    2016-04-01

    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 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 a litter 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). PMID:26661793

  17. Region-specific alterations in the corticotropin-releasing factor and glucocorticoid receptors in the postmortem brain of suicide victims

    OpenAIRE

    Pandey, Ghanshyam N.

    2012-01-01

    Rationale : Abnormalities of hypothalamic–pituitary–adrenal (HPA) axis in depression and suicide are among the most consistent findings in biological psychiatry. However, the specific molecular mechanism associated with HPA axis abnormality in the brain of depressed or suicidal subjects is not clear. It is believed that abnormal HPA axis is caused by increased levels of corticotropin-releasing factor (CRF) and decreased levels of glucocorticoid receptor (GR) in the brain of depr...

  18. Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

    Science.gov (United States)

    Weissmann, D; van der Laan, S; Underwood, M D; Salvetat, N; Cavarec, L; Vincent, L; Molina, F; Mann, J J; Arango, V; Pujol, J F

    2016-01-01

    Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide. PMID:27576167

  19. Enhanced specific immune responses by CpG DNA in mice immunized with recombinant hepatitis B surface antigen and HB vaccine

    Directory of Open Access Journals (Sweden)

    Wang Xingtai

    2011-02-01

    Full Text Available Abstract Background Hepatitis B vaccine adjuvant, alum, is generally used for vaccination although it does not stimulate Th1 immunity and 10% of the population has low or no antibody response. Efforts have been continued to find more efficient vaccine adjuvants for better antibody response as well as stimulation of Th1 immunity. Methods CpG DNA was used as an adjuvant for recombinant HBsAg to immunize 6- to 8-week-old female BALB/c mice with or without alum for different dosages. The production of HBsAb, CD80 and CD86 from dendritic cells, and cytokines IL-10, IL12, etc., were analyzed and compared for the performance of immunization. Results 5-20 μg CpG DNA had the best co-stimulation effect of HBsAb serum conversion for mice vaccinated with recombinant expressed HBsAg. The mice vaccinated with recombinant 20 μg CpG DNA and regular vaccine (containing alum adjuvant had the highest concentration of antibody production. IL-12b, IL-12a and IL10 mRNA reached to the peak level between 3 and 6 hours after the CpG DNA induction in splenocytes. The expression levels of CD80 and CD86 leucocyte surface molecules were increased with 20 μg CpG DNA alone or with 20 μg CpG DNA and 4 μg HBsAg. Conclusions Our results confirmed the adjuvant effect of CpG DNA for HBsAg in the mouse model. The increase of IL10 and IL12 production suggested the involvement of Th1 cell activation. The activation of CD80 and CD86 molecules by CpG-ODN might be part of the mechanism of T/B cells coordination and the enhancement of recombinant HBsAg induced immune response.

  20. Analysis of interchromosomal mitotic recombination.

    Science.gov (United States)

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

    1990-07-01

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

  1. Mitochondrial Alterations by PARKIN in Dopaminergic Neurons Using PARK2 Patient-Specific and PARK2 Knockout Isogenic iPSC Lines

    Directory of Open Access Journals (Sweden)

    Atossa Shaltouki

    2015-05-01

    Full Text Available In this study, we used patient-specific and isogenic PARK2-induced pluripotent stem cells (iPSCs to show that mutations in PARK2 alter neuronal proliferation. The percentage of TH+ neurons was decreased in Parkinson’s disease (PD patient-derived neurons carrying various mutations in PARK2 compared with an age-matched control subject. This reduction was accompanied by alterations in mitochondrial:cell volume fraction (mitochondrial volume fraction. The same phenotype was confirmed in isogenic PARK2 null lines. The mitochondrial phenotype was also seen in non-midbrain neurons differentiated from the PARK2 null line, as was the functional phenotype of reduced proliferation in culture. Whole genome expression profiling at various stages of differentiation confirmed the mitochondrial phenotype and identified pathways altered by PARK2 dysfunction that include PD-related genes. Our results are consistent with current model of PARK2 function where damaged mitochondria are targeted for degradation via a PARK2/PINK1-mediated mechanism.

  2. Genetic Exchange in an Arbuscular Mycorrhizal Fungus Results in Increased Rice Growth and Altered Mycorrhiza-Specific Gene Transcription▿†

    Science.gov (United States)

    Colard, Alexandre; Angelard, Caroline; Sanders, Ian R.

    2011-01-01

    Arbuscular mycorrhizal fungi (AMF) are obligate symbionts with most terrestrial plants. They improve plant nutrition, particularly phosphate acquisition, and thus are able to improve plant growth. In exchange, the fungi obtain photosynthetically fixed carbon. AMF are coenocytic, meaning that many nuclei coexist in a common cytoplasm. Genetic exchange recently has been demonstrated in the AMF Glomus intraradices, allowing nuclei of different Glomus intraradices strains to mix. Such genetic exchange was shown previously to have negative effects on plant growth and to alter fungal colonization. However, no attempt was made to detect whether genetic exchange in AMF can alter plant gene expression and if this effect was time dependent. Here, we show that genetic exchange in AMF also can be beneficial for rice growth, and that symbiosis-specific gene transcription is altered by genetic exchange. Moreover, our results show that genetic exchange can change the dynamics of the colonization of the fungus in the plant. Our results demonstrate that the simple manipulation of the genetics of AMF can have important consequences for their symbiotic effects on plants such as rice, which is considered the most important crop in the world. Exploiting natural AMF genetic variation by generating novel AMF genotypes through genetic exchange is a potentially useful tool in the development of AMF inocula that are more beneficial for crop growth. PMID:21784911

  3. Selected sperm traits are simultaneously altered after scrotal heat stress and play specific roles in in vitro fertilization and embryonic development.

    Science.gov (United States)

    Lucio, Aline C; Alves, Benner G; Alves, Kele A; Martins, Muller C; Braga, Lucas S; Miglio, Luisa; Alves, Bruna G; Silva, Thiago H; Jacomini, José O; Beletti, Marcelo E

    2016-09-01

    Improvements in the estimation of male fertility indicators require advances in laboratory tests for sperm assessment. The aims of the present work were (1) to apply a multivariate analysis to examine sperm set of alterations and interactions and (2) to evaluate the importance of sperm parameters on the outcome of standard IVF and embryonic development. Bulls (n = 3) were subjected to scrotal insulation, and ejaculates were collected before (preinsulation = Day 0) and through 56 days (Days 7, 14, 21, 28, 35, 42, 49, and 56) of the experimental period. Sperm head morphometry and chromatin variables were assessed by a computational image analysis, and IVF was performed. Scrotal heat stress induced alterations in all evaluated sperm head features, as well as cleavage and blastocyst rates. A principal component analysis revealed three main components (factors) that represented almost 89% of the cumulative variance. In addition, an association of factor scores with cleavage (factor 1) and blastocyst (factor 3) rates was observed. In conclusion, several sperm traits were simultaneously altered as a result of a thermal insult. These sperm traits likely play specific roles in IVF and embryonic development. PMID:27087533

  4. 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

    2015-04-01

    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.

  5. Mutation in the Pro-Peptide Region of a Cysteine Protease Leads to Altered Activity and Specificity-A Structural and Biochemical Approach.

    Science.gov (United States)

    Dutta, Sruti; Choudhury, Debi; Roy, Sumana; Dattagupta, Jiban Kanti; Biswas, Sampa

    2016-01-01

    Papain-like proteases contain an N-terminal pro-peptide in their zymogen form that is important for correct folding and spatio-temporal regulation of the proteolytic activity of these proteases. Catalytic removal of the pro-peptide is required for the protease to become active. In this study, we have generated three different mutants of papain (I86F, I86L and I86A) by replacing the residue I86 in its pro-peptide region, which blocks the specificity determining S2-subsite of the catalytic cleft of the protease in its zymogen form with a view to investigate the effect of mutation on the catalytic activity of the protease. Steady-state enzyme kinetic analyses of the corresponding mutant proteases with specific peptide substrates show significant alteration of substrate specificity-I86F and I86L have 2.7 and 29.1 times higher kcat/Km values compared to the wild-type against substrates having Phe and Leu at P2 position, respectively, while I86A shows lower catalytic activity against majority of the substrates tested. Far-UV CD scan and molecular mass analyses of the mature form of the mutant proteases reveal similar CD spectra and intact masses to that of the wild-type. Crystal structures of zymogens of I86F and I86L mutants suggest that subtle reorganization of active site residues, including water, upon binding of the pro-peptide may allow the enzyme to achieve discriminatory substrate selectivity and catalytic efficiency. However, accurate and reliable predictions on alteration of substrate specificity require atomic resolution structure of the catalytic domain after zymogen activation, which remains a challenging task. In this study we demonstrate that through single amino acid substitution in pro-peptide, it is possible to modify the substrate specificity of papain and hence the pro-peptide of a protease can also be a useful target for altering its catalytic activity/specificity. PMID:27352302

  6. 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.

  7. Altered target site specificity variants of the I-PpoI His-Cys box homing endonuclease

    OpenAIRE

    Eklund, Jennifer L.; Ulge, Umut Y.; Eastberg, Jennifer; Monnat, Raymond J.

    2007-01-01

    We used a yeast one-hybrid assay to isolate and characterize variants of the eukaryotic homing endonuclease I-PpoI that were able to bind a mutant, cleavage-resistant I-PpoI target or ‘homing’ site DNA in vivo. Native I-PpoI recognizes and cleaves a semi-palindromic 15-bp target site with high specificity in vivo and in vitro. This target site is present in the 28S or equivalent large subunit rDNA genes of all eukaryotes. I-PpoI variants able to bind mutant target site DNA had from 1 to 8 ami...

  8. 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

    2016-01-01

    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. PMID:26916151

  9. Perioperative prostate specific antigen levels among coronary artery bypass grafting patients: Does extracorporeal circulation and body temperature induce prostate specific antigen levels alterations?

    Science.gov (United States)

    Patris, Emmanuel; Giakoumidakis, Konstantinos; Patris, Vasileios; Kuduvalli, Manoj; Argiriou, Mihalis; Charitos, Christos; Kalaitzis, Christos; Touloupidis, Stavros

    2015-01-01

    Purpose: The purpose of this study is to compare the perioperative total prostate specific antigen (tPSA) levels among coronary artery bypass grafting (CABG) patients with and without extracorporeal circulation (ECC), to investigate the changes overtime of tPSA in each group separately and to determine the effect of body core temperature on tPSA levels. Materials and Methods: A prospective study was conducted. Our sample was allocated to: (a) Seven patients who underwent off pump CABG (Group I) and (b) 16 CABG patients with ECC (Group II). The levels of tPSA were measured preoperatively (baseline), intra-operatively and at the 4th postoperative day. We compared the two groups on their tPSA levels and we investigated the changes of tPSA overtime in each group separately. Results: Intra-operative serum samples were obtained in significantly lower body temperature in patients of Group II than in those of Group I (31°C vs. 36.9°C, P < 0.001). In each group separately, postoperative tPSA levels were increased significantly compared to the baseline values (2.55 ng/ml vs. 0.39 ng/ml for Group I, P = 0.005 and 4.36 ng/ml vs. 0.77 for Group II, P < 0.001). CABG patients with ECC had significantly lower intra-operative tPSA levels than the baseline values (0.67 ng/ml vs. 0.77 ng/ml, P = 0.008). We did not observe significant differences of tPSA levels between the two groups. Conclusions: CABG surgery affects similarly the perioperative tPSA independently the involvement of ECC. Although all patients had significantly higher early postoperative tPSA levels, only those who underwent CABG with ECC had exceeded normal values and significantly decreased intra-operative tPSA. Hypothermia seems to be the causal factor of tPSA reduction. PMID:25657546

  10. 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

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

    Science.gov (United States)

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    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 French Canadian families with genetic generalized epilepsy. We used a novel single-cell genetic approach, by preparing cortical organotypic cultures from GABRA1 (flox/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

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

    Directory of Open Access Journals (Sweden)

    Pamela Lachance-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

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

    Science.gov (United States)

    Lachance-Touchette, Pamela; Choudhury, Mayukh; Stoica, Ana; Di Cristo, Graziella; Cossette, Patrick

    2014-01-01

    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 French Canadian 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

  14. Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris.

    Science.gov (United States)

    You, Weon-Kyoo; Choi, Won-Seok; Koh, You-Seok; Shin, Hang-Cheol; Jang, Yangsoo; Chung, Kwang-Hoe

    2004-07-30

    A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent. PMID:15280019

  15. 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

  16. Carboxy-terminal mutations of bile acid CoA:N-acyltransferase alter activity and substrate specificity.

    Science.gov (United States)

    Styles, Nathan A; Shonsey, Erin M; Falany, Josie L; Guidry, Amber L; Barnes, Stephen; Falany, Charles N

    2016-07-01

    Bile acid CoA:amino acid N-acyltransferase (BAAT) is the terminal enzyme in the synthesis of bile salts from cholesterol and catalyzes the conjugation of taurine or glycine to bile acid CoA thioesters to form bile acid N-acylamidates. BAAT has a dual localization to the cytosol and peroxisomes, possibly due to an inefficient carboxy-terminal peroxisomal targeting signal (PTS), -serine-glutamine-leucine (-SQL). Mutational analysis was used to define the role of the carboxy terminus in peroxisomal localization and kinetic activity. Amidation activity of BAAT and BAAT lacking the final two amino acids (AAs) (BAAT-S) were similar, whereas the activity of BAAT with a canonical PTS sequence (BAAT-SKL) was increased >2.5-fold. Kinetic analysis of BAAT and BAAT-SKL showed that BAAT-SKL had a lower Km for taurine and glycine as well as a greater Vmax There was no difference in the affinity for cholyl-CoA. In contrast to BAAT, BAAT-SKL forms bile acid N-acylamidates with β-alanine. BAAT-S immunoprecipitated when incubated with peroxisomal biogenesis factor 5 (Pex5) and rabbit anti-Pex5 antibodies; however, deleting the final 12 AAs prevented coimmunoprecipitation with Pex5, indicating the Pex5 interaction involves more than the -SQL sequence. These results indicate that even small changes in the carboxy terminus of BAAT can have significant effects on activity and substrate specificity. PMID:27230263

  17. Short communication: Altered expression of specificity protein 1 impairs milk fat synthesis in goat mammary epithelial cells.

    Science.gov (United States)

    Zhu, J J; Luo, J; Xu, H F; Wang, H; Loor, J J

    2016-06-01

    Specificity protein 1 (encoded by SP1) is a novel transcription factor important for the regulation of lipid metabolism and the normal function of various hormones in model organisms. Its potential role, if any, on ruminant milk fat is unknown. Despite the lower expression of the lipolysis-related gene ATGL (by 44 and 37% respectively), both the adenoviral overexpression and the silencing of SP1 [via short interfering (si)RNA] markedly reduced cellular triacylglycerol (TAG) content (by 28 and 25%, respectively), at least in part by decreasing the expression of DGAT1 (-36% in adenovirus treatment) and DGAT2 (-81 and -87%, respectively) that are involved in TAG synthesis. Consistent with the markedly lower expression of genes related to lipid droplet formation and secretion (TIP47 by 19 and 32%, and ADFP by 25 and 25%, respectively), cellular lipid droplet content was also decreased sharply, by 9 and 8.5%, respectively, after adenoviral overexpression of SP1 or its silencing via siRNA. Overall, the results underscored a potentially important role of SP1 in maintaining milk-fat droplet synthesis in goat mammary epithelial cells.

  18. Infection with a Shoot-Specific Fungal Endophyte (Epichloë) Alters Tall Fescue Soil Microbial Communities.

    Science.gov (United States)

    Rojas, Xavier; Guo, Jingqi; Leff, Jonathan W; McNear, David H; Fierer, Noah; McCulley, Rebecca L

    2016-07-01

    Tall fescue (Schedonorus arundinaceus) is a widespread grass that can form a symbiotic relationship with a shoot-specific fungal endophyte (Epichloë coenophiala). While the effects of fungal endophyte infection on fescue physiology and ecology have been relatively well studied, less attention has been given to how this relationship may impact the soil microbial community. We used high-throughput DNA sequencing and phospholipid fatty acid analysis to determine the structure and biomass of microbial communities in both bulk and rhizosphere soils from tall fescue stands that were either uninfected with E. coenophiala or were infected with the common toxic strain or one of several novel strains of the endophyte. We found that rhizosphere and bulk soils harbored distinct microbial communities. Endophyte presence, regardless of strain, significantly influenced soil fungal communities, but endophyte effects were less pronounced in prokaryotic communities. E. coenophiala presence did not change total fungal biomass but caused a shift in soil and rhizosphere fungal community composition, increasing the relative abundance of taxa within the Glomeromycota phylum and decreasing the relative abundance of genera in the Ascomycota phylum, including Lecanicillium, Volutella, Lipomyces, Pochonia, and Rhizoctonia. Our data suggests that tripartite interactions exist between the shoot endophyte E. coenophiala, tall fescue, and soil fungi that may have important implications for the functioning of soils, such as carbon storage, in fescue-dominated grasslands.

  19. Select Prenatal Environmental Exposures and Subsequent Alterations of Gene-Specific and Repetitive Element DNA Methylation in Fetal Tissues.

    Science.gov (United States)

    Green, Benjamin B; Marsit, Carmen J

    2015-06-01

    Strong evidence implicates maternal environmental exposures in contributing to adverse outcomes during pregnancy and later in life through the developmental origins of health and disease hypothesis. Recent research suggests these effects are mediated through the improper regulation of DNA methylation in offspring tissues, specifically placental tissue, which plays a critical role in fetal development. This article reviews the relevant literature relating DNA methylation in multiple tissues at or near delivery to several prenatal environmental toxicants and stressors, including cigarette smoke, endocrine disruptors, heavy metals, as well as maternal diet. These human studies expand upon previously reported outcomes in animal model interventions and include effects on both imprinted and non-imprinted genes. We have also noted some of the strengths and limitations in the approaches used, and consider the appropriate interpretation of these findings in terms of their effect size and their relationship to differential gene expression and potential health outcomes. The studies suggest an important role of DNA methylation in mediating the effects of the intrauterine environment on children's health and a need for additional research to better clarify the role of this epigenetic mechanism as well as others. PMID:26231362

  20. Infection with a Shoot-Specific Fungal Endophyte (Epichloë) Alters Tall Fescue Soil Microbial Communities.

    Science.gov (United States)

    Rojas, Xavier; Guo, Jingqi; Leff, Jonathan W; McNear, David H; Fierer, Noah; McCulley, Rebecca L

    2016-07-01

    Tall fescue (Schedonorus arundinaceus) is a widespread grass that can form a symbiotic relationship with a shoot-specific fungal endophyte (Epichloë coenophiala). While the effects of fungal endophyte infection on fescue physiology and ecology have been relatively well studied, less attention has been given to how this relationship may impact the soil microbial community. We used high-throughput DNA sequencing and phospholipid fatty acid analysis to determine the structure and biomass of microbial communities in both bulk and rhizosphere soils from tall fescue stands that were either uninfected with E. coenophiala or were infected with the common toxic strain or one of several novel strains of the endophyte. We found that rhizosphere and bulk soils harbored distinct microbial communities. Endophyte presence, regardless of strain, significantly influenced soil fungal communities, but endophyte effects were less pronounced in prokaryotic communities. E. coenophiala presence did not change total fungal biomass but caused a shift in soil and rhizosphere fungal community composition, increasing the relative abundance of taxa within the Glomeromycota phylum and decreasing the relative abundance of genera in the Ascomycota phylum, including Lecanicillium, Volutella, Lipomyces, Pochonia, and Rhizoctonia. Our data suggests that tripartite interactions exist between the shoot endophyte E. coenophiala, tall fescue, and soil fungi that may have important implications for the functioning of soils, such as carbon storage, in fescue-dominated grasslands. PMID:26992401

  1. 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.

  2. 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.

  3. ALTERATIONS IN THE MICROBIAL LOAD AT CERTAIN NON-SPECIFIC IMMUNE SITES OF MACROBRACHIUM ROSENBERGII SUPPLEMENTED WITH CENTELLA ASIATICA

    Directory of Open Access Journals (Sweden)

    JASMINE ANAND

    2013-01-01

    Full Text Available Macrobrachium rosenbergii has been the focus of research in India in the past few years. As a negative impact tothe success of aquaculture due to intensification lead to higher disease outbreaks. The bacterial diseases are themost common due to intensification. In the present study the Macrobrachium rosenbergii fed with diet containingmedicinal plant, Centella asiatica to test the antimicrobial activity. The prawns were fed with diet containingC.asiatica (0.2%. After 4 months both control and the experimental prawns were examined for microbial flora.Isolation and identification were also done. The investigation showed a significant reduction in the pathogenicbacteria and also found an improvement in the probiotic bacteria in prawns fed with experimental diet containingC.asiatica than the control diet. The % survival is 75% in medicated diet, but it is only 40% in control diet. Themedicated diet also showed improved growth parameters. The total microbial load at non-specific immune sites,such as gill, gut and exoskeleton of both control and medicated feed were examined after the experiment. Thetotal microbial load at gill, gut,exoskeleton were 41.00 x 105,50.00 x 106,30.00 x 106 respectively in control feed,but total microbial load at gill, gut, exoskeleton were 34.00 x 105, 28.00 x 106, 22.00 x 106 respectively inmedicated feed. Major pathogenic bacteria found in culture were Vibrio type I, Staphylococcus type III, Micrococcustype I, Strepto coccus I, Acinetobacter type I, Acinetobacter typeIII, Arthrobacter type I, Enterobacteriacea,Flavobacterium Vibrio type II, Strepto coccus II, Pseudomonas. After treating with medicated diet, diversity andintensity of microbial flora get reduced and culture of medicated diet also showed presence of probiotic bacteriasuch as Bacillus

  4. 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.

  5. Daphnia magna's sense of competition: intra-specific interactions (ISI) alter life history strategies and increase metals toxicity.

    Science.gov (United States)

    Gust, Kurt A; Kennedy, Alan J; Melby, Nicolas L; Wilbanks, Mitchell S; Laird, Jennifer; Meeks, Barbara; Muller, Erik B; Nisbet, Roger M; Perkins, Edward J

    2016-08-01

    This work investigates whether the scale-up to multi-animal exposures that is commonly applied in genomics studies provides equivalent toxicity outcomes to single-animal experiments of standard Daphnia magna toxicity assays. Specifically, we tested the null hypothesis that intraspecific interactions (ISI) among D. magna have neither effect on the life history strategies of this species, nor impact toxicological outcomes in exposure experiments with Cu and Pb. The results show that ISI significantly increased mortality of D. magna in both Cu and Pb exposure experiments, decreasing 14 day LC50 s and 95 % confidence intervals from 14.5 (10.9-148.3) to 8.4 (8.2-8.7) µg Cu/L and from 232 (156-4810) to 68 (63-73) µg Pb/L. Additionally, ISI potentiated Pb impacts on reproduction eliciting a nearly 10-fold decrease in the no-observed effect concentration (from 236 to 25 µg/L). As an indication of environmental relevance, the effects of ISI on both mortality and reproduction in Pb exposures were sustained at both high and low food rations. Furthermore, even with a single pair of Daphnia, ISI significantly increased (p < 0.05) neonate production in control conditions, demonstrating that ISI can affect life history strategy. Given these results we reject the null hypothesis and conclude that results from scale-up assays cannot be directly applied to observations from single-animal assessments in D. magna. We postulate that D. magna senses chemical signatures of conspecifics which elicits changes in life history strategies that ultimately increase susceptibility to metal toxicity. PMID:27151402

  6. Enhanced specific immune responses by CpG DNA in mice immunized with recombinant hepatitis B surface antigen and HB vaccine

    OpenAIRE

    Wang Xingtai; Hu Zhongyu; He Peng; Zhang Xiancheng; Liang Zhenglun

    2011-01-01

    Abstract Background Hepatitis B vaccine adjuvant, alum, is generally used for vaccination although it does not stimulate Th1 immunity and 10% of the population has low or no antibody response. Efforts have been continued to find more efficient vaccine adjuvants for better antibody response as well as stimulation of Th1 immunity. Methods CpG DNA was used as an adjuvant for recombinant HBsAg to immunize 6- to 8-week-old female BALB/c mice with or without alum for different dosages. The producti...

  7. Species-Specific Antibody Responses to the Recombinant 53-Kilodalton Excretory and Secretory Proteins in Mice Infected with Trichinella spp.▿

    OpenAIRE

    Nagano, Isao; Wu, Zhiliang; Takahashi, Yuzo

    2008-01-01

    The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa reco...

  8. 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.

  9. Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy.

    Science.gov (United States)

    Mealey, Robert H; Littke, Matt H; Leib, Steven R; Davis, William C; McGuire, Travis C

    2008-01-15

    Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-challenged SCID foals, concurrent with low-dose aldesleukin (180,000U/m2), a modified recombinant human IL-2 (rhuIL-2) product. The dose was calculated based on the specific activity on equine PBMC in vitro, and resulted in plasma concentrations considered sufficient to saturate high affinity IL-2 receptors in humans. Despite specific activity on equine PBMC that was equivalent to recombinant equine IL-2 and another form of rhuIL-2, aldesleukin did not support the engraftment and expansion of infused CTL clones, and control of viral load and clinical disease did not occur. It was concluded that survival of Rev-specific CTL clones infused into EIAV-challenged SCID foals was not enhanced by aldesleukin at the doses used in this study, and that in vitro specific activity did not correlate with in vivo efficacy. Successful adoptive immunotherapy with CTL clones in immunodeficient horses will likely require higher doses of rhuIL-2, co-infusion of CD4+ T lymphocytes, or administration of equine IL-2. PMID:17727961

  10. 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)

    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

  11. 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.

  12. 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

    2011-08-01

    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.

  13. CALL interrupted in a patient with non-specific mental retardation: gene dosage-dependent alteration of murine brain development and behavior.

    Science.gov (United States)

    Frints, Suzanna G M; Marynen, Peter; Hartmann, Dieter; Fryns, Jean-Pierre; Steyaert, Jean; Schachner, Melitta; Rolf, Bettina; Craessaerts, Katleen; Snellinx, An; Hollanders, Karen; D'Hooge, Rudi; De Deyn, Peter P; Froyen, Guy

    2003-07-01

    Investigation of MR patients with 3p aberrations led to the identification of the translocation breakpoint in intron five of the neural Cell Adhesion L1-Like (CALL or CHL1) gene in a man with non-specific mental retardation and 46,Y, t(X;3)(p22.1;p26.3). The Xp breakpoint does not seem to affect a known or predicted gene. Moreover, a fusion transcript with the CALL gene could not be detected and no mutations were identified on the second allele. CALL is highly expressed in the central and peripheral nervous system, like the mouse ortholog 'close homolog to L1' (Chl1). Chl1 expression levels in the hippocampus of Chl1(+/-) mice were half of those obtained in wild-type littermates, reflecting a gene dosage effect. Timm staining and synaptophysin immunohistochemistry of the hippocampus showed focal groups of ectopic mossy fiber synapses in the lateral CA3 region, outside the trajectory of the infra-pyramidal mossy fiber bundle in Chl1(-/-) and Chl1(+/-) mice. Behavioral assessment demonstrated mild alterations in the Chl1(-/-) animals. In the probe trial of the Morris Water Maze test, Chl1(-/-) mice displayed an altered exploratory pattern. In addition, these mice were significantly more sociable and less aggressive as demonstrated in social exploration tests. The Chl1(+/-) mice showed a phenotypic spectrum ranging from wild-type to knockout behavior. We hypothesize that a 50% reduction of CALL expression in the developing brain results in cognitive deficits. This suggests that the CALL gene at 3p26.3 is a prime candidate for an autosomal form of mental retardation. So far, mutation analysis of the CALL gene in patients with non-specific MR did not reveal any disease-associated mutations.

  14. Osbpl8 Deficiency in Mouse Causes an Elevation of High-Density Lipoproteins and Gender-Specific Alterations of Lipid Metabolism

    Science.gov (United States)

    Béaslas, Olivier; Metso, Jari; Nissilä, Eija; Laurila, Pirkka-Pekka; Kaiharju, Essi; Batchu, Krishna Chaithanya; Kaipiainen, Leena; Mäyränpää, Mikko I.; Yan, Daoguang; Gylling, Helena; Jauhiainen, Matti; Olkkonen, Vesa M.

    2013-01-01

    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, demonstrating a

  15. 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

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    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.

  16. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed.

  17. Bacteriophage recombination systems and biotechnical applications.

    Science.gov (United States)

    Nafissi, Nafiseh; Slavcev, Roderick

    2014-04-01

    Bacteriophage recombination systems have been widely used in biotechnology for modifying prokaryotic species, for creating transgenic animals and plants, and more recently, for human cell gene manipulation. In contrast to homologous recombination, which benefits from the endogenous recombination machinery of the cell, site-specific recombination requires an exogenous source of recombinase in mammalian cells. The mechanism of bacteriophage evolution and their coexistence with bacterial cells has become a point of interest ever since bacterial viruses' life cycles were first explored. Phage recombinases have already been exploited as valuable genetic tools and new phage enzymes, and their potential application to genetic engineering and genome manipulation, vectorology, and generation of new transgene delivery vectors, and cell therapy are attractive areas of research that continue to be investigated. The significance and role of phage recombination systems in biotechnology is reviewed in this paper, with specific focus on homologous and site-specific recombination conferred by the coli phages, λ, and N15, the integrase from the Streptomyces phage, ΦC31, the recombination system of phage P1, and the recently characterized recombination functions of Yersinia phage, PY54. Key steps of the molecular mechanisms involving phage recombination functions and their application to molecular engineering, our novel exploitations of the PY54-derived recombination system, and its application to the development of new DNA vectors are discussed. PMID:24442504

  18. A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model

    Directory of Open Access Journals (Sweden)

    S. Gattenlöhner

    2010-01-01

    Full Text Available Rhabdomyosarcoma (RMS is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR γ-subunit, which defines the fetal AChR (fAChR isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA. While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms.

  19. 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

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    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.

  20. Prolonged agonist stimulation does not alter the protein composition of membrane domains in spite of dramatic changes induced in a specific signaling cascade.

    Science.gov (United States)

    Matousek, Petr; Novotny, Jirí; Rudajev, Vladimír; Svoboda, Petr

    2005-01-01

    Protein composition of membrane domains prepared by three different procedures (mechanical homogenization, alkaline treatment with 1 M Na2CO3 [pH 11.0], or extraction with nonionic detergent Triton X-100), and isolated from the bulk of plasma membranes by flotation on equilibrium sucrose density gradients, was analyzed by two-dimensional (2D) electrophoresis and compared in preparations from control (quiescent) and agonist-stimulated human embryonic kidney cells (HEK)293 or S49 cells. HEK293 cells (clone e2m11) stably expressing high levels of thyrotropin-releasing hormone receptor and G11alpha protein were stimulated by thyrotropin-releasing hormone and S49 lymphoma cells by the beta-adrenergic receptor agonist isoprenaline. Whereas sustained exposure (16 h) of both cell lines to the appropriate hormones led to substantial cellular redistribution and downregulation of the cognate G proteins (G(q)alpha/G11alpha and G(s)alpha, respectively), the distribution and levels of nonstimulated G(i) proteins remained unchanged. The 2D electrophoretic analysis of membrane domains distinguished approx 150-170 major proteins in these structures and none of these proteins was significantly altered by prolonged agonist stimulation. Furthermore, specific immunochemical determination of a number of plasma membrane markers, including transmembrane and glycosyl-phosphatidylinositol-anchored peripheral proteins, confirmed that their detergent-extractability/solubility was not influenced by hormone treatment. Collectively, our present data indicate that sustained hormone stimulation of target cells does not alter the basic protein composition of membrane domain/raft compartments of the plasma membrane in spite of marked changes proceeding in a given signaling cascade. PMID:15673926

  1. Controlled release from recombinant polymers.

    Science.gov (United States)

    Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

    2014-09-28

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

  2. 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.

  3. 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.

  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. 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.

  6. ERAP1 functions override the intrinsic selection of specific antigens as immunodominant peptides, thereby altering the potency of antigen-specific cytolytic and effector memory T-cell responses.

    Science.gov (United States)

    Rastall, David P W; Aldhamen, Yasser A; Seregin, Sergey S; Godbehere, Sarah; Amalfitano, Andrea

    2014-12-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a critical component of the adaptive immune system that has been shown to increase or decrease the presentation of specific peptides on MHC class I molecules. Here, we have demonstrated that ERAP1 functions are not only important during the presentation of antigen-derived peptides, but these functions can also completely change which antigen-derived peptides ultimately become selected as immunodominant T-cell epitopes. Our results suggest that ERAP1 may do this by destroying epitopes that would otherwise become immunodominant in the absence of adequate ERAP1 functionality. We further establish that ERAP1-mediated influences on T-cell functions are both qualitative and quantitative, by demonstrating that loss of ERAP1 function redirects CTL killing toward a different set of antigen-derived epitopes and increases the percent of antigen-specific memory T cells elicited by antigen exposure. As a result, our studies suggest that normal ERAP1 activity can act to suppress the numbers of T effector memory cells that respond to a given antigen. This unique finding may shed light on why certain ERAP1 single nucleotide polymorphisms are associated with several autoimmune diseases, for example, by significantly altering the robustness and quality of CD8+ T-cell memory responses to antigen-derived peptides. PMID:25087231

  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 brain development. PMID:26547318

  8. Sex Specific Estrogen Receptor beta (ERβ) mRNA Expression in the Rat Hypothalamus and Amygdala is Altered by Neonatal Bisphenol A (BPA) Exposure

    Science.gov (United States)

    Cao, Jinyan; Joyner, Linwood; Mickens, Jillian A.; Leyrer, Stephanie M; Patisaul, Heather B

    2014-01-01

    Perinatal life is a critical window for sexually dimorphic brain organization, and profoundly influenced by steroid hormones. Exposure to endocrine disrupting compounds (EDCs) may disrupt this process, resulting in compromised reproductive physiology and behavior. To test the hypothesis that neonatal BPA exposure can alter sex specific postnatal ERβ expression in brain regions fundamental to sociosexual behavior we mapped ERβ mRNA levels in the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), paraventricular nucleus (PVN), anterior portion of the medial amygdaloid nucleus (MeA), super optic nucleus (SON), suprachiasmic nucleus (SCN) and lateral habenula (LHb) across postnatal days (PNDs) 0 to 19. Next, rat pups of both sexes were subcutaneously injected over the first three days of life with 10 μg estradiol benzoate (EB), 50 μg/kg BPA (LBPA), or 50 mg/kg BPA (HBPA) and ERβ levels quantified in each region of interest (ROI) on PNDs 4 and 10. EB exposure decreased ERβ signal in most female ROIs, and in the male PVN. In the BNSTp, ERβ expression decreased in LBPA males and HBPA females on PND 10, thereby reversing the sex difference in expression. In the PVN, ERβ mRNA levels were elevated in LBPA females, also resulting in a reversal of sexually dimorphic expression. In the MeA, BPA decreased ERβ expression on PND 4. Collectively, these data demonstrate that region and sex specific ERβ expression is vulnerable to neonatal BPA exposure in regions of the developing rat brain critical to sociosexual behavior. PMID:24352099

  9. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

    Directory of Open Access Journals (Sweden)

    Zhongyang Zhang

    2015-11-01

    Full Text Available Cancer genomes exhibit profound somatic copy number alterations (SCNAs. Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1 extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2 performing joint segmentation on the two signal dimensions; 3 correcting the copy number baseline from which the SCNA state is determined; 4 calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  10. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data.

    Science.gov (United States)

    Zhang, Zhongyang; Hao, Ke

    2015-11-01

    Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity.

  11. 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.

  12. Embryonic GABAB 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 GABAB 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 GABAB receptor to a 7-day critical period (E11–E17) during embryonic development. Experiments tested the role of GABAB 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 GABAB receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABAB 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 GABAB receptor antagonist. Embryonic exposure to GABAB 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 GABAB 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. PMID:25162235

  13. 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. PMID:25162235

  14. Recombinant Technology and Probiotics

    Directory of Open Access Journals (Sweden)

    Icy D’Silva

    2011-09-01

    Full Text Available Recombinant technology has led the way to monumental advances in the development of useful molecules, including the development of safe probiotics. The development of novel approaches using recombinant technology and probiotics that allow accurate targeting of therapeutics to the mucosa is an interesting area of research. The creation and use of recombinant probiotics expressing recombinantovalbumin, recombinant ovalbumin mutants and yet-to-be-designed recombinant hypo/non-allergenic molecules offer the opportunity to further investigate their effects for food, nutrition, environment andhealth. This review highlights advances in native probiotics and recombinant probiotics expressing native and recombinant molecules for food, nutrition, environment and health.

  15. A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination

    OpenAIRE

    Choi, Sangdun; Begum, Dilara; Koshinsky, Heather; Ow, David W; Wing, Rod A

    2000-01-01

    With current plant transformation methods (Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombinati...

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

    OpenAIRE

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

    2015-01-01

    Background 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 sequen...

  17. Construction of a recombinant human insulin expression vector for mammary gland-specific expression in buffalo (Bubalus bubalis) mammary epithelial cell line.

    Science.gov (United States)

    Kaushik, Ramakant; Singh, Karn Pratap; Kumari, Archana; Rameshbabu, K; Singh, Manoj Kumar; Manik, Radhey Shyam; Palta, Prabhat; Singla, Suresh Kumar; Chauhan, Manmohan Singh

    2014-09-01

    The aim of the present study was construction of mammary gland specific expression vector for high level of human insulin (hINS) expression in transgenic buffalo for therapeutic use. We have constructed mammary gland specific vector containing human insulin gene and there expression efficiency was checked into in vitro cultured buffalo mammary epithelial cells (BuMECs). Human pro-insulin coding region was isolated from human genomic DNA by intron skipping PCR primer and furin cleavage site was inserted between B-C and C-A chain of human insulin by overlap extension PCR. A mammary gland-specific buffalo beta-lactoglobulin promoter was isolated from buffalo DNA and used for human insulin expression in BuMEC cells. The construct was transfected into BuMECs by lipofection method and positive transgene cell clones were obtained by G418 selection after 3 weeks. Expression of hINS in transfected cells were confirmed by RT-PCR, Immunocytochemistry, Western Blotting and ELISA. The pAcISUBC insulin-expressing clones secreted insulin at varying levels between 0.18 - 1.43 ng/ml/24 h/2.0 × 10(6) cells. PMID:24969480

  18. Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

    Science.gov (United States)

    Kornberger, Petra; Skerra, Arne

    2014-01-01

    We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. PMID:24492291

  19. Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin.

    Science.gov (United States)

    Kornberger, Petra; Skerra, Arne

    2014-01-01

    We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.

  20. Recombinant HPV16 E7 assembled into particles induces an immune response and specific tumour protection administered without adjuvant in an animal model

    Directory of Open Access Journals (Sweden)

    Giorgi Colomba

    2011-05-01

    Full Text Available Abstract Background The HPV16 E7 protein is both a tumour-specific and a tumour-rejection antigen, the ideal target for developing therapeutic vaccines for the treatment of HPV16-associated cancer and its precursor lesions. E7, which plays a key role in virus-associated carcinogenesis, contains 98 amino acids and has two finger-type structures which bind a Zn++ ion. The ability of an Escherichia coli-produced E7-preparation, assembled into particles, to induce protective immunity against a HPV16-related tumour in the TC-1-C57BL/6 mouse tumour model, was evaluated. Methods E7 was expressed in E. coli, purified via a one-step denaturing protocol and prepared as a soluble suspension state after dialysis in native buffer. The presence in the E7 preparation of particulate forms was analysed by non-reducing SDS-PAGE and negative staining electron microscopy (EM. The Zn++ ion content was analysed by mass-spectrometry. Ten μg of protein per mouse was administered to groups of animals, once, twice or three times without adjuvant. The E7-specific humoral response was monitored in mice sera using an E7-based ELISA while the cell-mediated immune response was analysed in mice splenocytes with lymphoproliferation and IFN-γ ELISPOT assays. The E7 immunized mice were challenged with TC-1 tumour cells and the tumour growth monitored for two months. Results In western blot analysis E7 appears in multimers and high molecular mass oligomers. The EM micrographs show the protein dispersed as aggregates of different shape and size. The protein appears clustered in micro-, nano-aggregates, and structured particles. Mice immunised with this protein preparation show a significant E7-specific humoral and cell-mediated immune response of mixed Th1/Th2 type. The mice are fully protected from the tumour growth after vaccination with three E7-doses of 10 μg without any added adjuvant. Conclusions This report shows that a particulate form of HPV16 E7 is able to induce

  1. Comprehensive genetic testing identifies targetable genomic alterations in most patients with non-small cell lung cancer, specifically adenocarcinoma, single institute investigation

    Science.gov (United States)

    Won, Brian M.; Patton, Kathryn Alexa; Villaflor, Victoria M.; Hoffman, Philip C.; Hensing, Thomas; Hogarth, D. Kyle; Malik, Renuka; MacMahon, Heber; Mueller, Jeffrey; Simon, Cassie A.; Vigneswaran, Wickii T.; Wigfield, Christopher H.; Ferguson, Mark K.; Husain, Aliya N.; Vokes, Everett E.; Salgia, Ravi

    2016-01-01

    This study reviews extensive genetic analysis in advanced non-small cell lung cancer (NSCLC) patients in order to: describe how targetable mutation genes interrelate with the genes identified as variants of unknown significance; assess the percentage of patients with a potentially targetable genetic alterations; evaluate the percentage of patients who had concurrent alterations, previously considered to be mutually exclusive; and characterize the molecular subset of KRAS. Thoracic Oncology Research Program Databases at the University of Chicago provided patient demographics, pathology, and results of genetic testing. 364 patients including 289 adenocarcinoma underwent genotype testing by various platforms such as FoundationOne, Caris Molecular Intelligence, and Response Genetics Inc. For the entire adenocarcinoma cohort, 25% of patients were African Americans; 90% of KRAS mutations were detected in smokers, including current and former smokers; 46% of EGFR and 61% of ALK alterations were detected in never smokers. 99.4% of patients, whose samples were analyzed by next-generation sequencing (NGS), had genetic alterations identified with an average of 10.8 alterations/tumor throughout different tumor subtypes. However, mutations were not mutually exclusive. NGS in this study identified potentially targetable genetic alterations in the majority of patients tested, detected concurrent alterations and provided information on variants of unknown significance at this time but potentially targetable in the future. PMID:26934441

  2. 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. PMID:27156997

  3. The generation and evaluation of recombinant human IgA specific for Plasmodium falciparum merozoite surface protein 1-19 (PfMSP119

    Directory of Open Access Journals (Sweden)

    Corran Patrick H

    2011-07-01

    Full Text Available Abstract Background Human immunoglobulin G (IgG plays an important role in mediating protective immune responses to malaria. Although human serum immunoglobulin A (IgA is the second most abundant class of antibody in the circulation, its contribution, if any, to protective responses against malaria is not clear. Results To explore the mechanism(s by which IgA may mediate a protective effect, we generated fully human IgA specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP119, a major target of protective immune responses. This novel human IgA bound antigen with an affinity comparable to that seen for an epitope-matched protective human IgG1. Furthermore, the human IgA induced significantly higher NADPH-mediated oxidative bursts and degranulation from human neutrophils than the epitope-matched human IgG1 from which it was derived. Despite showing efficacy in in vitro functional assays, the human IgA failed to protect against parasite challenge in vivo in mice transgenic for the human Fcα receptor (FcαRI/CD89. A minority of the animals treated with IgA, irrespective of FcαRI expression, showed elevated serum TNF-α levels and concomitant mouse anti-human antibody (MAHA responses. Conclusions The lack of protection afforded by MSP119-specific IgA against parasite challenge in mice transgenic for human FcαRI suggests that this antibody class does not play a major role in control of infection. However, we cannot exclude the possibility that protective capacity may have been compromised in this model due to rapid clearance and inappropriate bio-distribution of IgA, and differences in FcαRI expression profile between humans and transgenic mice.

  4. Progress in Research of FLP/FRT Site-Specific Recombination System in Higher Eukaryotes%FLP/FRT位点特异性重组系统在高等真核生物中的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵爱春; 龙定沛; 谭兵; 许龙霞; 向仲怀

    2011-01-01

    来源于酵母2 μm质粒的FLP/FRT位点特异性重组系统已经被广泛应用于拟南芥、水稻、小鼠、果蝇、线虫等高等真核模式生物,正逐渐成为转基因动植物研究领域进行基因操作的强有力工具.笔者综述了FLP/FRT系统的重组原理及其在高等真核生物中的应用,系统地概述了该系统在转墓因植物、哺乳动物、昆虫以及其它相关高等真核模式生物中的研究现状,讨论了FLP/FRT系统在研究中存在的主要问题、应用前景和发展方向.%FLP/FRT site-specific recombination system derived from 2 μm plasmid of yeast has been widely used in Arabidopsis thaliana, Oryza sativa Linnaeus, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans and other higher eukaryotic organisms, and gradually become one of the powerful tools of genetic manipulation in transgenic animals and plants research areas. This review introduced the recombination principles of FLP/FRT system and its application in higher eukaryotes, and systematically summarized the main achievements of the system in transgenic plants, mammals, insects and other higher eukaryotic model organisms. In addition, the main problem, application prospect and developmental trend of the FLP/FRT system were discussed in this review.

  5. Maternal immune activation by LPS selectively alters specific gene expression profiles of interneuron migration and oxidative stress in the fetus without triggering a fetal immune response

    OpenAIRE

    Oskvig, Devon B.; Elkahloun, Abdel G.; Johnson, Kory R.; Phillips, Terry M.; Herkenham, Miles

    2012-01-01

    Maternal immune activation (MIA) is a risk factor for the development of schizophrenia and autism. Infections during pregnancy activate the mother’s immune system and alter the fetal environment, with consequential effects on CNS function and behavior in the offspring, but the cellular and molecular links between infection-induced altered fetal development and risk for neuropsychiatric disorders are unknown. We investigated the immunological, molecular, and behavioral effects of MIA in the of...

  6. Sex-specific alterations in mRNA level of key lipid metabolism enzymes in skeletal muscle of overweight and obese subjects following endurance exercise

    OpenAIRE

    Smith, Ira J.; Huffman, Kim M.; Durheim, Michael T.; Duscha, Brian D.; Kraus, William E.

    2008-01-01

    Endurance exercise (EE) leads to beneficial alterations in skeletal muscle lipid metabolism in overweight and obese individuals; however, the mechanisms of these improvements are poorly understood. The primary goal of the current investigation was to test the hypothesis that long-term EE training (6 mo) leads to alterations in the mRNA abundance of key lipid metabolism enzymes in skeletal muscle of overweight and obese middle-aged women and men. A secondary aim of this study was to investigat...

  7. Gigantoxin-4-4D5 scFv is a novel recombinant immunotoxin with specific toxicity against HER2/neu-positive ovarian carcinoma cells.

    Science.gov (United States)

    Lv, Xinxin; Zhang, Jian; Xu, Rui; Dong, Yuguo; Sun, Aiyou; Shen, Yaling; Wei, Dongzhi

    2016-07-01

    Immunotoxins are a new class of antibody-targeted therapy in clinical development. Traditional immunotoxins that are constructed from the toxins of plants or bacteria need to be internalized to the cytoplasm and thus have limited antitumor efficacy. In the present study, we combined a recently reported sea anemone cytolysin Gigantoxin-4 with an anti-HER2/neu single-chain variable fragment 4D5 scFv to construct a novel immunotoxin. We fused a SUMO tag to the N-terminus of Gigantoxin-4-4D5 scFv and it was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. After purification, the purity of Gigantoxin-4-4D5 scFv reached 96 % and the yield was 14.3 mg/L. Our results demonstrated that Gigantoxin-4-4D5 scFv exerted a highly cytotoxic effect on the HER2/neu-positive ovarian carcinoma SK-OV-3 cell line. And the hemolytic activity was weaker, making it safe for normal cells. The results of immunofluorescence analysis showed that this novel immunotoxin could specifically bind to SK-OV-3 cells with no recognition of human embryonic kidney 293 cells. Scanning electron microscope observations and extracellular lactate dehydrogenase activity indicated that it could induce necrosis in SK-OV-3 cells by disrupting the cell membrane. Moreover, it could also mediate apoptosis of SK-OV-3 cells. PMID:27063011

  8. Systemic lupus erythematosus: molecular cloning and analysis of 22 individual recombinant monoclonal kappa light chains specifically hydrolyzing human myelin basic protein.

    Science.gov (United States)

    Timofeeva, Anna M; Buneva, Valentina N; Nevinsky, Georgy A

    2015-10-01

    Antibodies hydrolyzing myelin basic protein (MBP) can play an important role in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). An immunoglobulin light chain phagemid library derived from peripheral blood lymphocytes of patients with SLE was used. Small pools of phage particles displaying light chains with different affinities for MBP were isolated by affinity chromatography on MBP-Sepharose, and the fraction eluted with 0.5 M NaCl was used for preparation of individual monoclonal light chains (MLChs, 26-27 kDa). Seventy-two of 440 individual colonies were randomly chosen, expressed in Escherichia coli in a soluble form, and MLChs were purified by metal chelating chromatography. Twenty-two of 72 MLChs have high affinity and efficiently hydrolyze only MBP (not other control proteins) demonstrating various pH optima in a 5.7-9.0 range and different substrate specificity in the hydrolysis of four different MBP oligopeptides. Four MLChs demonstrated serine protease-like and three thiol protease-like activities, while 11 MLChs were metalloproteases. The activity of three MLChs was inhibited by both phenylmethylsulfonyl fluoride (PMSF) and Ethylenediaminetetraacetic acid (EDTA), two other by EDTA and iodoacetamide, and one by PMSF, EDTA, and iodoacetamide. The ratio of relative activity in the presence of Ca(2+), Mg(2+), Mn(2+), Ni(2+), Zn(2+), Cu(2+), and Co(2+) was individual for each of 22 MLCh preparations. It is the first examples of human MLChs, which probably can possess two or even three different proteolytic activities. These observations suggest an extreme diversity of anti-MBP abzymes in SLE patients. The immune systems of individual SLE patients can generate a variety of anti-MBP abzymes, which can attack MBP of myelin-proteolipid sheath of axons and play an important role in MS and SLE pathogenesis.

  9. Recombinant DNA in Medicine

    OpenAIRE

    Cederbaum, Stephen D.; Fareed, George C.; Lovett, Michael A.; Shapiro, Larry J.

    1984-01-01

    Studies in bacteria and bacterial viruses have led to methods to manipulate and recombine DNA in unique and reproducible ways and to amplify these recombined molecules millions of times. Once properly identified, the recombinant DNA molecules can be used in various ways useful in medicine and human biology. There are many applications for recombinant DNA technology. Cloned complementary DNA has been used to produce various human proteins in microorganisms. Insulin and growth hormone have been...

  10. Improving baculovirus recombination

    OpenAIRE

    Zhao, Yuguang; Chapman, David A. G.; Jones, Ian M.

    2003-01-01

    Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins. The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments. Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination. Viral DNA prepared from the knockout fails to initiate an infecti...

  11. The unconventional xer recombination machinery of Streptococci/Lactococci

    NARCIS (Netherlands)

    Le Bourgeois, Pascal; Bugarel, Marie; Campo, Nathalie; Daveran-Mingot, Marie-Line; Labonte, Jessica; Lanfranchi, Daniel; Lautier, Thomas; Pages, Carine; Ritzenthaler, Paul

    2007-01-01

    Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In Escherichia coli, dimer resolution is achieved by site-specific recombination, Xer recombination, involving tw

  12. 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...... (Bcl-2 and Bax) were analyzed in selected brain regions. CLPE showed significant improvement in survival compared with CLPV 18 h postinduction of sepsis (P ... induced by cecal ligation alters cerebral redox status and supports a proapoptotic phenotype. The free radical scavenger edavarone reduces mortality of septic mice and protects against sepsis-induced neuronal cell death....

  13. Xylose reductase from the thermophilic fungus Talaromyces emersonii: cloning and heterologous expression of the native gene (Texr) and a double mutant (TexrK271R+N273D) with altered coenzyme specificity

    Indian Academy of Sciences (India)

    Sara Fernandes; Maria G Tuohy; Patrick G Murray

    2009-12-01

    Xylose reductase is involved in the first step of the fungal pentose catabolic pathway. The gene encoding xylose reductase (Texr) was isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli and purified to homogeneity. Texr encodes a 320 amino acid protein with a molecular weight of 36 kDa, which exhibited high sequence identity with other xylose reductase sequences and was shown to be a member of the aldoketoreductase (AKR) superfamily with a preference for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as coenzyme. Given the potential application of xylose reductase enzymes that preferentially utilize the reduced form of nicotinamide adenine dinucleotide (NADH) rather than NADPH in the fermentation of five carbon sugars by genetically engineered microorganisms, the coenzyme selectivity of TeXR was altered by site-directed mutagenesis. The TeXRK271R+N273D double mutant displayed an altered coenzyme preference with a 16-fold improvement in NADH utilization relative to the wild type and therefore has the potential to reduce redox imbalance of xylose fermentation in recombinant S. cerevisiae strains. Expression of Texr was shown to be inducible by the same carbon sources responsible for the induction of genes encoding enzymes relevant to lignocellulose hydrolysis, suggesting a coordinated expression of intracellular and extracellular enzymes relevant to hydrolysis and metabolism of pentose sugars in T. emersonii in adaptation to its natural habitat. This indicates a potential advantage in survival and response to a nutrient-poor environment.

  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 (Ab)

    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. 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.

    Directory of Open Access Journals (Sweden)

    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.

  16. Prime-boost vaccination with Bacillus Calmette Guerin and a recombinant adenovirus co-expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis induces robust antigen-specific immune responses in mice.

    Science.gov (United States)

    Li, Wu; Li, Min; Deng, Guangcun; Zhao, Liping; Liu, Xiaoming; Wang, Yujiong

    2015-08-01

    Tuberculosis (TB) remains to be a prevalent health issue worldwide. At present, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the singular anti-TB vaccine available for the prevention of disease in humans; however, this vaccine only provides limited protection against Mycobacterium tuberculosis (Mtb) infection. Therefore, the development of alternative vaccines and strategies for increasing the efficacy of vaccination against TB are urgently required. The present study aimed to evaluate the ability of a recombinant adenoviral vector (Ad5-CEAB) co-expressing 10-kDa culture filtrate protein, 6-kDa early-secreted antigenic target, antigen 85 (Ag85)A and Ag85B of Mtb to boost immune responses following primary vaccination with BCG in mice. The mice were first subcutaneously primed with BCG and boosted with two doses of Ad5-CEAB via an intranasal route. The immunological effects of Ad5-CEAB boosted mice primed with BCG were then evaluated using a series of immunological indexes. The results demonstrated that the prime-boost strategy induced a potent antigen-specific immune response, which was primarily characterized by an enhanced T cell response and increased production of cytokines, including interferon-γ, tumor necrosis factor-α and interleukin-2, in mice. In addition, this vaccination strategy was demonstrated to have an elevated humoral response with increased concentrations of antigen-specific bronchoalveolar lavage secretory immunoglobulin (Ig)A and serum IgG in mice compared with those primed with BCG alone. These data suggested that the regimen of subcutaneous BCG prime and mucosal Ad5-CEAB boost was a novel strategy for inducing a broad range of antigen-specific immune responses to Mtb antigens in vivo, which may provide a promising strategy for further development of adenoviral-based vaccine against Mtb infection.

  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.

    Science.gov (United States)

    Krupka, Michal; Masek, Josef; Barkocziova, Lucia; Turanek Knotigova, Pavlina; Kulich, Pavel; Plockova, Jana; Lukac, Robert; Bartheldyova, Eliska; Koudelka, Stepan; Chaloupkova, Radka; Sebela, Marek; Zyka, Daniel; Droz, Ladislav; Effenberg, Roman; Ledvina, Miroslav; Miller, Andrew D; Turanek, Jaroslav; Raska, Milan

    2016-01-01

    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. PMID:26848589

  18. 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.

  19. Vaccination with a recombinant protein encoding the tumor-specific antigen NY-ESO-1 elicits an A2/157-165-specific CTL repertoire structurally distinct and of reduced tumor reactivity than that elicited by spontaneous immune responses to NY-ESO-1-expressing Tumors.

    Science.gov (United States)

    Bioley, Gilles; Guillaume, Philippe; Luescher, Immanuel; Bhardwaj, Nina; Mears, Gregory; Old, Lloyd; Valmori, Danila; Ayyoub, Maha

    2009-01-01

    In a recent vaccination trial assessing the immunogenicity of an NY-ESO-1 (ESO) recombinant protein administered with Montanide and CpG, we have obtained evidence that this vaccine induces specific cytolytic T lymphocytes (CTL) in half of the patients. Most vaccine-induced CTLs were directed against epitopes located in the central part of the protein, between amino acids 81 and 110. This immunodominant region, however, is distinct from another ESO CTL region, 157-165, that is a frequent target of spontaneous CTL responses in A2+ patients bearing ESO tumors. In this study, we have investigated the CTL responses to ESO 157-165 in A2+ patients vaccinated with the recombinant protein. Our data indicate that after vaccination with the protein, CTL responses to ESO 157-165 are induced in some, but not all, A2+ patients. ESO 157-165-specific CTLs induced by vaccination with the ESO protein were functionally heterogeneous in terms of tumor recognition and often displayed decreased tumor reactivity as compared with ESO 157-165-specific CTLs isolated from patients with spontaneous immune responses to ESO. Remarkably, protein-induced CTLs used T-cell receptors similar to those previously isolated from patients vaccinated with synthetic ESO peptides (Vbeta4.1) and distinct from those used by highly tumor-reactive CTLs isolated from patients with spontaneous immune responses (Vbeta1.1, Vbeta8.1, and Vbeta13.1). Together, these results demonstrate that vaccination with the ESO protein elicits a repertoire of ESO 157-165-specific CTLs bearing T-cell receptors that are structurally distinct from those of CTLs found in spontaneous immune responses to the antigen and that are heterogeneous in terms of tumor reactivity, being often poorly tumor reactive.

  20. 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, inclu

  1. BAY 50-4798, a novel, high-affinity receptor-specific recombinant interleukin-2 analog, induces dose-dependent increases in CD25 expression and proliferation among unstimulated, human peripheral blood mononuclear cells in vitro.

    Science.gov (United States)

    Matthews, Lynn; Chapman, Sherita; Ramchandani, Meena S; Lane, H Clifford; Davey, Richard T; Sereti, Irini

    2004-12-01

    Interleukin-2 administration induces CD4 T cell expansion in HIV-infected patients, however, toxicity can limit dosing. BAY 50-4798 is a recombinant IL-2 analog with >1000-fold specificity for the high-affinity IL-2 receptor. The effects of this compound on unstimulated human PBMC were evaluated. PBMC from HIV(-) and HIV(+) donors were cultured in vitro with incremental doses of BAY 50-4798 or aldesleukin. CD25 expression and proliferation were evaluated with flow cytometry. Cytokine levels were measured by ELISA in culture supernatants. BAY 50-4798 induced dose-dependent increases in CD25 expression and proliferation of T cells, NK, and B cells and showed selectivity for CD4 T cells expressing CD25. Induction of pro-inflammatory cytokines was also dose-dependent and was observed at the concentrations of BAY 50-4798 with the highest biologic activity. These data suggest that BAY 50-4798 can induce proliferation of unstimulated T cells but loss of T cell selectivity and induction of pro-inflammatory cytokines occur at concentrations exerting the highest biologic activity. PMID:15507389

  2. pKBuS13, a KPC-2-encoding plasmid from Klebsiella pneumoniae sequence type 833, carrying Tn4401b inserted into an Xer site-specific recombination locus.

    Science.gov (United States)

    Garbari, Luigi; Busetti, Marina; Dolzani, Lucilla; Petix, Vincenzo; Knezevich, Anna; Bressan, Raffaela; Gionechetti, Fabrizia; Tonin, Enrico A; Lagatolla, Cristina

    2015-09-01

    Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported. PMID:26077252

  3. Recombineering Homologous Recombination Constructs in Drosophila

    OpenAIRE

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

    2013-01-01

    The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineeri...

  4. 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.

  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

    OpenAIRE

    Pamela Lachance-Touchette; Graziella Dicristo

    2014-01-01

    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 ...

  6. Relationship between cognitive impairment and white-matter alteration in Parkinson’s disease with dementia: tract-based spatial statistics and tract-specific analysis

    OpenAIRE

    Kamagata, Koji; Motoi, Yumiko; Tomiyama, Hiroyuki; Abe, Osamu; Ito, Kenji; Shimoji, Keigo; Suzuki, Michimasa; Hori, Masaaki; Nakanishi, Atsushi; Sano, Tamotsu; Kuwatsuru, Ryohei; Sasai, Keisuke; Aoki, Shigeki; Hattori, Nobutaka

    2013-01-01

    Objectives We investigated the relationship between white-matter alteration and cognitive status in Parkinson’s disease (PD) with and without dementia by using diffusion tensor imaging. Methods Twenty PD patients, 20 PDD (Parkinson’s disease with dementia) patients and 20 age-matched healthy controls underwent diffusion tensor imaging. The mean diffusivity and fractional anisotropy (FA) map of each patient group were compared with those of the control group by using tract-based spatial statis...

  7. Molecular mechanisms of DNA recombination: testing mitotic and meiotic models

    International Nuclear Information System (INIS)

    A hyperhaploid n + 1 strain of Saccharomyces cerevisiae (LBL1) disomic for chromosome VII was employed to isolate hyper-rec and hypo-rec mutations affecting spontaneous mitotic gene conversion and intergenic recombination. The genotype of LBL1 permits simultaneous and independent identification of rec mutations that enhance or diminish gene conversion and those that enhance or diminish intergenic recombination. Five phenotypic groups of rec mutants were isolated following ultraviolet light mutagenesis. Rec mutations that simultaneously abolish or enhance both classes of recombinational events were detected. These results demonstrate that gene conversion and intergenic recombination are under joint genetic control in mitotic cells. Conversion-specific and intergenic recombination-specific rec mutants were also recovered. Their properties indicate that conversion and intergenic recombination are separable pheonomena dependent upon discrete REC genes. The rec mutants isolated in LBL1 provide a method to test molecular models of mitotic and meiotic recombination

  8. Recombinant allergens: what does the future hold?

    Science.gov (United States)

    Valenta, Rudolf; Niespodziana, Katarzyna; Focke-Tejkl, Margit; Marth, Katharina; Huber, Hans; Neubauer, Angela; Niederberger, Verena

    2011-04-01

    This year we are celebrating not only the centenary of allergen-specific immunotherapy but also the 10-year anniversary of the first administration of recombinant allergen-based vaccines to allergic patients. By using recombinant DNA technology, defined and safe allergy vaccines can be produced that allow us to overcome many, if not all, of the problems associated with the use of natural allergen extracts, such as insufficient quality, allergenic activity, and poor immunogenicity. Here we provide an update of clinical studies with recombinant allergen-based vaccines, showing that some of these vaccines have undergone successful clinical evaluation up to phase III studies. Furthermore, we introduce a strategy for allergen-specific immunotherapy based on recombinant fusion proteins consisting of viral carrier proteins and allergen-derived peptides without allergenic activity, which holds the promise of being free of side effects and eventually being useful for prophylactic vaccination.

  9. Construction and identification of the dual specific antitumor recombinant adeno- virus expressing apoptin%表达Apoptin基因抗肿瘤双特异性重组腺病毒的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王卓越; 李霄; 王浩然; 阚式绂; 王宇航; 齐延新; 吴娜; 刘燕瑜; 金宁一

    2011-01-01

    为研发一种安全、特异、高效的抗肿瘤基因治疗候选药物,本试验基于人端粒酶启动子(hTERTp)、人5型腺病毒复制必需基N(Ela)和特异性抑癌基N(Apoption),利用RAPAd.I腺病毒载体系统,构建了一种具有肿瘤特异性杀伤和特异性复制能力的双特异性抗肿瘤重组腺病毒Ad—VT。并利用人肝癌细胞(HepCr2)、人肺癌细胞(A549)、人宫颈癌细胞(Hela)、人胚胎肺细胞(WI-38)和非洲绿猿肾细胞(Vero)对hTERTp的肿瘤特异性进行鉴定。结果显示,hTERTp可在HepC-2、A549和Hela等肿瘤细胞内特异性启动外源基因的表达,而在WI-38、Vero等正常细胞内不能启动相关基因表达。用RT—PCR及Western—blot等方法对Apoptin基因和Ela基因的转录和表达进行了鉴定。结果表明,Ad—VT能够有效表达Apoptin和Ela等外源基因。%To develop a safe, specific and effective succedaneous praeparatum in cancer gene therapy, a dual specific anti-tumoral recombinant adenovirus, Ad-VT, which has both tumor specific restrain and tumor specific replication a- bilities,was constructed by using RAPAd. i system basing on human telomerase reverse transcriptase core promot- er (hTERTp), replicate indispensable gene El a and specific anti-tumor gene apoptin. The specificity of hTERTp was identified in human hepatoma carcinoma (HepG-2) cells,human lung carcinoma (A549) cells, human cervical carci- noma (Hela) cells,human embryonic lung (WI-38) cells and african green monkey kidney (Vero) cells. The expres- sion of apoptin and Ela of Ad-VT was identified by RT-PCR and Western-blot. The results showed that hTERTp had a promoter acitivity specifically in HepG-2, A549 and Hela cells, whereas not in WI 38 and Vero, and the foreign genes of Ad-VT were expressed effectively in target ceils.

  10. Prenatal Exposure to Autism-Specific Maternal Autoantibodies Alters Proliferation of Cortical Neural Precursor Cells, Enlarges Brain, and Increases Neuronal Size in Adult Animals.

    Science.gov (United States)

    Martínez-Cerdeño, Verónica; Camacho, Jasmin; Fox, Elizabeth; Miller, Elaine; Ariza, Jeanelle; Kienzle, Devon; Plank, Kaela; Noctor, Stephen C; Van de Water, Judy

    2016-01-01

    Autism spectrum disorders (ASDs) affect up to 1 in 68 children. Autism-specific autoantibodies directed against fetal brain proteins have been found exclusively in a subpopulation of mothers whose children were diagnosed with ASD or maternal autoantibody-related autism. We tested the impact of autoantibodies on brain development in mice by transferring human antigen-specific IgG directly into the cerebral ventricles of embryonic mice during cortical neurogenesis. We show that autoantibodies recognize radial glial cells during development. We also show that prenatal exposure to autism-specific maternal autoantibodies increased stem cell proliferation in the subventricular zone (SVZ) of the embryonic neocortex, increased adult brain size and weight, and increased the size of adult cortical neurons. We propose that prenatal exposure to autism-specific maternal autoantibodies directly affects radial glial cell development and presents a viable pathologic mechanism for the maternal autoantibody-related prenatal ASD risk factor.

  11. Prenatal Exposure to Autism-Specific Maternal Autoantibodies Alters Proliferation of Cortical Neural Precursor Cells, Enlarges Brain, and Increases Neuronal Size in Adult Animals.

    Science.gov (United States)

    Martínez-Cerdeño, Verónica; Camacho, Jasmin; Fox, Elizabeth; Miller, Elaine; Ariza, Jeanelle; Kienzle, Devon; Plank, Kaela; Noctor, Stephen C; Van de Water, Judy

    2016-01-01

    Autism spectrum disorders (ASDs) affect up to 1 in 68 children. Autism-specific autoantibodies directed against fetal brain proteins have been found exclusively in a subpopulation of mothers whose children were diagnosed with ASD or maternal autoantibody-related autism. We tested the impact of autoantibodies on brain development in mice by transferring human antigen-specific IgG directly into the cerebral ventricles of embryonic mice during cortical neurogenesis. We show that autoantibodies recognize radial glial cells during development. We also show that prenatal exposure to autism-specific maternal autoantibodies increased stem cell proliferation in the subventricular zone (SVZ) of the embryonic neocortex, increased adult brain size and weight, and increased the size of adult cortical neurons. We propose that prenatal exposure to autism-specific maternal autoantibodies directly affects radial glial cell development and presents a viable pathologic mechanism for the maternal autoantibody-related prenatal ASD risk factor. PMID:25535268

  12. Selection of Recombinant Human Antibodies.

    Science.gov (United States)

    Tomszak, Florian; Weber, Susanne; Zantow, Jonas; Schirrmann, Thomas; Hust, Michael; Frenzel, André

    2016-01-01

    Since the development of therapeutic antibodies the demand of recombinant human antibodies is steadily increasing. Traditionally, therapeutic antibodies were generated by immunization of rat or mice, the generation of hybridoma clones, cloning of the antibody genes and subsequent humanization and engineering of the lead candidates. In the last few years, techniques were developed that use transgenic animals with a human antibody gene repertoire. Here, modern recombinant DNA technologies can be combined with well established immunization and hybridoma technologies to generate already affinity maturated human antibodies. An alternative are in vitro technologies which enabled the generation of fully human antibodies from antibody gene libraries that even exceed the human antibody repertoire. Specific antibodies can be isolated from these libraries in a very short time and therefore reduce the development time of an antibody drug at a very early stage.In this review, we describe different technologies that are currently used for the in vitro and in vivo generation of human antibodies. PMID:27236551

  13. 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.

  14. 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.

  15. Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

    OpenAIRE

    Höllsberg, P; Weber, W E; Dangond, F; Batra, V; Sette, A.; Hafler, D A

    1995-01-01

    Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substitute...

  16. 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

    2014-02-01

    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

  17. Genetic background alters the severity and onset of neuromuscular disease caused by the loss of ubiquitin-specific protease 14 (usp14.

    Directory of Open Access Journals (Sweden)

    Andrea G Marshall

    Full Text Available In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU induced mutation in Usp14 (nmf375 that leads to adult-onset neurological disease. The nmf375 mutation causes aberrant splicing of Usp14 mRNA, resulting in a 95% reduction in USP14. We previously showed that loss of USP14 in ataxia (ax (J mice results in reduced ubiquitin levels, motor endplate disease, Purkinje cell axonal dystrophy and decreased hippocampal paired pulse facilitation (PPF during the first 4-6 weeks of life, and early postnatal lethality by two months of age. Although the loss of USP14 is comparable between the nmf375 and ax (J mice, the nmf375 mice did not exhibit these ax (J developmental abnormalities. However, by 12 weeks of age the nmf375 mutants present with ubiquitin depletion and motor endplate disease, indicating a continual role for USP14-mediated regulation of ubiquitin pools and neuromuscular junction (NMJ structure in adult mice. The observation that motor endplate disease was only seen after ubiquitin depletion suggests that the preservation of NMJ structure requires the stable maintenance of synaptic ubiquitin pools. Differences in genetic background were shown to affect ubiquitin expression and dramatically alter the phenotypes caused by USP14 deficiency.

  18. PCBs alter gene expression of nuclear transcription factors and other heart-specific genes in cultures of primary cardiomyocytes: possible implications for cardiotoxicity.

    Science.gov (United States)

    Borlak, J; Thum, T

    2002-12-01

    1. Polychlorinated biphenyls (PCBs) are well-known environmental pollutants that bioaccumulate mainly in the fatty tissue of animals and humans. Although contamination occurs primarily via the food chain, waste combustion leads to airborne PCBs. From epidemiological studies, there is substantial evidence that cardiovascular disease is linked to air pollution, but little is known about the underlying molecular events. 2. We investigated the effects of Aroclor 1254, a complex mixture of >80 PCB isomers and congeners, on the expression of nuclear transcription factors (GATA-4, Nkx-2.5, MEF-2c, OCT-1) and of downstream target genes (atrial and brain natriuretic peptide, alpha- and beta-myosin heavy chain, alpha-cardiac and alpha-skeletal actin), which play an important role in cardiac biology. 3. We treated cultures of primary cardiomyocytes of adult rats with Aroclor 1254 (10.0 micro M) and found significant induction of the transcription factor genes GATA-4 and MEF-2c and of genes regulated by these factors, i.e. atrial natriuretic peptide, brain-type natriuretic peptide, alpha- and beta-myosin heavy chain, and skeletal alpha actin. 4. We have shown PCBs to modulate expression of genes coding for programmes of cellular differentiation and stress (e.g. atrial natriuretic peptide, brain-type natriuretic peptide) and these alterations may be important in the increase of cardiovascular disease in polluted areas.

  19. 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.

  20. Metabolomic Analyses of Brain Tissue in Sepsis Induced by Cecal Ligation Reveal Specific Redox Alterations--Protective Effects of the Oxygen Radical Scavenger Edaravone.

    Science.gov (United States)

    Hara, Naomi; Chijiiwa, Miyuki; Yara, Miki; Ishida, Yusuke; Ogiwara, Yukihiko; Inazu, Masato; Kuroda, Masahiko; Karlsson, Michael; Sjovall, Fredrik; Elmér, Eskil; Uchino, Hiroyuki

    2015-12-01

    The pathophysiology of sepsis-associated encephalopathy (SAE) is complex and remains incompletely elucidated. Dysregulated reactive oxygen species (ROS) production and mitochondrial-mediated necrotic-apoptotic pathway have been proposed as part of the pathogenesis. The present study aimed 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), and sham-operated (Sham). Mice in CLPV and CLPE were injected with saline or edaravone intraperitoneally at a dose of 10 mg/kg twice daily. The treatments were initiated 4 days prior to the surgical procedure. Mortality, histological changes, electron microscopy (EM), and expression of Bcl-2 family genes (Bcl-2 and Bax) were analyzed in selected brain regions. CLPE showed significant improvement in survival compared with CLPV 18 h postinduction of sepsis (P free radical scavenger edavarone reduces mortality of septic mice and protects against sepsis-induced neuronal cell death.

  1. Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism.

    Science.gov (United States)

    Karim-Silva, Sabrina; Moura, Juliana de; Noiray, Magali; Minozzo, Joao Carlos; Aubrey, Nicolas; Alvarenga, Larissa M; Billiald, Philippe

    2016-08-01

    Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms. PMID:27288291

  2. Recombinant human erythropoietin in sports: a review

    OpenAIRE

    Rafael Maia de Almeida Bento; Lúcia Menezes Pinto Damasceno; Francisco Radler Aquino Neto

    2003-01-01

    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 ox...

  3. SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

    Science.gov (United States)

    Friend, Samantha F; Peterson, Lisa K; Kedl, Ross M; Dragone, Leonard L

    2013-03-01

    How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vβ5 mice, expressing only the β-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vβ5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vβ5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vβ5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vβ5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

  4. Resistance to high-fat diet-induced obesity and altered expression of adipose-specific genes in HSL-deficient mice.

    Science.gov (United States)

    Harada, Kenji; Shen, Wen-Jun; Patel, Shailja; Natu, Vanita; Wang, Jining; Osuga, Jun-ichi; Ishibashi, Shun; Kraemer, Fredric B

    2003-12-01

    To elucidate the role of hormone-sensitive lipase (HSL) in diet-induced obesity, HSL-deficient (HSL-/-) and wild-type mice were fed normal chow or high-fat diets. HSL-/- mice were resistant to diet-induced obesity showing higher core body temperatures. Weight and triacylglycerol contents were decreased in white adipose tissue (WAT) but increased in both brown adipose tissue (BAT) and liver of HSL-/- mice. Serum insulin levels in the fed state and tumor necrosis factor-alpha mRNA levels in adipose tissues were higher, whereas serum levels of adipocyte complement-related protein of 30 kDa (ACRP30)/adiponectin and leptin, as well as mRNA levels of ACRP30/adiponectin, leptin, resistin, and adipsin in WAT, were lower in HSL-/- mice than in controls. Expression of transcription factors associated with adipogenesis (peroxisome proliferator-activated receptor-gamma, CAAT/enhancer-binding protein-alpha) and lipogenesis (carbohydrate response element-binding protein, adipocyte determination- and differentiation-dependent factor-1/sterol regulatory element-binding protein-1c), as well as of adipose differentiation markers (adipocyte lipid-binding protein, perilipin, lipoprotein lipase), lipogenic enzymes (glycerol-3-phosphate acyltransferase, acyl-CoA:diacylglycerol acyltransferase-1 and -2, fatty acid synthase, ATP citrate lyase) and insulin signaling proteins (insulin receptor, insulin receptor substrate-1, GLUT4), was suppressed in WAT but not in BAT of HSL-/- mice. In contrast, expression of genes associated with cholesterol metabolism (sterol-regulatory element-binding protein-2, 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA:cholesterol acyltransferase-1) and thermogenesis (uncoupling protein-2) was upregulated in both WAT and BAT of HSL-/- mice. Our results suggest that impaired lipolysis in HSL deficiency affects lipid metabolism through alterations of adipose differentiation and adipose-derived hormone levels.

  5. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay.

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    Jacqueline M Leung

    Full Text Available T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58 ± 0.07 µm/s and 2.01 ± 0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.

  6. Disruption of TgPHIL1 alters specific parameters of Toxoplasma gondii motility measured in a quantitative, three-dimensional live motility assay.

    Science.gov (United States)

    Leung, Jacqueline M; Rould, Mark A; Konradt, Christoph; Hunter, Christopher A; Ward, Gary E

    2014-01-01

    T. gondii uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. T. gondii engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58 ± 0.07 µm/s and 2.01 ± 0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δphil1 parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δphil1 mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δphil1 mutant. These results show that alterations in morphology may have a significant impact on T. gondii motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δphil1 parasites in vivo, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility. PMID:24489670

  7. S1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids.

    Science.gov (United States)

    Alves, L C; Melo, R L; Sanderson, S J; Mottram, J C; Coombs, G H; Caliendo, G; Santagada, V; Juliano, L; Juliano, M A

    2001-03-01

    We have explored the substrate specificity of a recombinant cysteine proteinase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basic group at the P1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115--122], but we have also observed high affinity for peptides with hydrophobic residues at this position. In order to have substrates containing both features, we synthesized one series of internally quenched fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P1 position with the following non-natural basic amino acids: 4-aminomethyl-phenylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopropyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Ppa), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine (Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. The peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 12,000 mM(-1) x s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2,4-dinitrophenyl]-ethylenediamine (k(cat)/K(m) = 27,000 mM(-1) x s(-1)) were the best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FAmaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ-N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited this enzyme with K(i) values of 23 nM and 30 nM, respectively. Cruzain hydrolyzed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and

  8. Skin-specific Deletion of Stearoyl-CoA Desaturase-1 Alters Skin Lipid Composition and Protects Mice from High Fat Diet-induced Obesity*

    OpenAIRE

    Sampath, Harini; Flowers, Matthew T; Liu, Xueqing; Chad M Paton; Sullivan, Ruth; Chu, Kiki; Zhao, Minghui; Ntambi, James M.

    2009-01-01

    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 en...

  9. 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.

  10. 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)

    Lei, Shulei; Zavala-Flores, Laura; Garcia-Garcia, Aracely; Nandakumar, Renu; Huang, Yuting; Madayiputhiya, Nandakumar; Stanton, Robert C; Dodds, Eric D; Powers, Robert; Franco, Rodrigo

    2014-09-19

    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 in

  11. Colony mutants of compatible nocardiae displaying variations in recombining capacity.

    Science.gov (United States)

    Brownell, G H; Walsh, R S

    1972-03-01

    Colonial morphology mutants of Nocardia erythropolis were isolated following ultraviolet (UV) irradiation. The alleles rou-1/smo-1 were located by recombinant analysis and found to be linked to previously mapped characters. On the basis of recombinant class type patterns obtained from various selective characters it was postulated that the rou-1 allele may span a region of unique nucleotides in the Mat-Ce genome. Recombination frequencies of rou-1 and smo-2 bearing mutants of the Mat-Ce mating type were found to differ by over 1000 fold. Attempts to demonstrate that low recombination frequencies produced by the Smo mutants were due to Rec(-) genes were unsuccessful. No increased sensitivity to either UV or X irradiation was observed by the Smo mutants. Acriflavine treatment of either Rou or Smo colony mutants failed to accelerate reversion or to alter the recombining potentials of the mutants.

  12. [The structure of the O-specific polysaccharide from a mutant of nitrogen-fixing rhizobacterium Azospirillum brasilense Sp245 with an altered plasmid content].

    Science.gov (United States)

    Fedonenko, Iu P; Katsy, E I; Petrova, L P; Boĭko, A S; Zdorovenko, E L; Kachala, V V; Shashkov, A S; Knirel', Iu A

    2010-01-01

    The rhizobacteria Azospirillum brasilense Sp245 produce antigenically different lipopolysaccharides LPSI and LPSII, both containing identical pentasaccharides built from D-rhamnose residues as the repeated chains of O-specific oligosaccharides (OPS). In this study, we report the structure of the OPS from A. brasilense LPSI(-)LPSII(-)-mutant Sp245.5, which spontaneously lost the p85 and p120 plasmids upon the formation of a new 300-MDa megaplasmid after the long-term storage of the bacteria in a rich medium. The repeating unit of the A. brasilense mutant Sp245.5 appeared to be a disaccharide consisting of residues of N-acetyl-D-galactosamine and N-acetyl-D-mannosaminuronic acid: [Formula: see text].

  13. 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

  14. Dissociation of recombinant prion autocatalysis from infectivity.

    Science.gov (United States)

    Noble, Geoffrey P; Supattapone, Surachai

    2015-01-01

    Within the mammalian prion field, the existence of recombinant prion protein (PrP) conformers with self-replicating (ie. autocatalytic) activity in vitro but little to no infectious activity in vivo challenges a key prediction of the protein-only hypothesis of prion replication--that autocatalytic PrP conformers should be infectious. To understand this dissociation of autocatalysis from infectivity, we recently performed a structural and functional comparison between a highly infectious and non-infectious pair of autocatalytic recombinant PrP conformers derived from the same initial prion strain. (1) We identified restricted, C-terminal structural differences between these 2 conformers and provided evidence that these relatively subtle differences prevent the non-infectious conformer from templating the conversion of native PrP(C) substrates containing a glycosylphosphatidylinositol (GPI) anchor. (1) In this article we discuss a model, consistent with these findings, in which recombinant PrP, lacking post-translational modifications and associated folding constraints, is capable of adopting a wide variety of autocatalytic conformations. Only a subset of these recombinant conformers can be adopted by post-translationally modified native PrP(C), and this subset represents the recombinant conformers with high specific infectivity. We examine this model's implications for the generation of highly infectious recombinant prions and the protein-only hypothesis of prion replication.

  15. Establishment of ELISA techniques to detect Der f 1-specific IgE based on recombinant proteins%Der f1血清特异性IgE ELISA检测方法的建立

    Institute of Scientific and Technical Information of China (English)

    王运刚; 周鹰; 马桂芳; 杨李; 崔玉宝

    2012-01-01

    Objectives To establish an enzyme-linked immunosorbent assay (ELISA) technique to detect Der f 1-specific IgE based on recombinant proteins of group 1 allergens from Dermatophagoides farinae (rDer f 1) and to detect that IgE in sera from patients with asthma. Methods Indirect ELISA and ABC-ELISA techniques were developed using rDer f 1 as a capture antigen, and the techniques were then used to detect Der f 1-specific IgE in sera from 39 patients with asthma to analyze the sensitivity and specificity of the two techniques. Results With rDer f 1 as a coating antigen, the optimum concentration of coating antigen was 15 fig/ml and the proper dilution of serum was 1 : 2 for both indirect ELISA and ABC-ELISA to detect Der f 1-specific IgE. The proper dilution of horseradish peroxidase labeling anti-human IgE antibody (anti-human IgE/HRP) in indirect ELISA was 1 : 1000, and the proper dilution of anti-human IgE/Biotin and Avidin/HRP in ABC-ELISA was 1 : 1000 and 1 : 2000 , respectively. Indirect ELISA and ABC-ELISA had a sensitivity of 0. 68 U/ml and 0. 73 U/ml, respectively. Patients tested positive for Der f 1-specific IgE according to indirect ELISA and ABC-ELISA at rates of 92. 3% and 97. 4% respectively, and the rates did not differ significantly (P>0. 05). Conclusions Both ELISA techniques were highly sensitive at detecting rDer f 1 and should be used in laboratory diagnosis of allergic diseases associated with mites.%目的 利用粉尘螨变应原第1组分重组蛋白rDer f 1建立特异性IgE ELISA检测方法,并对哮喘患者血清进行检测.方法 以rDer f 1为包被抗原,分别建立间接ELISA法和亲和素(一)生物素复合ELISA (ABC-ELISA)法,检测Der f 1特异性IgE阳性标准血清和39例哮喘患者血清中的Der f 1特异性IgE,并分析检测结果.结果 间接ELISA法和ABC-ELISA法检测Der f 1特异性IgE的最适抗原包被浓度和血清稀释倍数均为15 μg/ml和1:2,间接ELISA法辣根过氧化物酶(HRP)标记抗人IgE

  16. 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.

  17. Neuroprotection by Exendin-4 Is GLP-1 Receptor Specific but DA D3 Receptor Dependent, Causing Altered BrdU Incorporation in Subventricular Zone and Substantia Nigra

    Directory of Open Access Journals (Sweden)

    A. Harkavyi

    2013-01-01

    Full Text Available Glucagon-like peptide-1 receptor (GLP-1R activation by exendin-4 (EX-4 is effective in preclinical models of Parkinson’s disease (PD and appears to promote neurogenesis even in severely lesioned rats. In the present study, we determined the effects of EX-4 on cellular BrdU incorporation in the rat subventricular zone (SVZ and substantia nigra (SN. We also determined the specificity of this effect with the GLP-1R antagonist EX-(9-39 as well as the potential role of dopamine (DA D3 receptors. Rats were administered 6-OHDA and 1 week later given EX-4 alone, with EX-(9-39 or nafadotride (D3 antagonist and BrdU. Seven days later, rats were challenged with apomorphine to evaluate circling. Extracellular DA was measured using striatal microdialysis and subsequently tissue DA measured. Tyrosine hydroxylase and BrdU were verified using immunohistochemistry. Apomorphine circling was reversed by EX-4 in lesioned rats, an effect reduced by EX-4, while both EX-(9-39 and NAF attenuated this. 6-OHDA decreased extracellular and tissue DA, both reversed by EX-4 but again attenuated by EX-(9-39 or NAF. Analysis of BrdU+ cells in the SVZ revealed increases in 6-OHDA-treated rats which were reversed by EX-4 and antagonised by either EX-(9-39 or NAF, while in the SN the opposite profile was seen.

  18. 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.

  19. MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

    Science.gov (United States)

    Paugh, Steven W; Coss, David R; Bao, Ju; Laudermilk, Lucas T; Grace, Christy R; Ferreira, Antonio M; Waddell, M Brett; Ridout, Granger; Naeve, Deanna; Leuze, Michael; LoCascio, Philip F; Panetta, John C; Wilkinson, Mark R; Pui, Ching-Hon; Naeve, Clayton W; Uberbacher, Edward C; Bonten, Erik J; Evans, William E

    2016-02-01

    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, ptriplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription. PMID:26844769

  20. RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence.

    Science.gov (United States)

    Xiao, Yinghong; Rouzine, Igor M; Bianco, Simone; Acevedo, Ashley; Goldstein, Elizabeth Faul; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

    2016-04-13

    Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations. PMID:27078068

  1. 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.

  2. 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.

  3. 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

  4. Specific gene mutations induced by heavy ions

    International Nuclear Information System (INIS)

    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

  5. Photoionization and electron-ion recombination of Ti I

    Science.gov (United States)

    Nahar, Sultana N.

    2016-07-01

    Study of the inverse processes of photoionization and electron-ion recombination of (Ti I + h ν ⇋ Ti II + e) using the unified method is reported. The method, based on close coupling (CC) approximation and R-matrix method, subsumes both the radiative recombination (RR) and dielectronic recombination (DR) in a unified manner and provides state-specific and total electron-ion recombination rate coefficients which are self-consistent with the state-specific photoionization cross sections. The present results include state-specific electron-ion recombination rates (αRC(i))and partial photoionization cross sections (σPI(i)) leaving the ion in the ground state of 813 bound states with n ≤ 10 and l ≤ 9 of Ti I. Various features of state-specific and total electron-ion recombination with temperature, and the corresponding photoionization cross sections with energies are discussed with illustrations. Due to closely lying excited states near the ground state of the core, photoionization cross sections show presence of narrow Rydberg resonances in low energy region near the ionization threshold. Many excited states also show broad and enhanced Seaton resonances due to PEC (photo-excitation-of-core) which contribute to the high temperature recombination. The total recombination rate coefficient is found to show a low hump around temperature 280 K and a high dielectronic recombination peak at temperature 25,000 K. Total spectrum of recombination cross sections and rates with photoelectron energy are also presented for experimental observation. Calculations were carried out using a CC wave function expansion of 36 states of the core ion Ti II. The large set of data for recombination rates and partial photoionization cross sections with resonances should provide a complete and accurate modelings of plasmas.

  6. Recombinant Helicobacter pylori catalase

    Institute of Scientific and Technical Information of China (English)

    Yang Bai; Ya-Li Zhang; Jian-Feng Jin; Ji-De Wang; Zhao-Shan Zhang

    2003-01-01

    AIM: To construct a recombinant strain which highly expresses catalase of Helicobacter pylori(H.pylori) and assay the activity of H. pylori catalase.METHODS: The catalase DNA was amplified from H. pylori chromosomal DNA with PCR techniques and inserted into the prokaryotie expression vector pET-22b (+), and then was transformed into the BL21 (DE3) E. coli strain which expressed catalase recombinant protein. The activity of H.pylori catalase was assayed by the Beers & Sizers.RESULTS: DNA sequence analysis showed that the sequence of catalase DNA was the same as GenBank's research. The catalase recombinant protein amounted to 24.4 % of the total bacterial protein after induced with IPTG for 3 hours at 37 ℃ and the activity of H. pylori catalase was high in the BL21 (DE3) E. coli strain.CONCLUSION: A clone expressing high activity H. pylori catalase is obtained, laying a good foundation for further studies.

  7. 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 in...

  8. 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...... 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...

  9. Sex, not genotype, determines recombination levels in mice.

    Science.gov (United States)

    Lynn, Audrey; Schrump, Stefanie; Cherry, Jonathan; Hassold, Terry; Hunt, Patricia

    2005-10-01

    Recombination, the precise physical breakage and rejoining of DNA between homologous chromosomes, plays a central role in mediating the orderly segregation of meiotic chromosomes in most eukaryotes. Despite its importance, the factors that control the number and placement of recombination events within a cell remain poorly defined. The rate of recombination exhibits remarkable species specificity, and, within a species, recombination is affected by the physical size of the chromosome, chromosomal location, proximity to other recombination events (i.e., chiasma interference), and, intriguingly, the sex of the transmitting parent. To distinguish between simple genetic and nongenetic explanations of sex-specific recombination differences in mammals, we compared recombination in meiocytes from XY sex-reversed and XO females with that in meiocytes from XX female and XY male mice. The rate and pattern of recombination in XY and XO oocytes were virtually identical to those in normal XX females, indicating that sex, not genotype, is the primary determinant of meiotic recombination patterns in mammals.

  10. Recombination in ionized gases

    International Nuclear Information System (INIS)

    In this paper it is shown how capture-stabilized methodology (both macroscopic and microscopic) can provide a generic basis for a unified treatment of all of the above recombination mechanisms. A new semiclassical theory of dissociative recombination is also presented in an effort to gain further insight into the physics not included in the first-order treatment and difficult to extract from numerical quantal treatments based on configuration mixing and on multichannel quantum defect theory. A simple analytical expression more accurate than the standard first-order result is obtained for the cross section σ and rate coefficient α. (author)

  11. Smectite alteration

    International Nuclear Information System (INIS)

    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)

  12. Performance testing of passive autocatalytic recombiners (PARs)

    Energy Technology Data Exchange (ETDEWEB)

    Blanchat, T. [Sandia National Laboratories, Albuquerque, NM (United States); Malliakos, A. [U.S. Nuclear Regulatory Commission, Washington, DC (United States)

    1997-03-01

    Passive autocatalytic recombiners (PARs) have been under consideration in the U.S. as a combustible gas control system in advanced light water reactor (ALWR) containments for design basis and severe accidents. PARs do not require a source of power. Instead they use palladium or platinum as a catalyst to recombine hydrogen and oxygen gases into water vapor upon contact with the catalyst. Energy from the recombination of hydrogen with oxygen is released at a relatively slow but continuous rate into the containment which prevents the pressure from becoming too high. The heat produced creates strong buoyancy effects which increases the influx of the surrounding gases to the recombiner. These natural convective flow currents promote mixing of combustible gases in the containment. PARs are self-starting and self-feeding under a very wide range of conditions. The recombination rate of the PAR system needs to be great enough to keep the concentration of hydrogen (or oxygen) below acceptable limits. There are several catalytic recombiner concepts under development worldwide. The USNRC is evaluating a specific design of a PAR which is in an advanced stage of engineering development and has been proposed for ALWR designs. Sandia National laboratories (SNL), under the sponsorship and the direction of the USNRC, is conducting an experimental program to evaluate the performance of PARs. The PAR will be tested at the SURTSEY facility at SNL. The test plan currently includes the following experiments: experiments will be conducted to define the startup characteristics of PARs (i.e., to define what is the lowest hydrogen concentration that the PAR starts recombining the hydrogen with oxygen); experiments will be used to define the hydrogen depletion rate of PARs as a function of hydrogen concentration; and experiments will be used to define the PAR performance in the presence of high concentrations of steam. (author)

  13. Recombinant DNA for Teachers.

    Science.gov (United States)

    Duvall, James G., III

    1992-01-01

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

  14. Recombineering Pseudomonas syringae

    Science.gov (United States)

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

  15. Productive homologous and non-homologous recombination of hepatitis C virus in cell culture

    DEFF Research Database (Denmark)

    Scheel, Troels K H; Galli, Andrea; Li, Yi-Ping;

    2013-01-01

    . In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a...... incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter......-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed...

  16. Fine-scale recombination and adaptive radiation could be linked.

    Science.gov (United States)

    Bodilis, Josselin

    2013-09-15

    The difficult reconstruction of the evolutionary history of the major surface protein gene oprF highlighted an adaptive radiation in the Pseudomonas fluorescens group. The recent work of Hao (2013) showed that partial recombination events in oprF gene occurred specifically in a P. fluorescens lineage under ecological niche segregation. So, I suggest that identification of lineage-specific fine-scale recombination may be a way to detect putative adaptive radiation in bacteria. PMID:23774687

  17. Metabolic footprint analysis of recombinant Escherichia coli strains during fed-batch fermentations.

    Science.gov (United States)

    Carneiro, Sónia; Villas-Bôas, Silas G; Ferreira, Eugénio C; Rocha, Isabel

    2011-03-01

    Metabolic footprinting has become a valuable analytical approach for the characterization of phenotypes and the distinction of specific metabolic states resulting from environmental and/or genetic alterations. The metabolic impact of heterologous protein production in Escherichia coli cells is of particular interest, since there are numerous cellular stresses triggered during this process that limit the overall productivity, e.g. the stringent response. Because the knowledge on the metabolic responses in recombinant bioprocesses is still scarce, metabolic footprinting can provide relevant information on the intrinsic metabolic adjustments. Thus, the metabolic footprints generated by E. coli W3110 and the ΔrelA mutant strain during recombinant fed-batch fermentations at different experimental conditions were measured and interpreted. The IPTG-induction of the heterologous protein expression resulted in the rapid accumulation of inhibitors of the glyoxylate shunt in the culture broth, suggesting the clearance of this anaplerotic route to replenish the TCA intermediaries withdrawn for the additional formation of the heterologous protein. Nutritional shifts were also critical in the recombinant cellular metabolism, indicating that cells employ diverse strategies to counteract imbalances in the cellular metabolism, including the secretion of certain metabolites that are, most likely, used as a metabolic relief to survival processes. PMID:21152511

  18. Ablation of the ID2 gene results in altered circadian feeding behavior, and sex-specific enhancement of insulin sensitivity and elevated glucose uptake in skeletal muscle and brown adipose tissue.

    Directory of Open Access Journals (Sweden)

    Deepa Mathew

    Full Text Available Inhibitor of DNA binding 2 (ID2 is a helix-loop-helix transcriptional repressor rhythmically expressed in many adult tissues. Our earlier studies have demonstrated a role for ID2 in the input pathway, core clock function and output pathways of the mouse circadian system. We have also reported that Id2 null (Id2-/- mice are lean with low gonadal white adipose tissue deposits and lower lipid content in the liver. These results coincided with altered or disrupted circadian expression profiles of liver genes including those involved in lipid metabolism. In the present phenotypic study we intended to decipher, on a sex-specific basis, the role of ID2 in glucose metabolism and in the circadian regulation of activity, important components of energy balance. We find that Id2-/- mice exhibited altered daily and circadian rhythms of feeding and locomotor activity; activity profiles extended further into the late night/dark phase of the 24-hr cycle, despite mice showing reduced total locomotor activity. Also, male Id2-/- mice consumed a greater amount of food relative to body mass, and displayed less weight gain. Id2-/- females had smaller adipocytes, suggesting sexual-dimorphic programing of adipogenesis. We observed increased glucose tolerance and insulin sensitivity in male Id2-/- mice, which was exacerbated in older animals. FDG-PET analysis revealed increased glucose uptake by skeletal muscle and brown adipose tissue of male Id2-/- mice, suggesting increased glucose metabolism and thermogenesis in these tissues. Reductions in intramuscular triacylglycerol and diacylglycerol were detected in male Id2-/- mice, highlighting its possible mechanistic role in enhanced insulin sensitivity in these mice. Our findings indicate a role for ID2 as a regulator of glucose and lipid metabolism, and in the circadian control of feeding/locomotor behavior; and contribute to the understanding of the development of obesity and diabetes, particularly in shift work

  19. Gestational and Lactational Exposure to Atrazine via the Drinking Water Causes Specific Behavioral Deficits and Selectively Alters Monoaminergic Systems in C57BL/6 Mouse Dams, Juvenile and Adult Offspring

    Science.gov (United States)

    Krishna, Saritha; Ye, Xiaoqin; Filipov, Nikolay M.

    2014-01-01

    Atrazine (ATR) is one of the most frequently detected pesticides in the U.S. water supply. This study aimed to investigate neurobehavioral and neurochemical effects of ATR in C57BL/6 mouse offspring and dams exposed to a relatively low (3 mg/l, estimated intake 1.4 mg/kg/day) concentration of ATR via the drinking water (DW) from gestational day 6 to postnatal day (PND) 23. Behavioral tests included open field, pole, grip strength, novel object recognition (NOR), forced swim, and marble burying tests. Maternal weight gain and offspring (PND21, 35, and 70) body or brain weights were not affected by ATR. However, ATR-treated dams exhibited decreased NOR performance and a trend toward hyperactivity. Juvenile offspring (PND35) from ATR-exposed dams were hyperactive (both sexes), spent less time swimming (males), and buried more marbles (females). In adult offspring (PND70), the only behavioral change was a sex-specific (females) decreased NOR performance by ATR. Neurochemically, a trend toward increased striatal dopamine (DA) in dams and a significant increase in juvenile offspring (both sexes) was observed. Additionally, ATR exposure decreased perirhinal cortex serotonin in the adult female offspring. These results suggest that perinatal DW exposure to ATR targets the nigrostriatal DA pathway in dams and, especially, juvenile offspring, alters dams’ cognitive performance, induces sex-selective changes involving motor and emotional functions in juvenile offspring, and decreases cognitive ability of adult female offspring, with the latter possibly associated with altered perirhinal cortex serotonin homeostasis. Overall, ATR exposure during gestation and lactation may cause adverse nervous system effects to both offspring and dams. PMID:24913803

  20. 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.

  1. AECL passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, L.B.; Marcinkowska, K. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2012-03-15

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  2. AECL passive autocatalytic recombiners

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, L.B.; Marcinkowska, K. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2011-07-01

    Atomic Energy of Canada Limited's (AECL) Passive Autocatalytic Recombiner (PAR) is a passive device used for hydrogen mitigation under post-accident conditions in nuclear reactor containment. The PAR employs a proprietary AECL catalyst which promotes the exothermal reaction between hydrogen and oxygen to form water vapour. The heat of reaction combined with the PAR geometry establishes a convective flow through the recombiner, where ambient hydrogen-rich gas enters the PAR inlet and hot, humid, hydrogen-depleted gas exits the outlet. AECL's PAR has been extensively qualified for CANDU and light water reactors (LWRs), and has been supplied to France, Finland, Ukraine, South Korea and is currently being deployed in Canadian nuclear power plants. (author)

  3. RECOMBINANT INFLUENZA VACCINES

    OpenAIRE

    Sedova, E.; Shcherbinin, D.; Migunov, A.; Smirnov, Iu; Logunov, D.; Shmarov, M.; Tsybalova, L.; Naroditskiĭ, B.; O. Kiselev; Gintsburg, A.

    2012-01-01

    This review covers the problems encountered in the construction and production of new recombinant influenza vaccines. New approaches to the development of influenza vaccines are investigated; they include reverse genetics methods, production of virus-like particles, and DNA- and viral vector-based vaccines. Such approaches as the delivery of foreign genes by DNA- and viral vector-based vaccines can preserve the native structure of antigens. Adenoviral vectors are a promising gene-delivery pla...

  4. Soluble recombinant influenza vaccines.

    OpenAIRE

    Fiers, W; Neirynck, S; Deroo, T; Saelens, X; Jou, W M

    2001-01-01

    Soluble, recombinant forms of influenza A virus haemagglutinin and neuraminidase have been produced in cells of lower eukaryotes, and shown in a mouse model to induce complete protective immunity against a lethal virus challenge. Soluble neuraminidase, produced in a baculovirus system, consisted of tetramers, dimers and monomers. Only the tetramers were enzymatically active. The immunogenicity decreased very considerably in the order tetra > di > mono. Therefore, we fused the head part of the...

  5. Introduction of restriction enzyme sites in protein-coding DNA sequences by site-specific mutagenesis not affecting the amino acid sequence: a computer program.

    OpenAIRE

    Arentzen, R; Ripka, W. C.

    1984-01-01

    Structure/function relationship studies of proteins are greatly facilitated by recombinant DNA technology which allows specific amino acid mutations to be made at the DNA sequence level by site-specific mutagenesis employing synthetic oligonucleotides. This technique has been successfully used to alter one or two amino acids in a protein. Replacement of existing DNA sequence coding for several amino acids with new synthetic DNA fragments would be facilitated by the presence of unique restrict...

  6. 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

  7. Zinc effect on the structure of recombinant human growth hormone

    International Nuclear Information System (INIS)

    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

  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. Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.

    Directory of Open Access Journals (Sweden)

    Troels K H Scheel

    2013-03-01

    Full Text Available Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a lacking functional envelope genes and strain J6 (2a, which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5' end to the NS2-NS3 region followed by JFH1 sequence from Core to the 3' end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural

  10. A Heritable Recombination system for synthetic Darwinian evolution in yeast.

    Science.gov (United States)

    Romanini, Dante W; Peralta-Yahya, Pamela; Mondol, Vanessa; Cornish, Virginia W

    2012-12-21

    Genetic recombination is central to the generation of molecular diversity and enhancement of evolutionary fitness in living systems. Methods such as DNA shuffling that recapitulate this diversity mechanism in vitro are powerful tools for engineering biomolecules with useful new functions by directed evolution. Synthetic biology now brings demand for analogous technologies that enable the controlled recombination of beneficial mutations in living cells. Thus, here we create a Heritable Recombination system centered around a library cassette plasmid that enables inducible mutagenesis via homologous recombination and subsequent combination of beneficial mutations through sexual reproduction in Saccharomyces cerevisiae. Using repair of nonsense codons in auxotrophic markers as a model, Heritable Recombination was optimized to give mutagenesis efficiencies of up to 6% and to allow successive repair of different markers through two cycles of sexual reproduction and recombination. Finally, Heritable Recombination was employed to change the substrate specificity of a biosynthetic enzyme, with beneficial mutations in three different active site loops crossed over three continuous rounds of mutation and selection to cover a total sequence diversity of 10(13). Heritable Recombination, while at an early stage of development, breaks the transformation barrier to library size and can be immediately applied to combinatorial crossing of beneficial mutations for cell engineering, adding important features to the growing arsenal of next generation molecular biology tools for synthetic biology. PMID:23412545

  11. Primordial magnetogenesis before recombination

    CERN Document Server

    Fabre, Ophélia

    2015-01-01

    The origin of large magnetic fields in the Universe remains currently unknown. We investigate here a mechanism before recombination based on known physics. The source of the vorticity is due to the changes in the photon distribution function caused by the fluctuations in the background photons. We show that the magnetic field generated in the MHD limit, due to the Coulomb scattering, is of the order $10^{-49}$ G. We explicitly show that the magnetic fields generated from this process are sustainable and are not erased by resistive diffusion. We compare the results with current observations and discuss the implications.

  12. Recombinant CBM-fusion technology : applications overview

    OpenAIRE

    Oliveira, Carla Cristina Marques de; Carvalho, Vera; Domingues, Lucília; Gama, F. M.

    2015-01-01

    Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for...

  13. Production of recombinant antibodies using bacteriophages

    OpenAIRE

    Shukra, A. M.; Sridevi, N. V.; Dev Chandran,; Kapil Maithal,

    2014-01-01

    Recombinant antibody fragments such as Fab, scFv, diabodies, triabodies, single domain antibodies and minibodies have recently emerged as potential alternatives to monoclonal antibodies, which can be engineered using phage display technology. These antibodies match the strengths of conventionally produced monoclonal antibodies and offer advantages for the development of immunodiagnostic kits and assays. These fragments not only retain the specificity of the whole monoclonal ...

  14. Fundamental Studies of Recombinant Hydrogenases

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Michael W

    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.

  15. Epigenetic alterations in gastric carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    In-Seon CHOI; Tsung-Teh WU

    2005-01-01

    Gastric cancer is believed to result in part from the accumulation of multiple genetic alterations leading to oncogene overexpression and tumor suppressor loss. Epigenetic alterations as a distinct and crucial mechanism to silence a variety of methylated tissue-specific and imprinted genes, have been extensively studied in gastric carcinoma and play important roles in gastric carcinogenesis. This review will briefly discuss the basic aspects of DNA methylation and CpG island methylation, in particular the epigenetic alterations of certain critical genes implicated in gastric carcinogenesis and its relevance of clinical implications.

  16. 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.

  17. Linker-Mediated Recombinational Subcloning of Large DNA Fragments Using Yeast

    OpenAIRE

    Raymond, Christopher K.; Sims, Elizabeth H.; Olson, Maynard V.

    2002-01-01

    The homologous recombination pathway in yeast is an ideal tool for the sequence-specific assembly of plasmids. Complementary 80-nucleotide oligonucleotides that overlap a vector and a target fragment were found to serve as efficient recombination linkers for fragment subcloning. Using electroporation, single-stranded 80-mers were adequate for routine plasmid construction. A cycloheximide-based counterselection was introduced to increase the specificity of cloning by homologous recombination r...

  18. E. coli Tarafından Sentezlenen Recombinant Soyacystatinin Karakterizasyonu

    OpenAIRE

    AKPINAR, Özlem; AN, Haejung

    2004-01-01

    Recombinant (r-) soyacystatin was characterized for their inhibitory activity against papain and compared to egg white cystatin. r-Soyacystatin expressed in E. coli was purified 4.33 fold as a recombinant protein with phenyl-Sepharose and DEAE. Egg white cystatin was purified by using affinity chromatography on CM-papain-Sepharose. The specific interaction of r-soyacystatin and papain was detected on isoelectric focusing gel. Papain and r-soyacystatin formed a complex and the complex was res...

  19. The next wave of recombinant and synthetic anticancer vaccines

    OpenAIRE

    Irvine, Kari R.; Restifo, Nicholas P

    1995-01-01

    The identification of tumor-associated antigens (TAA) recognized by T lymphocytes makes the development of antigen-specific synthetic and recombinant vaccines possible. The expression of TAA within a recombinant vector increases control over the kinetics and quantity, the molecular form, and the subcellular location of the immunogen delivered. The next generation of antitumor vaccines employs cytokines and costimulatory molecules expressed in concert with TAA that are capable of augmenting th...

  20. 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

    OpenAIRE

    Zhang, Jianhua; Jiang, Bingfu; Xu, Mingjie; Dai, Xing; Purdy, Michael A.; Meng, Jihong

    2014-01-01

    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 hum...

  1. Neonatal Maternal Separation Alters, in a Sex-Specific Manner, the Expression of TRH, of TRH-Degrading Ectoenzyme in the Rat Hypothalamus, and the Response of the Thyroid Axis to Starvation.

    Science.gov (United States)

    Jaimes-Hoy, Lorraine; Gutiérrez-Mariscal, Mariana; Vargas, Yamili; Pérez-Maldonado, Adrián; Romero, Fidelia; Sánchez-Jaramillo, Edith; Charli, Jean-Louis; Joseph-Bravo, Patricia

    2016-08-01

    Hypothalamic-pituitary-thyroid (HPT) axis activity is important for energy homeostasis, and is modified by stress. Maternal separation (MS) alters the stress response and predisposes to metabolic disturbances in the adult. We therefore studied the effect of MS on adult HPT axis activity. Wistar male and female pups were separated from their mothers 3 h/d during postnatal day (PND)2-PND21 (MS), or left nonhandled (NH). Open field and elevated plus maze tests revealed increased locomotion in MS males and anxiety-like behavior in MS females. At PND90, MS females had increased body weight gain, Trh expression in the hypothalamic paraventricular nucleus, and white adipose tissue mass. MS males had increased expression of TRH-degrading enzyme in tanycytes, reduced TSH and T3, and enhanced corticosterone serum concentrations. MS stimulated brown adipose tissue deiodinase 2 activity in either sex. Forty-eight hours of fasting (PND60) augmented serum corticosterone levels similarly in MS or NH females but more in MS than in NH male rats. MS reduced the fasting-induced drop in hypothalamic paraventricular nucleus-Trh expression of males but not of females and abolished the fasting-induced increase in Trh expression in both sexes. Fasting reduced serum concentrations of TSH, T4, and T3, less in MS than in NH males, whereas in females, TSH decreased in MS but not in NH rats, but T4 and T3 decreased similarly in NH and MS rats. In conclusion, MS produced long-term changes in the activity of the HPT axis that were sex specific; response to fasting was partially blunted in males, which could affect their adaptive response to negative energy balance. PMID:27323240

  2. Primordial magnetogenesis before recombination

    Science.gov (United States)

    Fabre, Ophélia; Shankaranarayanan, S.

    2016-04-01

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

  3. Detection of specific antibody IgM of Lyme disease by ELISA using recombinant outer surface protein C of borrelia burgdorferi%用基因工程抗原建立ELISA检测莱姆病特异IgM抗体

    Institute of Scientific and Technical Information of China (English)

    贾月萍; 周国萍; 曾丽苹; 高峰

    2001-01-01

    目的利用伯氏疏螺旋体基因工程抗原外膜蛋白C(OspC)建立间接ELISA,检测莱姆病特异性抗体IgM。方法基因工程抗原OspC的包被浓度和酶标抗μ链单抗所用浓度及血清稀释倍数,均由方阵滴定法确定,并进行精密度、特异性试验、阻断试验和干扰试验。结果 OspC最佳浓度为150 μg/L,批内平均变异系数4.6%,批间平均变异系数14.2%,用ELISA测定临床已确诊莱姆病33例,57例正常体检者,同时与进口ELISA试剂盒比较,两方法符合率97.8%。结论该方法特异性强、敏感性高、实验结果可靠,是莱姆病早期诊断的好方法。%Objective To detect specific IgM of Lyme disease indirect ELISA using recombinant outer surface protein C(OpsC)of Borrelia burgdorferi in vitro was established. Methods Coated concentration of recombinant OspC and dilution multiple of serum anol concentration of enzyme secondary antibody were determined by block,and degree of percision.specificity interference and interruption test were performed. Results Best concentration of OspC was 150 μg/L.within-run CV was 6.3% between-run CV was 11.8%.Confimred 33 Lyme patients in clinic and 57 controls were examined meanwhile by this method and import ELISA kit,coincidena rate was 97.8%.Conclusion This ELISA using recombinant OspC was a good method for early diagnostic of Lyme disease.

  4. 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 w...

  5. A whole recombinant yeast-based therapeutic vaccine elicits HBV X, S and Core specific T cells in mice and activates human T cells recognizing epitopes linked to viral clearance.

    Directory of Open Access Journals (Sweden)

    Thomas H King

    Full Text Available Chronic hepatitis B infection (CHB is characterized by sub-optimal T cell responses to viral antigens. A therapeutic vaccine capable of restoring these immune responses could potentially improve HBsAg seroconversion rates in the setting of direct acting antiviral therapies. A yeast-based immunotherapy (Tarmogen platform was used to make a vaccine candidate expressing hepatitis B virus (HBV X, surface (S, and Core antigens (X-S-Core. Murine and human immunogenicity models were used to evaluate the type and magnitude of HBV-Ag specific T cell responses elicited by the vaccine. C57BL/6J, BALB/c, and HLA-A*0201 transgenic mice immunized with yeast expressing X-S-Core showed T cell responses to X, S and Core when evaluated by lymphocyte proliferation assay, ELISpot, intracellular cytokine staining (ICS, or tumor challenge assays. Both CD4+ and CD8+ T cell responses were observed. Human T cells transduced with HBc18-27 and HBs183-91 specific T cell receptors (TCRs produced interferon gamma (IFNγ following incubation with X-S-Core-pulsed dendritic cells (DCs. Furthermore, stimulation of peripheral blood mononuclear cells (PBMCs isolated from CHB patients or from HBV vaccine recipients with autologous DCs pulsed with X-S-Core or a related product (S-Core resulted in pronounced expansions of HBV Ag-specific T cells possessing a cytolytic phenotype. These data indicate that X-S-Core-expressing yeast elicit functional adaptive immune responses and supports the ongoing evaluation of this therapeutic vaccine in patients with CHB to enhance the induction of HBV-specific T cell responses.

  6. Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: lack of correlation between in vitro activity and in vivo efficacy

    OpenAIRE

    Mealey, Robert H.; Littke, Matt H.; Leib, Steven R.; Davis, William C.; McGuire, Travis C.

    2007-01-01

    Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient ...

  7. Bimolecular recombination in organic photovoltaics.

    Science.gov (United States)

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

    2014-01-01

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

  8. Initiation of V(D)J recombination in a cell-free system

    NARCIS (Netherlands)

    D.C. van Gent (Dik); J.F. McBlane; D.A. Ramsden; M.J. Sadokfsky; J.E. Hesse (Joanne); M. Gellert

    1995-01-01

    textabstractCells performing V(D)J recombination make specific cuts in DNA at recombination signal sequences. Here, we show that nuclear extracts of pre-B cell lines carry out this specific cleavage. The products of cleavage are the same as found previously in thymocytes: full-leng

  9. Numerical Study of Passive Catalytic Recombiner for Hydrogen Mitigation

    Directory of Open Access Journals (Sweden)

    Pavan K Sharma

    2010-10-01

    Full Text Available A significant amount of hydrogen is expected to be released within the containment of a water cooled power reactor after a severe accident. To reduce the risk of deflagration/detonation various means for hydrogen control have been adopted all over the world. Passive catalytic recombiner with vertical flat catalytic plate is one of such hydrogen mitigating device. Passive catalytic recombiners are designed for the removal of hydrogen generated in order to limit the impact of possible hydrogen combustion. Inside a passive catalytic recombiner, numerous thin steel sheets coated with catalyst material are vertically arranged at the bottom opening of a sheet metal housing forming parallel flow channels for the surrounding gas atmosphere. Already below conventional flammability limits, hydrogen and oxygen react exothermally on the catalytic surfaces forming harmless steam. Detailed numerical simulations and experiments are required for an in-depth knowledge of such plate type catalytic recombiners. Specific finite volume based in-house CFD code has been developed to model and analyse the working of these recombiner. The code has been used to simulate the recombiner device used in the Gx-test series of Battelle-Model Containment (B-MC experiments. The present paper briefly describes the working principle of such passive catalytic recombiner and salient feature of the CFD model developed at Bhabha Atomic Research Centre (BARC. Finally results of the calculations and comparison with existing data are discussed.

  10. Suppression of Recj Exonuclease Mutants of Escherichia Coli by Alterations in DNA Helicases II (Uvrd) and IV (Held)

    OpenAIRE

    Lovett, S T; Sutera-Jr., V. A.

    1995-01-01

    The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, ``uvrD517(am),'' at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517(am) mutation does not eliminate ...

  11. The Contribution of Genetic Recombination to CRISPR Array Evolution.

    Science.gov (United States)

    Kupczok, Anne; Landan, Giddy; Dagan, Tal

    2015-06-16

    CRISPR (clustered regularly interspaced short palindromic repeats) is a microbial immune system against foreign DNA. Recognition sequences (spacers) encoded within the CRISPR array mediate the immune reaction in a sequence-specific manner. The known mechanisms for the evolution of CRISPR arrays include spacer acquisition from foreign DNA elements at the time of invasion and array erosion through spacer deletion. Here, we consider the contribution of genetic recombination between homologous CRISPR arrays to the evolution of spacer repertoire. Acquisition of spacers from exogenic arrays via recombination may confer the recipient with immunity against unencountered antagonists. For this purpose, we develop a novel method for the detection of recombination in CRISPR arrays by modeling the spacer order in arrays from multiple strains from the same species. Because the evolutionary signal of spacer recombination may be similar to that of pervasive spacer deletions or independent spacer acquisition, our method entails a robustness analysis of the recombination inference by a statistical comparison to resampled and perturbed data sets. We analyze CRISPR data sets from four bacterial species: two Gammaproteobacteria species harboring CRISPR type I and two Streptococcus species harboring CRISPR type II loci. We find that CRISPR array evolution in Escherichia coli and Streptococcus agalactiae can be explained solely by vertical inheritance and differential spacer deletion. In Pseudomonas aeruginosa, we find an excess of single spacers potentially incorporated into the CRISPR locus during independent acquisition events. In Streptococcus thermophilus, evidence for spacer acquisition by recombination is present in 5 out of 70 strains. Genetic recombination has been proposed to accelerate adaptation by combining beneficial mutations that arose in independent lineages. However, for most species under study, we find that CRISPR evolution is shaped mainly by spacer acquisition and

  12. Aspergillus: sex and recombination.

    Science.gov (United States)

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

    2014-12-01

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

  13. 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.

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

  16. Cell encoding recombinant human erythropoietin

    Energy Technology Data Exchange (ETDEWEB)

    Beck, A.K.; Withy, R.M.; Zabrecky, J.R.; Masiello, N.C.

    1990-09-04

    This patent describes a C127 cell transformed with a recombinant DNA vector. It comprises: a DNA sequence encoding human erythropoietin, the transformed cell being capable of producing N-linked and O-linked glycosylated human erythropoietin.

  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. Progenitors of Recombining Supernova Remnants

    OpenAIRE

    Moriya, Takashi J.

    2012-01-01

    Usual supernova remnants have either ionizing plasma or plasma in collisional ionization equilibrium, i.e., the ionization temperature is lower than or equal to the electron temperature. However, the existence of recombining supernova remnants, i.e., supernova remnants with the ionization temperature higher than the electron temperature, is recently confirmed. One suggested way to have recombining plasma in a supernova remnant is to have a dense circumstellar medium at the time of the superno...

  19. Recombinant snake venom prothrombin activators

    OpenAIRE

    Lövgren, 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 ...

  20. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  1. Use of Recombinant Envelope Proteins for Serological Diagnosis of Dengue Virus Infection in an Immunochromatographic Assay

    OpenAIRE

    Cuzzubbo, Andrea J.; Endy, Timothy P.; Nisalak, Ananda; Kalayanarooj, Siripen; Vaughn, David W.; Ogata, Steven A.; Clements, David E.; Devine, Peter L.

    2001-01-01

    An immunochromatographic test that incorporates recombinant antigens (Dengue Duo Rapid Strip Test; PanBio, Brisbane, Australia) has recently become commercially available. This assay is performed in 15 min and detects both immunoglobulin M (IgM) and IgG in a capture format. The four recombinant proteins used represent the N-terminal 80% of the viral envelope glycoproteins of dengue viruses 1, 2, 3, and 4, respectively. The sensitivity and specificity of the recombinant-antigen-based assay wer...

  2. A genetic system for direct selection of gene-positive clones during recombinational cloning in yeast

    OpenAIRE

    Noskov, Vladimir; Kouprina, Natalay; Leem, Sun-Hee; Koriabine, Maxim; Barrett, J. Carl; Larionov, Vladimir

    2002-01-01

    Transformation-associated recombination (TAR) is a cloning technique that allows specific chromosomal regions or genes to be isolated directly from genomic DNA without prior construction of a genomic library. This technique involves homologous recombination during spheroplast transformation between genomic DNA and a TAR vector that has 5′ and 3′ gene targeting sequences (hooks). Typically, TAR cloning produces positive YAC recombinants at a frequency of ∼0.5%; the positive clones are identifi...

  3. Critical Role of RecN in Recombinational DNA Repair and Survival of Helicobacter pylori▿

    OpenAIRE

    Wang, Ge; Maier, Robert J.

    2007-01-01

    Homologous recombination is one of the key mechanisms responsible for the repair of DNA double-strand breaks. Recombinational repair normally requires a battery of proteins, each with specific DNA recognition, strand transfer, resolution, or other functions. Helicobacter pylori lacks many of the proteins normally involved in the early stage (presynapsis) of recombinational repair, but it has a RecN homologue with an unclear function. A recN mutant strain of H. pylori was shown to be much more...

  4. Horizontal Transmissible Protection against Myxomatosis and Rabbit Hemorrhagic Disease by Using a Recombinant Myxoma Virus

    OpenAIRE

    Bárcena, Juan; Morales, Mónica; Vázquez, Belén; Boga, José A.; Parra, Francisco; Lucientes, Javier; Pagès-Manté, Albert; Sánchez-Vizcaíno, José M.; Blasco, Rafael; Torres, Juan M.

    2000-01-01

    We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RH...

  5. Hyperglycaemia Alters Thymic Epithelial Cell Function

    Directory of Open Access Journals (Sweden)

    Vera Alexandrovna Abramova

    2013-07-01

    Full Text Available Insulin-dependent diabetes mellitus (IDDM is considered to be a consequence of unchecked auto-immune processes. Alterations in immune system responses are thought to be the cause of the disease, but the possibility that altered metabolite levels (glucose can establish the disease by specifically acting on and altering thymus stroma functions has not been investigated. Therefore, the direct effect of hyperglycaemia (HG on central tolerance mechanisms as a causative agent needs to be investigated.

  6. Homologous recombination in DNA repair and DNA damage tolerance

    Institute of Scientific and Technical Information of China (English)

    Xuan Li; Wolf-Dietrich Heyer

    2008-01-01

    Homologous recombination (HR) comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks (DSBs) and interstrand crosslinks (ICLs). In addition, recombination provides critical sup-port for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR: homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modaUties of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.

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

    Science.gov (United States)

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

    2013-01-01

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

  8. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    Science.gov (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  9. Mechanisms underlying allergy vaccination with recombinant hypoallergenic allergen derivatives.

    Science.gov (United States)

    Linhart, Birgit; Valenta, Rudolf

    2012-06-19

    Hundred years ago therapeutic vaccination with allergen-containing extracts has been introduced as a clinically effective, disease-modifying, allergen-specific and long-lasting form of therapy for allergy, a hypersensitivity disease affecting more than 25% of the population. Today, the structures of most of the disease-causing allergens have been elucidated and recombinant hypoallergenic allergen derivatives with reduced allergenic activity have been engineered to reduce side effects during allergen-specific immunotherapy (SIT). These recombinant hypoallergens have been characterized in vitro, in experimental animal models and in clinical trials in allergic patients. This review provides a summary of the molecular, immunological and preclinical evaluation criteria applied for this new generation of allergy vaccines. Furthermore, we summarize the mechanisms underlying SIT with recombinant hypoallergens which are thought to be responsible for their therapeutic effect.

  10. 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.

  11. Molecular recognition between ligands and nucleic acids: Novel pyridine- and benzoxazole-containing agents related to Hoechst 33258 that exhibit altered DNA sequence specificity deduced from footprinting analysis and spectroscopic studies

    Energy Technology Data Exchange (ETDEWEB)

    Bathini, Y.; Rao, K.E.; Shea, R.G.; Lown, J.W. (Univ. of Alberta, Edmonton (Canada))

    The syntheses of certain analogues of the DNA minor groove binding agent Hoechst 33258 designed to exhibit altered sequence recognition are described. The structural modifications include the following: substitution of pyridine for the benzene ring of the benzimidazole moiety, replacement of one benzimidazole unit by a benzoxazole in the two possible orientations with respect to the DNA receptor, and a synthesis of 2,2{prime}-m-phenylenebis(6-(4-methyl-1-piperazinyl)benzimidazole). Sequence recognition of these agents on a HindIII/EcoRI fragment of pBR322 DNA was determined by MPE footprinting procedures. Some of the analogues exhibited altered DNA sequence preference compared with Hoechst 33258. In particular, a structure bearing a benzoxazole moiety with the oxygen oriented inward to the minor groove together with an inward-directed pyridine nitrogen appears to confer the property of recognition of a GC base pair within the binding sequence. The possible factors, structural, stereochemical, and electrostatic, contributing to the altered DNA sequence recognition properties are discussed.

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

    Science.gov (United States)

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

    2005-07-07

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

  13. Recombinant Human DFF45 Inhibits Apoptosis-specific Endonuclease in a Cell-free System of Xenopus Egg Extracts%重组人DFF45能抑制爪蟾卵非细胞体系中的凋亡特异核酸酶

    Institute of Scientific and Technical Information of China (English)

    杨巍; 卢智刚; 张岚; 翟中和

    2001-01-01

    利用RT-PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了人DNA断裂因子(DFF)45的cDNA, 将其克隆到pET-28a(+)表达载体中, 经IPTG诱导在大肠杆菌BL21(DE3)中获得了高效表达。 表达的DFF45重组蛋白占菌体总蛋白质的56.6%, 并位于细胞的可溶性组分中。 利用融合在DFF45 N端的His*Tag, 采用亲和层析的方法纯化重组蛋白, 再经热变性处理去除杂蛋白后, 获得了电泳纯的DFF45。 将纯化的重组人DFF45加入到爪蟾卵非细胞凋亡体系中, 可有效地抑制外源λDNA的水解, 并可抑制外源鸡红细胞核形成凋亡特有的DNA梯。 实验结果说明爪蟾卵非细胞体系中确实存在凋亡特异核酸酶, 而且其活性可为重组人DFF45所抑制。%The human DNA fragmentation factor (DFF) is a heterodimer of 40 kD and 45 kD subunits. The 40 kD subunit (DFF40) has an intrinsic DNase activity responsible for the genomic DNA degradation into nucleosomal fragments during apoptosis. As an inhibitor for DFF40, the 45 kD subunit (DFF45) complexes with DFF40, inhibiting DNase activity until certain apoptosis signals are received. In cells undergoing apoptosis, the cleavage of DFF45 by activated caspase-3 frees DFF40 from the complex and initiates the apoptosis-specific DNA fragmentation. In this report, the coding region of human DFF45 gene was amplified from the total RNA of HeLa cells by RT-PCR. The resulting 1 kb DNA fragment was cloned into the bacterial expression vector pET-28a(+) with a 6×histidine tag fused to the N-terminus of DFF45, generating plasmid pET28a-DFF45, which was then used to transform E.coli BL21(DE3). Induced by IPTG, the recombinant DFF45 was expressed efficiently with a yield of 56.6% of total bacterial proteins. The product was purified to homogeneity through a nickel affinity column, followed by heat treatment, and approximately 4—6 mg of DFF was purified from 100 ml culture. Purified recombinant human DFF45, added into the apoptotic cell

  14. 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

  15. Fine scale analysis of crossover and non-crossover and detection of recombination sequence motifs in the honeybee (Apis mellifera.

    Directory of Open Access Journals (Sweden)

    Nadia Bessoltane

    Full Text Available BACKGROUND: Meiotic exchanges are non-uniformly distributed across the genome of most studied organisms. This uneven distribution suggests that recombination is initiated by specific signals and/or regulations. Some of these signals were recently identified in humans and mice. However, it is unclear whether or not sequence signals are also involved in chromosomal recombination of insects. METHODOLOGY: We analyzed recombination frequencies in the honeybee, in which genome sequencing provided a large amount of SNPs spread over the entire set of chromosomes. As the genome sequences were obtained from a pool of haploid males, which were the progeny of a single queen, an oocyte method (study of recombination on haploid males that develop from unfertilized eggs and hence are the direct reflect of female gametes haplotypes was developed to detect recombined pairs of SNP sites. Sequences were further compared between recombinant and non-recombinant fragments to detect recombination-specific motifs. CONCLUSIONS: Recombination events between adjacent SNP sites were detected at an average distance of 92 bp and revealed the existence of high rates of recombination events. This study also shows the presence of conversion without crossover (i. e. non-crossover events, the number of which largely outnumbers that of crossover events. Furthermore the comparison of sequences that have undergone recombination with sequences that have not, led to the discovery of sequence motifs (CGCA, GCCGC, CCGCA, which may correspond to recombination signals.

  16. Novel Heterotypic Rox Sites for Combinatorial Dre Recombination Strategies

    Directory of Open Access Journals (Sweden)

    Katherine Chuang

    2016-03-01

    Full Text Available Site-specific recombinases (SSRs such as Cre are widely used in gene targeting and genetic approaches for cell labeling and manipulation. They mediate DNA strand exchange between two DNA molecules at dedicated recognition sites. Precise understanding of the Cre recombination mechanism, including the role of individual base pairs in its loxP target site, guided the generation of mutant lox sites that specifically recombine with themselves but not with the wild type loxP. This has led to the development of a variety of combinatorial Cre-dependent genetic strategies, such as multicolor reporters, irreversible inversions, or recombination-mediated cassette exchange. Dre, a Cre-related phage integrase that recognizes roxP sites, does not cross-react with the Cre-loxP system, but has similar recombination efficiency. We have previously described intersectional genetic strategies combining Dre and Cre. We now report a mutagenesis screen aimed at identifying roxP base pairs critical for self-recognition. We describe several rox variant sites that are incompatible with roxP, but are able to efficiently recombine with themselves in either purified systems or bacterial and eukaryotic tissue culture systems. These newly identified rox sites are not recognized by Cre, thus enabling potential combinatorial strategies involving Cre, Dre, and target loci including multiple loxP and roxP variants.

  17. 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.

  18. Recombinant antigens for immunodiagnosis of cystic echinococcosis

    Directory of Open Access Journals (Sweden)

    Li Jun

    2004-01-01

    Full Text Available Three cDNAs, termed EpC1, TPxEg and EgG5, were isolated by immunoscreening from an Echinococcus granulosus cDNA library. The recombinant phages exhibited strong reactivity with sera from humans with confirmed cystic echinococcosis (CE and with sera from mice infected with E. granulosus oncospheres. The cDNAs were subcloned into a pET vector, expressed as fusion proteins tagged with GST and affinity purified against the GST tag. Of the three recombinant proteins, EpC1 achieved the highest performance for serodiagnosis of CE in Western blot analysis using a panel of clinically defined human sera to initially address the sensitivity and specificity of the molecules. The protein yielded an overall sensitivity of 92.2% and specificity of 95.6%, levels unprecedented taking into account the large panel of 896 human sera that were tested. The strategy used may also prove suitable for improved immunodiagnosis of other parasitic infections.

  19. Recombinant snake venom prothrombin activators.

    Science.gov (United States)

    Lövgren, Ann

    2013-01-01

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

  20. Heterogeneity in recombinant protein production

    DEFF Research Database (Denmark)

    Schalén, Martin; Johanson, Ted; Lundin, Luisa;

    2012-01-01

    . the cell physiology is affected. Cells are stressed, and this may severely affect growth, by-product accumulation, biomass yield and recombinant product yield. The stress caused by exposure to divergent microenvironments, genetic differences of individual cells, differing cell cycle stage and cell age, all...... 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...... 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....

  1. Review of literature on catalytic recombination of hydrogen--oxygen

    International Nuclear Information System (INIS)

    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

  2. Androgen and taxol cause cell type-specific alterations of centrosome and DNA organization in androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cells

    Science.gov (United States)

    Schatten, H.; Ripple, M.; Balczon, R.; Weindruch, R.; Chakrabarti, A.; Taylor, M.; Hueser, C. N.

    2000-01-01

    We investigated the effects of androgen and taxol on the androgen-responsive LNCaP and androgen-independent DU145 prostate cancer cell lines. Cells were treated for 48 and 72 h with 0.05-1 nM of the synthetic androgen R1881 and with 100 nM taxol. Treatment of LNCaP cells with 0.05 nM R1881 led to increased cell proliferation, whereas treatment with 1 nM R1881 resulted in inhibited cell division, DNA cycle arrest, and altered centrosome organization. After treatment with 1 nM R1881, chromatin became clustered, nuclear envelopes convoluted, and mitochondria accumulated around the nucleus. Immunofluorescence microscopy with antibodies to centrosomes showed altered centrosome structure. Although centrosomes were closely associated with the nucleus in untreated cells, they dispersed into the cytoplasm after treatment with 1 nM R1881. Microtubules were only faintly detected in 1 nM R1881-treated LNCaP cells. The effects of taxol included microtubule bundling and altered mitochondria morphology, but not DNA organization. As expected, the androgen-independent prostate cancer cell line DU145 was not affected by R1881. Treatment with taxol resulted in bundling of microtubules in both cell lines. Additional taxol effects were seen in DU145 cells with micronucleation of DNA, an indication of apoptosis. Simultaneous treatment with R1881 and taxol had no additional effects on LNCaP or DU145 cells. These results suggest that LNCaP and DU145 prostate cancer cells show differences not only in androgen responsiveness but in sensitivity to taxol as well. Copyright 2000 Wiley-Liss, Inc.

  3. 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...

  4. 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..

  5. Preparing Recombinant Gonad Organ Cultures

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Blanche Capel and Jordan Batchvarov Corresponding author ([]()) ### INTRODUCTION It can be useful to assay migration between any two adjacent tissues during development. This protocol assays cell migration between the gonad and mesonephros using tissue recombination between genetically marked and unmarked tissue, combined with an organ culture technique. First, agar blocks are prepared in a custom-built mold. The size and sh...

  6. Controlled Release from Recombinant Polymers

    OpenAIRE

    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 tempor...

  7. 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.

  8. 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

  9. High-Resolution Mapping of Homologous Recombination Events in rad3 Hyper-Recombination Mutants in Yeast.

    Directory of Open Access Journals (Sweden)

    Sabrina L Andersen

    2016-03-01

    Full Text Available The Saccharomyces cerevisae RAD3 gene is the homolog of human XPD, an essential gene encoding a DNA helicase of the TFIIH complex involved in both nucleotide excision repair (NER and transcription. Some mutant alleles of RAD3 (rad3-101 and rad3-102 have partial defects in DNA repair and a strong hyper-recombination (hyper-Rec phenotype. Previous studies showed that the hyper-Rec phenotype associated with rad3-101 and rad3-102 can be explained as a consequence of persistent single-stranded DNA gaps that are converted to recombinogenic double-strand breaks (DSBs by replication. The systems previously used to characterize the hyper-Rec phenotype of rad3 strains do not detect the reciprocal products of mitotic recombination. We have further characterized these events using a system in which the reciprocal products of mitotic recombination are recovered. Both rad3-101 and rad3-102 elevate the frequency of reciprocal crossovers about 100-fold. Mapping of these events shows that three-quarters of these crossovers reflect DSBs formed at the same positions in both sister chromatids (double sister-chromatid breaks, DSCBs. The remainder reflects DSBs formed in single chromatids (single chromatid breaks, SCBs. The ratio of DSCBs to SCBs is similar to that observed for spontaneous recombination events in wild-type cells. We mapped 216 unselected genomic alterations throughout the genome including crossovers, gene conversions, deletions, and duplications. We found a significant association between the location of these recombination events and regions with elevated gamma-H2AX. In addition, there was a hotspot for deletions and duplications at the IMA2 and HXT11 genes near the left end of chromosome XV. A comparison of these data with our previous analysis of spontaneous mitotic recombination events suggests that a sub-set of spontaneous events in wild-type cells may be initiated by incomplete NER reactions, and that DSCBs, which cannot be repaired by sister

  10. CRISPR-Cas9-assisted recombineering in Lactobacillus reuteri.

    Science.gov (United States)

    Oh, Jee-Hwan; van Pijkeren, Jan-Peter

    2014-01-01

    Clustered regularly interspaced palindromic repeats (CRISPRs) and the CRISPR-associated (Cas) nuclease protect bacteria and archeae from foreign DNA by site-specific cleavage of incoming DNA. Type-II CRISPR-Cas systems, such as the Streptococcus pyogenes CRISPR-Cas9 system, can be adapted such that Cas9 can be guided to a user-defined site in the chromosome to introduce double-stranded breaks. Here we have developed and optimized CRISPR-Cas9 function in the lactic acid bacterium Lactobacillus reuteri ATCC PTA 6475. We established proof-of-concept showing that CRISPR-Cas9 selection combined with single-stranded DNA (ssDNA) recombineering is a realistic approach to identify at high efficiencies edited cells in a lactic acid bacterium. We show for three independent targets that subtle changes in the bacterial genome can be recovered at efficiencies ranging from 90 to 100%. By combining CRISPR-Cas9 and recombineering, we successfully applied codon saturation mutagenesis in the L. reuteri chromosome. Also, CRISPR-Cas9 selection is critical to identify low-efficiency events such as oligonucleotide-mediated chromosome deletions. This also means that CRISPR-Cas9 selection will allow identification of recombinant cells in bacteria with low recombineering efficiencies, eliminating the need for ssDNA recombineering optimization procedures. We envision that CRISPR-Cas genome editing has the potential to change the landscape of genome editing in lactic acid bacteria, and other Gram-positive bacteria. PMID:25074379

  11. Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

    Institute of Scientific and Technical Information of China (English)

    ZOU Hong-yun; MA Li; MENG Min-jie; YAO Xin-sheng; LIN Ying; WU Zhen-qiang; HE Xiao-wei; WANG Ju-fang; WANG Xiao-ning

    2007-01-01

    Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes (RAG) and RAG-mediated V(D)J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor (TCR) gene recombination.Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles (sjTRECs) were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences (RSSs) in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 (CDR3) size of the TCRVβ chain was examined by the TCR GeneScan technique.Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining (NHEJ) pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ25' and Dβ 23' sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation.Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire. However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V(D)J recombination and as a "special" model of the coexistence of TCR gene rearrangements and "negative" receptor revision.

  12. 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 protect

  13. Recombinant innovation and endogenous technological transitions

    NARCIS (Netherlands)

    K. Frenken; L.R. Izquierdo; P. Zeppini

    2012-01-01

    We propose a model of technological transitions based on two different types of innovations. Branching innovations refer to technological improvements along a particular path, while recombinant innovations represent fusions of multiple paths. Recombinant innovations create "short-cuts" which reduce

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

    Science.gov (United States)

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

    2014-01-01

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

  15. Sorbitol production using recombinant Zymomonas mobilis strain.

    Science.gov (United States)

    Liu, Changjun; Dong, Hongwei; Zhong, Jianjiang; Ryu, Dewey D Y; Bao, Jie

    2010-07-20

    A recombinant Zymomonas mobilis strain harboring the plasmid pHW20a-gfo for over-expression of glucose-fructose oxidoreductase (GFOR) was constructed. The specific activity of GFOR enzyme in the new recombinant strain was at least two folds greater than that in the wild strain. The maximum GFOR activity achieved in terms of the volumetric, and the cellular were 2.59 U ml(-1), and 0.70 U mg(-1), respectively, in the batch cultures. A significant improvement of the bioconversion process for the production of sorbitol and gluconic acid from glucose and fructose was made using divalent metal ions which drastically reduced the ethanol yield and significantly increased the yield of target product. Among several divalent metal ions evaluated, Zn(2+) was found to be most effective by inhibiting the Entner-Doudoroff pathway enzymes. The yield of the byproduct ethanol was reduced from 16.7 to 1.8 gl(-1) and the sorbitol yield was increased to almost 100% from 89%. The Ca(2+) enhanced the sorbitol yield and the formation of calcium gluconate salt made the separation of gluconate from the reaction system easier.

  16. Pesquisa de IgA contra o antígeno recombinante HspX de Mycobacterium tuberculosis no diagnóstico de tuberculose pleural Determination of levels of specific IgA to the HspX recombinant antigen of Mycobacterium tuberculosis for the diagnosis of pleural tuberculosis

    Directory of Open Access Journals (Sweden)

    Loanda Carvalho Sant' Ana Limongi

    2011-06-01

    Full Text Available OBJETIVO: Avaliar a acurácia da dosagem de IgA contra o antígeno recombinante HspX no líquido pleural e no soro de pacientes com derrame pleural para o diagnóstico de tuberculose pleural. MÉTODOS: Estudo transversal de teste diagnóstico. Amostras de líquido pleural e de soro de pacientes com derrame pleural e suspeita de tuberculose pleural foram avaliadas para a determinação da densidade óptica de IgA contra HspX utilizando ELISA indireto. RESULTADOS: Foram avaliadas amostras de líquido pleural e de soro de 132 pacientes: 97 com tuberculose pleural (grupo de estudo e 35 com derrame pleural por outras causas (grupo controle. A dosagem de IgA em líquido pleural foi capaz de discriminar os pacientes com tuberculose pleural dos controles. A sensibilidade do teste em líquido pleural e em soro foi, respectivamente, de 69% e 30%, enquanto a especificidade foi de 83% e 84%, respectivamente. CONCLUSÕES: Os dados sugerem o potencial da utilização deste teste no diagnóstico de tuberculose pleural. Estudos com amostras maiores e em diferentes cenários epidemiológicos são necessáriosOBJECTIVE: To evaluate the accuracy of determining specific IgA to HspX recombinant antigen in pleural fluid and serum samples for the diagnosis of pleural tuberculosis in patients with pleural effusion. METHODS: This was a cross-sectional study. Serum and pleural fluid samples of patients with pleural effusion and suspected of having pleural tuberculosis were tested with indirect ELISA in order to determine the optical density of specific IgA to HspX. RESULTS: We evaluated serum and pleural fluid samples from 132 patients: 97 diagnosed with pleural tuberculosis (study group and 35 diagnosed with pleural effusion due to other causes (control group. The determination of IgA in pleural fluid satisfactorily discriminated between pleural tuberculosis patients and control patients. The sensitivity of the test in pleural fluid and in serum was 69% and 30

  17. 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,

  18. 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,

  19. hotspot: software to support sperm-typing for investigating recombination hotspots

    Science.gov (United States)

    Dutheil, Julien Y.; Klötzl, Fabian; Haubold, Bernhard

    2016-01-01

    Motivation: In many organisms, including humans, recombination clusters within recombination hotspots. The standard method for de novo detection of recombinants at hotspots is sperm typing. This relies on allele-specific PCR at single nucleotide polymorphisms. Designing allele-specific primers by hand is time-consuming. We have therefore written a package to support hotspot detection and analysis. Results: hotspot consists of four programs: asp looks up SNPs and designs allele-specific primers; aso constructs allele-specific oligos for mapping recombinants; xov implements a maximum-likelihood method for estimating the crossover rate; six, finally, simulates typing data. Availability and Implementation: hotspot is written in C. Sources are freely available under the GNU General Public License from http://github.com/evolbioinf/hotspot/ Contact: haubold@evolbio.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27153632

  20. AID to overcome the limitations of genomic information by introducing somatic DNA alterations.

    Science.gov (United States)

    Honjo, Tasuku; Muramatsu, Masamichi; Nagaoka, Hitoshi; Kinoshita, Kazuo; Shinkura, Reiko

    2006-05-01

    The immune system has adopted somatic DNA alterations to overcome the limitations of the genomic information. Activation induced cytidine deaminase (AID) is an essential enzyme to regulate class switch recombination (CSR), somatic hypermutation (SHM) and gene conversion (GC) of the immunoglobulin gene. AID is known to be required for DNA cleavage of S regions in CSR and V regions in SHM. However, its molecular mechanism is a focus of extensive debate. RNA editing hypothesis postulates that AID edits yet unknown mRNA, to generate specific endonucleases for CSR and SHM. By contrast, DNA deamination hypothesis assumes that AID deaminates cytosine in DNA, followed by DNA cleavage by base excision repair enzymes. We summarize the basic knowledge for molecular mechanisms for CSR and SHM and then discuss the importance of AID not only in the immune regulation but also in the genome instability. PMID:25873751

  1. Expression and purification of recombinant polyomavirus VP2 protein and its interactions with polyomavirus proteins

    Science.gov (United States)

    Cai, X.; Chang, D.; Rottinghaus, S.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Recombinant polyomavirus VP2 protein was expressed in Escherichia coli (RK1448), using the recombinant expression system pFPYV2. Recombinant VP2 was purified to near homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroelution, and Extracti-Gel chromatography. Polyclonal serum to this protein which reacted specifically with recombinant VP2 as well as polyomavirus virion VP2 and VP3 on Western blots (immunoblots) was produced. Purified VP2 was used to establish an in vitro protein-protein interaction assay with polyomavirus structural proteins and purified recombinant VP1. Recombinant VP2 interacted with recombinant VP1, virion VP1, and the four virion histones. Recombinant VP1 coimmunoprecipitated with recombinant VP2 or truncated VP2 (delta C12VP2), which lacked the carboxy-terminal 12 amino acids. These experiments confirmed the interaction between VP1 and VP2 and revealed that the carboxyterminal 12 amino acids of VP2 and VP3 were not necessary for formation of this interaction. In vivo VP1-VP2 interaction study accomplished by cotransfection of COS-7 cells with VP2 and truncated VP1 (delta N11VP1) lacking the nuclear localization signal demonstrated that VP2 was capable of translocating delta N11VP1 into the nucleus. These studies suggest that complexes of VP1 and VP2 may be formed in the cytoplasm and cotransported to the nucleus for virion assembly to occur.

  2. 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...

  3. Sleep deprivation alters choice strategy without altering uncertainty or loss aversion preferences.

    Science.gov (United States)

    Mullette-Gillman, O'Dhaniel A; Kurnianingsih, Yoanna A; Liu, Jean C J

    2015-01-01

    Sleep deprivation alters decision making; however, it is unclear what specific cognitive processes are modified to drive altered choices. In this manuscript, we examined how one night of total sleep deprivation (TSD) alters economic decision making. We specifically examined changes in uncertainty preferences dissociably from changes in the strategy with which participants engage with presented choice information. With high test-retest reliability, we show that TSD does not alter uncertainty preferences or loss aversion. Rather, TSD alters the information the participants rely upon to make their choices. Utilizing a choice strategy metric which contrasts the influence of maximizing and satisficing information on choice behavior, we find that TSD alters the relative reliance on maximizing information and satisficing information, in the gains domain. This alteration is the result of participants both decreasing their reliance on cognitively-complex maximizing information and a concomitant increase in the use of readily-available satisficing information. TSD did not result in a decrease in overall information use in either domain. These results show that sleep deprivation alters decision making by altering the informational strategies that participants employ, without altering their preferences.

  4. Sleep Deprivation Alters Choice Strategy Without Altering Uncertainty or Loss Aversion Preferences

    Directory of Open Access Journals (Sweden)

    O'Dhaniel A Mullette-Gillman

    2015-10-01

    Full Text Available Sleep deprivation alters decision making; however, it is unclear what specific cognitive processes are modified to drive altered choices. In this manuscript, we examined how one night of total sleep deprivation (TSD alters economic decision making. We specifically examined changes in uncertainty preferences dissociably from changes in the strategy with which participants engage with presented choice information. With high test-retest reliability, we show that TSD does not alter uncertainty preferences or loss aversion. Rather, TSD alters the information the participants rely upon to make their choices. Utilizing a choice strategy metric which contrasts the influence of maximizing and satisficing information on choice behavior, we find that TSD alters the relative reliance on maximizing information and satisficing information, in the gains domain. This alteration is the result of participants both decreasing their reliance on cognitively-complex maximizing information and a concomitant increase in the use of readily-available satisficing information. TSD did not result in a decrease in overall information use in either domain. These results show that sleep deprivation alters decision making by altering the informational strategies that participants employ, without altering their preferences.

  5. Construction and characterization of recombinant Japanese encephalitis virus carrying brainspecific miRNA target sequences

    OpenAIRE

    Cao, Wen-Yuan; Ye, Qing; Li, Xiao-Feng; Wang, Hong-jiang; Zhu, Shun-Ya; Qin, E-De; Jiang, Zhen-You; Qin, Cheng-Feng

    2014-01-01

    Objective To construct the recombinant Japanese encephalitis virus ( JEV) carrying brain-specific miRNA targeting sequences. Methods The target sequences of brain-specific miR-124 and miR-125 were introduced into the infectious cDNA clone of JEV to generate recombinant plasmids based on reverse genetics technology. The recombinant plasmids were linearized with Xho Ⅰ and served as templates of transcription with SP6 RNA polymerase to generate infectious viral RNA. The RNA transcripts were then...

  6. Ocular infection of mice with an avirulent recombinant HSV-1 expressing IL-4 and an attenuated HSV-1 strain generates virulent recombinants in vivo

    OpenAIRE

    Mott, Kevin R; Wechsler, Steven L.; Ghiasi, Homayon

    2010-01-01

    Purpose To assess the relative impact of overexpression of interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN-γ) expressing recombinant herpes simplex virus type 1 (HSV-1) on altering immune responses in ocularly infected mice. Methods BALB/c mice were co-infected ocularly with avirulent HSV-1 strain KOS and avirulent recombinant HSV-1 expressing murine IL-4 (HSV-IL-4). Controls mice were co-infected with KOS + HSV-IL-2 or KOS + HSV-IFNγ. Following ocular infection, virus r...

  7. Oral alterations among chemical dependents

    Directory of Open Access Journals (Sweden)

    Estela Vanessa COLODEL

    2009-03-01

    Full Text Available Introduction: It has been daily observed a significant increase ofchemical dependent individuals, as well as the lack of depth on thisissue in the dentistry area. Nevertheless many times the dentalclinicians are the first professionals to diagnose possible alterations,which appear due to the consumption of tobacco, alcohol and other illicit drugs. Objectives: To make a literature review of oral alterations and to identify them on a specific group of persons, which are addicted to different types of drugs. Material and methods: The clinical history of the selected individuals was added to the answers of a questionnaire,comprising the data of the present research. Results: Besides other minor soft tissue alterations, a high prevalence of caries and periodontal diseases were found in the studied population. Conclusion: It was concluded that the role of the dental clinician is very important to the health rehabilitation of drug addicts, individuals with physical and mental disorders that need specific oral care, which sometimes is neglected.

  8. Mechanisms of sister chromatid recombination

    International Nuclear Information System (INIS)

    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-G2 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.)

  9. Structural characterization of recombinant therapeutic proteins by circular dichroism.

    Science.gov (United States)

    Bertucci, Carlo; Pistolozzi, Marco; De Simone, Angela

    2011-10-01

    Most of the protein therapeutics are now produced by recombinant DNA technology. The advantages of recombinant proteins are related to their higher specificity and to their safety as exposure to animal or human diseases. However, several problems are still present in development of recombinant proteins as therapeutics, such as low bioavailability, short serum half-life, and immune response. Their successful application hinges on the protein stereochemical stability, and on the folding and the tendency to aggregate induced by purification steps and storage. All these aspects determine the failure of many potential protein therapies, and limitations in the development of the formulation. The application of multiple analytical techniques is important in order to obtain a detailed product profile and to understand how manufacturing can influence product structure and activity. Surely the protein conformation is a key aspect to be assessed, because a specific conformation is often essential for the biological function of the protein. Thus, there is a growing need to perform structural studies under the conditions in which the proteins operate, and to monitor the structural changes of the protein. Circular dichroism has been increasingly recognised as a valuable and reliable technique to get this information. In particular, examples will be here reported on the use of circular dichroism spectroscopy in the structural characterization of free and formulated recombinant proteins, looking at the prediction of the secondary structure, propensity to conformational changes, stability, and tendency to aggregate.

  10. Recombinant erythropoietin in clinical practice

    OpenAIRE

    Ng, T; Marx, G.; Littlewood, T; Macdougall, I

    2003-01-01

    The introduction of recombinant human erythropoietin (RHuEPO) has revolutionised the treatment of patients with anaemia of chronic renal disease. Clinical studies have demonstrated that RHuEPO is also useful in various non-uraemic conditions including haematological and oncological disorders, prematurity, HIV infection, and perioperative therapies. Besides highlighting both the historical and functional aspects of RHuEPO, this review discusses the applications of RHuEPO in clinical practice a...

  11. Recombinant antibodies and tumor targeting

    OpenAIRE

    Sheikholvaezin, Ali

    2006-01-01

    Different antibody derived constructs are rapidly advancing as putative tools for treatment of malignant diseases. Antibody engineering has added significant new technologies to modify size, affinities, solubility, stability and biodistribution properties for immunoconjugates. In the present thesis, the aim was to increase our knowledge on how new recombinant antibodies could be tailored to optimize localization to experimental tumors in mice. One hybridoma, producing the monoclonal antibody ...

  12. Alterations in p53-specific T cells and other lymphocyte subsets in breast cancer patients during vaccination with p53-peptide loaded dendritic cells and low-dose interleukin-2

    DEFF Research Database (Denmark)

    Svane, Inge Marie; Pedersen, Anders E; Nikolajsen, Kirsten;

    2008-01-01

    We have previously established a cancer vaccine using autologous DCs, generated by in vitro stimulation with IL-4 and GM-CSF, and pulsed with six HLA-A*0201 binding wild-type p53 derived peptides. This vaccine was used in combination with low-dose interleukin-2 in a recently published clinical...... Phase II trial where 26 HLA-A2+ patients with progressive late-stage metastatic breast cancer (BC) were included. Almost 1/3rd of the patients obtained stable disease or minor regression during treatment with a positive correlation to tumour over-expression of p53. In the present study, we performed...... a comprehensive analysis of the effector stage of the p53-specific CD8+ T cells by the use of Dextramer Technology and multicolour FACS. Pre- and post-treatment blood samples from eight BC patients were analysed. Independent of clinical outcome p53-specific T cells were phenotypic distinctly antigen experienced...

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

    Science.gov (United States)

    Kaiser, R; Rauer, S

    1999-01-01

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

  14. 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...

  15. Consistency and stability of recombinant fermentations.

    Science.gov (United States)

    Wiebe, M E; Builder, S E

    1994-01-01

    Production of proteins of consistent quality in heterologous, genetically-engineered expression systems is dependent upon identifying the manufacturing process parameters which have an impact on product structure, function, or purity, validating acceptable ranges for these variables, and performing the manufacturing process as specified. One of the factors which may affect product consistency is genetic instability of the primary product sequence, as well as instability of genes which code for proteins responsible for post-translational modification of the product. Approaches have been developed for mammalian expression systems to assure that product quality is not changing through mechanisms of genetic instability. Sensitive protein analytical methods, particularly peptide mapping, are used to evaluate product structure directly, and are more sensitive in detecting genetic instability than is direct genetic analysis by nucleotide sequencing of the recombinant gene or mRNA. These methods are being employed to demonstrate that the manufacturing process consistently yields a product of defined structure from cells cultured through the range of cell ages used in the manufacturing process and well beyond the maximum cell age defined for the process. The combination of well designed validation studies which demonstrate consistent product quality as a function of cell age, and rigorous quality control of every product lot by sensitive protein analytical methods provide the necessary assurance that product structure is not being altered through mechanisms of mutation and selection.

  16. Specific anti-tumor immune responses of dendritic cells pulsed with recombinant human rhHSP70 and freeze-thaw cellular lysates derived from breast cancer%rhHSP70联合冻融抗原修饰树突状细胞诱导的抗乳腺癌作用*

    Institute of Scientific and Technical Information of China (English)

    李斌; 陈鹏; 郑建云

    2013-01-01

      目的:利用rhHSP70联合树突状细胞递呈肿瘤抗原的特性提高细胞毒T淋巴细胞(CTLs)对乳腺癌细胞的杀伤活性。方法:外周血单个核细胞体外经GM-CSF和IL-4诱导产生树突状细胞,负载冻融抗原肽的同时加入新型热休克蛋白(rhHSP70),不同分组分别诱导自体CTLs产生。ELISA测定CTLs杀伤活性和细胞因子的分泌。结果:冻融抗原肽致敏的DCs促进CTLs增殖,上调CTLs中CD3+和CD8+T细胞群及Th1型细胞因子的分泌;体外实验中具有对人乳腺癌细胞MCF-7的杀伤活性,在加入rhHSP70后效果更加明显,并能显著增强CTLs对肿瘤细胞的杀伤率。结论:rhHSP70联合肝癌冻融抗原修饰DCs,能够促进DCs的成熟,增强DCs刺激淋巴细胞增殖的能力,诱导的CTLs在体外对乳腺癌细胞能产生高效杀伤力。rhHSP70增强DCs抗肿瘤能力的机制可能与其促进DCs成熟有关。%Objective:This work aims to use the characteristics of dendritic cells (DCs) pulsed with recombinant human HSP70, which can present and process tumor antigens, to enhance the killing activity of cytotoxic t lymphocytes (CTLs) against breast neoplasms. Methods:Autologous DCs were isolated from peripheral blood mononuclear cells and then stimulated in vitro with granulocyte macrophage-colony stimulating factor and interleukin-4. The DCs were loaded with A549 tumor cell freeze-thaw lysate, and rhHSP70 was added as an immune adjuvant. The specific groups were subjected to tumor-specific cytotoxic assay, enzyme-linked immunosorbent assay, and fluores-cence-activated cell sorting. Results:DCs pulsed with A549 tumor cell lysate enhanced the growth expansion of CTLs, upregulated CD40 and CD80 populations in CTLs, and augmented Th1 cytokines. In addition, the cytotoxicity of specific CTLs against A549 was highly enhanced. The above indications became more obvious after the addition of rhHSP70. Conclusion:DCs pulsed with freeze-thaw cell

  17. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

    OpenAIRE

    Firmenich, A A; Elias-Arnanz, M; Berg, P

    1995-01-01

    To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endon...

  18. Genetic Confirmation of Mungbean (Vigna radiata) and Mashbean (Vigna mungo) Interspecific Recombinants using Molecular Markers

    Science.gov (United States)

    Abbas, Ghulam; Hameed, Amjad; Rizwan, Muhammad; Ahsan, Muhammad; Asghar, Muhammad J.; Iqbal, Nayyer

    2015-01-01

    Molecular confirmation of interspecific recombinants is essential to overcome the issues like self-pollination, environmental influence, and inadequacy of morphological characteristics during interspecific hybridization. The present study was conducted for genetic confirmation of mungbean (female) and mashbean (male) interspecific crosses using molecular markers. Initially, polymorphic random amplified polymorphic DNA (RAPD), universal rice primers (URP), and simple sequence repeats (SSR) markers differentiating parent genotypes were identified. Recombination in hybrids was confirmed using these polymorphic DNA markers. The NM 2006 × Mash 88 was most successful interspecific cross. Most of true recombinants confirmed by molecular markers were from this cross combination. SSR markers were efficient in detecting genetic variability and recombination with reference to specific chromosomes and particular loci. SSR (RIS) and RAPD identified variability dispersed throughout the genome. In conclusion, DNA based marker assisted selection (MAS) efficiently confirmed the interspecific recombinants. The results provided evidence that MAS can enhance the authenticity of selection in mungbean improvement program. PMID:26697053

  19. Genetic confirmation of mungbean (Vigna radiata and mashbean (Vigna mungo interspecific recombinants using molecular markers

    Directory of Open Access Journals (Sweden)

    Ghulam eAbbas

    2015-12-01

    Full Text Available The present study was conducted with the aim to investigate recombination between mungbean (female and mashbean (male interspecific crosses using molecular markers i.e., URP (Universal Rice Primers, RAPD (Random Amplified Polymorphic DNA and SSR (Simple Sequence Repeats. As a first step parental screening was performed and polymorphic markers differentiating parent genotypes were identified. Recombinations were then confirmed through polymorphic DNA markers in many of the hybrids. The NM 2006 × Mash 88 was found to be most successful interspecific cross as many of true recombinants, confirmed by molecular markers, belonged to this cross combination. The SSR markers were more efficient in detecting genetic variability and recombinations with reference to specific chromosomes and particular loci, while SSR (RIS and RAPD identified variability dispersed throughout the genome. The DNA based marker assisted approach provided evidence for genetic confirmation of mungbean and mashbean interspecific recombinants and escalated the authenticity of selection in mungbean improvement programme.

  20. Genetic Confirmation of Mungbean (Vigna radiata) and Mashbean (Vigna mungo) Interspecific Recombinants using Molecular Markers.

    Science.gov (United States)

    Abbas, Ghulam; Hameed, Amjad; Rizwan, Muhammad; Ahsan, Muhammad; Asghar, Muhammad J; Iqbal, Nayyer

    2015-01-01

    Molecular confirmation of interspecific recombinants is essential to overcome the issues like self-pollination, environmental influence, and inadequacy of morphological characteristics during interspecific hybridization. The present study was conducted for genetic confirmation of mungbean (female) and mashbean (male) interspecific crosses using molecular markers. Initially, polymorphic random amplified polymorphic DNA (RAPD), universal rice primers (URP), and simple sequence repeats (SSR) markers differentiating parent genotypes were identified. Recombination in hybrids was confirmed using these polymorphic DNA markers. The NM 2006 × Mash 88 was most successful interspecific cross. Most of true recombinants confirmed by molecular markers were from this cross combination. SSR markers were efficient in detecting genetic variability and recombination with reference to specific chromosomes and particular loci. SSR (RIS) and RAPD identified variability dispersed throughout the genome. In conclusion, DNA based marker assisted selection (MAS) efficiently confirmed the interspecific recombinants. The results provided evidence that MAS can enhance the authenticity of selection in mungbean improvement program.

  1. Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization

    Energy Technology Data Exchange (ETDEWEB)

    Batenburg, A.M.; Demel, R.A.; Verkleij, A.J.; de Kruijff, B.

    1988-07-26

    In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary /sup 31/P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure.

  2. Penetration of the signal sequence of Escherichia coli PhoE protein into phospholipid model membranes leads to lipid-specific changes in signal peptide structure and alterations of lipid organization

    International Nuclear Information System (INIS)

    In order to obtain more insight in the initial steps of the process of protein translocation across membranes, biophysical investigations were undertaken on the lipid specificity and structural consequences of penetration of the PhoE signal peptide into lipid model membranes and on the conformation of the signal peptide adopted upon interaction with the lipids. When the monolayer technique and differential scanning calorimetry are used, a stronger penetration is observed for negatively charged lipids, significantly influenced by the physical state of the lipid but not by temperature or acyl chain unsaturation as such. Although the interaction is principally electrostatic, as indicated also by the strong penetration of N-terminal fragments into negatively charged lipid monolayers, the effect of ionic strength suggests an additional hydrophobic component. Most interestingly with regard to the mechanism of protein translocation, the molecular area of the peptide in the monolayer also shows lipid specificity: the area in the presence of PC is consistent with a looped helical orientation, whereas in the presence of cardiolipin a time-dependent conformational change is observed, most likely leading from a looped to a stretched orientation with the N-terminus directed toward the water. This is in line also with the determined peptide-lipid stoichiometry. Preliminary 31P NMR and electron microscopy data on the interaction with lipid bilayer systems indicate loss of bilayer structure

  3. Recombinant phage probes for Listeria monocytogenes

    International Nuclear Information System (INIS)

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products

  4. Recombinant phage probes for Listeria monocytogenes

    Energy Technology Data Exchange (ETDEWEB)

    Carnazza, S; Gioffre, G; Felici, F; Guglielmino, S [Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Messina (Italy)

    2007-10-03

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 10{sup 4} cells ml{sup -1}. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  5. Recombinant phage probes for Listeria monocytogenes

    Science.gov (United States)

    Carnazza, S.; Gioffrè, G.; Felici, F.; Guglielmino, S.

    2007-10-01

    Monitoring of food and environmental samples for biological threats, such as Listeria monocytogenes, requires probes that specifically bind biological agents and ensure their immediate and efficient detection. There is a need for robust and inexpensive affinity probes as an alternative to antibodies. These probes may be recruited from random peptide libraries displayed on filamentous phage. In this study, we selected from two phage peptide libraries phage clones displaying peptides capable of specific and strong binding to the L. monocytogenes cell surface. The ability of isolated phage clones to interact specifically with L. monocytogenes was demonstrated using enzyme-linked immunosorbent assay (ELISA) and confirmed by co-precipitation assay. We also assessed the sensitivity of phage-bacteria binding by PCR on phage-captured Listeria cells, which could be detected at a concentration of 104 cells ml-1. In addition, as proof-of-concept, we tested the possibility of immobilizing the affinity-selected phages to a putative biosensor surface. The quality of phage deposition was monitored by ELISA and fluorescent microscopy. Phage-bacterial binding was confirmed by high power optical phase contrast microscopy. Overall, the results of this work validate the concept of affinity-selected recombinant filamentous phages as probes for detecting and monitoring bacterial agents under any conditions that warrant their recognition, including in food products.

  6. Meiotic recombination, synapsis, meiotic inactivation and sperm aneuploidy in a chromosome 1 inversion carrier.

    Science.gov (United States)

    Kirkpatrick, Gordon; Chow, Victor; Ma, Sai

    2012-01-01

    Disrupted meiotic behaviour of inversion carriers may be responsible for suboptimal sperm parameters in these carriers. This study investigated meiotic recombination, synapsis, transcriptional silencing and chromosome segregation effects in a pericentric inv(1) carrier. Recombination (MLH1), synapsis (SYCP1, SYCP3) and transcriptional inactivation (γH2AX, BRCA1) were examined by fluorescence immunostaining. Chromosome specific rates of recombination were determined by fluorescence in-situ hybridization. Furthermore, testicular sperm was examined for aneuploidy and segregation of the inv(1). Our findings showed that global recombination rates were similar to controls. Recombination on the inv(1) and the sex chromosomes were reduced. The inv(1) associated with the XY body in 43.4% of cells, in which XY recombination was disproportionately absent, and 94.3% of cells displayed asynapsed regions which displayed meiotic silencing regardless of their association with the XY body. Furthermore, a low frequency of chromosomal imbalance was observed in spermatozoa (3.4%). Our results suggest that certain inversion carriers may display unimpaired global recombination and impaired recombination on the involved and the sex chromosomes during meiosis. Asynapsis or inversion-loop formation in the inverted region may be responsible for impaired spermatogenesis and may prevent sperm-chromosome imbalance.

  7. [Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

    Science.gov (United States)

    Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

    2016-01-01

    We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

  8. Buccal alterations in diabetes mellitus

    Directory of Open Access Journals (Sweden)

    Negrato Carlos

    2010-01-01

    Full Text Available Abstract Long standing hyperglycaemia besides damaging the kidneys, eyes, nerves, blood vessels, heart, can also impair the function of the salivary glands leading to a reduction in the salivary flow. When salivary flow decreases, as a consequence of an acute hyperglycaemia, many buccal or oral alterations can occur such as: a increased concentration of mucin and glucose; b impaired production and/or action of many antimicrobial factors; c absence of a metalloprotein called gustin, that contains zinc and is responsible for the constant maturation of taste papillae; d bad taste; e oral candidiasis f increased cells exfoliation after contact, because of poor lubrication; g increased proliferation of pathogenic microorganisms; h coated tongue; i halitosis; and many others may occur as a consequence of chronic hyperglycaemia: a tongue alterations, generally a burning mouth; b periodontal disease; c white spots due to demineralization in the teeth; d caries; e delayed healing of wounds; f greater tendency to infections; g lichen planus; h mucosa ulcerations. Buccal alterations found in diabetic patients, although not specific of this disease, have its incidence and progression increased when an inadequate glycaemic control is present.

  9. CRMAGE: CRISPR Optimized MAGE Recombineering

    DEFF Research Database (Denmark)

    Ronda, Carlotta; Pedersen, Lasse Ebdrup; Sommer, Morten Otto Alexander;

    2016-01-01

    A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli...... that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red...

  10. Studies of recombinant forms of Aleuria aurantia lectin

    OpenAIRE

    Olausson, Johan

    2009-01-01

    The presented work describes construction and analysis of recombinantly produced forms of Aleuria aurantia lectin (AAL). The binding properties of the produced AAL forms were studied using techniques such as tryptophan fluorescence, hemagglutination analysis, ELISA and surface plasmon resonance analysis. Lectins are proteins that are ubiquitous in nature with the ability to bind specifically to different types of carbohydrates. The physiological function of different lectins is not always kno...

  11. A multi-Fc-species system for recombinant antibody production

    OpenAIRE

    Nizak Clément; Vielemeyer Ole; El Marjou Ahmed; Moutel Sandrine; Benaroch Philippe; Dübel Stefan; Perez Franck

    2009-01-01

    Abstract Background Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural anti...

  12. Genetically engineered poxviruses for recombinant gene expression, vaccination, and safety.

    OpenAIRE

    B. Moss

    1996-01-01

    Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure-function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of...

  13. A recombinant vaccinia virus expressing human carcinoembryonic antigen (CEA).

    Science.gov (United States)

    Kaufman, H; Schlom, J; Kantor, J

    1991-07-30

    Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein expressed on most gastrointestinal carcinomas. A 2.4-kb cDNA clone, containing the complete coding sequence, was isolated from a human colon tumor cell library and inserted into a vaccinia virus genome. This newly developed construct was characterized by Southern blotting, DNA hybridization studies, and polymerase chain reaction analysis. The CEA gene was stably integrated into the vaccinia virus thymidine kinase gene. The recombinant was efficiently replicated upon serial passages in cell cultures and in animals. The recombinant virus expresses on the surface of infected cells a protein product recognized by a monoclonal antibody (COL-I) directed against CEA. Immunization of mice with the vaccinia construct elicited a humoral immune response against CEA. Pilot studies also showed that administration of the recombinant CEA vaccinia construct was able to greatly reduce the growth in mice of a syngeneic murine colon adenocarcinoma which had been transduced with the human CEA gene. The use of this new recombinant CEA vaccinia construct may thus provide an approach in the specific active immunotherapy of human GI cancer and other CEA expressing carcinoma types.

  14. Studies of the cytosolic thymidine kinase in human cells and comparison to the recombinantly expressed enzyme

    DEFF Research Database (Denmark)

    Kock Jensen, Helle

    Thymidine kinase (TK) is a key enzyme in the salvage pathway of the nucleoside metabolism catalyzing the first phosphorylation step in TTP synthesis. Human cytosolic TK (TKl) is highly cell cycle regulated. TKl is regulated on many different levels of expression and isoforms with altered enzymatic...... identical but further investigations showed some interesting differences. Recombinant TKl is about 10 fold more sensitive towards TTP as inhibitor. Furthermore the effect of removal of ATP from the native TKl on the enzyme kinetics and native molecular weight was not found for recombinant TKl. Native TKl...

  15. Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

    OpenAIRE

    Chen-Ji Huang; Hwei-Ling Peng; Chih-Yu Cheng

    2011-01-01

    In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M ure...

  16. Comparison of 2 synthetically generated recombinant prions

    OpenAIRE

    Zhang, Yi; Wang, Fei; Wang, Xinhe; Zhang, Zhihong; Xu, Yuanyuan; Yu, Guohua; Yuan, Chonggang; Ma, Jiyan

    2014-01-01

    Prion is a protein-conformation-based infectious agent causing fatal neurodegenerative diseases in humans and animals. Our previous studies revealed that in the presence of cofactors, infectious prions can be synthetically generated in vitro with bacterially expressed recombinant prion protein (PrP). Once initiated, the recombinant prion is able to propagate indefinitely via serial protein misfolding cyclic amplification (sPMCA). In this study, we compared 2 separately initiated recombinant p...

  17. How well do we understand cosmological recombination?

    OpenAIRE

    Wong, Wan Yan; Moss, Adam; Scott, Douglas

    2007-01-01

    The major theoretical limitation for extracting cosmological parameters from the CMB sky lies in the precision with which we can calculate the cosmological recombination process. Uncertainty in the details of hydrogen and helium recombination could effectively increase the errors or bias the values of the cosmological parameters derived from the Planck satellite, for example. Here we modify the cosmological recombination code RECFAST by introducing one more parameter to reproduce the recent n...

  18. Role of ubiquitination in meiotic recombination repair

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Programmed and unprogrammed double-strand breaks (DSBs) often arise from such physiological requirements as meiotic recombination, and exogenous insults, such as ionizing radiation (IR). Due to deleterious impacts on genome stability, DSBs must be appropriately processed and repaired in a regulatory manner. Recent investigations have indicated that ubiquitination is a critical factor in DNA damage response and meiotic recombination repair. This review summarizes the effects of proteins and complexes associated with ubiquitination with regard to homologous recombination (HR)-dependent DSB repair.

  19. Rapid purification of recombinant histones.

    Directory of Open Access Journals (Sweden)

    Henrike Klinker

    Full Text Available The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.

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

    DEFF Research Database (Denmark)

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas Salhøj;

    2014-01-01

    -erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers...... of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens....

  1. A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

    OpenAIRE

    Peter J Bredenbeek; Molenkamp, Richard; Spaan, Willy J. M.; Deubel, Vincent; Marianneau, Phillippe; Salvato, Maria S.; Moshkoff, Dmitry; Zapata, Juan; Tikhonov, Ilia; Patterson, Jean; Carrion, Ricardo; Ticer, Anysha; Brasky, Kathleen; Lukashevich, Igor S.

    2006-01-01

    The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa F...

  2. Human Insulin from Recombinant DNA Technology

    Science.gov (United States)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  3. Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea.

    Science.gov (United States)

    Tonin, Fabio; Rosini, Elena; Piubelli, Luciano; Sanchez-Amat, Antonio; Pollegioni, Loredano

    2016-07-01

    Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications. PMID:27050199

  4. Attention Alters Perceived Attractiveness.

    Science.gov (United States)

    Störmer, Viola S; Alvarez, George A

    2016-04-01

    Can attention alter the impression of a face? Previous studies showed that attention modulates the appearance of lower-level visual features. For instance, attention can make a simple stimulus appear to have higher contrast than it actually does. We tested whether attention can also alter the perception of a higher-order property-namely, facial attractiveness. We asked participants to judge the relative attractiveness of two faces after summoning their attention to one of the faces using a briefly presented visual cue. Across trials, participants judged the attended face to be more attractive than the same face when it was unattended. This effect was not due to decision or response biases, but rather was due to changes in perceptual processing of the faces. These results show that attention alters perceived facial attractiveness, and broadly demonstrate that attention can influence higher-level perception and may affect people's initial impressions of one another. PMID:26966228

  5. Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning

    OpenAIRE

    Leem, Sun-Hee; Noskov, Vladimir N.; Park, Jung-Eun; Kim, Seung Il; Larionov, Vladimir; Kouprina, Natalay

    2003-01-01

    Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5′ and 3′ gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ∼10-f...

  6. SERUM ANTIBODIES TO WHOLE-CELL AND RECOMBINANT ANTIGENS OF BORRELIA BURGDORFERI IN COTTONTAIL RABBITS

    OpenAIRE

    Magnarelli, Louis A.; Norris, Steven J; Fikrig, Erol

    2012-01-01

    Archived serum samples, from 95 eastern cottontail rabbits (Sylvilagus floridanus) captured in New York, New York, USA and Millbrook, New York, USA, during 1985–86, were analyzed in solid-phase enzyme-linked immunosorbent assays (ELISA) for total and class-specific immunoglobulin (Ig) M antibodies to whole-cell or recombinant antigens of Borrelia burgdorferi sensu stricto. Using a polyvalent conjugate, rabbit sera contained antibodies to whole-cell and recombinant antigens (protein [p]35, p37...

  7. Recombinant Hepatitis A Virus Antigen: Improved Production and Utility in Diagnostic Immunoassays

    OpenAIRE

    LaBrecque, F. D.; LaBrecque, D. R.; Klinzman, D.; Perlman, S; Cederna, J. B.; Winokur, P. L.; Han, J.-Q.; Stapleton, J. T.

    1998-01-01

    Hepatitis A virus (HAV) immunoassays use cell culture-derived HAV antigen to detect HAV-specific antibodies. The current method of production of HAV antigen in tissue culture is time-consuming and expensive. We previously expressed the HAV open reading frame in recombinant vaccinia viruses (rV-ORF). The recombinant HAV polyprotein was accurately processed and was assembled into subviral particles. These particles were bound by HAV-neutralizing antibodies and were able to elicit antibodies whi...

  8. The production of recombinant single chain antibody fragments for the detection of illicit drug residues

    OpenAIRE

    Brennan, Joanne

    2005-01-01

    Recombinant antibodies represent a more sensitive and specific detection tool for immunoanalysis. The research carried out for this thesis describes the production of genetically-derived single chain antibody fragments to detect illicit drugs. A variety of novel recombinant antibody fragments against morphine-3-glucuronide, a metabolite of heroin has been produced. A monomeric, dimeric and enzyme-labelled scFv were characterised with respect to their binding abilities and cross reactiviti...

  9. Construction, transfection and production of recombinant vigilin in mammalian expression system

    OpenAIRE

    Sayed Kamel Areida

    2006-01-01

    Vigilin is an abundant, highly conserved, ubiquitous protein containing 15 related, but non-identical, K-homolous nucleic acid binding domains. The construction, transfection and production of recombinant vigilin in mammalian expression system were investigated. The whole length of vigilin was amplified by polymerase chain reaction (PCR) and ligated to pCEP-PU vector. The recombinant construct pCEP-PU with vigilin was produced and transfected into Human embryonic kidney cells in a specific cu...

  10. Multiple genomic recombination events in the evolution of saffold cardiovirus.

    Directory of Open Access Journals (Sweden)

    Lili Ren

    Full Text Available BACKGROUND: Saffold cardiovirus (SAFV is a new human cardiovirus with 11 identified genotypes. Little is known about the natural history and pathogenicity of SAFVs. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced the genome of five SAFV-1 strains which were identified from fecal samples taken from children with viral diarrhea in Beijing, China between March 2006 and November 2007, and analyzed the phylogenetic and phylodynamic properties of SAFVs using the genome sequences of every known SAFV genotypes. We identified multiple recombination events in our SAFV-1 strains, specifically recombination between SAFV-2, -3, -4, -9, -10 and the prototype SAFV-1 strain in the VP4 region and recombination between SAFV-4, -6, -8, -10, -11 and prototype SAFV-1 in the VP1/2A region. Notably, recombination in the structural gene VP4 is a rare event in Cardiovirus. The ratio of nonsynonymous substitutions to synonymous substitutions indicates a purifying selection of the SAFV genome. Phylogenetic and molecular clock analysis indicates the existence of at least two subclades of SAFV-1 with different origins. Subclade 1 includes two strains isolated from Pakistan, whereas subclade 2 includes the prototype strain and strains isolated in China, Pakistan, and Afghanistan. The most recent common ancestor of all SAFV genotypes dates to the 1710s, and SAFV-1, -2, and -3 to the 1940s, 1950s, and 1960s, respectively. No obvious relationship between variation and pathogenicity exists in the critical domains of the CD and EF loops of viral capsid proteins or the multi-functional proteins L based on amino acid sequence identity comparison between SAFV genotypes. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that intertypic recombination plays an important role in the diversity of SAFVs, highlighting the diversity of the five strains with the previously described SAFV-1 strains.

  11. 添加重组人生长激素的营养支持对烧伤脓毒症危重病人营养状况的影响%Altered nutrition state in the severe burn sepsis patients with adjuvant recombinant human growth hormone nutritional support therapy

    Institute of Scientific and Technical Information of China (English)

    吴祖煌; 宋斌; 黄永新; 陈建崇; 陈国养

    2011-01-01

    Objective: To observe the nutrition state in the severely burn sepsis patients with adjuvant recombinant human growth hormone (rhGH) nutritional support therapy. Methods: 42 cases were randomized into three groups. All the three groups had been supplied with nutrition according to the need of patients. Besides parenteral nutrition, rhGH was given subcutaneously in the dosage of 0.2 U/( kg · d) and 0.4 U/( kg · d) in two treatment groups respectively. The plasma concentration levels of prealbumin ( PA ), procalcitonin (PCT), tunmor necrosis factor-α ( TNF-α ), fasting blood glucose (FBG),glycated hemoglob (HbAIC), nitrogen balance (NB) were measured after burn injury. Results: ①The serum PA and NB were decreased three groups ( P < 0.01 ~ 0.05 ). ②Length of hospital stay and the healing time of donor site and burn wound were significantly shortened in two groups of rhGH compared with control group( P <0.05 ). ③There was no significant difference of HbAIC between treatment group Ⅰ and treatment group Ⅱ. ④The serum levels of TNF-α, FBG, PCT in treatment group Ⅰ and treatment group Ⅱ were markedly lower than those of control group ( P < 0.01 ~ 0.05 ). Conclusion: The nutrition support therapy with adjuvant rhGH is beneficial to the nutrition state of the patients with burn sepsis. It is safe for severe burn sepsis patients who treated with rhGH at the dose of 0.2 ~ 0.4 U/( kg · d).%目的:观察添加重组人生长激素(rhGH)的营养支持对烧伤脓毒症危重病人营养状况的影响,探讨rhGH在烧伤脓毒症治疗中的作用.方法:42例烧伤脓毒症危重病人[烧伤面积(TBSA)≥30%]随机分为治疗Ⅰ组、治疗Ⅱ组和对照组,每组各14例.三组病人除实施相同的脓毒症综合治疗方案和标准营养支持外,其中两组皮下注射thGH为0.2和0.4 U/(kg·d);对照组皮下注射等量的等渗盐水,每天1次,连续10 d.在给药前后不同时相点取血测定并对比分析三组间血浆

  12. Meiotic recombination initiated by a double-strand break in rad50{Delta} yeast cells otherwise unable to initiate meiotic recombination

    Energy Technology Data Exchange (ETDEWEB)

    Malkova, A.; Haber, J.E. [Brandeis Univ., Waltham, MA (United States); Dawson, D. [Tufts Univ., Boston, MA (United States)] [and others

    1996-06-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand breaks (DSBs). We have developed a system to compare the properties of meiotic DSBs with those created by the site-specific HO endonuclease. HO endonuclease was expressed under the control of the meiotic-specific SPO13 promoter, creating a DSB at a single site on one of yeast`s 16 chromosomes. In Rad{sup +} strains the times of appearance of the HO-induced DSBs and of subsequent recombinants are coincident with those induced by normal meiotic DSBs. Physical monitoring of DNA showed that SPO13::HO induced gene conversions both in Rad{sup +} and in rad50{Delta} cells that cannot initiate normal meiotic DSBs. We find that the RAD50 gene is important, but not essential, for recombination even after a DSB has been created in a meiotic cell. In rad50{Delta} cells, some DSBs are not repaired until a broken chromosome has been packaged into a spore and is subsequently germinated. This suggests that a broken chromosome does not signal an arrest of progression through meiosis. The recombination defect in rad50{Delta} diploids is not, however, meiotic specific, as mitotic rad50 diploids, experiencing an HO-induced DSB, exhibit similar departures from wild-type recombination. 57 refs., 5 figs., 3 tabs.

  13. Detection of TGEV Antibody by Enzyme-Linked Immunosorbent Assay Using Recombinant Nucleocapsid Proteins

    Institute of Scientific and Technical Information of China (English)

    YU Li-yun; HOU Xi-lin

    2005-01-01

    An enzyme linked immunosorbent assays (ELISA) based on recombinant nucleocapsid (N) protein generated in Escherichia coli was evaluated for its sensitivity and specificity for diagnosis of transmissible gastroenteritis virus (TGEV) infection.The N gene encoding the N protein was cloned and expressed as a fusion protein with His tag protein in E. coli. The recombinant N protein migrated at 42 kDa and reacted with His6 tag specific monoclonal antibody by immunoblotting.Recombinant N protein ELISA (rnELISA) demonstrated 97.5% specificity among 80 TGEV-free individuals, and 97.3%sensitivity ranging among 110 clinical samples with TGEV. Taken together, these results indicated that nucleocapsid may be a useful antigen for the sera-diagnosis of TGEV and it was also suggested that the ELISA is a highly sensitive and specific test for detecting antibodies against TGEV.

  14. Assembly of recombinant Israeli Acute Paralysis Virus capsids.

    Directory of Open Access Journals (Sweden)

    Junyuan Ren

    Full Text Available The dicistrovirus Israeli Acute Paralysis Virus (IAPV has been implicated in the worldwide decline of honey bees. Studies of IAPV and many other bee viruses in pure culture are restricted by available isolates and permissive cell culture. Here we show that coupling the IAPV major structural precursor protein ORF2 to its cognate 3C-like processing enzyme results in processing of the precursor to the individual structural proteins in a number of insect cell lines following expression by a recombinant baculovirus. The efficiency of expression is influenced by the level of IAPV 3C protein and moderation of its activity is required for optimal expression. The mature IAPV structural proteins assembled into empty capsids that migrated as particles on sucrose velocity gradients and showed typical dicistrovirus like morphology when examined by electron microscopy. Monoclonal antibodies raised to recombinant capsids were configured into a diagnostic test specific for the presence of IAPV. Recombinant capsids for each of the many bee viruses within the picornavirus family may provide virus specific reagents for the on-going investigation of the causes of honeybee loss.

  15. Titania Photocatalysis beyond Recombination: A Critical Review

    Directory of Open Access Journals (Sweden)

    Bunsho Ohtani

    2013-11-01

    Full Text Available This short review paper shows the significance of recombination of a photoexcited electron and a hole in conduction and valence bands, respectively, of a titania photocatalyst, since recombination has not yet been fully understood and has not been evaluated adequately during the past several decades of research on heterogeneous photocatalysis.