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Sample records for antibody expressing pea

  1. Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens

    Directory of Open Access Journals (Sweden)

    Macek Jeanette

    2009-09-01

    Full Text Available Abstract Background Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Results Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability. Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. Conclusion The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

  2. Antibody expressing pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens.

    Science.gov (United States)

    Zimmermann, Jana; Saalbach, Isolde; Jahn, Doreen; Giersberg, Martin; Haehnel, Sigrun; Wedel, Julia; Macek, Jeanette; Zoufal, Karen; Glünder, Gerhard; Falkenburg, Dieter; Kipriyanov, Sergey M

    2009-09-11

    Coccidiosis caused by protozoans of genus Eimeria is a chicken parasitic disease of great economical importance. Conventional disease control strategies depend on vaccination and prophylactic use of anticoccidial drugs. Alternative solution to prevent and treat coccidiosis could be provided by passive immunization using orally delivered neutralizing antibodies. We investigated the possibility to mitigate the parasitic infection by feeding poultry with antibody expressing transgenic crop seeds. Using the phage display antibody library, we generated a panel of anti-Eimeria scFv antibody fragments with high sporozoite-neutralizing activity. These antibodies were expressed either transiently in agrobacteria-infiltrated tobacco leaves or stably in seeds of transgenic pea plants. Comparison of the scFv antibodies purified either from tobacco leaves or from the pea seeds demonstrated no difference in their antigen-binding activity and molecular form compositions. Force-feeding experiments demonstrated that oral delivery of flour prepared from the transgenic pea seeds had higher parasite neutralizing activity in vivo than the purified antibody fragments isolated from tobacco. The pea seed content was found to protect antibodies against degradation by gastrointestinal proteases (>100-fold gain in stability). Ad libitum feeding of chickens demonstrated that the transgenic seeds were well consumed and not shunned. Furthermore, feeding poultry with shred prepared from the antibody expressing pea seeds led to significant mitigation of infection caused both by high and low challenge doses of Eimeria oocysts. The results suggest that our strategy offers a general approach to control parasitic infections in production animals using cost-effective antibody expression in crop seeds affordable for the animal health market.

  3. Transient protein expression in three Pisum sativum (green pea) varieties.

    Science.gov (United States)

    Green, Brian J; Fujiki, Masaaki; Mett, Valentina; Kaczmarczyk, Jon; Shamloul, Moneim; Musiychuk, Konstantin; Underkoffler, Susan; Yusibov, Vidadi; Mett, Vadim

    2009-02-01

    The expression of proteins in plants both transiently and via permanently transformed lines has been demonstrated by a number of groups. Transient plant expression systems, due to high expression levels and speed of production, show greater promise for the manufacturing of biopharmaceuticals when compared to permanent transformants. Expression vectors based on a tobacco mosaic virus (TMV) are the most commonly utilized and the primary plant used, Nicotiana benthamiana, has demonstrated the ability to express a wide range of proteins at levels amenable to purification. N. benthamiana has two limitations for its use; one is its relatively slow growth, and the other is its low biomass. To address these limitations we screened a number of legumes for transient protein expression. Using the alfalfa mosaic virus (AMV) and the cucumber mosaic virus (CMV) vectors, delivered via Agrobacterium, we were able to identify three Pisum sativum varieties that demonstrated protein expression transiently. Expression levels of 420 +/- 26.24 mg GFP/kgFW in the green pea variety speckled pea were achieved. We were also able to express three therapeutic proteins indicating promise for this system in the production of biopharmaceuticals.

  4. Enhancing Neoplasm Expression in Field Pea (Pisum sativum via Intercropping and its Significance to Pea Weevil (Bruchus pisorum Management

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    Abel eTeshome

    2016-05-01

    Full Text Available Neoplasm formation, a non-meristematic tissue growth on young field pea (Pisum sativum L. pods is triggered in the absence of UV light and/or in response to oviposition by pea weevil (Bruchus pisorum L.. This trait is expressed in some genotypes (Np genotypes of P. sativum and has the capacity to obstruct pea weevil larval entry into developing seeds. In the present study, 26% of the tested accessions depicted the trait when grown under greenhouse conditions. However, UV light inhibits full expression of this trait and subsequently it is inconspicuous at the field level. In order to investigate UV light impact on the expression of neoplasm, particular Np genotypes were subjected to UV lamp light exposure in the greenhouse and sunlight at the field level. Under these different growing conditions, the highest mean percentage of neoplastic pods was in the control chamber in the greenhouse (36% whereas in single and double UV lamp chambers, the percentage dropped to 10% and 15%, respectively. Furthermore, when the same Np genotypes were grown in the field, the percentage of neoplastic pods dropped significantly (7%. In order to enhance neoplastic expression at the field level, intercropping of Np genotypes with sorghum was investigated. As result, the percentage of neoplastic pods was threefold in intercropped Np genotypes as compared to those without intercropping. Therefore, intercropping neoplastic genotypes with other crops such as sorghum and maize can facilitate neoplasm formation, which in turn can minimize the success rate of pea weevil larvae entry into developing seeds. Greenhouse artificial infestation experiments showed that pea weevil damage in neoplastic genotypes is lower in comparison to wild type genotypes. Therefore, promoting neoplastic formation under field conditions via intercropping can serve as part of an integrated pea weevil management strategy especially for small scale farming systems.

  5. Transient expression of acidic fibroblast growth factor in pea (Pisum ...

    African Journals Online (AJOL)

    Yomi

    2012-03-15

    Mar 15, 2012 ... Nowadays, there are many therapeutic proteins produced in different host plants in transient easy to perform, short production cycle, efficient and inexpensive. In this study, the modified pea early browning virus (PEBV) vector containing GFP and acidic fibroblast growth factor (aFGF) was introduced into pea ...

  6. Transient expression of acidic fibroblast growth factor in pea ( Pisum ...

    African Journals Online (AJOL)

    Nowadays, there are many therapeutic proteins produced in different host plants in transient easy to perform, short production cycle, efficient and inexpensive. In this study, the modified pea early browning virus (PEBV) vector containing GFP and acidic fibroblast growth factor (aFGF) was introduced into pea plants by leave ...

  7. Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology

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    Cubero José I

    2011-01-01

    Full Text Available Abstract Background Ascochyta blight, caused by Mycosphaerella pinodes is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against M. pinodes in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in Pisum relatives. The identification of the genes underlying resistance would increase our knowledge about M. pinodes-pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant P. sativum ssp. syriacum accession P665 comparing to the susceptible pea cv. Messire after inoculation with M. pinodes have been identified using a M. truncatula microarray. Results Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR proteins and detoxification processes. Genes associated with jasmonic acid (JA and ethylene signal transduction pathways were induced suggesting that the response to M. pinodes in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b, glutathione S-transferase (GST and 6a-hydroxymaackiain methyltransferase. Conclusions Through this study a global view of genes expressed during resistance to M. pinodes has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the M. truncatula microarray represents an efficient tool to

  8. Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology.

    Science.gov (United States)

    Fondevilla, Sara; Küster, Helge; Krajinski, Franziska; Cubero, José I; Rubiales, Diego

    2011-01-13

    Ascochyta blight, caused by Mycosphaerella pinodes is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against M. pinodes in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in Pisum relatives. The identification of the genes underlying resistance would increase our knowledge about M. pinodes-pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant P. sativum ssp. syriacum accession P665 comparing to the susceptible pea cv. Messire after inoculation with M. pinodes have been identified using a M. truncatula microarray. Of the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to M. pinodes in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase. Through this study a global view of genes expressed during resistance to M. pinodes has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the M. truncatula microarray represents an efficient tool to identify candidate genes controlling resistance to M

  9. [Digestion of pea and soya proteins in the preruminant calf. I. Circulating levels of nutrients, antibody formation and intestinal permeability to macromolecules].

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    Nunes do Prado, I; Toullec, R; Lallès, J P; Guéguen, J; Hingand, J; Guilloteau, P

    1989-01-01

    Three milk-substitutes (control, pea and soya-bean)were given to 6 preruminant calves. In the control diet protein was almost entirely provided by skim milk powder. In the pea diet a pregelatinized dehulled pea flour provided 33.5% of the protein, the remainder being supplied by skim milk powder. In the soya-bean diet, 73.2% of the protein were provided by a soya-bean isolate and the remainder by whey powder. The concentrations of plasma triglycerides and the free alpha-amino nitrogen in the peripheral blood were lower with the pea and soya-bean diets than with the control diet before the morning meal, but became higher after feeding. That suggested a faster abomasal emptying of fat and protein with the pea and especially the soya-bean diet. Systemic antibody responses were induced against pea protein but not against soya-bean protein. No effect of the diet on the plasma concentration of immunoreactive beta-lactoglobulin was apparent after 6 days, suggesting there was no important change in gut permeability at least at that time.

  10. Improving nutritional quality and fungal tolerance in soya bean and grass pea by expressing an oxalate decarboxylase.

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    Kumar, Vinay; Chattopadhyay, Arnab; Ghosh, Sumit; Irfan, Mohammad; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis

    2016-06-01

    Soya bean (Glycine max) and grass pea (Lathyrus sativus) seeds are important sources of dietary proteins; however, they also contain antinutritional metabolite oxalic acid (OA). Excess dietary intake of OA leads to nephrolithiasis due to the formation of calcium oxalate crystals in kidneys. Besides, OA is also a known precursor of β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), a neurotoxin found in grass pea. Here, we report the reduction in OA level in soya bean (up to 73%) and grass pea (up to 75%) seeds by constitutive and/or seed-specific expression of an oxalate-degrading enzyme, oxalate decarboxylase (FvOXDC) of Flammulina velutipes. In addition, β-ODAP level of grass pea seeds was also reduced up to 73%. Reduced OA content was interrelated with the associated increase in seeds micronutrients such as calcium, iron and zinc. Moreover, constitutive expression of FvOXDC led to improved tolerance to the fungal pathogen Sclerotinia sclerotiorum that requires OA during host colonization. Importantly, FvOXDC-expressing soya bean and grass pea plants were similar to the wild type with respect to the morphology and photosynthetic rates, and seed protein pool remained unaltered as revealed by the comparative proteomic analysis. Taken together, these results demonstrated improved seed quality and tolerance to the fungal pathogen in two important legume crops, by the expression of an oxalate-degrading enzyme. © 2016 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  11. Role of plant expression systems in antibody production for passive immunization.

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    Virdi, Vikram; Depicker, Ann

    2013-01-01

    Passive immunization is a method to achieve immediate protection against infectious agents by administering pathogen-specific antibodies. It has proven to be lifesaving for many acute infections, and it is now also used for cancer treatment. Passive immunization therapies, however, are extremely expensive because they require large amounts of specific antibodies that are produced predominantly in mammalian expression systems. The cost for manufacturing plant-made antibodies is estimated to be comparatively low since plant production systems require relatively less capital investments. In addition, they are not prone to mammalian pathogens, which also eases downstream processing along with making it a safe expression system. Moreover, some of the recent developments in transient expression have enabled rapid, cGMP (current Good Manufacturing Practices) compliant manufacturing of antibodies. Whether lower production costs will be reflected in a lower market price for purified antibodies will be known when more plant-produced antibodies come to the market. Promisingly, the current molecular techniques in the field of in planta expression have enabled high-level production of a variety of antibodies in different plant organs, like roots/tubers/fruits, leaves and seeds, of a variety of plants, like potato, tobacco, maize, rice, tomato and pea, providing a very wide range of possible plant-based passive immunization therapies. For instance, the production of antibodies in edible tissues would allow for a unique, convenient, needle-less, oral passive immunization at the gastric mucosal surface. The technological advances, together with the innate capacity of plant tissues to assemble complex antibodies, will enable carving a niche in the antibody market. This non-exhaustive review aims to shed light on the role of plants as a flexible expression system for passive immunotherapy, which we envisage to progress alongside the conventional production platforms to manufacture

  12. Does pea lectin expressed transgenically in oilseed rape (Brassica napus) influence honey bee (Apis mellifera) larvae?

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    Lehrman, Anna

    2007-01-01

    The European honey bee (Apis mellifera) is important both for pollination and for honey production. Pollen is the major protein source for bees, which exposes them directly to changes in pollen quality e.g. through genetic engineering. In order to create a worst case scenario regarding pea lectin (PSL) expressed transgenically in oilseed rape anthers and pollen, the maximum amount of dried pollen that could be mixed in an artificial diet without negatively affecting larval performance (1.5% w/w) was fed to bee larvae. Pollen from two transgenic plant lines expressing PSL up to 1.2% of total soluble protein and pollen from one non-transgenic line was added to the same diet and used as a pollen control. When these three pollen diets and the control diet (without added pollen) were compared, no negative effect from the pollen of the transgenic plants could be detected on larval mortality, weight, or development time. An increased weight and a reduced developmental time were recorded for larvae on all diets containing pollen when compared to the diet without pollen.

  13. Antifungal genes expressed in transgenic pea (Pisum sativum L.) do not affect root colonization of arbuscular mycorrhizae fungi.

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    Kahlon, Jagroop Gill; Jacobsen, Hans-Jörg; Cahill, James F; Hall, Linda M

    2017-10-01

    Genetically modified crops have raised concerns about unintended consequences on non-target organisms including beneficial soil associates. Pea transformed with four antifungal genes 1-3 β glucanase, endochitinase, polygalacturonase-inhibiting proteins, and stilbene synthase is currently under field-testing for efficacy against fungal diseases in Canada. Transgenes had lower expression in the roots than leaves in greenhouse experiment. To determine the impact of disease-tolerant pea or gene products on colonization by non-target arbuscular mycorrhizae and nodulation by rhizobium, a field trial was established. Transgene insertion, as single gene or stacked genes, did not alter root colonization by arbuscular mycorrhiza fungus (AMF) or root nodulation by rhizobium inoculation in the field. We found no effect of transgenes on the plant growth and performance although, having a dual inoculant with both AMF and rhizobium yielded higher fresh weight shoot-to-root ratio in all the lines tested. This initial risk assessment of transgenic peas expressing antifungal genes showed no deleterious effect on non-target organisms.

  14. Altered life cycle in Arabidopsis plants expressing PsUGT1, a UDP-glucuronosyltransferase-encoding gene from pea.

    Science.gov (United States)

    Woo, Ho-Hyung; Faull, Kym F; Hirsch, Ann M; Hawes, Martha C

    2003-10-01

    Alfalfa (Medicago sativa) and Arabidopsis were used as model systems to examine molecular mechanisms underlying developmental effects of a microsomal UDP-glucuronosyltransferase-encoding gene from pea (Pisum sativum; PsUGT1). Alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited delayed root emergence, reduced root growth, and increased lateral root development. The timing of root emergence in wild-type and antisense plants was correlated with the transient accumulation of auxin at the site of root emergence. Cell suspension cultures derived from the antisense alfalfa plants exhibited a delay in cell cycle from 24-h in the wild-type plants to 48-h in the antisense plants. PsUGT1::uidA was introduced into Arabidopsis to demonstrate that, as in alfalfa and pea, PsUGT1 expression occurs in regions of active cell division. This includes the root cap and root apical meristems, leaf primordia, tips of older leaves, and the transition zone between the hypocotyl and the root. Expression of PsUGT1::uidA colocalized with the expression of the auxin-responding reporter DR5::uidA. Co-expression of DR5::uidA in transgenic Arabidopsis lines expressing CaMV35S::PsUGT1 revealed that ectopic expression of CaMV35S::PsUGT1 is correlated with a change in endogenous auxin gradients in roots. Roots of ecotype Columbia expressing CaMV35S::PsUGT1 exhibited distinctive responses to exogenous naphthalene acetic acid. Completion of the life cycle occurred in 4 to 6 weeks compared with 6 to 7 weeks for wild-type Columbia. Inhibition of endogenous ethylene did not correct this early senescence phenotype.

  15. Reactive astrocytes associated with plaques in TgCRND8 mouse brain and in human Alzheimer brain express phosphoprotein enriched in astrocytes (PEA-15).

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    Thomason, Lynsie A M; Smithson, Laura J; Hazrati, Lili-Naz; McLaurin, JoAnne; Kawaja, Michael D

    2013-08-02

    To identify potential biomarkers associated with Alzheimer's disease (AD)-like neuropathologies in the murine brain, we conducted proteomic analyses of neocortices from TgCRND8 mice. Here we found that phosphoprotein enriched in astrocytes 15 kDa (PEA-15) is expressed at higher levels in the neocortical proteomes from 6-month old TgCRND8 mice, as compared to non-transgenic mice. Immunostaining for PEA-15 revealed reactive astrocytes associated with the neocortical amyloid plaques in TgCRND8 mice and in post-mortem human AD brains. This is the first report of increased phosphoprotein enriched in astrocytes (PEA-15) expression in reactive astrocytes of an AD mouse model and human AD brains. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. Effect of antifungal genes expressed in transgenic pea (Pisum sativum L.) on root colonization with Glomus intraradices.

    Science.gov (United States)

    Hassan, Fathi; Noorian, Mojgan Sharifi; Jacobsen, Hans-Jörg

    2012-01-01

    Pathogenic fungi have always been a major problem in agriculture. One of the effective methods for controlling pathogen fungi to date is the introduction of resistance genes into the genome of crops. It is interesting to find out whether the induced resistance in crops will have a negative effect on non-target organisms such as root colonization with the AM fungi.   The objective of the present research was to study the influence of producing antifungal molecules by four transgenic pea (Pisum sativum L.) lines expressing PGIP gene from raspberry, VST-stilbene synthase from vine, a hybrid of PGIP/VST and bacterial Chitinase gene (Chit30) from Streptomyces olivaceoviridis respectively on the colonization potential of Glomus intraradices. Four different experiments were done in greenhouse and climate chamber, colonization was observed in all replications. The following parameters were used for evaluation: frequency of mycorrhization, the intensity of mycorrhization, the average presence of arbuscules within the colonized areas and the presence of arbuscules in the whole root system which showed insignificant difference between transgenic and non-transgenic plants. The root/shoot ratio exhibited different values according to the experiment condition. Compared with negative non-transgenic control all transgenic lines showed the ability to establish symbiosis and the different growth parameters had insignificant effect due to mycorrhization. The results of the present study proved that the introduced pathogen resistance genes did not affect the mycorrhization allocations in pea.

  17. Phytochrome-regulated expression of the genes encoding the small GTP-binding proteins in peas.

    OpenAIRE

    Yoshida, K; Nagano, Y; Murai, N; Sasaki, Y

    1993-01-01

    We examined the effect of light on the mRNA levels of 11 genes (pra1-pra9A, pra9B, and pra9C) encoding the small GTP-binding proteins that belong to the ras superfamily in Pisum sativum. When the dark-grown seedlings were exposed to continuous white light for 24 hr, the levels of several pra mRNAs in the pea buds decreased: pra2 and pra3 mRNAs decreased markedly; pra4, pra6, and pra9A mRNAs decreased slightly; the other 6 pra mRNAs did not decrease. We studied the kinetics of mRNA accumulatio...

  18. Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera).

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    Richter, A; Jacobsen, H-J; de Kathen, A; de Lorenzo, G; Briviba, K; Hain, R; Ramsay, G; Kiesecker, H

    2006-11-01

    The pea (Pisum sativum L.) varieties Baroness (United Kingdome) and Baccara (France) were transformed via Agrobacterium tumefaciens-mediated gene transfer with pGPTV binary vectors containing the bar gene in combination with two different antifungal genes coding for polygalacturonase-inhibiting protein (PGIP) from raspberry (Rubus idaeus L.) driven by a double 35S promoter, or the stilbene synthase (Vst1) from grape (Vitis vinifera L.) driven by its own elicitor-inducible promoter. Transgenic lines were established and transgenes combined via conventional crossing. Resveratrol, produced by Vst1 transgenic plants, was detected using HPLC and the PGIP expression was determined in functional inhibition assays against fungal polygalacturonases. Stable inheritance of the antifungal genes in the transgenic plants was demonstrated.

  19. Gene Expression Analysis of Parthenogenetic Embryonic Development of the Pea Aphid, Acyrthosiphon pisum, Suggests That Aphid Parthenogenesis Evolved from Meiotic Oogenesis

    Science.gov (United States)

    Srinivasan, Dayalan G.; Abdelhady, Ahmed; Stern, David L.

    2014-01-01

    Aphids exhibit a form of phenotypic plasticity, called polyphenism, in which genetically identical females reproduce sexually during one part of the life cycle and asexually (via parthenogenesis) during the remainder of the life cycle. The molecular basis for aphid parthenogenesis is unknown. Cytological observations of aphid parthenogenesis suggest that asexual oogenesis evolved either through a modification of meiosis or from a mitotic process. As a test of these alternatives, we assessed the expression levels and expression patterns of canonical meiotic recombination and germline genes in the sexual and asexual ovaries of the pea aphid, Acyrthosiphon pisum. We observed expression of all meiosis genes in similar patterns in asexual and sexual ovaries, with the exception that some genes encoding Argonaute-family members were not expressed in sexual ovaries. In addition, we observed that asexual aphid tissues accumulated unspliced transcripts of Spo11, whereas sexual aphid tissues accumulated primarily spliced transcripts. In situ hybridization revealed Spo11 transcript in sexual germ cells and undetectable levels of Spo11 transcript in asexual germ cells. We also found that an obligately asexual strain of pea aphid produced little spliced Spo11 transcript. Together, these results suggest that parthenogenetic oogenesis evolved from a meiosis-like, and not a mitosis-like, process and that the aphid reproductive polyphenism may involve a modification of Spo11 gene activity. PMID:25501006

  20. Gene expression analysis of parthenogenetic embryonic development of the pea aphid, Acyrthosiphon pisum, suggests that aphid parthenogenesis evolved from meiotic oogenesis.

    Science.gov (United States)

    Srinivasan, Dayalan G; Abdelhady, Ahmed; Stern, David L

    2014-01-01

    Aphids exhibit a form of phenotypic plasticity, called polyphenism, in which genetically identical females reproduce sexually during one part of the life cycle and asexually (via parthenogenesis) during the remainder of the life cycle. The molecular basis for aphid parthenogenesis is unknown. Cytological observations of aphid parthenogenesis suggest that asexual oogenesis evolved either through a modification of meiosis or from a mitotic process. As a test of these alternatives, we assessed the expression levels and expression patterns of canonical meiotic recombination and germline genes in the sexual and asexual ovaries of the pea aphid, Acyrthosiphon pisum. We observed expression of all meiosis genes in similar patterns in asexual and sexual ovaries, with the exception that some genes encoding Argonaute-family members were not expressed in sexual ovaries. In addition, we observed that asexual aphid tissues accumulated unspliced transcripts of Spo11, whereas sexual aphid tissues accumulated primarily spliced transcripts. In situ hybridization revealed Spo11 transcript in sexual germ cells and undetectable levels of Spo11 transcript in asexual germ cells. We also found that an obligately asexual strain of pea aphid produced little spliced Spo11 transcript. Together, these results suggest that parthenogenetic oogenesis evolved from a meiosis-like, and not a mitosis-like, process and that the aphid reproductive polyphenism may involve a modification of Spo11 gene activity.

  1. PEA-CLARITY: 3D molecular imaging of whole plant organs.

    Science.gov (United States)

    Palmer, William M; Martin, Antony P; Flynn, Jamie R; Reed, Stephanie L; White, Rosemary G; Furbank, Robert T; Grof, Christopher P L

    2015-09-02

    Here we report the adaptation of the CLARITY technique to plant tissues with addition of enzymatic degradation to improve optical clearing and facilitate antibody probe penetration. Plant-Enzyme-Assisted (PEA)-CLARITY, has allowed deep optical visualisation of stains, expressed fluorescent proteins and IgG-antibodies in Tobacco and Arabidopsis leaves. Enzyme treatment enabled penetration of antibodies into whole tissues without the need for any sectioning of the material, thus facilitating protein localisation of intact tissue in 3D whilst retaining cellular structure.

  2. Expression of recombinant vaccines and antibodies in plants.

    Science.gov (United States)

    Ko, Kisung

    2014-06-01

    Plants are able to perform post-translational maturations of therapeutic proteins required for their functional biological activity and suitable in vivo pharmacokinetics. Plants can be a low-cost, large-scale production platform of recombinant biopharmaceutical proteins such as vaccines and antibodies. Plants, however, lack mechanisms of processing authentic human N-glycosylation, which imposes a major limitation in their use as an expression system for therapeutic glycoproducts. Efforts have been made to circumvent plant-specific N-glycosylation, as well as to supplement the plant's endogenous system with human glycosyltransferases for non-immunogenic and humanized N-glycan production. Herein we review studies on the potential of plants to serve as production systems for therapeutic and prophylactic biopharmaceuticals. We have especially focused on recombinant vaccines and antibodies and new expression strategies to overcome the existing problems associated with their production in plants.

  3. Downregulation of transferrin receptor surface expression by intracellular antibody

    International Nuclear Information System (INIS)

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin

    2007-01-01

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 ± 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors

  4. Meristem-localized inducible expression of a UDP-glycosyltransferase gene is essential for growth and development in pea and alfalfa.

    Science.gov (United States)

    Woo, H H; Orbach, M J; Hirsch, A M; Hawes, M C

    1999-12-01

    PsUGT1, which encodes a microsomal UDP-glucuronosyltransferase, was cloned from root tips of Pisum sativum. PsUGT1 expression is correlated with mitosis and strongly induced in dividing cells. A region at the C terminus of the encoded protein is closely related to the UDP-glucuronic acid binding site consensus sequence, and the protein encoded by PsUGT1 catalyzes conjugation of UDP-glucuronic acid to an unknown compound. Overexpression of PsUGT1 sense mRNA has no detectable effect on transgenic pea hairy root cultures or regenerated alfalfa. However, inhibiting PsUGT1 expression by the constitutive expression of antisense mRNA (under the control of the cauliflower mosaic virus 35S promoter) markedly retards growth and development of transgenic alfalfa. Cell structure and organization in the antisense plants are similar to those of controls, but plant growth is reduced and development is delayed. This inhibition in growth is correlated with a twofold delay in the time required for completion of a cell cycle and with a >99% inhibition of border cell production. Inhibition of PsUGT1 expression by meristem-localized inducible expression of PsUGT1 antisense mRNA (under the control of its own promoter) is lethal both in pea hairy roots and in transgenic alfalfa plants. These results indicate that PsUGT1 expression is required for normal plant growth and development, and they are consistent with the hypothesis that this UDP-glycosyltransferase regulates activity of a ligand(s) needed for cell division.

  5. Covalent and Oriented Surface Immobilization of Antibody Using Photoactivatable Antibody Fc-Binding Protein Expressed in Escherichia coli.

    Science.gov (United States)

    Lee, Yeolin; Jeong, Jiyun; Lee, Gabi; Moon, Jeong Hee; Lee, Myung Kyu

    2016-10-04

    Fc-specific antibody binding proteins (FcBPs) with the minimal domain of protein G are widely used for immobilization of well-oriented antibodies onto solid surfaces, but the noncovalently bound antibodies to FcBPs are unstable in sera containing large amounts of antibodies. Here we report novel photoactivatable FcBPs with photomethionine (pMet) expressed in E. coli, which induce Fc-specific photo-cross-linking with antibodies upon UV irradiation. Unfortunately, pMet did not support protein expression in the native E. coli system, and therefore we also developed an engineered methionyl tRNA synthetase (MRS5m). Coexpression of MRS5m proteins successfully induced photoactivatable FcBP overexpression in methionine-auxotroph E. coli cells. The photoactivatable FcBPs could be easily immobilized on beads and slides via their N-terminal cysteine residues and 6xHis tag. The antibodies photo-cross-linked onto the photoactivatable FcBP-beads were resistant from serum-antibody mediated dissociation and efficiently captured antigens in human sera. Furthermore, photo-cross-linked antibody arrays prepared using this system allowed sensitive detection of antigens in human sera by sandwich immunoassay. The photoactivatable FcBPs will be widely applicable for well-oriented antibody immobilization on various surfaces of microfluidic chips, glass slides, and nanobeads, which are required for development of sensitive immunosensors.

  6. Expansion of genes encoding piRNA-associated argonaute proteins in the pea aphid: diversification of expression profiles in different plastic morphs.

    Directory of Open Access Journals (Sweden)

    Hsiao-Ling Lu

    Full Text Available Piwi-interacting RNAs (piRNAs are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are "specialised" in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction.

  7. Cloning of Single-Chain Antibody Variants by Overlap-Extension PCR for Evaluation of Antibody Expression in Transient Gene Expression.

    Science.gov (United States)

    Mayrhofer, Patrick; Kunert, Renate

    2017-01-01

    Single-chain fragment variable-fragment crystallizable antibody constructs (scFv-Fc) are homodimeric proteins representing valuable alternatives to heterotetrameric full-length IgG molecules to study protein properties and product-dependent cellular behavior. In contrast to naturally occurring antibodies, these artificial molecules are assembled from functional antibody domains to reduce molecule complexity and enhance antibody expression levels. The scFv-Fc format retains critical antibody functions such as antigen binding affinity and antibody effector functions. Here, we present a protocol to convert the full-length anti-HIV-1 IgG1 antibody 2F5 into a scFv-Fc construct. Variable and constant regions are amplified by conventional PCR reactions and assembled by a single overlap-extension PCR reaction. The amplified product is then cloned into a mammalian expression vector suitable for high-titer transient gene expression. This workflow can be applied to any antibody sequence by adapting the specific primer sequences to the antibody of choice.

  8. Males without apparent alloimmunization could have HLA antibodies that recognize target HLA specificities expressed on cells.

    Science.gov (United States)

    Nakamura, J; Nakajima, F; Kamada, H; Tadokoro, K; Nagai, T; Satake, M

    2017-05-01

    Human leukocyte antigen (HLA) antibodies, which are involved in the development of transfusion-related side effects such as transfusion-related lung injury, are sometimes found in males without a history of alloimmunization (eg, transplantation and transfusion). Whether HLA antibodies in male donors can interact with their target HLA specificities expressed on cells have not been completely investigated. The HLA antibodies detected in 7 male donors were characterized. Flow cytometry and immunocomplex capture fluorescence analysis were performed to evaluate the ability of these antibodies to bind with target HLA specificities expressed on cells. The association of these antibodies with complement was examined using anti-C1q antibody. Sustainability of HLA antibodies over time was compared in 26 male vs 57 female donors. The antibodies from all 7 donors recognized intact HLA molecules coated onto microbeads. The antibodies in 2 of 7 donors also recognized their target HLA specificities expressed on cells. Furthermore, the antibodies in one of these 2 donors showed HLA specificities that involved complement binding. Twenty-one of 26 initially positive male donors had turned negative for HLA antibody at least 1 year after their initial positive screening, whereas HLA antibody positivity was maintained for a long time in most female donors. Males without apparent alloimmunization could have HLA antibodies that recognize their target HLA specificities on cells and that could potentially modify molecular events in affected cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Unwinding after high salinity stress: Pea DNA helicase 45 over- expression in tobacco confers high salinity tolerance without affecting yield (abstract)

    International Nuclear Information System (INIS)

    Tuteja, N.

    2005-01-01

    Soil salinity is an increasing threat for agriculture and is a major factor in reducing plant productivity; therefore, it is necessary to obtain salinity-tolerant varieties. A typical characteristic of soil salinity is the induction of multiple stress- inducible genes. Some of the genes encoding osmolytes, ion channels or enzymes are able to confer salinity-tolerant phenotypes when transferred to sensitive plants. As salinity stress affects the cellular gene-expression machinery, it is evident that molecules involved in nucleic acid processing including helicases, are likely to be affected as well. DNA helicases unwind duplex DNA and are involved in replication, repair, recombination and transcription while RNA helicases unfold the secondary structures in RNA and are involved in transcription, ribosome biogenesis and translation initiation. We have earlier reported the isolation of a pea DNA helicase 45 (PDH45) that exhibits striking homology with eIF-4A (Plant J. 24:219-230,2000). Here we report that PDH45 mRNA is induced in pea seedlings in response to high salt and its over- expression driven by a constitutive CAMV-355-promoter in tobacco plants confers salinity tolerance, thus suggesting a new pathway for manipulating stress tolerance in crop plants. The T0 transgenic plants showed high-levels of PDH45 protein in normal and stress conditions, as compared to wild type (WT) plants. The T0 transgenics also showed tolerance to high salinity as tested by a leaf disc senescence assay. The T1 transgenics were able to grow to maturity and set normal viable seeds under continuous salinity stress, without any reduction in plant yield, in terms of seed weight. Measurement of Na/sup +/ ions in different parts of the plant showed higher accumulation in the old leaves and negligible in seeds of T1 transgenic lines as compared with the WT plants. The possible mechanism of salinity tolerance will be discussed. Over-expression of PDH45 provides a possible example of the

  10. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus.

    Science.gov (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S; Jia, Letong; Lee, Peter P; Fouts, Timothy R; Parks, Thomas P; Lagenaur, Laurel A

    Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1 BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission.

  11. Antibodies against HLA-DP recognize broadly expressed epitopes.

    Science.gov (United States)

    Simmons, Daimon P; Kafetzi, Maria L; Wood, Isabelle; Macaskill, Peter C; Milford, Edgar L; Guleria, Indira

    2016-12-01

    HLA matching and avoidance of pre-transplant donor-specific antibodies are important in selection of donors for solid organ transplant. Solid phase testing with single antigen beads allows resolution of antibody reactivity to the level of the allele. Single antigen bead testing results at a large transplant center were reviewed to identify selective reactivity patterns of anti-HLA antibodies. Many HLA-DP antibodies were identified in the context of other HLA antibodies, but some sera had antibodies against only HLA-DP. B cell flow crossmatch testing was positive for 2 out of 9 sera with HLA-DP antibodies. Many patterns of reactivity corresponded to epitopes in hypervariable regions C and F of DPB1, but some matched epitopes in other regions or DPA1. Through analysis of single antigen bead testing from a large number of patients, we report that anti-HLA-DP antibodies predominantly recognize broadly cross-reactive epitopes. The United Network for Organ Sharing has mandated HLA-DP typing on all deceased kidney donors, and HLA-DP epitopes should be considered as the major antigens for avoidance of pre-transplant donor-specific antibodies. Published by Elsevier Inc.

  12. Identification of the prothoracicotropic hormone (Ptth) coding gene and localization of its site of expression in the pea aphid Acyrthosiphon pisum.

    Science.gov (United States)

    Barberà, M; Martínez-Torres, D

    2017-10-01

    Insect hormones control essential aspects of physiology, behaviour and development in insects. The majority of insect hormones are peptide hormones that perform a highly diverse catalogue of functions. Prothoracicotropic hormone (PTTH) is a brain neuropeptide hormone whose main function is to stimulate the secretion of ecdysone (the moulting hormone) by the prothoracic glands in insect larvae thus playing a key role in the control of moulting and metamorphosis. Moreover, both PTTH release or blockade have been reported to act as a switch to terminate or initiate larval and pupal diapauses. In insects, diapause is a prevalent response often regulated by the photoperiod. It has been shown that PTTH participates as an output of the circadian clock and a role in photoperiodic processes is suggested in some insect species. Aphids (Hemiptera: Aphididae) reproduce by cyclical parthenogenesis with a sexual phase, induced by short photoperiods, that leads to the production of diapausing eggs. With the availability of the pea aphid (Acyrthosiphon pisum) genome, efforts to identify and characterize genes relevant to essential aspects of aphid biology have multiplied. In spite of its relevance, several genomic and transcriptomic studies on aphid neuropeptides failed to detect aphid PTTH amongst them. Here we report on the first identification of the aphid PTTH coding gene and the neuroanatomical localization of its expression in the aphid brain. © 2017 The Royal Entomological Society.

  13. Introgression, Generational Expression and Salinity Tolerance Conferred by the Pea DNA Helicase 45 Transgene into Two Commercial Rice Genotypes, BR28 and BR47.

    Science.gov (United States)

    Biswas, Sudip; Amin, U S Mahzabin; Sarker, Sarah; Rahman, M Sazzadur; Amin, Ruhul; Karim, Rezaul; Tuteja, Narendra; Seraj, Zeba I

    2018-02-01

    DNA helicase (PDH45) from the pea plant (Pisum sativum) is a member of the DEAD box protein family and plays a vital regulatory role in saline stress tolerance in plants. We previously reported that over-expression of PDH45 gene confers both seedling and reproductive stage salinity tolerance to a Bangladeshi rice landrace, Binnatoa (BA). In this study, transgenic BA-containing PDH45 (♂) was crossed with two different farmer-popular BRRI rice varieties (♀), BR28 and BR47, in a contained net house. F 1 plants positive for the transgene and having recipient phenotype were advanced from F 1 to F 5 . Expression of the PDH45 gene was detected in all generations. The expression level of PDH45 was 200-fold higher in the donor compared to the two recipient genotypes but without any effect on their salt stress tolerance ability in various assays. Under 120 mM NaCl stress at seedling stage, all rice genotypes showed vigorous growth, higher chlorophyll content, lower electrolyte leakage and lower LDS (Leaf Damage Score) compared to their corresponding wild types. At the reproductive stage under continuous salinity stress at 80 mM NaCl, the cross-bred lines BR28 and BR47 showed significantly better spikelet fertility and yield per plant, which were two- and 2.5-folds, respectively, than their corresponding wild types. The PDH45 transgene was observed to increase the expression of 6 salt stress-related downstream genes at 150 mM NaCl stress to similar differential degrees in the donor and recipient genotypes. However, the expression of OsLEA was significantly higher in transgenic BR28 compared to transgenic BR47, where the latter shows comparatively higher salt tolerance. The study shows stability of transgene expression across generations. It also demonstrates that there may be an effect of background genotype on transgene expression. Moreover, some downstream effects of the transgene may also be genotype-specific.

  14. Mutation breeding in peas

    International Nuclear Information System (INIS)

    Jaranowski, J.; Micke, A.

    1985-01-01

    The pea as an ancient crop plant still today has wide uses and is an import source of food protein. It is also an important object for genetic studies and as such has been widely used in mutation induction experiments. However, in comparison with cereals this ancient crop plant (like several other grain legumes) has gained relatively little from advances in breeding. The review focuses on the prospects of genetic improvement of pea by induced mutations, discusses principles and gives methodological information. (author)

  15. Comparative transcriptomic analyses of vegetable and grain pea (Pisum sativum L. seed development

    Directory of Open Access Journals (Sweden)

    Na eLiu

    2015-11-01

    Full Text Available Understanding the molecular mechanisms regulating pea seed developmental process is extremely important for pea breeding. In this study, we used high-throughput RNA-Seq and bioinformatics analyses to examine the changes in gene expression during seed development in vegetable pea and grain pea, and compare the gene expression profiles of these two pea types. RNA-Seq generated 18.7 G of raw data, which were then de novo assembled into 77,273 unigenes with a mean length of 930 bp. Our results illustrate that transcriptional control during pea seed development is a highly coordinated process. There were 459 and 801 genes differentially expressed at early and late seed maturation stages between vegetable pea and grain pea, respectively. Soluble sugar and starch metabolism related genes were significantly activated during the development of pea seeds coinciding with the onset of accumulation of sugar and starch in the seeds. A comparative analysis of genes involved in sugar and starch biosynthesis in vegetable pea (high seed soluble sugar and low starch and grain pea (high seed starch and low soluble sugar revealed that differential expression of related genes at late development stages results in a negative correlation between soluble sugar and starch biosynthetic flux in vegetable and grain pea seeds. RNA-Seq data was validated by using real-time quantitative RT-PCR analysis for 30 randomly selected genes. To our knowledge, this work represents the first report of seed development transcriptomics in pea. The obtained results provide a foundation to support future efforts to unravel the underlying mechanisms that control the developmental biology of pea seeds, and serve as a valuable resource for improving pea breeding.

  16. Expression of functional recombinant antibody molecules in insect cell expression systems.

    Science.gov (United States)

    Reavy, B; Ziegler, A; Diplexcito, J; Macintosh, S M; Torrance, L; Mayo, M

    2000-03-01

    Recombinant single-chain variable-fragment molecules (scFv) were constructed from a cell line expressing a monoclonal antibody against African cassava mosaic virus (ACMV) and expressed in Escherichia coli. DNA sequences that encoded the scFv were manipulated to allow scFv expression in insect cell lines. A recombinant baculovirus containing the scFv cDNA was constructed and large amounts of scFv were produced in each of three insect cell lines infected with the baculovirus. However, the scFv were not secreted into the medium by any of the cell lines despite the scFv having been linked to a honeybee melittin leader sequence. The same scFv cDNA construct was introduced into Drosophila DS2 cells and a stable recombinant cell line was obtained that produced scFv that was secreted into the medium. Culture medium containing the scFv was used directly in enzyme-linked immunosorbent assay (ELISA) tests to detect ACMV in plant tissues. Another construct that encoded the Ckappa domain of human IgG was fused to the C-terminus of the scFv that was produced and expressed in Drosophila cells. This scFv derivative also accumulated in the medium and was more active in ELISA than scFv lacking the Ckappa domain. Copyright 2000 Academic Press.

  17. INCREASING OF THE EXPRESSION OF RECOMBINANT scFv-ANTIBODIES EFFICIENCY

    Directory of Open Access Journals (Sweden)

    O.V. Galkin

    2017-10-01

    Full Text Available Obtaining single-chain variable fragments (scFv of recombinant antibodies in E. coli cells is often associated with numerous problems causing low yields or inactive conformation of the product. The aim of this work was to study the influence of staphylococcal protein A fragment fused with scFv antibodies (SpA-tag on the efficiency of expression of final product. Examination of scFv antibodies of different origin and specificity has shown that in similar expression systems fused scFv is synthesized in much higher quantities than free scFv. Furthermore, the scFv antibodies in fused form retained their antigen-binding properties and the SpA fragment the ability to bind other immunoglobulins. Thus, the proposed strategy can be considered effective in improving the efficiency of scFv-antibodies production in E. coli cells.

  18. Expression of biological active VHH camelid single domain antibody ...

    African Journals Online (AJOL)

    Transgenic tobacco plants were then developed by introducing VHH gene under the control of CaMV 35S promoter. The presence of the VHH antibody gene in the plant genome was verified by PCR analysis and Southern hybridization experiments. Northern blot analysis showed that the genes coding for the VHH could be ...

  19. Expression of Rabies antibodies in tobacco and maize

    CSIR Research Space (South Africa)

    Zungu, N

    2008-01-01

    Full Text Available Rabies, an important disease in Asia and Africa, is an acute viral disease of the central nervous system that affects humans and other mammals. Upon bites or contact with rabid animals patients are immediately immunized with antibodies followed...

  20. Plant expression of chicken secretory antibodies derived from combinatorial libraries

    NARCIS (Netherlands)

    Wieland, W.H.; Lammers, A.; Schots, A.; Orzaéz, D.V.

    2006-01-01

    Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA

  1. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

    Science.gov (United States)

    Gaynor, J. J.

    1984-01-01

    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  2. In Situ Liver Expression of HBsAg/CD3-Bispecific Antibodies for HBV Immunotherapy

    Directory of Open Access Journals (Sweden)

    Robert L. Kruse

    2017-12-01

    Full Text Available Current therapies against hepatitis B virus (HBV do not reliably cure chronic infection, necessitating new therapeutic approaches. The T cell response can clear HBV during acute infection, and the adoptive transfer of antiviral T cells during bone marrow transplantation can cure patients of chronic HBV infection. To redirect T cells to HBV-infected hepatocytes, we delivered plasmids encoding bispecific antibodies directed against the viral surface antigen (HBsAg and CD3, expressed on almost all T cells, directly into the liver using hydrodynamic tail vein injection. We found a significant reduction in HBV-driven reporter gene expression (184-fold in a mouse model of acute infection, which was 30-fold lower than an antibody only recognizing HBsAg. While bispecific antibodies triggered, in part, antigen-independent T cell activation, antibody production within hepatocytes was non-cytotoxic. We next tested the bispecific antibodies in a different HBV mouse model, which closely mimics the transcriptional template for HBV, covalently closed circular DNA (cccDNA. We found that the antiviral effect was noncytopathic, mediating a 495-fold reduction in HBsAg levels at day 4. At day 33, bispecific antibody-treated mice exhibited 35-fold higher host HBsAg immunoglobulin G (IgG antibody production versus untreated groups. Thus, gene therapy with HBsAg/CD3-bispecific antibodies represents a promising therapeutic strategy for patients with HBV.

  3. Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

    NARCIS (Netherlands)

    Joosten, V.; Gouka, R.J.; Hondel, C.A.M.J.J. van den; Verrips, C.T.; Lokman, B.C.

    2005-01-01

    We report the expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori. Fragments encoding VHHs were cloned in a suitable Aspergillus expression vector and transformants secreting VHH fragments were analysed for integrated gene copy-numbers, mRNA

  4. Murine carcinoma expressing carcinoembryonic antigen-like protein is restricted by antibody against neem leaf glycoprotein.

    Science.gov (United States)

    Das, Arnab; Barik, Subhasis; Bose, Anamika; Roy, Soumyabrata; Biswas, Jaydip; Baral, Rathindranath; Pal, Smarajit

    2014-11-01

    We have generated a polyclonal antibody against a novel immunomodulator, neem leaf glycoprotein (NLGP) that can react to a specific 47 kDa subunit of NLGP. Generated anti-NLGP antibody (primarily IgG2a) was tested for its anti-tumor activity in murine carcinoma (EC, CT-26), sarcoma (S180) and melanoma (B16Mel) tumor models. Surprisingly, tumor growth restriction was only observed in CT-26 carcinoma models, without any alteration in other tumor systems. Comparative examination of antigenicity between four different tumor models revealed high expression of CEA-like protein on the surface of CT-26 tumors. Subsequent examination of the cross-reactivity of anti-NLGP antibody with purified or cell bound CEA revealed prominent recognition of CEA by anti-NLGP antibody, as detected by ELISA, Western Blotting and immunohistochemistry. This recognition seems to be responsible for anti-tumor function of anti-NLGP antibody only on CEA-like protein expressing CT-26 tumor models, as confirmed by ADCC reaction in CEA(+) tumor systems where dependency to anti-NLGP antibody is equivalent to anti-CEA antibody. Obtained result with enormous therapeutic potential for CEA(+) tumors may be explained in view of the epitope spreading concept, however, further investigation is crucial. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Antibody-bound amyloid precursor protein upregulates ornithine decarboxylase expression

    DEFF Research Database (Denmark)

    Nilsson, Tatjana; Malkiewicz, Katarzyna; Gabrielsson, Maria

    2006-01-01

    on the protein level. APP knockdown by RNAi verified that upregulation of ODC was APP-mediated. This APP signalling event did not require gamma-secretase cleavage, as it was independent of the presence of presenilin-1 or -2. The induced ODC expression was rapid and biphasic, resembling growth-factor stimulated...

  6. High-level expression, purification, polyclonal antibody preparation ...

    African Journals Online (AJOL)

    OprD is a specific porin which can binds imipenem and carbapenems in Pseudomonas aeruginosa. OprD loss plays a central role in mediating carbapenem resistance. Therefore, purification of oprD protein lays a pavement for the study in vivo and in vitro. In our study, the oprD gene was cloned into pQE30 expression ...

  7. Glatiramer acetate antibodies, gene expression and disease activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn Thorup; Hedegaard, Chris Juul; Krakauer, M

    2011-01-01

    . Objectives: We studied the immunological response to GA and its relationship with disease activity. Methods: Anti-GA antibodies in plasma and the expression of genes encoding cytokines and T-cell-polarizing transcription factors in blood cells were analysed by flow cytometric bead array and polymerase chain...

  8. Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

    Directory of Open Access Journals (Sweden)

    Markiv Anatoliy

    2011-11-01

    trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.

  9. Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein.

    Science.gov (United States)

    Torres, J M; Sánchez, C; Suñé, C; Smerdou, C; Prevec, L; Graham, F; Enjuanes, L

    1995-11-10

    Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebauer et al., 1991, Virology 183, 225-238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5-TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.

  10. Extracellular Expression in Aspergillus niger of an Antibody Fused to Leishmania sp. Antigens.

    Science.gov (United States)

    Magaña-Ortíz, Denis; Fernández, Francisco; Loske, Achim M; Gómez-Lim, Miguel A

    2018-01-01

    Nucleoside hydrolase and sterol 24-c-methyltransferase, two antigenic proteins of Leishmania sp., were expressed in Aspergillus niger. Genetic transformation of conidia was achieved using underwater shock waves. scFv antibody addressed to DEC205, a receptor of dendritic cells, was fused to two proteins of Leishmania sp. Receptor 205 has a relevant role in the immune system in mammals; it can modulate T cell response to different antigens. Extracellular expression strategy of recombinant antibody was achieved using a fragment of native glucoamylase A (514 aa) as a carrier. Fermentations in shake flasks showed that the recombinant protein (104 kDa) was expressed and secreted only when maltose was used as carbon source; on the contrary, the expression was highly repressed in presence of xylose. Noteworthy, recombinant protein was secreted without glucoamylase-carrier and accumulation at intracellular level was not observed. The results presented here demonstrate the high value of Aspergillus niger as biotechnological platform for recombinant antibodies against Leishmania sp. at low cost. To the best of our knowledge, this is the first report about the recombinant expression of antigenic proteins of Leishmania sp. in filamentous fungi. The protein obtained can be used to explore novel strategies to induce immunity against Leishmania sp. or it can be employed in diagnostic kits to detect this neglected disease.

  11. Upregulation of HLA Class I Expression on Tumor Cells by the Anti-EGFR Antibody Nimotuzumab

    Directory of Open Access Journals (Sweden)

    Greta Garrido

    2017-10-01

    Full Text Available Defining how epidermal growth factor receptor (EGFR-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune regulator genes that govern tumor – T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR, we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression in vivo and tumors treated with this antibody display higher susceptibility to CD8+ T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.

  12. ( Voandze subterranean ) and pigeon pea

    African Journals Online (AJOL)

    Formulation and evaluation of complementary food based on bambara nut ( Voandze subterranean ) and pigeon pea ( Cajanus cajan ) ... Nigerian Journal of Nutritional Sciences ... Objectives: The study formulated and evaluated complementary food made from composites of maize, bambara nut and pigeon pea. Materials ...

  13. PEA3 activates VEGF transcription in T47D and SKBR3 breast cancer cells.

    Science.gov (United States)

    Hua, Dong; Chen, Bobin; Bai, Mei; Yu, Hao; Wu, Xiaohong; Jin, Wei

    2009-01-01

    Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and a prognostic factor for many tumors, including those of endocrine-responsive tissues such as the breast and uterus. In this study, we found that overexpression of PEA3 could increase VEGF mRNA levels and VEGF promoter activity in human T47D and SKBR3 breast cancer cells. Chromatin immunoprecipitation assay demonstrated that PEA3 could bind to the VEGF promoter in the cells transfected with PEA3 expression vector. PEA3 small interfering RNA attenuated VEGF promoter activity and the binding of PEA3 to the VEGF promoter in T47D and SKBR3 cells. These results indicated that PEA3 could activate VEGF promoter transcription.

  14. Expression characteristics and specific antibody reactivity of diverse cathepsin F members of Paragonimus westermani.

    Science.gov (United States)

    Ahn, Chun-Seob; Na, Byoung-Kuk; Chung, Dong-Ll; Kim, Jeong-Geun; Kim, Jin-Taek; Kong, Yoon

    2015-02-01

    Paragonimiasis, caused by the lung fluke Paragonimus, is a major food-borne helminthic disease. Differential diagnosis of paragonimiasis from tuberculosis and other infectious granulomas in the lung is a prerequisite to proper management of patients. Cysteine proteases of Paragonimus westermani (PwCPs) invoke specific antibody responses against patient sera, while antibody capturing activity of different PwCPs has not been comparatively analyzed. In this study, we observed the expressional regulation of 11 species of different PwCPs (PwCP1-11). We expressed recombinant PwCPs and assessed diagnostic reliability employing sera from patients with P. westermani (n=138), other trematodiases (n=80), cestodiases (n=60) and pulmonary tuberculosis (n=20), and those of normal controls (n=20). PwCPs formed a monophyletic clade into cathepsin F and showed differential expression patterns along with developmental stages of worm. Bacterially expressed recombinant PwCPs (rPwCPs) exhibited variable sensitivity of 38.4-84.5% and specificity of 87.2-100% in diagnosing homologous infection. rPwCPs recognized specific antibodies of experimental cat sera as early as 3 or 6weeks after infection. Patient sera of fascioliasis, Schistosomiasis japonicum and clonorchiasis demonstrated weak cross-reactions. Our results demonstrate that diverse PwCPs of the cathepsin F family participate in inducing specific antibody responses. Most P. westermani cathepsin F, except for PwCP2 (AAF21461), which showed negligible antibody responses, might be applicable for paragonimiasis serodiagnosis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. Cloning, reformatting, and small-scale expression of monoclonal antibody isolated from mouse, rat, or hamster hybridoma.

    Science.gov (United States)

    Loyau, Jeremy; Rousseau, François

    2014-01-01

    The hybridoma technology, first described in 1975 by Milstein and Köhler, is still to date one of the most commonly used approaches to produce monoclonal antibodies. However, despite many advantages, this approach suffers from limitations like limited antibody productivity. Here, we describe a method for efficient cloning of antibody VH and VL produced by mouse, rat, or hamster hybridoma before reformatting in full-length IgG and small-scale expression in mammalian cell line.

  16. Evaluation of {sup 111}In labeled antibodies for SPECT imaging of mesothelin expressing tumors

    Energy Technology Data Exchange (ETDEWEB)

    Misri, Ripen; Saatchi, Katayoun [Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3 (Canada); Ng, Sylvia S.W. [Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3 (Canada); Advanced Therapeutics, British Columbia Cancer Agency, Vancouver BC V5Z 1G1 (Canada); Kumar, Ujendra [Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3 (Canada); Haefeli, Urs O., E-mail: uhafeli@interchange.ubc.ca [Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3 (Canada)

    2011-08-15

    Introduction: Mesothelin is expressed in many cancers, especially in mesothelioma and lung, pancreatic and ovarian cancers. In the present study, we evaluate {sup 111}In labeled antimesothelin antibodies as an imaging bioprobe for the SPECT imaging of mesothelin-expressing tumors. Methods: We radiolabeled the antimesothelin antibodies mAbMB and mAbK1 with {sup 111}In using the p-SCN-bn-DTPA chelator. The immunoreactivity, affinity (K{sub d}) and internalization properties of the resulting two {sup 111}In labeled antibodies were evaluated in vitro using mesothelin-expressing A431K5 cells. The biodistribution and microSPECT/CT imaging studies with {sup 111}In labeled antibodies were performed in mice bearing both mesothelin positive (A431K5) and mesothelin negative (A431) tumors. Results: In vitro studies demonstrated that {sup 111}In-mAbMB bound with a higher affinity (K{sub d}=3.6{+-}1.7 nM) to the mesothelin-expressing A431K5 cells than did the {sup 111}In-mAbK1 (K{sub d}=29.3{+-}2.3 nM). {sup 111}In-mAbMB was also internalized at a greater rate and extent into the A431K5 cells than was the {sup 111}In-mAbK1. Biodistribution studies showed that {sup 111}In-mAbMB was preferentially localized in A431K5 tumors when compared to A431 tumors. At the low dose, the peak A431K5 tumor uptake of 9.65{+-}2.65% ID/g (injected dose per gram) occurred at 48 h, while at high dose tumor uptake peaked with 14.29{+-}6.18% ID/g at 72 h. Non-specific localization of {sup 111}In-mAbMB was mainly observed in spleen.{sup 111}In-mAbK1 also showed superior localization in A431K5 tumors than in A431 tumors, but the peak uptake was only 3.04{+-}0.68% ID/g at 24 h. MicroSPECT/CT studies confirmed better visualization of A431K5 tumors with {sup 111}In-mAbMB, than with {sup 111}In-mAbK1. Conclusion: SPECT imaging of mesothelin expressing tumors was demonstrated successfully. Our findings indicate that the antimesothelin antibody mAbMB has the potential to be developed into a diagnostic agent

  17. Scintigraphic imaging of P-glycoprotein expression with a radiolabelled antibody

    International Nuclear Information System (INIS)

    Eerd, Julliette E.M. van; Geus-Oei, Lioe-Fee de; Oyen, Wim J.G.; Corstens, Frans H.M.; Boerman, Otto C.

    2006-01-01

    P-glycoprotein (P-gp) is a membrane efflux pump protein that is involved in multidrug resistance (MDR). Tumour cells with high P-gp expression show poor response to cancer treatment with several chemotherapeutics. In vivo targeting and visualisation of P-gp expression would allow MDR to be evaluated non-invasively prior to treatment. The aim of this study was to investigate the feasibility of visualising P-gp expression in tumours using a monoclonal anti-P-gp antibody, 15D3. Nude BALB/c mice with subcutaneously growing human uterine sarcoma cell tumours with either high (MES-SA/D x 5 1977) or low (MES-SA 1976) P-gp expression were used. When tumours were 0.2-0.4 g, mice received 131 I-15D3 or 111 In-DTPA-15D3 monoclonal anti-P-gp antibody intravenously. Images were acquired up to 3 days p.i. and radioactivity concentration in various tissues was determined after euthanisation of the animals. The images demonstrated that radioactivity accumulated to a higher concentration in high P-gp expressing tumours than in the low P-gp expressing MES-SA 1976 tumour. Furthermore, visualisation of the P-gp expressing tumours was superior with 111 In-DTPA-15D3 than with 131 I-15D3. After injection of 111 In-DTPA-15D3, the high P-gp expressing MES-SA/D x 5 1977 tumours were clearly visualised at 3 days p.i. The biodistribution data indicated that radioactivity concentration in the high P-gp expressing tumours was higher than in the tumours with low P-gp expression (20.78±1.42 %ID/g for MES-SA/Dx5 1977 tumours and 8.39±3.78 %ID/g for MES-SA 1976 tumours for 111 In-DTPA-15D3). The 111 In-labelled monoclonal anti-P-gp antibody clearly visualised P-gp expression in a human uterine sarcoma tumour in nude mice. (orig.)

  18. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  19. Matrix metalloproteinases in the mouse retina: a comparative study of expression patterns and MMP antibodies.

    Science.gov (United States)

    De Groef, Lies; Andries, Lien; Lemmens, Kim; Van Hove, Inge; Moons, Lieve

    2015-12-29

    Matrix metalloproteinases (MMPs), a family of Zn(2+)-dependent endoproteases, have been shown to act as fine regulators of both health and disease. Limited research revealed that they are essential to maintaining ocular physiology and inordinate MMP activities have been linked to several neurodegenerative disorders of the retina, including age-related macular degeneration, proliferative diabetic retinopathy and glaucomatous optic neuropathies (GONs). Nevertheless, a clear definition of their pathology-exacerbating and/or -resolving actions is lacking, especially in the context of GONs, as most studies thus far merely focused on expression profiling in human patients. Therefore, in an initial step towards an improved understanding of MMP functions in the retina, we studied the spatial expression pattern of MMP-2, -3, -9 and MT1-MMP in the healthy mouse retina. The spatial expression pattern of MMP-2, -3, -9 and MT1-MMP was studied in the healthy mouse retina via immunohistochemical stainings, and immunoreactivity profiles were compared to existing literature. Moreover, we considered sensitivity and specificity issues with commercially available MMP antibodies via Western blot. Basal expression of MMP-2,-3, -9 and MT1-MMP was found in the retina of healthy, adult mice. MMP-2 expression was seen in Müller glia, predominantly in their end feet, which is in line with available literature. MMP-3 expression was described for the first time in the retina, and was observed in vesicle-like structures along the radial fibers of Müller glia. MMP-9 expression, about which still discords exists, was seen in microglia and in a sparse subset of (apoptosing) RGCs. MT1-MMP localization was for the first time studied in adult mice and was found in RGC axons and Müller glia, mimicking the MT1-MMP expression pattern seen in rabbits and neonatal mice. Moreover, one antibody was selected for each MMP, based on its staining pattern in Western blot. The present MMP immunoreactivity

  20. Study of Pea Accessions for Development of an Oilseed Pea

    Directory of Open Access Journals (Sweden)

    Ehsan Khodapanahi

    2012-09-01

    Full Text Available Global interest in stable energy resources coupled with growing demand for bio-oils in various conventional and arising industries has renewed the importance of vegetable oil production. To address this global interest, oilseed production has been increased in recent decades by different approaches, such as extending the cultivation area of oil crops, or breeding and growing genetically modified plants. In this study, pea (Pisum sativum L. accessions were screened for lipid content using a rapid extraction method. This method quantifies lipid concentration in pea seeds and was developed by assessing and comparing the results of existing extraction methods used for canola and soybean, the top two Canadian oilseeds. Seeds of 151 field pea accessions were grown to maturity in 2009 and 2010 at McGill University (Quebec, Canada. Overall, lipid concentration in pea seeds ranged from 0.9 to 5.0%. Among several seed characteristics, only seed shape (wrinkled verses round had a significant effect on the total lipid production in the seeds. Peas are a valuable source of protein and starch, but the lipid concentration in their seeds has been undervalued. This research supports the idea of developing a novel dual-purpose oilseed pea that emulates the protein and oil production in soybean seeds while being conveniently adapted to a colder climate.

  1. Design of an Expression System for Rapid Production of Tri-Functional Antibody Substitution of Hybrid Hybridoma Technology

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Dehghani

    2018-03-01

    Full Text Available Tri-functional antibodies, as an effective novel tumor targeting agents, are composed of anti-CD3 rat IgG2b and an anti-tumor antigen antibody. We have intended to develop a novel drug system to induce the apoptosis of HER2-expressing tumor cells, activate and engage cytotoxic T lymphocytes, natural killer cells, and macrophages. In addition, the drug can  inhibit programmed death ligand 1 (PDL-1-expressing tumor cells. We have designed a mammalian vector system suitable to express the trifunctional antibody composed of antiHER2×human-CD80: human-IgG1Fc antibody. This antibody contains four chains of anti-HER2/CL, anti-HER2/CH1-3, B7.1/CL, and B7.1/CH1-3 within the human IgG1 framework and so the vector system will simultaneously express the four chains. The amino acid/nucleotide sequences datasets were retrieved from the GenBank, UniProtKB and PDB databases. The heavy and light chains variable domain framework regions and complementarity determining regions of scA21 antibody were determined by IMGT/V-QUEST and Paratome software. The amino acid sequences of tri-functional antibody chains manually were assembled and converted to DNA sequences by sequence manipulation suite software. The adapting codon usage of these DNA sequences was performed by JCAT software. Finally, the secondary structures of obtained RNAs from the DNAs were individually analyzed by RNAfold program. The Prodigy equilibrium dissociation constant, a ratio of koff/kon, between the antibody and its antigen for hantiHER2VHCH-HER2, hantiHER2VLCL-HER2, CD80CH-CD28, and CD80CL-CD28 were equal to 3.60E-10, 3.10E-9, 1.10E-8 and 2.70E-10; respectively. These findings were confirmed the composition and nano-molar affinity of the respective constructs. A single specific no-cloning expression vector, pHuchiTriomAb, was designed in silico with a desirable length of 8.292 Kbps and bidirectional expression potential for the four chimeric antibody chains. This construct was designed, and

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

    Science.gov (United States)

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

    2015-02-05

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

  3. Rapid transcriptome characterization and parsing of sequences in a non-model host-pathogen interaction; pea-Sclerotinia sclerotiorum

    Directory of Open Access Journals (Sweden)

    Zhuang Xiaofeng

    2012-11-01

    Full Text Available Abstract Background White mold, caused by Sclerotinia sclerotiorum, is one of the most important diseases of pea (Pisum sativum L., however, little is known about the genetics and biochemistry of this interaction. Identification of genes underlying resistance in the host or pathogenicity and virulence factors in the pathogen will increase our knowledge of the pea-S. sclerotiorum interaction and facilitate the introgression of new resistance genes into commercial pea varieties. Although the S. sclerotiorum genome sequence is available, no pea genome is available, due in part to its large genome size (~3500 Mb and extensive repeated motifs. Here we present an EST data set specific to the interaction between S. sclerotiorum and pea, and a method to distinguish pathogen and host sequences without a species-specific reference genome. Results 10,158 contigs were obtained by de novo assembly of 128,720 high-quality reads generated by 454 pyrosequencing of the pea-S. sclerotiorum interactome. A method based on the tBLASTx program was modified to distinguish pea and S. sclerotiorum ESTs. To test this strategy, a mixture of known ESTs (18,490 pea and 17,198 S. sclerotiorum ESTs from public databases were pooled and parsed; the tBLASTx method successfully separated 90.1% of the artificial EST mix with 99.9% accuracy. The tBLASTx method successfully parsed 89.4% of the 454-derived EST contigs, as validated by PCR, into pea (6,299 contigs and S. sclerotiorum (2,780 contigs categories. Two thousand eight hundred and forty pea ESTs and 996 S. sclerotiorum ESTs were predicted to be expressed specifically during the pea-S. sclerotiorum interaction as determined by homology search against 81,449 pea ESTs (from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings and 57,751 S. sclerotiorum ESTs (from mycelia at neutral pH, developing apothecia and developing sclerotia. Among those ESTs specifically expressed

  4. Rapid transcriptome characterization and parsing of sequences in a non-model host-pathogen interaction; pea-Sclerotinia sclerotiorum.

    Science.gov (United States)

    Zhuang, Xiaofeng; McPhee, Kevin E; Coram, Tristan E; Peever, Tobin L; Chilvers, Martin I

    2012-11-26

    White mold, caused by Sclerotinia sclerotiorum, is one of the most important diseases of pea (Pisum sativum L.), however, little is known about the genetics and biochemistry of this interaction. Identification of genes underlying resistance in the host or pathogenicity and virulence factors in the pathogen will increase our knowledge of the pea-S. sclerotiorum interaction and facilitate the introgression of new resistance genes into commercial pea varieties. Although the S. sclerotiorum genome sequence is available, no pea genome is available, due in part to its large genome size (~3500 Mb) and extensive repeated motifs. Here we present an EST data set specific to the interaction between S. sclerotiorum and pea, and a method to distinguish pathogen and host sequences without a species-specific reference genome. 10,158 contigs were obtained by de novo assembly of 128,720 high-quality reads generated by 454 pyrosequencing of the pea-S. sclerotiorum interactome. A method based on the tBLASTx program was modified to distinguish pea and S. sclerotiorum ESTs. To test this strategy, a mixture of known ESTs (18,490 pea and 17,198 S. sclerotiorum ESTs) from public databases were pooled and parsed; the tBLASTx method successfully separated 90.1% of the artificial EST mix with 99.9% accuracy. The tBLASTx method successfully parsed 89.4% of the 454-derived EST contigs, as validated by PCR, into pea (6,299 contigs) and S. sclerotiorum (2,780 contigs) categories. Two thousand eight hundred and forty pea ESTs and 996 S. sclerotiorum ESTs were predicted to be expressed specifically during the pea-S. sclerotiorum interaction as determined by homology search against 81,449 pea ESTs (from flowers, leaves, cotyledons, epi- and hypocotyl, and etiolated and light treated etiolated seedlings) and 57,751 S. sclerotiorum ESTs (from mycelia at neutral pH, developing apothecia and developing sclerotia). Among those ESTs specifically expressed, 277 (9.8%) pea ESTs were predicted to be

  5. Quality Control of Canned Peas

    Directory of Open Access Journals (Sweden)

    Lucica Nistor

    2010-05-01

    Full Text Available Vegetables are food of vegetable origin which has an important place in human nutrition because of their complex chemical composition. With the discovery of causes that cause alteration of food (the vital action of microorganisms, the problem of food conservation began to be substantiated in terms scientific, in that way that the present conservation means not only to avoid microbial spoilage of food, but also keeping most of the nutritional and organoleptic properties of the product under conservation. The paper is structured in six chapters that have followed all the stages of pea to preserve the finished product-peas canned, and changes occurring in the process of preservation by analyzing four different kinds of peas. In this paper work is has been showed fat content determination, determination of sodium chloride and microbiological control of canned peas.

  6. A Comparative Antibody Analysis of Pannexin1 Expression in Four Rat Brain Regions Reveals Varying Subcellular Localizations

    OpenAIRE

    Cone, Angela C.; Ambrosi, Cinzia; Scemes, Eliana; Martone, Maryann E.; Sosinsky, Gina E.

    2013-01-01

    Pannexin1 (Panx1) channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding p...

  7. [Expression of recombinant rubella virus E1 protein and initial application for detecting of antibody].

    Science.gov (United States)

    Yi, Yao; Guo, Min-zhuo; Yu, Tao; Xu, Wen-bo; Yang, Jin-ye; Chen, Si-yong

    2008-10-01

    To apply recombinant rubella virus envelope protein-1 (E1) to detect human rubella virus IgG antibody. Rubella virus E1 protein was expressed in E. coli, purified E1 protein was used as the antigen for the detecting of anti rubella in human sera in the way of enzyme linked Immunosorbant assay (ELISA). The antigenicity of the recombinant protein was checked by WHO rubella sera panel. We detected 200 sera samples, which came from Guangxi Guilin. 93% of these samples were positive. The antigenicity of recombinant E1 is a satisfied candidate antigen for the detecting of human rubella virus antibody. The prevalence of anti rubella virus IgG in Guangxi is 93%. It is at the some level compared with other provinces in China.

  8. Intracellular reprogramming of expression, glycosylation, and function of a plant-derived antiviral therapeutic monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Jeong-Hwan Lee

    Full Text Available Plant genetic engineering, which has led to the production of plant-derived monoclonal antibodies (mAb(Ps, provides a safe and economically effective alternative to conventional antibody expression methods. In this study, the expression levels and biological properties of the anti-rabies virus mAb(P SO57 with or without an endoplasmic reticulum (ER-retention peptide signal (Lys-Asp-Glu-Leu; KDEL in transgenic tobacco plants (Nicotiana tabacum were analyzed. The expression levels of mAb(P SO57 with KDEL (mAb(PK were significantly higher than those of mAb(P SO57 without KDEL (mAb(P regardless of the transcription level. The Fc domains of both purified mAb(P and mAb(PK and hybridoma-derived mAb (mAb(H had similar levels of binding activity to the FcγRI receptor (CD64. The mAb(PK had glycan profiles of both oligomannose (OM type (91.7% and Golgi type (8.3%, whereas the mAb(P had mainly Golgi type glycans (96.8% similar to those seen with mAb(H. Confocal analysis showed that the mAb(PK was co-localized to ER-tracker signal and cellular areas surrounding the nucleus indicating accumulation of the mAb(P with KDEL in the ER. Both mAb(P and mAb(PK disappeared with similar trends to mAb(H in BALB/c mice. In addition, mAb(PK was as effective as mAb(H at neutralizing the activity of the rabies virus CVS-11. These results suggest that the ER localization of the recombinant mAb(P by KDEL reprograms OM glycosylation and enhances the production of the functional antivirus therapeutic antibody in the plant.

  9. A comparative antibody analysis of Pannexin1 expression in four rat brain regions reveals varying subcellular localizations

    Directory of Open Access Journals (Sweden)

    Angela C Cone

    2013-02-01

    Full Text Available Pannexin1 (Panx1 channels release cytosolic ATP in response to signaling pathways. Panx1 is highly expressed in the central nervous system. We used four antibodies with different Panx1 anti-peptide epitopes to analyze four regions of rat brain. These antibodies labeled the same bands in Western blots and had highly similar patterns of immunofluorescence in tissue culture cells expressing Panx1, but Western blots of brain lysates from Panx1 knockout and control mice showed different banding patterns. Localizations of Panx1 in brain slices were generated using automated wide-field mosaic confocal microscopy for imaging large regions of interest while retaining maximum resolution for examining cell populations and compartments. We compared Panx1 expression over the cerebellum, hippocampus with adjacent cortex, thalamus and olfactory bulb. While Panx1 localizes to the same neuronal cell types, subcellular localizations differ. Two antibodies with epitopes against the intracellular loop and one against the carboxy terminus preferentially labeled cell bodies, while an antibody raised against an N-terminal peptide highlighted neuronal processes more than cell bodies. These labeling patterns may be a reflection of different cellular and subcellular localizations of full-length and/or modified Panx1 channels where each antibody is highlighting unique or differentially accessible Panx1 populations. However, we cannot rule out that one or more of these antibodies have specificity issues. All data associated with experiments from these four antibodies are presented in a manner that allows them to be compared and our claims thoroughly evaluated, rather than eliminating results that were questionable. Each antibody is given a unique identifier through the NIF Antibody Registry that can be used to track usage of individual antibodies across papers and all image and metadata are made available in the public repository, the Cell Centered Database, for on

  10. [Gene cloning, expression and polyclonal antibody preparation and application of translocated intimin receptor-cytoskeleton coupling protein].

    Science.gov (United States)

    Yang, Qin; Cao, Jun-hao; Ding, Jin-ya; Huang, Qian-chuan

    2012-12-01

    To prepare translocated intimin receptor-cytoskeleton coupling protein (TccP) of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 and its polyclonal antibody. TccP was amplified from the genome of EHEC O157:H7 Sakai strain by PCR and used to construct the recombinant prokaryotic expression vector pET28a-TccP. The recombinant vector was transformed into E.coli BL21( DE3) to express the protein in the bacteria under the induction of isopropy-D-thiogalactoside (IPTG). After purification, the protein was injected into New Zealand rabbits to prepare polyclonal antibody. Then the antibody was tested by ELISA and Western blotting for its sensitivity and specificity. The rabbit anti-TccP polyclonal antibody was then applied in the study on the localization of TccP within the host cells adhered by EHEC O157:H7. The sequence of TccP cDNA we amplified was the same as reported by GenBank. The recombinant prokaryotic expression vector pET28a-TccP was constructed successfully. Western blotting revealed that M(r); of the target protein expressed in E.coli BL21(DE3) was 37 000 and the rabbit anti-TccP polyclonal antibody had a specific reaction with the target protein, which demonstrated that the recombinant protein and its polyclonal antibody were prepared successfully. Immunofluorescence detection using rabbit anti-TccP polyclonal antibody showed that TccP aggregated in the cell membrane of the host cell adhered by EHEC O157:H7. We successfully prepared the recombinant vector pET28a-TccP and the anti-TccP polyclonal antibody and applied the antibody to confirm the localization of TccP in EHEC O157:H7 adhesion host cells.

  11. The ERK MAP kinase-PEA3/ETV4-MMP-1 axis is operative in oesophageal adenocarcinoma

    LENUS (Irish Health Repository)

    Keld, Richard

    2010-12-09

    Abstract Background Many members of the ETS-domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-domain transcription factors. The PEA3 subfamily of ETS-domain transcription factors have been implicated in tumour metastasis in several different cancers. Results Here, we have studied the expression of the PEA3 subfamily members PEA3\\/ETV4 and ER81\\/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas. Conclusions This study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.

  12. Pichia pastoris-Expressed Bivalent Virus-Like Particulate Vaccine Induces Domain III-Focused Bivalent Neutralizing Antibodies without Antibody-Dependent Enhancement in Vivo

    Directory of Open Access Journals (Sweden)

    Rahul Shukla

    2018-01-01

    Full Text Available Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4. Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE. A recently launched live attenuated vaccine (LAV for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII. Recently, we found that Pichia pastoris-expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in P. pastoris. The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs designated as mE1E2bv VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus

  13. Deletion of PEA-15 in mice is associated with specific impairments of spatial learning abilities

    Directory of Open Access Journals (Sweden)

    Hale Gregory

    2009-11-01

    Full Text Available Abstract Background PEA-15 is a phosphoprotein that binds and regulates ERK MAP kinase and RSK2 and is highly expressed throughout the brain. PEA-15 alters c-Fos and CREB-mediated transcription as a result of these interactions. To determine if PEA-15 contributes to the function of the nervous system we tested mice lacking PEA-15 in a series of experiments designed to measure learning, sensory/motor function, and stress reactivity. Results We report that PEA-15 null mice exhibited impaired learning in three distinct spatial tasks, while they exhibited normal fear conditioning, passive avoidance, egocentric navigation, and odor discrimination. PEA-15 null mice also had deficient forepaw strength and in limited instances, heightened stress reactivity and/or anxiety. However, these non-cognitive variables did not appear to account for the observed spatial learning impairments. The null mice maintained normal weight, pain sensitivity, and coordination when compared to wild type controls. Conclusion We found that PEA-15 null mice have spatial learning disabilities that are similar to those of mice where ERK or RSK2 function is impaired. We suggest PEA-15 may be an essential regulator of ERK-dependent spatial learning.

  14. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    International Nuclear Information System (INIS)

    Bujak, Emil; Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah; Neri, Dario

    2014-01-01

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  15. Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

    Energy Technology Data Exchange (ETDEWEB)

    Bujak, Emil [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland); Pretto, Francesca; Ritz, Danilo; Gualandi, Laura; Wulhfard, Sarah [Philochem AG, Libernstrasse 3, CH-8112 Otelfingen (Switzerland); Neri, Dario, E-mail: neri@pharma.ethz.ch [Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zürich), Vladimir-Prelog-Weg 2, CH-8093 Zurich (Switzerland)

    2014-09-10

    There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer. - Highlights: • High affinity monoclonal antibodies to murine and human TSP1 and 2 were raised. • Both antigens are virtually undetectable in normal mouse tissues. • Strong positivity of human tumor xenografts for TSP1 was detected. • Study revealed much lower level of TSP2 expression in cancer specimens

  16. PEA-15 (Phosphoprotein Enriched in Astrocytes 15) Is a Protective Mediator in the Vasculature and Is Regulated During Neointimal Hyperplasia.

    Science.gov (United States)

    Greig, Fiona H; Kennedy, Simon; Gibson, George; Ramos, Joe W; Nixon, Graeme F

    2017-09-11

    Neointimal hyperplasia following angioplasty occurs via vascular smooth muscle cell proliferation. The mechanisms involved are not fully understood but include mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2). We recently identified the intracellular mediator PEA-15 (phosphoprotein enriched in astrocytes 15) in vascular smooth muscle cells as a regulator of ERK1/2-dependent proliferation in vitro. PEA-15 acts as a cytoplasmic anchor for ERK1/2, preventing nuclear localization and thereby reducing ERK1/2-dependent gene expression. The aim of the current study was to examine the role of PEA-15 in neointimal hyperplasia in vivo. Mice deficient in PEA-15 or wild-type mice were subjected to wire injury of the carotid artery. In uninjured arteries from PEA-15-deficient mice, ERK1/2 had increased nuclear translocation and increased basal ERK1/2-dependent transcription. Following wire injury, arteries from PEA-15-deficient mice developed neointimal hyperplasia at an increased rate compared with wild-type mice. This occurred in parallel with an increase in a proliferative marker and vascular smooth muscle cell proliferation. In wild-type mice, PEA-15 expression was decreased in vascular smooth muscle cells at an early stage before any increase in intima:media ratio. This regulation of PEA-15 expression following injury was also observed in an ex vivo human model of hyperplasia. These results indicate, for the first time, a novel protective role for PEA-15 against inappropriate vascular proliferation. PEA-15 expression may also be repressed during vascular injury, suggesting that maintenance of PEA-15 expression is a novel therapeutic target in vascular disease. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  17. Expression and detection of ScFvB9 and its mutant in recombinant phage antibody system.

    Science.gov (United States)

    Yang, E J; Kwak, J W; Chang, H C

    2001-01-01

    Recombinant single-chain antibody (ScFvB9) and its mutant (ScFvB9-6) were generated by using a polymerase chain reaction (PCR) from the Fab fragment of the murine monoclonal antibody (MAb) B9, MabB9 (gamma2b,kappa), which is specific for human plasma apolipoprotein (apo) B-100 of low density lipopreotein (LDL). In the recombinant phage antibody system (RPAS), the constructed ScFvB9 and ScFvB9-6 antibody genes were cloned into the pCANTAB5E phagemid vector and expressed in E. coli. The active forms of single-chain antibodies (ScFvB9 and ScFvB9-6) were produced as phage-displayed recombinant antibodies or soluble antibody forms in E. coli. The activities of ScFvB9 and ScFvB9-6 were confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting analysis; the generated mutant ScFvB9-6 showed slightly higher antigen binding activity than native ScFvB9 as a soluble antibody in this RPAS.

  18. Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-08-01

    Single domain antibodies (sdAbs) are small antigen-binding domains derived from naturally occurring, heavy chain-only immunoglobulins isolated from camelid and sharks. They maintain the same binding capability of full-length IgGs but with improved thermal stability and permeability, which justifies their scientific, medical and industrial interest. Several described recombinant forms of sdAbs have been produced in different hosts and with different strategies. Here we present an optimized method for a time-saving, high yield production and extraction of a poly-histidine-tagged sdAb from Escherichia coli classical inclusion bodies. Protein expression and extraction were attempted using 4 different methods (e.g. autoinducing or IPTG-induced soluble expression, non-classical and classical inclusion bodies). The best method resulted to be expression in classical inclusion bodies and urea-mediated protein extraction which yielded 60-70 mg/l bacterial culture. The method we here describe can be of general interest for an enhanced and efficient heterologous expression of sdAbs for research and industrial purposes. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. De Novo Assembly of the Pea (Pisum sativum L. Nodule Transcriptome

    Directory of Open Access Journals (Sweden)

    Vladimir A. Zhukov

    2015-01-01

    Full Text Available The large size and complexity of the garden pea (Pisum sativum L. genome hamper its sequencing and the discovery of pea gene resources. Although transcriptome sequencing provides extensive information about expressed genes, some tissue-specific transcripts can only be identified from particular organs under appropriate conditions. In this study, we performed RNA sequencing of polyadenylated transcripts from young pea nodules and root tips on an Illumina GAIIx system, followed by de novo transcriptome assembly using the Trinity program. We obtained more than 58,000 and 37,000 contigs from “Nodules” and “Root Tips” assemblies, respectively. The quality of the assemblies was assessed by comparison with pea expressed sequence tags and transcriptome sequencing project data available from NCBI website. The “Nodules” assembly was compared with the “Root Tips” assembly and with pea transcriptome sequencing data from projects indicating tissue specificity. As a result, approximately 13,000 nodule-specific contigs were found and annotated by alignment to known plant protein-coding sequences and by Gene Ontology searching. Of these, 581 sequences were found to possess full CDSs and could thus be considered as novel nodule-specific transcripts of pea. The information about pea nodule-specific gene sequences can be applied for gene-based markers creation, polymorphism studies, and real-time PCR.

  20. FX knockout CHO hosts can express desired ratios of fucosylated or afucosylated antibodies with high titers and comparable product quality.

    Science.gov (United States)

    Louie, Salina; Haley, Benjamin; Marshall, Brett; Heidersbach, Amy; Yim, Mandy; Brozynski, Martina; Tang, Danming; Lam, Cynthia; Petryniak, Bronislawa; Shaw, David; Shim, Jeongsup; Miller, Aaron; Lowe, John B; Snedecor, Brad; Misaghi, Shahram

    2017-03-01

    During antibody dependent cell cytotoxicity (ADCC) the target cells are killed by monocytes and natural killer cells. ADCC is enhanced when the antibody heavy chain's core N-linked glycan lacks the fucose molecule(s). Several strategies have been utilized to generate fully afucosylated antibodies. A commonly used and efficient approach has been knocking out the FUT8 gene of the Chinese hamster ovary (CHO) host cells, which results in expression of antibody molecules with fully afucosylated glycans. However, a major drawback of the FUT8-KO host is the requirement for undertaking two separate cell line development (CLD) efforts in order to obtain both primarily fucosylated and fully afucosylated antibody species for comparative studies in vitro and in vivo. Even more challenging is obtaining primarily fucosylated and FUT8-KO clones with similar enough product quality attributes to ensure that any observed ADCC advantage(s) can be strictly attributed to afucosylation. Here, we report generation and use of a FX knockout (FXKO) CHO host cell line that is capable of expressing antibody molecules with either primarily fucosylated or fully afucosylated glycan profiles with otherwise similar product quality attributes, depending on addition of fucose to the cell culture media. Hence, the FXKO host not only obviates the requirement for undertaking two separate CLD efforts, but it also averts the need for screening many colonies to identify clones with comparable product qualities. Finally, FXKO clones can express antibodies with the desired ratio of primarily fucosylated to afucosylated glycans when fucose is titrated into the production media, to allow achieving intended levels of FcγRIII-binding and ADCC for an antibody. Biotechnol. Bioeng. 2017;114: 632-644. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Characterization of mechanical properties of transgenic tobacco roots expressing a recombinant monoclonal antibody against tooth decay.

    Science.gov (United States)

    Hassan, Sally; Liu, Wei; Ma, Julian K-C; Thomas, Colin R; Keshavarz-Moore, Eli

    2008-07-01

    In this article, we describe a new approach that allows the determination of the magnitude of force required to break single plant roots. Roots were taken from transgenic tobacco plants, expressing a secreted monoclonal antibody. They were divided into four key developmental stages. A novel micromanipulation technique was used to pull to breakage, single tobacco roots in buffer in order to determine their breaking force. A characteristic uniform step-wise increase in the force up to a peak force for breakage was observed. The mean breaking force and mean work done were 101mN and 97microJ per root respectively. However, there was a significant increase in breaking force from the youngest white roots to the oldest, dark red-brown roots. We speculate that this was due to increasing lignin deposition with root stage of development (shown by phloroglucinol staining). No significant differences between fresh root mass, original root length, or mean root diameter for any of the root categories were found, displaying their uniformity, which would be beneficial for bioprocessing. In addition, no significant difference in antibody yield from the different root categories was found. These data show that it is possible to characterise the force requirements for root breakage and should assist in the optimisation of recombinant protein extraction from these roots. (c) 2008 Wiley Periodicals, Inc.

  2. Expression of basal cell marker revealed by RAM11 antibody during epithelial regeneration in rabbits.

    Directory of Open Access Journals (Sweden)

    Tadeusz Cichocki

    2010-06-01

    Full Text Available RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular macrophages. Our previous report showed that RAM11 reacted with basal cells of stratified squamous epithelia of rabbit skin, oral mucosa and esophagus. The aim of the present study was to follow the appearance of RAM11 immunoreactivity in basal cells of regenerating oral epithelium in rabbits. No RAM11 immunostaining was observed in the regenerating epithelium examined on days 1 and 3 of wound healing. A weak immunofluorescence first appeared on day 7 in single basal cells and 32% of RAM11- positive basal cells were observed on day 14. These findings indicate that expression of the antigen recognized by RAM11 antibody is a transient event in the differentiation of oral keratinocytes which not always occurs during epithelial repair, although it is a constant feature of epithelial turnover in mature epithelium. Therefore this antigen can be regarded as basal cell marker only in mature stratified squamous epithelia.

  3. Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Imanaka Tadayuki

    2002-09-01

    Full Text Available Abstract Background Hep27 monoclonal (Hep27 Mab is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102. We attempted to produce a single-chain fragment (scFv, a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. Results The sequences encoding the variable regions of heavy (VH and light (VL chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa. Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab. Conclusion This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.

  4. Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma.

    Science.gov (United States)

    Sandee, Duanpen; Tungpradabkul, Sumalee; Tsukio, Manae; Imanaka, Tadayuki; Takagi, Masahiro

    2002-09-12

    Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. The sequences encoding the variable regions of heavy (VH) and light (VL) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)3 and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab). This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy.

  5. Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

    Science.gov (United States)

    Yu, Yongjiao; Fu, Lu; Jiang, Xiaoyu; Guan, Shanshan; Kuai, Ziyu; Kong, Wei; Shi, Yuhua; Shan, Yaming

    2016-12-01

    Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Secretory signal peptide modification for optimized antibody-fragment expression-secretion in Leishmania tarentolae

    Directory of Open Access Journals (Sweden)

    Klatt Stephan

    2012-07-01

    Full Text Available Abstract Background Secretory signal peptides (SPs are well-known sequence motifs targeting proteins for translocation across the endoplasmic reticulum membrane. After passing through the secretory pathway, most proteins are secreted to the environment. Here, we describe the modification of an expression vector containing the SP from secreted acid phosphatase 1 (SAP1 of Leishmania mexicana for optimized protein expression-secretion in the eukaryotic parasite Leishmania tarentolae with regard to recombinant antibody fragments. For experimental design the online tool SignalP was used, which predicts the presence and location of SPs and their cleavage sites in polypeptides. To evaluate the signal peptide cleavage site as well as changes of expression, SPs were N-terminally linked to single-chain Fragment variables (scFv’s. The ability of L. tarentolae to express complex eukaryotic proteins with highly diverse post-translational modifications and its easy bacteria-like handling, makes the parasite a promising expression system for secretory proteins. Results We generated four vectors with different SP-sequence modifications based on in-silico analyses with SignalP in respect to cleavage probability and location, named pLTEX-2 to pLTEX-5. To evaluate their functionality, we cloned four individual scFv-fragments into the vectors and transfected all 16 constructs into L. tarentolae. Independently from the expressed scFv, pLTEX-5 derived constructs showed the highest expression rate, followed by pLTEX-4 and pLTEX-2, whereas only low amounts of protein could be obtained from pLTEX-3 clones, indicating dysfunction of the SP. Next, we analysed the SP cleavage sites by Edman degradation. For pLTEX-2, -4, and -5 derived scFv’s, the results corresponded to in-silico predictions, whereas pLTEX-3 derived scFv’s contained one additional amino-acid (AA. Conclusions The obtained results demonstrate the importance of SP-sequence optimization for efficient

  7. Reduction of malaria transmission by transgenic mosquitoes expressing an antisporozoite antibody in their salivary glands.

    Science.gov (United States)

    Sumitani, M; Kasashima, K; Yamamoto, D S; Yagi, K; Yuda, M; Matsuoka, H; Yoshida, S

    2013-02-01

    We have previously developed a robust salivary gland-specific expression system in transgenic Anopheles stephensi mosquitoes. To establish transgenic mosquito lines refractory to Plasmodium falciparum using this system, we generated a transgenic mosquito harbouring the gene encoding an anti-P. falciparum circumsporozoite protein (PfCSP) single-chain antibody (scFv) fused to DsRed in a secretory form (mDsRed-2A10 scFv). Fluorescence microscopy showed that the mDsRed-2A10 scFv was localized in the secretory cavities and ducts of the salivary glands in a secreted form. To evaluate P. falciparum transmission-blocking in a rodent malaria model, a transgenic Plasmodium berghei line expressing PfCSP in place of PbCSP (PfCSP/Pb) was constructed. The PfCSP/Pb parasites were able to bind to the mDsRed-2A10 scFv in the salivary glands of the transgenic mosquitoes. Importantly, the infectivity of the transgenic mosquitoes to mice was strongly impaired, indicating that the parasites had been inactivated. These results suggest that salivary gland-specific expression of antisporozoite molecules could be a promising strategy for blocking malaria transmission to humans. © 2012 Royal Entomological Society.

  8. Efficient production of antibody Fab fragment by transient gene expression in insect cells.

    Science.gov (United States)

    Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

    2017-08-01

    Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  9. In vitro functional test of two subclasses of an anti-RhD antibody produced by transient expression in COS cells

    DEFF Research Database (Denmark)

    Nielsen, Leif Kofoed; Norderhaug, Lars; Sandlie, Inger

    2006-01-01

    that other sources of anti-RhD will be needed. One such source is recombinant human antibodies. Here we describe the construction of plasmids encoding two subclasses (IgG1 and IgG3) of an anti-RhD antibody, their transient expression in COS cells, and subsequent functional characterization of the antibodies...

  10. Detection of MHC class II expression on human basophils is dependent on antibody specificity but independent of atopic disposition

    DEFF Research Database (Denmark)

    Poulsen, Britta Cathrina; Poulsen, Lars K.; Jensen, Bettina M

    2012-01-01

    A debate has recently arisen as to whether murine basophils can function as antigen presenting cells in allergic inflammation. However, mouse and human basophils differ considerably, and the expression of MHC class II on human basophils has been investigated as a proxy for their capability...... of antigen presentation but conflicting results have emerged. In this technical note, we show that an antibody specific for all three MHC class II subtypes (HLA-DR, -DP, and -DQ), leads to a significantly higher amount of MHC class II+ basophils compared to antibodies specific for HLA-DR only. A significant...... difference was also observed between the HLA-DR specific antibodies, indicating that the choice of antibody is crucial. Furthermore, critical compensation was essential to avoid false HLA-DR+ basophils. Finally, we found that detection of MHC class II on human basophils was independent of atopic disposition....

  11. Mutant genes in pea breeding

    International Nuclear Information System (INIS)

    Swiecicki, W.K.

    1990-01-01

    Full text: Mutations of genes Dpo (dehiscing pods) and A (anthocyanin synthesis) played a role in pea domestication. A number of other genes were important in cultivar development for 3 types of usage (dry seeds, green vegetable types, fodder), e.g. fn, fna, le, p, v, fas and af. New genes (induced and spontaneous), are important for present ideotypes and are registered by the Pisum Genetics Association (PGA). Comparison of a pea variety ideotype with the variation available in gene banks shows that breeders need 'new' features. In mutation induction experiments, genotype, mutagen and method of treatment (e.g. combined or fractionated doses) are varied for broadening the mutation spectrum and selecting more genes of agronomic value. New genes are genetically analysed. In Poland, some mutant varieties with the gene afila were registered, controlling lodging by a shorter stem and a higher number of internodes. Really non-lodging pea varieties could strongly increase seed yield. But the probability of detecting a major gene for lodging resistance is low. Therefore, mutant genes with smaller influence on plant architecture are sought, to combine their effect by crossing. Promising seem to be the genes rogue, reductus and arthritic as well as a number of mutant genes not yet genetically identified. The gene det for terminal inflorescence - similarly to Vicia faba - changes plant development. Utilisation of assimilates and ripening should be better. Improvement of harvest index should give higher seed yield. A number of genes controlling disease resistance are well known (eg. Fw, Fnw, En, mo and sbm). Important in mass screening of resistance are closely linked gene markers. Pea gene banks collect respective lines, but mutants induced in highly productive cultivars would be better. Inducing gene markers sometimes seems to be easier than transfer by crossing. Mutation induction in pea breeding is probably more important because a high number of monogenic features are

  12. Expression cloning and production of human heavy-chain-only antibodies from murine transgenic plasma cells

    NARCIS (Netherlands)

    D.D. Drabek (Dubravka); R. Janssens (Rick); Boer, E. (Ernie de); Rademaker, R. (Rik); Kloess, J. (Johannes); J.J. Skehel (John ); Grosveld, F. (Frank)

    2016-01-01

    textabstractSeveral technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies, this involves either random pairing of VH and

  13. A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and nod factor induced expression

    DEFF Research Database (Denmark)

    Vijn, I; Christiansen, H; Lauridsen, P

    1995-01-01

    previously described. The isolation and characterization of a PsENOD12A genomic clone is presented in this paper. By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V. hirsuta root nodules. Promoter analyses...

  14. Adaptation of Pea to contrasted French regions : simulation of pea varieties with the AZODYN-Pea crop

    OpenAIRE

    Benezit, Maud; Larmure, Annabelle; Munier-Jolain, Nathalie; Jeuffroy, Marie-Helene

    2016-01-01

    Pea (Pisum sativum L.) is a particularly sensitive crop regarding abiotic stress throughout its cycle (Schneider and Huyghe 2015). Climate change results in the increase of the unpredictability of both the frequency and the intensity of these stresses (Stocker et al. 2013). To avoid abiotic stress, breeders have been developing, for the last fifteen years, winter pea varieties sown during autumn, more resistant to frost with an earlier flowering date than spring pea, as well as varieties call...

  15. Surface expression of protein A on magnetosomes and capture of pathogenic bacteria by magnetosome/ antibody complexes

    Directory of Open Access Journals (Sweden)

    Jun eXu

    2014-04-01

    Full Text Available Magnetosomes are membrane-enclosed magnetite nanocrystals synthesized by magnetotactic bacteria (MTB. They display chemical purity, narrow size ranges, and species-specific crystal morphologies. Specific transmembrane proteins are sorted to the magnetosome membrane (MM. MamC is the most abundant MM protein of Magnetospirillum gryphiswaldense strain MSR-1. MamF is the second most abundant MM protein of MSR-1 and forms stable oligomers. We expressed staphylococcal protein A (SPA, an immunoglobulin-binding protein from the cell wall of Staphylococcus aureus, on MSR-1 magnetosomes by fusion with MamC or MamF. The resulting recombinant magnetosomes were capable of self-assembly with the Fc region of mammalian antibodies (Abs and were therefore useful for functionalization of magnetosomes. Recombinant plasmids pBBR-mamC-spa and pBBR-mamF-spa were constructed by fusing spa (the gene that encodes SPA with mamC and mamF, respectively. Recombinant magnetosomes with surface expression of SPA were generated by introduction of these fusion genes into wild-type MSR-1 or a mamF mutant strain. Studies with a Zeta Potential Analyzer showed that the recombinant magnetosomes had hydrated radii significantly smaller than those of WT magnetosomes and zeta potentials less than -30 mV, indicating that the magnetosome colloids were relatively stable. Observed conjugation efficiencies were as high as 71.24 µg Ab per mg recombinant magnetosomes, and the conjugated Abs retained most of their activity. Numbers of Vibrio parahaemolyticus (a common pathogenic bacterium in seafood captured by recombinant magnetosome/ Ab complexes were measured by real-time fluorescence-based quantitative PCR. One mg of complex was capable of capturing as many as 1.74×107 Vibrio cells. The surface expression system described here will be useful for design of functionalized magnetosomes from MSR-1 and other MTB.

  16. Data on enhanced expression and purification of camelid single domain antibodies from Escherichia coli classical inclusion bodies.

    Science.gov (United States)

    Maggi, Maristella; Scotti, Claudia

    2017-06-01

    Heterologous expression of high amounts of recombinant proteins is a milestone for research and industrial purposes. Single domain antibodies (sdAbs) are heavy-chain only antibody fragments with applications in the biotechnological, medical and industrial fields. The simple nature and small size of sdAbs allows for efficient expression of the soluble molecule in different hosts. However, in some cases, it results in low functional protein yield. To overcome this limitation, expression of a 6xHistag sdAb was attempted in different conditions in Escherichia coli BL21(DE3) cells. Data showed that high amount of sdAb can be expressed in E. coli classical inclusion bodies, efficiently extracted by urea in a short-time, and properly purified by metal ion affinity chromatography. These data originate from the research article "Enhanced expression and purification of camelid single domain VHH antibodies from classical inclusion bodies" Maggi and Scotti (2017) [1] (DOI: http://dx.doi.org/10.1016/j.pep.2017.02.007).

  17. Prokaryotic Expression and Purification of Human TLE1 N-terminal Q Domain Fragment and Production of its Polyclonal Antibody

    Directory of Open Access Journals (Sweden)

    Su WANG

    2010-11-01

    Full Text Available Background and objective TLE1 is an important protein in regulating Wnt, Notch and EGFR signaling pathways. The TLE1 N-terminal Q domain regulates the pathways by mediating its oligomerization and interaction with LEF1. The aim of this study is to construct the human TLE1 N-terminal Q domain fragment in prokaryotic expression system, express and purify protein TLE1 N-terminal Q domain and prepare its polyclonal antibody. Methods The sequence of TLE1 N-terminal Q domain obtained by PCR from human lung adenocarcinoma cDNA, was cloned into the prokaryotic expression vector pGEX-4T-1 containing Glutathione S-transferase (GST. Vector pGEX-4T1-TLE1-Q was transformed into E.coli BL21 condon plus. The GST-TLE1-Q(1-136 fusion protein was induced by IPTG, digested by Thrombin, purified with glutathione-sepharose beads and FPLC, identified by SDS-PAGE. Then rabbits were immunized with the purified protein TLE1-Q(1-136 for obtaining the antiserum. The titers and specificity of antibodies were measured by ELISA and Western blot. Results The PCR identification and the sequencing of recombinant plasmid demonstrated that vector pGEX-4T1-TLE1-Q was successfully constructed. The SDS-PAGE shows target protein (14 000 Da is the interest protein TLE1-Q(1-136. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired, with an antibody titer of 1:20 000. Conclusion Expression vector pGEX-4T1-TLE1-Q is correctly constructed. The TLE1 N-terminal Q domain fragment TLE1-Q(1-136 and its polyclonal antibody have been acquired. These work established the foundation for further biological study between TLE1 and lung cancers.

  18. Immunohistochemical antigenic expression and in vivo tumor uptake of monoclonal antibodies with specificity for tumors of the gastrointestinal tract

    International Nuclear Information System (INIS)

    Douillard, J.Y.; Lehur, P.A.; Aillet, G.; Kremer, M.; Bianco-Arco, A.; Peltier, P.; Chatal, J.F.

    1986-01-01

    Two monoclonal antibodies with specificity for carcinoembryonic antigen and Ca 19-9 gastrointestinal tract tumor associated antigens were infused after iodination with 125 I and 131 I, respectively, in six patients 3 days and in one patient 4 days before radical surgery for colon or rectal carcinoma. Biopsy specimens from tumor, normal colon, fat, muscle, and skin along with a blood sample were excised at surgery and counting was performed for gamma emission. Fragments were then studied by two independent pathologists for immunohistochemical expression of corresponding antigens using the avidin-biotin peroxidase complex. A correlation study was thereafter performed between the amount of antibody bound in vivo, expressed as the percentage of injected dose per gram of tissue and the quantitative expression of tumor associated antigens, taking into account both the percentage of cells expressing the antigen and intensity of staining. For this limited number of patients a good correlation was found between amount of targeted antibodies and amount of expressed antigens. For carcinoembryonic antigen, r values were 0.69 and 0.90 for each pathologist (with an r value of interobserver correlation of 0.74); for Ca 19-9, values of 0.78 and 0.84 were obtained for each observer, with an interobserver r value of 0.97. Based on this limited study, it may be assumed that the possibility of imaging a given tumor is in part correlated to intensity of antigenic expression at the tumor site; other parameters, like tumor vascularization and blood flow for instance, are, however, to be considered for accessibility of antibodies to corresponding antigens

  19. Spatiotemporal Expression Patterns and Antibody Reactivity of Taeniidae Endophilin B1

    Science.gov (United States)

    Ahn, Chun-Seob; Bae, Young-An; Kim, Seon-Hee; Kim, Jeong-Geun; Yu, Jae-Ran; Yang, Hyun-Jong; Eom, Keeseon S.; Wang, Hu; Kang, Insug; Yang, Yichao

    2016-01-01

    Larval Taeniidae, such as metacestodes of Taenia solium, Echinococcus granulosus, and Echinococcus multilocularis, produce chronic and fatal helminthic diseases. Proper identification of these zoonotic cestodiases is often challenging and is hampered in some clinical settings. Endophilin B1 plays critical roles in the maintenance of membrane contours and endocytosis. We isolated proteins homologous to endophilin B1 from T. solium, Taenia saginata, and Taenia asiatica. The three Taeniidae endophilin B1 proteins shared 92.9 to 96.6% sequence identity. They harbored a Bin1/amphiphysin/Rvs (BAR) domain and residues for a dimeric interface but lacked a SRC homology 3 (SH3) domain. Endophilin B1 showed a unique immunological profile and was abundantly expressed in the tegumental syncytium of Taeniidae metacestodes and adults. Bacterially expressed recombinant T. solium endophilin B1 (rTsMEndoB1) demonstrated a sensitivity of 79.7% (345/433 cases) for serodiagnosis of larval Taeniidae infections. The protein showed strong immune recognition patterns against sera from patients with chronic neurocysticercosis, cystic echinococcosis, or advanced-stage alveolar echinococcosis. Adult Taeniidae infections exhibited moderate degrees of positive antibody responses (65.7% [23/35 samples]). rTsMEndoB1 showed some cross-reactivity with sera from patients infected with Diphyllobothriidae (23.6% [25/106 samples]) but not with sera from patients with other parasitic diseases or normal controls. The specificity was 91.7% (256/301 samples). The positive and negative predictive values were 93.6% and 73.4%, respectively. Our results demonstrate that Taeniidae endophilin B1 may be involved in the control of membrane dynamics, thus contributing to shaping and maintaining the tegumental curvature. rTsMEndoB1 may be useful for large-scale screening, as well as for individual diagnosis and follow-up surveillance of Taeniidae infections. PMID:27487955

  20. An anti-HIV-1 gp120 antibody expressed as an endocytotic transmembrane protein mediates internalization of HIV-1

    International Nuclear Information System (INIS)

    Tan, Yee-Joo; Lim, S.-P.; Ting, Anthony E.; Goh, Phuay-Yee; Tan, Y.H.; Lim, Seng Gee; Hong Wanjin

    2003-01-01

    In this study, we used HIV-1 as a model to demonstrate a novel approach for receptor-independent cell entry of virus. The heavy chain of an anti-HIV-1 gp120 antibody was engineered with endocytotic and transmembrane motifs from either the cation-independent mannose 6-phospate receptor or the low-density lipoprotein receptor. Flow cytometry and immunofluorescence studies showed that the chimeric antibodies were expressed on the cell surface and can undergo rapid internalization. Furthermore, one of the chimeric antibodies was able to bind and internalize HIV-1. Using a luciferase reporter HIV-1, we further showed that internalized viruses could undergo replication. Therefore, we have demonstrated a proof-of-principle of a novel method that can be used to internalize virus into cells, without prior knowledge of the cellular receptor for the virus. We propose that this approach would be particularly useful for studying viruses whose cellular receptor(s) is not known

  1. Protein expression profiling by antibody array analysis with use of dried blood spot samples on filter paper.

    Science.gov (United States)

    Jiang, Weidong; Mao, Ying Qing; Huang, Ruochun; Duan, Chaohui; Xi, Yun; Yang, Kai; Huang, Ruo-Pan

    2014-01-31

    Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Optimization of ELISA Conditions to Quantify Colorectal Cancer Antigen-Antibody Complex Protein (GA733-FcK) Expressed in Transgenic Plant

    OpenAIRE

    Ahn, Junsik; Lee, Kyung Jin; Ko, Kisung

    2014-01-01

    The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-F...

  3. Expression and purification of swine RAG2 in E. coli for production of porcine RAG2 polyclonal antibodies.

    Science.gov (United States)

    Jin, Yu-Bei; Yang, Wen-Tao; Huang, Ke-Yan; Chen, Hong-Liang; Shonyela, Seria-Masole; Liu, Jing; Liu, Qiong; Feng, Bo; Zhou, You; Zhi, Shu-Li; Jiang, Yan-Long; Wang, Jian-Zhong; Huang, Hai-Bin; Shi, Chun-Wei; Yang, Gui-Lian; Wang, Chun-Feng

    2017-08-01

    Recombination activating gene 2 (RAG2) is necessary for immature B cell differentiation. Antibodies to human and rabbit RAG2 are currently commercially available, but antibodies to swine RAG remain unavailable to date. In this study, the swine RAG2 genes sequence was synthesized and then cloned into a pET-28a vector. The recombinant fusion protein was successfully expressed in E. coli, purified through nickel column chromatography, and further digested with Tobacco Etch Virus protease. The cleaved protein was purified by molecular-exclusion chromatography and named pRAG2. We used pRAG2 to immunize rabbits, collected the serum and purified rabbit anti-pRAG2 polyclonal antibodies. The rabbit anti-pRAG2 polyclonal antibodies were tested via immunofluorescence on eukaryotic cells overexpressing pRAG2 and also able to recognize pig natural RAG2 and human RAG2 protein in western blotting. These results indicated that the prepared rabbit anti-pRAG2 polyclonal antibodies may serve as a tool to detect immature B cell differentiation of swine.

  4. Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

    Science.gov (United States)

    Meagher, Richard B.; Laterza, Vince

    2006-12-12

    The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

  5. Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

    Directory of Open Access Journals (Sweden)

    Rongzhi eWang

    2013-11-01

    Full Text Available Single-chain variable fragment (scFv is a class of engineered antibodies generated by the fusion of the heavy (VH and light chains (VL of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

  6. Transgenic expression in citrus of single-chain antibody fragments specific to Citrus tristeza virus confers virus resistance.

    Science.gov (United States)

    Cervera, Magdalena; Esteban, Olga; Gil, Maite; Gorris, M Teresa; Martínez, M Carmen; Peña, Leandro; Cambra, Mariano

    2010-12-01

    Citrus tristeza virus (CTV) causes one of the most destructive viral diseases of citrus worldwide. Generation of resistant citrus genotypes through genetic engineering could be a good alternative to control CTV. To study whether production of single-chain variable fragment (scFv) antibodies in citrus could interfere and immunomodulate CTV infection, transgenic Mexican lime plants expressing two different scFv constructs, separately and simultaneously, were generated. These constructs derived from the well-referenced monoclonal antibodies 3DF1 and 3CA5, specific against CTV p25 major coat protein, whose mixture is able to detect all CTV isolates characterized so far. ScFv accumulation levels were low and could be readily detected just in four transgenic lines. Twelve homogeneous and vigorous lines were propagated and CTV-challenged by graft inoculation with an aggressive CTV strain. A clear protective effect was observed in most transgenic lines, which showed resistance in up to 40-60% of propagations. Besides, both a delay in symptom appearance and attenuation of symptom intensity were observed in infected transgenic plants compared with control plants. This effect was more evident in lines carrying the 3DF1scFv transgene, being probably related to the biological functions of the epitope recognized by this antibody. This is the first report describing successful protection against a pathogen in woody transgenic plants by ectopic expression of scFv recombinant antibodies.

  7. Assessment varieties garden pea (Pisum sativum L. in terms of quality green peas and seeds

    Directory of Open Access Journals (Sweden)

    В. М. Стригун

    2014-03-01

    Full Text Available To obtain a high-quality source material for breeding new varieties of garden pea studied biochemical and technological properties of 450 sample grades collection. As a result, selected varieties, sources that had green peas in a high content of individual chemicals and combine incorporates several of these substances. These varieties have different duration of the growing season. Among them, identified those with green peas excessive ripeness not for a long time. Such quality varieties are especially important because provides over a longer period of receipt of a high quality green peas in a cannery. In addition, the collection highlighted garden pea varieties of high quality seed in biological maturity. The selected varieties were included in the selection process and, based on a number of new varieties garden pea, providing conveyor receipt green peas for processing plants.

  8. The transferrin receptor of Actinobacillus pleuropneumoniae: Quantitation of expression and structural characterization using a peptide-specific monoclonal antibody

    DEFF Research Database (Denmark)

    Bøg, Yang S.; Andresen, Lars Ole; Bastholm, L.

    2001-01-01

    antigens were detected with the Mab in iron-starved Actinobacillus lignieresii, Actinobacillus porcinus, Actinobacillus minor Haemophilus influenzae. and Haemophilus parasuis. Using an enzyme-linked immunosorbent assay (ELISA) based on the Mab 1.48, Tbp2 could be detected in both recombinant E. coli......-restricted culture. It was found that Tbp2 was not expressed in iron replete medium by any serotype except serotypes 5a, 5b and 6 where a weak expression was seen. There was a weak expression of related antigens in Actinobacillus indolicus and Actinobacillus suis under iron-depleted conditions while no similar...... expressing Tbp2 and in wild type A. pp grown under iron restricted conditions. The subcellular location of Tbp2 in A. pp was studied by immunoelectron microscopy using the Mab 1.48. Interestingly, all antibody binding was found inside the A. pp cells, while Tbp2 expressed in recombinant E. coli was found...

  9. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics.

    Science.gov (United States)

    Fagerberg, Linn; Hallström, Björn M; Oksvold, Per; Kampf, Caroline; Djureinovic, Dijana; Odeberg, Jacob; Habuka, Masato; Tahmasebpoor, Simin; Danielsson, Angelika; Edlund, Karolina; Asplund, Anna; Sjöstedt, Evelina; Lundberg, Emma; Szigyarto, Cristina Al-Khalili; Skogs, Marie; Takanen, Jenny Ottosson; Berling, Holger; Tegel, Hanna; Mulder, Jan; Nilsson, Peter; Schwenk, Jochen M; Lindskog, Cecilia; Danielsson, Frida; Mardinoglu, Adil; Sivertsson, Asa; von Feilitzen, Kalle; Forsberg, Mattias; Zwahlen, Martin; Olsson, IngMarie; Navani, Sanjay; Huss, Mikael; Nielsen, Jens; Ponten, Fredrik; Uhlén, Mathias

    2014-02-01

    Global classification of the human proteins with regards to spatial expression patterns across organs and tissues is important for studies of human biology and disease. Here, we used a quantitative transcriptomics analysis (RNA-Seq) to classify the tissue-specific expression of genes across a representative set of all major human organs and tissues and combined this analysis with antibody-based profiling of the same tissues. To present the data, we launch a new version of the Human Protein Atlas that integrates RNA and protein expression data corresponding to ∼80% of the human protein-coding genes with access to the primary data for both the RNA and the protein analysis on an individual gene level. We present a classification of all human protein-coding genes with regards to tissue-specificity and spatial expression pattern. The integrative human expression map can be used as a starting point to explore the molecular constituents of the human body.

  10. AAVrh.10-Mediated Expression of an Anti-Cocaine Antibody Mediates Persistent Passive Immunization That Suppresses Cocaine-Induced Behavior

    OpenAIRE

    Rosenberg, Jonathan B.; Hicks, Martin J.; De, Bishnu P.; Pagovich, Odelya; Frenk, Esther; Janda, Kim D.; Wee, Sunmee; Koob, George F.; Hackett, Neil R.; Kaminsky, Stephen M.; Worgall, Stefan; Tignor, Nicole; Mezey, Jason G.; Crystal, Ronald G.

    2012-01-01

    Cocaine addiction is a major problem affecting all societal and economic classes for which there is no effective therapy. We hypothesized an effective anti-cocaine vaccine could be developed by using an adeno-associated virus (AAV) gene transfer vector as the delivery vehicle to persistently express an anti-cocaine monoclonal antibody in vivo, which would sequester cocaine in the blood, preventing access to cognate receptors in the brain. To accomplish this, we constructed AAVrh.10antiCoc.Mab...

  11. 21 CFR 155.172 - Canned dry peas.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Canned dry peas. 155.172 Section 155.172 Food and... peas. (a) Identity. Canned dry peas conforms to the definition and standard of identity, and is subject to the requirements for label declaration of ingredients, prescribed for canned peas by § 155.170(a...

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

    Science.gov (United States)

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

    2014-07-01

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

  13. Sonoporation delivery of monoclonal antibodies against human papillomavirus 16 E6 restores p53 expression in transformed cervical keratinocytes.

    Directory of Open Access Journals (Sweden)

    Melissa Togtema

    Full Text Available High-risk types of human papillomavirus (HPV, such as HPV16, have been found in nearly all cases of cervical cancer. Therapies targeted at blocking the HPV16 E6 protein and its deleterious effects on the tumour suppressor pathways of the cell can reverse the malignant phenotype of affected keratinocytes while sparing uninfected cells. Through a strong interdisciplinary collaboration between engineering and biology, a novel, non-invasive intracellular delivery method for the HPV16 E6 antibody, F127-6G6, was developed. The method employs high intensity focused ultrasound (HIFU in combination with microbubbles, in a process known as sonoporation. In this proof of principle study, it was first demonstrated that sonoporation antibody delivery into the HPV16 positive cervical carcinoma derived cell lines CaSki and SiHa was possible, using chemical transfection as a baseline for comparison. Delivery of the E6 antibody using sonoporation significantly restored p53 expression in these cells, indicating the antibody is able to enter the cells and remains active. This delivery method is targeted, non-cytotoxic, and non-invasive, making it more easily translatable for in vivo experiments than other transfection methods.

  14. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

  15. CACNA1S expression in mouse retina: Novel isoforms and antibody cross-reactivity with GPR179.

    Science.gov (United States)

    Hasan, Nazarul; Ray, Thomas A; Gregg, Ronald G

    2016-01-01

    Cacna1s encodes the α1S subunit (Cav1.1) of voltage-dependent calcium channels, and is required for normal skeletal and cardiac muscle function, where it couples with the ryanodine receptor to regulate muscle contraction. Recently CACNA1S was reported to be expressed on the tips of retinal depolarizing bipolar cells (DBCs) and colocalized with metabotropic glutamate receptor 6 (mGluR6), which is critical to DBC signal transduction. Further, in mGluR6 knockout mice, expression at this location is down regulated. We examined RNAseq data from mouse retina and found expression of a novel isoform of Cacna1s. To determine if CACNA1S was a functional component of the DBC signal transduction cascade, we performed immunohistochemistry to visualize its expression in several mouse lines that lack DBC function. Immunohistochemical staining with antibodies to CACNA1S show punctate labeling at the tips of DBCs in wild type (WT) retinas that are absent in Gpr179 nob5 mutant retinas and decreased in Grm6 -/- mouse retinas. CACNA1S and transient receptor potential cation channel, subfamily M, member 1 (TRPM1) staining also colocalized in WT retinas. Western blot analyses for CACNA1S of either retinal lysates or proteins after immunoprecipitation with the CACNA1S antibody failed to show the presence of bands expected for CACNA1S. Mass spectrometric analysis of CACNA1S immunoprecipitated proteins also failed to detect any peptides matching CACNA1S. Immunohistochemistry and western blotting after expression of GPR179 in HEK293T cells indicate that the CACNA1S antibody used here and in the retinal studies published to date, cross-reacts with GPR179. These data suggest caution should be exercised in conferring a role for CACNA1S in DBC signal transduction based solely on immunohistochemical staining.

  16. Optimized expression of full-length IgG1 antibody in a common E. coli strain.

    Directory of Open Access Journals (Sweden)

    Conrad En Zuo Chan

    Full Text Available Multi-polypeptide proteins such as antibodies are difficult to express in prokaryotic systems such as E. coli due to the complexity of protein folding plus secretion. Thus far, proprietary strains or fermenter cultures have been required for appreciable yields. Previous studies have shown that expression of heterologous proteins in E. coli can be enhanced by the reduction of protein translation rates. In this paper, we demonstrate that useful quantities of full-length IgG can be expressed and purified from the common E. coli laboratory strain HB2151 in standard shaking culture using a simple strategy of reduced inducer concentration combined with delayed induction times to modulate translation rates. Purified IgG had only marginally reduced avidity compared to mammalian derived IgG. This indicates that this technique can be used to derive antibodies of potentially equal utility as those expressed in mammalian cell culture, particularly for applications where effector functions mediated by the glycosylated residues in the Fragment Crystallizable (Fc portion of the immunoglobulin are not required.

  17. Intracytoplasmic stable expression of IgG1 antibody targeting NS3 helicase inhibits replication of highly efficient hepatitis C Virus 2a clone

    Directory of Open Access Journals (Sweden)

    Clementi Massimo

    2010-06-01

    Full Text Available Abstract Background Hepatitis C virus (HCV infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone. Results The antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA. Conclusion Recombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular

  18. Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

    Science.gov (United States)

    Morino, K; Katsumi, H; Akahori, Y; Iba, Y; Shinohara, M; Ukai, Y; Kohara, Y; Kurosawa, Y

    2001-11-01

    We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

  19. B7h-expressing dendritic cells and plasma B cells mediate distinct outcomes of ICOS costimulation in T cell-dependent antibody responses

    Directory of Open Access Journals (Sweden)

    Larimore Kevin

    2012-06-01

    Full Text Available Abstract Background The ICOS-B7h costimulatory receptor-ligand pair is required for germinal center formation, the production of isotype-switched antibodies, and antibody affinity maturation in response to T cell-dependent antigens. However, the potentially distinct roles of regulated B7h expression on B cells and dendritic cells in T cell-dependent antibody responses have not been defined. Results We generated transgenic mice with lineage-restricted B7h expression to assess the cell-type specific roles of B7h expression on B cells and dendritic cells in regulating T cell-dependent antibody responses. Our results show that endogenous B7h expression is reduced on B cells after activation in vitro and is also reduced in vivo on antibody-secreting plasma B cells in comparison to both naïve and germinal center B cells from which they are derived. Increasing the level of B7h expression on activated and plasma B cells in B-B7hTg mice led to an increase in the number of antibody-secreting plasma cells generated after immunization and a corresponding increase in the concentration of antigen-specific high affinity serum IgG antibodies of all isotypes, without affecting the number of responding germinal center B cells. In contrast, ICOS costimulation mediated by dendritic cells in DC-B7hTg mice contributed to germinal center formation and selectively increased IgG2a production without affecting the overall magnitude of antibody responses. Conclusions Using transgenic mice with lineage-restricted B7h expression, we have revealed distinct roles of ICOS costimulation mediated by dendritic cells and B cells in the regulation of T cell-dependent antibody responses.

  20. A rat monoclonal antibody that recognizes pro- and active MMP-7 indicates polarized expression in vivo

    DEFF Research Database (Denmark)

    Fingleton, Barbara; Powell, William C; Crawford, Howard C

    2007-01-01

    Matrix metalloproteinases (MMPs) are a family of enzymes named for their ability to degrade proteins of the extracellular matrix. Here we describe the characterization of a rat monoclonal antibody specifically recognizing one member of this enzyme family, MMP-7. This antibody has been tested....... The localization pattern would suggest that in normal or early stage tumors, MMP-7 is most likely not directly involved in extracellular matrix degradation. In contrast, advanced colon tumors show MMP-7 in invading cells at the advancing edge of the tumor....

  1. Improved production of single domain antibodies with two disulfide bonds by co-expression of chaperone proteins in the Escherichia coli periplasm.

    Science.gov (United States)

    Shriver-Lake, Lisa C; Goldman, Ellen R; Zabetakis, Daniel; Anderson, George P

    2017-04-01

    Single domain antibodies are recombinantly expressed variable domains derived from camelid heavy chain antibodies. Natural single domain antibodies can have noncanonical disulfide bonds between their complementarity-determining regions that help position the binding site. In addition, engineering a second disulfide bond serves to tie together β-sheets thereby inhibiting unfolding. Unfortunately, the additional disulfide bond often significantly decreases yield, presumably due to formation of incorrect disulfide bonds during the folding process. Here, we demonstrate that inclusion of the helper plasmid pTUM4, which results in the expression of four chaperones, DsbA, DsbC, FkpA, and SurA, increased yield on average 3.5-fold for the nine multi-disulfide bond single domain antibodies evaluated. No increase in production was observed for single domain antibodies containing only the canonical disulfide bond. Published by Elsevier B.V.

  2. Expression of Early Light Induced Protein in Grapevine and Pea, under Different Conditions and its Relation with Photoinhibition Expresión de una Proteína inducida Tempranamente por Luz en Vid y Arveja Bajo Diferentes Condiciones y su Relación con la Fotoinhibición

    Directory of Open Access Journals (Sweden)

    Maritza Berti

    2012-09-01

    Full Text Available Early light induced proteins (ELIP are a type of proteins which are expressed before than other chloroplast proteins in the presence of light. These proteins have been studied in a large number of annual species such as pea (Pisum sativum L., barley (Hordeum vulgare L. and Arabidopsis sp. In perennials plants the studies about ELIPs are very scarce. The possible photoprotective function of the ELIPs has motivated the interest in investigating the presence of this type of proteins in a perennial plant such as grapevine (Vitis vinifera L. and if their characteristics differ from those found in annual plants. In this paper a comparative study was conducted on the ELIPs expression in grapevine and pea to investigate whether there are differences regarding to temperature and light intensity conditions necessary for maximum ELIP expression in each species and for studying in each case the relationship between ELIP expression and photoinhibition degree. The results of this study showed that the maximum ELIPs expression was reached from 25 °C and 1000 µmol PAR m-2 s-1 in both species. Above these values the expression remained constant. Regarding the temperature and light intensity effect on the photoinhibition degree, it was observed that temperature produced inverse relation in grapevine but no relation with pea. On the other hand, the light intensity produced direct relation in both grapevine and pea. The light intensity effect on ELIP expression suggests that these proteins may have a photorepairing role of the photosynthetic system, but the effect of temperature on the ELIP expression in short-term stress may be associated rather to the optimum conditions for their synthesis.Las proteínas tempranamente inducidas por luz (ELIP se expresan antes que otras proteínas del cloroplasto en presencia de luz. Estas proteínas han sido estudiadas en un gran número de especies anuales tales como arveja (Pisum sativum L., cebada (Hordeum bulgare L. y

  3. Circulating microRNA expression pattern separates patients with anti-neutrophil cytoplasmic antibody-associated vasculitis from healthy controls

    DEFF Research Database (Denmark)

    Skoglund, C.; Carlsen, A.; Weiner, M.

    2015-01-01

    patients from healthy subjects as well as from renal transplant recipients. Loadings plots indicated similar contribution of the same miRNAs in both cohorts to the PCA. Renal engagement was important for miRNA expression but consistent correlations between estimated glomerular filtration rate and mi......Objective. Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) has an unpredictable course and better biomarkers are needed. Micro-RNAs in body fluids are protected from degradation and might be used as biomarkers for diagnosis and prognosis, here we explore the potential in AAV...... individual miRNAs were differently expressed compared to controls in both cohorts; miR-29a, -34a, -142-3p and -383 were up-regulated and miR-20a, -92a and -221 were down-regulated. Cluster analysis as well as principal component analysis (PCA) indicated that patterns of miRNA expression differentiate AAV...

  4. Expression of Recombinant Potato leafroll virus Structural and Non-structural Proteins for Antibody Production

    Czech Academy of Sciences Publication Activity Database

    Plchová, Helena; Moravec, Tomáš; Dědič, P.; Čeřovská, Noemi

    2011-01-01

    Roč. 159, č. 2 (2011), s. 130-132 ISSN 0931-1785 R&D Projects: GA MŠk 1M06030; GA MZe QH71123 Institutional research plan: CEZ:AV0Z50380511 Keywords : Potato leafroll virus * recombinant viral antigen * antibody production Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.791, year: 2011

  5. Viral gene expression, antibody production and immune complex formation in human immunodeficiency virus infection

    NARCIS (Netherlands)

    Lange, J. M.; Paul, D. A.; de Wolf, F.; Coutinho, R. A.; Goudsmit, J.

    1987-01-01

    Human immunodeficiency virus (HIV) antigen (HIV-Ag) in polyethylene glycol (PEG) precipitates and supernatants and HIV antibodies (HIV-Ab) to core and envelope antigens were studied in serial serum samples of three HIV-Ab seroconverters and 11 HIV-Ab seropositive men with a mean follow-up time of

  6. Detection of cytokine expression patterns in the peripheral blood of patients with acute leukemia by antibody microarray analysis.

    Science.gov (United States)

    Li, Qing; Li, Mei; Wu, Yao-hui; Zhu, Xiao-jian; Zeng, Chen; Zou, Ping; Chen, Zhi-chao

    2014-04-01

    The cytokines of acute leukemia (AL) patients have certain expression patterns, forming a complex network involved in diagnosis, progression, and prognosis. We collected the serum of different AL patients before and after complete remission (CR) for detection of cytokines by using an antibody chip. The expression patterns of cytokines were determined by using bioinformatics computational analysis. The results showed that there were significant differences in the cytokine expression patterns between AL patients and normal controls, as well as between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). In confirmatory test, ELISA revealed the expression of uPAR in AL. Moreover, the bioinformatic analysis showed that the differentially expressed cytokines among the AL groups were involved in different biological behaviors and were closely related with the development of the disease. It was concluded that the cytokine expression pattern of AL patients is significantly different from that of healthy volunteers. Also, differences of cytokine expression patterns exist between AML and ALL, and between before and after CR in the same subtype of AL, which holds important clinical significance for revealing disease progression.

  7. Transcriptome sequencing of field pea and faba bean for discovery and validation of SSR genetic markers

    Directory of Open Access Journals (Sweden)

    Kaur Sukhjiwan

    2012-03-01

    Full Text Available Abstract Background Field pea (Pisum sativum L. and faba bean (Vicia faba L. are cool-season grain legume species that provide rich sources of food for humans and fodder for livestock. To date, both species have been relative 'genomic orphans' due to limited availability of genetic and genomic information. A significant enrichment of genomic resources is consequently required in order to understand the genetic architecture of important agronomic traits, and to support germplasm enhancement, genetic diversity, population structure and demographic studies. Results cDNA samples obtained from various tissue types of specific field pea and faba bean genotypes were sequenced using 454 Roche GS FLX Titanium technology. A total of 720,324 and 304,680 reads for field pea and faba bean, respectively, were de novo assembled to generate sets of 70,682 and 60,440 unigenes. Consensus sequences were compared against the genome of the model legume species Medicago truncatula Gaertn., as well as that of the more distantly related, but better-characterised genome of Arabidopsis thaliana L.. In comparison to M. truncatula coding sequences, 11,737 and 10,179 unique hits were obtained from field pea and faba bean. Totals of 22,057 field pea and 18,052 faba bean unigenes were subsequently annotated from GenBank. Comparison to the genome of soybean (Glycine max L. resulted in 19,451 unique hits for field pea and 16,497 unique hits for faba bean, corresponding to c. 35% and 30% of the known gene space, respectively. Simple sequence repeat (SSR-containing expressed sequence tags (ESTs were identified from consensus sequences, and totals of 2,397 and 802 primer pairs were designed for field pea and faba bean. Subsets of 96 EST-SSR markers were screened for validation across modest panels of field pea and faba bean cultivars, as well as related non-domesticated species. For field pea, 86 primer pairs successfully obtained amplification products from one or more template

  8. MOLECULAR MARKERS FOR VEGETABLE PEA SAMPLES

    Directory of Open Access Journals (Sweden)

    V. S. Anokhina

    2014-01-01

    Full Text Available The paper presents the results of research on the presence in genomes of pea hybrid materials of the DNA regions complementary to the primers that are associated with the biochemical characteristics and resistance to powdery mildew.

  9. Use of the uteroglobin platform for the expression of a bivalent antibody against oncofetal fibronectin in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Elisa Ventura

    Full Text Available Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284∶26646-26654. Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19 toward the oncofetal fibronectin (B-FN, a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG, 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.

  10. Optimization of ELISA conditions to quantify colorectal cancer antigen-antibody complex protein (GA733-FcK) expressed in transgenic plant.

    Science.gov (United States)

    Ahn, Junsik; Lee, Kyung Jin; Ko, Kisung

    2014-02-01

    The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 μg/well). Among the amounts of the capture antibody, 1 and 0.5 μg/well (capture antibody) showed similar absorbance, whereas 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R(2) between the ELISA quantified value and protein density was 0.85 (pFcK expression in transgenic plant.

  11. Antibody Microarray Analyses of Signal Transduction Protein Expression and Phosphorylation during Porcine Oocyte Maturation

    Czech Academy of Sciences Publication Activity Database

    Pelech, S.; Jelínková, Lucie; Šušor, Andrej; Zhang, H.; Shi, X.; Pavlok, Antonín; Kubelka, Michal; Kovářová, Hana

    2008-01-01

    Roč. 7, č. 7 (2008), s. 2860-2871 ISSN 1535-3893 R&D Projects: GA ČR GA204/06/1297 Grant - others:GA AV ČR(CZ) 1QS500450568 Program:1Q Institutional research plan: CEZ:AV0Z50450515 Keywords : antibody microarray * pig * frog Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.684, year: 2008

  12. Target antigens for Hs-14 monoclonal antibody and their various expression in normozoospermic and asthenozoospermic men

    Czech Academy of Sciences Publication Activity Database

    Čapková, Jana; Margaryan, Hasmik; Kubátová, Alena; Novák, Petr; Pěknicová, Jana

    2015-01-01

    Roč. 25, č. 11 (2015) ISSN 2051-4190 R&D Projects: GA ČR(CZ) GAP503/12/1834; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:86652036 ; RVO:61388971 Keywords : acrosome * human sperm atozoa * monoclonal antibody * asthenozoospermia * transitional endoplasmic reticulum ATPase Subject RIV: CE - Biochemistry http://link.springer.com/article/10.1186/s12610-015-0025-0

  13. Novel polyclonal antibodies as a useful tool for expression studies in amphioxus embryos

    Czech Academy of Sciences Publication Activity Database

    Bozzo, M.; Pergner, Jiří; Kozmik, Zbyněk; Kozmiková, Iryna

    2017-01-01

    Roč. 61, č. 10-12 (2017), s. 793-800 ISSN 0214-6282 R&D Projects: GA ČR GC15-21285J; GA MŠk(CZ) LM2015062; GA MŠk LO1419 Institutional support: RVO:68378050 Keywords : Branchiostoma * amphioxus * antibody * espression pattern Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biology (theoretical, mathematical, thermal, cryobiology, biological rhythm), Evolutionary biology Impact factor: 1.981, year: 2016

  14. Expression cloning and production of Human Heavy Chain Only antibodies from murine transgenic plasma cells

    Directory of Open Access Journals (Sweden)

    Dubravka Drabek

    2016-12-01

    Full Text Available Several technologies have been developed to isolate human antibodies against different target antigens as a source of potential therapeutics, including hybridoma technology, phage and yeast display systems. For conventional antibodies this involves either random pairing of VH and VL domains in combinatorial display libraries, or isolation of cognate pairs of VH and VL domains from human B cells or from transgenic mice carrying human immunoglobulin loci followed by single cell sorting, single cell RT-PCR and bulk cloning of isolated natural VH-VL pairs. Heavy chain only antibodies (HCAbs that naturally occur in camelids require only heavy immunoglobulin chain cloning. Here, we present an automatable novel, high-throughput technology, for rapid direct cloning and production of fully human HCAbs from sorted population of transgenic mouse plasma cells carrying a human HCAb locus. Utility of the technique is demonstrated by isolation of diverse sets of sequence unique, soluble, high affinity influenza A strain X-31 hemagglutinin (HA specific HCAbs

  15. Deciphering common and specific transcriptional immune responses in pea towards the oomycete pathogens Aphanomyces euteiches and Phytophthora pisi.

    Science.gov (United States)

    Hosseini, Sara; Elfstrand, Malin; Heyman, Fredrik; Funck Jensen, Dan; Karlsson, Magnus

    2015-08-21

    Root rot caused by Aphanomyces euteiches is one of the most destructive pea diseases while a distantly related species P. pisi has been recently described as the agent of pea and faba bean root rot. These two oomycete pathogens with different pathogenicity factor repertories have both evolved specific mechanisms to infect pea. However, little is known about the genes and mechanisms of defence against these pathogens in pea. In the present study, the transcriptomic response of pea to these two pathogens was investigated at two time points during early phase of infection using a Medicago truncatula microarray. Of the 37,976 genes analysed, 574 and 817 were differentially expressed in response to A. euteiches at 6 hpi and 20 hpi, respectively, while 544 and 611 genes were differentially regulated against P. pisi at 6 hpi and 20 hpi, respectively. Differentially expressed genes associated with plant immunity responses were involved in cell wall reinforcement, hormonal signalling and phenylpropanoid metabolism. Activation of cell wall modification, regulation of jasmonic acid biosynthesis and induction of ethylene signalling pathway were among the common transcriptional responses to both of these oomycetes. However, induction of chalcone synthesis and the auxin pathway were specific transcriptional changes against A. euteiches. Our results demonstrate a global view of differentially expressed pea genes during compatible interactions with P. pisi and A. euteiches at an early phase of infection. The results suggest that distinct signalling pathways are triggered in pea by these two pathogens that lead to common and specific immune mechanisms in response to these two oomycetes. The generated knowledge may eventually be used in breeding pea varieties with resistance against root rot disease.

  16. Effects of a concentrate of pea antinutritional factors on pea protein digestibility in piglets

    NARCIS (Netherlands)

    Guen, M.P. Le; Huisman, J.; Guéguen, J.; Beelen, G.; Verstegen, M.W.A.

    1995-01-01

    Four experiments were designed to investigate the apparent ileal digestibility of raw pea (Pisum sativum) and two of its components - an isolate of its proteins and a concentrate of its proteinaceous antinutritional factors (ANFs). Three varieties of peas were used: spring varieties Finale and

  17. Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna *

    Science.gov (United States)

    Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

    2016-01-01

    To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as

  18. Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna.

    Science.gov (United States)

    Liu, Hong-cui; Yuan, Bing-qiang; Li, Shao-nan

    2016-02-01

    To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as

  19. Specific Detection of Dog Podoplanin Expressed in Renal Glomerulus by a Novel Monoclonal Antibody PMab-38 in Immunohistochemistry.

    Science.gov (United States)

    Honma, Ryusuke; Kaneko, Mika K; Ogasawara, Satoshi; Fujii, Yuki; Konnai, Satoru; Takagi, Michiaki; Kato, Yukinari

    2016-08-01

    Podoplanin (PDPN) is expressed in several normal tissues including podocytes of renal glomerulus, lymphatic endothelial cells (LECs), and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelets. Many monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, and bovine PDPN have been established; antidog PDPN (dPDPN) mAbs have not been developed. Herein, we immunized mice with the recombinant proteins of dPDPN and developed anti-dPDPN mAbs. One of the clones, PMab-38, is useful for detecting podocytes in immunohistochemical analysis; in contrast, it did not react with LECs or type I alveolar cells. PMab-38 also detected dPDPN specifically in flow cytometry and Western blot analysis. PMab-38 is expected to be useful for investigating the function of dPDPN, which is expressed in podocytes.

  20. Peas in a Pod: Environment and Ionization in Green Pea Galaxies

    Science.gov (United States)

    Kurtz, Heather; Jaskot, Anne; Drew, Patrick; Pare, Dylan; Griffin, Jon; Petersen, Michael

    2016-01-01

    The Green Peas are extreme, highly ionized, starburst galaxies with strong [OIII] 5007 emission. Using the Sloan Digital Sky Survey, we present statistics on the environment of Green Peas and investigate its effects on their ionized gas properties. Although most dwarf starburst galaxies are in low-density environments, we identify a sample of Green Peas in dense environments. Emission line observations with the WIYN 0.9-meter telescope at Kitt Peak reveal that one cluster Green Pea is more highly ionized in the direction of the cluster center. Ram pressure stripping likely generates this ionization gradient. We explore the role of the environment in enhancing star formation rates and ionization, and we compare the nebular properties of Green Peas in high-density environments to those in low-density environments.

  1. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

    DEFF Research Database (Denmark)

    Lavrsen, Kirstine; Madsen, Caroline B; Rasch, Morten G

    2013-01-01

    not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed...... to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis...... is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies....

  2. Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation

    Directory of Open Access Journals (Sweden)

    Minerva Camacho-Nuez

    2017-11-01

    Full Text Available Abstract Background Bovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future. Results To isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro. Conclusion Babesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.

  3. Hap2, a novel gene in Babesia bigemina is expressed in tick stages, and specific antibodies block zygote formation.

    Science.gov (United States)

    Camacho-Nuez, Minerva; Hernández-Silva, Diego Josimar; Castañeda-Ortiz, Elizabeth Jacqueline; Paredes-Martínez, María Elena; Rocha-Martínez, Marisol Karina; Alvarez-Sánchez, María Elizbeth; Mercado-Curiel, Ricardo Francisco; Aguilar-Tipacamu, Gabriela; Mosqueda, Juan

    2017-11-13

    Bovine babesiosis is a tick-borne disease caused by the protozoan parasites of the genus Babesia. In their host vector, Babesia spp. undergo sexual reproduction. Therefore, the development of sexual stages and the subsequent formation of the zygote are essential for the parasite to invade the intestinal cells of the vector tick and continue its life-cycle. HAP2/GCS1 is a protein identified in plants, protozoan parasites and other organisms that has an important role during membrane fusion in fertilization processes. The identification and characterization of HAP-2 protein in Babesia would be very significant to understand the biology of the parasite and to develop a transmission-blocking vaccine in the future. To isolate and sequence the hap2 gene DNA from an infected bovine with Babesia bigemina was purified. The hap2 gene was amplified, cloned and sequenced. The sequences of hap2 from four geographically different strains showed high conservation at the amino acid level, including the typical structure with a signal peptide and the HAP2/GSC domain. Antisera anti-HAP2 against the conserved extracellular region of the HAP2 amino acid sequence were obtained from rabbits. The expression of hap2 in the host and vector tissues was analyzed by using semi-quantitative RT-PCR, and the protein was examined by western blot and immunofluorescence. Based on the RT-PCR and WB results, HAP2 is expressed in both, sexual stages induced in vitro, and in infected ticks as well. We did not detect any expression in asexual erythrocytic stages of B. bigemina, relevantly anti-HAP2 specific antibodies were able to block zygotes formation in vitro. Babesia bigemina HAP2 is expressed only in tick-infecting stages, and specific antibodies block zygote formation. Further studies regarding the function of HAP2 during tick infection may provide new insights into the molecular mechanisms of sexual reproduction of the parasite.

  4. Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice.

    Science.gov (United States)

    Su, Jin; Sherman, Alexandra; Doerfler, Phillip A; Byrne, Barry J; Herzog, Roland W; Daniell, Henry

    2015-10-01

    Deficiency of acid alpha glucosidase (GAA) causes Pompe disease in which the patients systemically accumulate lysosomal glycogen in muscles and nervous systems, often resulting in infant mortality. Although enzyme replacement therapy (ERT) is effective in treating patients with Pompe disease, formation of antibodies against rhGAA complicates treatment. In this report, we investigated induction of tolerance by oral administration of GAA expressed in chloroplasts. Because full-length GAA could not be expressed, N-terminal 410-amino acids of GAA (as determined by T-cell epitope mapping) were fused with the transmucosal carrier CTB. Tobacco transplastomic lines expressing CTB-GAA were generated through site-specific integration of transgenes into the chloroplast genome. Homoplasmic lines were confirmed by Southern blot analysis. Despite low-level expression of CTB-GAA in chloroplasts, yellow or albino phenotype of transplastomic lines was observed due to binding of GAA to a chloroplast protein that has homology to mannose-6 phosphate receptor. Oral administration of the plant-made CTB-GAA fusion protein even at 330-fold lower dose (1.5 μg) significantly suppressed immunoglobulin formation against GAA in Pompe mice injected with 500 μg rhGAA per dose, with several-fold lower titre of GAA-specific IgG1 and IgG2a. Lyophilization increased CTB-GAA concentration by 30-fold (up to 190 μg per g of freeze-dried leaf material), facilitating long-term storage at room temperature and higher dosage in future investigations. This study provides the first evidence that oral delivery of plant cells is effective in reducing antibody responses in ERT for lysosomal storage disorders facilitating further advances in clinical investigations using plant cell culture system or in vitro propagation. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Effect of Pigeon pea and Cow pea on the performance and gut immunity of broiler chicks

    International Nuclear Information System (INIS)

    Yagoub, Yagoub Magboul

    1998-03-01

    two experiments were conducted to examine the effect of pigeon pea and cow pea on the performance and gut immunity of broiler chicks. In experiment 1, 3 experimental diets were formulated containing graded levels of cow pea were maintained. Diets were prepared containing 18.21, 18.25 and 18.25% crude protein and 3076.41, 3062 Kel/Kg metabolizable energy for experiment 1, while diets of experiment 11 were prepared containing 18.21, 18.22, and 18.22% crude protein and 3076.41, 3080.5 and 3055.89 KEl/Kg metabolized energy. 120 Loghmann broiler chicks were equally allocated into 15 pens (8 chicks/pen). Then the experimental diets were randomly assigned to the pens. feed and water were provided ad libitum in both experiments. In experiment 1, the results showed no significant difference were found in chick performance at day 45. The feed conversation ratio increased with the level of pigeon pea used. The pancreas mass was increased as the level of pigeon pea increase. In experiment 2 the results showed significant decrease in the body weight and feed intake at day 45, while the pancreas mass tend to increase with increasing level of cow pea in the diet. Histological examination of small intestine slides showed no histopathological differences between the control and chicks fed cow pea and/or pigeon pea. Immunological test of the serum and mucous samples using ELISA techniques revealed no significant difference between the control and chicks given cow pea and / or pigeon pea

  6. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    Science.gov (United States)

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  7. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    International Nuclear Information System (INIS)

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

    2004-01-01

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

  8. Evaluation of the expression of sperm proteins in normozoospermic and asthenozoospermic men using monoclonal antibodies

    Czech Academy of Sciences Publication Activity Database

    Čapková, Jana; Kubátová, Alena; Děd, Lukáš; Teplá, O.; Pěknicová, Jana

    2016-01-01

    Roč. 18, č. 1 (2016), s. 108-113 ISSN 1008-682X R&D Projects: GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GAP503/12/1834 Institutional support: RVO:86652036 Keywords : asthenozoospermia * flow cytometry * fluorescence microscopy * monoclonal antibodies * sperm proteins Subject RIV: CE - Biochemistry Impact factor: 2.996, year: 2016 http://www.ajandrology.com/article.asp?issn=1008-682X;year=2016;volume=18;issue=1;spage=108;epage=113;aulast=Capkova

  9. Antibodies Expressed by Intratumoral B Cells as the Basis for a Diagnostic Test for Lung Cancer

    Science.gov (United States)

    2015-06-01

    G., Montefiori, D. C., Voss, G., Liao, H. X., Tomaras, G. D., and Haynes, B. F. (2012) HIV-1 gp120 vaccine induces affinity maturation in both new...reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination . PLoS One 6, e25797 32. Chen, Z. J., Wheeler, C. J., Shi, W...A., Alam, S. M., Tomaras, G. D., Kepler, T. B., Kelsoe, G., Liao, H. X., and Haynes, B. F. (2011) H3N2 influenza infection elicits more cross

  10. Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

    OpenAIRE

    Sandee, Duanpen; Tungpradabkul, Sumalee; Tsukio, Manae; Imanaka, Tadayuki; Takagi, Masahiro

    2002-01-01

    Abstract Background Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. Results The sequences encoding the variable regions of heavy (VH) and light (VL) chains of a murine Hep27 Mab were linked together by a linker pepti...

  11. Expression of class 5 antigens by meningococcal strains obtained from patients in Brazil and evaluation of two new monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Elizabeth N. De Gaspari

    Full Text Available Determining the profile of antigen expression among meningococci is important for epidemiologic surveillance and vaccine development. To this end, two new mouse monoclonal antibodies (MAbs have been derived against Neisseria meningitidis proteins (class 5. The MAbs were reactive against outer membrane antigens and were bactericidal. Selected anti-class 5 MAbs [(5.1-3E6-2; (5.3-3BH4-C7; (5.4-1BG11-C7; (5.5-3DH-F5G9 also 5F1F4-T3(5.c], and the two new monoclonal antibodies C14F10Br2 (5.8 and 7F11B5Br3 (5.9, were then tested against different meningococcal strains, (63 strains of serogroup A, 60 strains of serogroup C (from 1972 to 1974; and 136 strains of serogroup B (from 1992 meningococci. Our results demonstrated that the expression of class 5 proteins in the N. meningitidis B Brazilian strains studied is highly heterogeneous. The serotypes and subtypes of B:4:P1.15, B:4:P1.9, B:4:P1.7, B:4:P1.3, B:4:P1.14, B:4:P1.16, B:4:NT, and B:NT:NT were detected in N. meningitidis B serogroups.The strains C:2a:P1.2 and A:4.21:P1.9 were dominant in the C and A serogroups, respectively. Serogroup B organisms expressed the class 5 epitopes 5.4 (18%, 5.5 (22%, 5.8 (3.6%, 5.9 (8.0% and 5c (38%. Serogroup C expressed class 5 epitopes 5.1 (81%, 5.4 (35%, 5.5 (33% and 5.9 (5.0%; and serogroup A showed reactivity directed at the class 5 protein 5c (47%; and reactivity was present with the new monoclonal antibody, 5.9 (5.5%. We conclude that the two new MAbs are useful in detecting important group B, class 5 antigens, and that a broad selection of serogroup B, class 5 proteins would be required for an effective vaccine based on the class 5 proteins.

  12. Expression of 12 rabbit IgA C alpha genes as chimeric rabbit-mouse IgA antibodies.

    Science.gov (United States)

    Schneiderman, R D; Hanly, W C; Knight, K L

    1989-10-01

    Serologic analysis of rabbit secretory IgA initially identified two subclasses of IgA, IgA-f and IgA-g. Recent molecular genetic studies have resulted in the identification and cloning of 13 genes encoding the constant region (C) of rabbit IgA heavy chains. Each of these 13 C alpha genes, C alpha 1-C alpha 13, was subcloned into an expression vector containing the VDJ (V, variable; D, diversity; J, joining) gene of a dansyl (DNS)-binding hybridoma antibody. The alpha heavy-chain constructs were transfected into SP2/0 cells producing murine light chains with specificity for DNS. Of the 13 resulting transfectomas, 12 were shown by ELISA to secrete DNS-binding chimeric rabbit-mouse IgA molecules. By immunoblot analysis, the 12 IgA-producing transfectomas were shown to secrete alpha chains ranging in size from 60 to 72 kDa. These data suggest that rabbit IgA may be composed of as many as 12 IgA isotypes. This is in marked contrast to mouse and human, in which only 1 and 2 IgA isotypes, respectively, are found. Serologic analyses, using anti-IgA-f and anti-IgA-g alloantisera, revealed that 11 of the 12 transfectoma IgAs reacted with anti-IgA-f and not with anti-IgA-g antibodies and that one reacted with anti-IgA-g and not with anti-IgA-f antibodies. Each of the IgA-producing transfectomas was cocultured with a Madin-Darby canine kidney cell line expressing the rabbit polymeric immunoglobulin receptor, and the transcytosed IgA antibodies were analyzed by immunoblots to determine whether they associated with secretory component (SC) through covalent or noncovalent interactions. Each of the 11 IgA-f isotypes was shown to bind SC by a disulfide linkage, whereas the single IgA-g isotype appeared to bind SC through noncovalent interactions only.

  13. An EGP-2/Ep-CAM-expressing transgenic rat model to evaluate antibody-mediated immunotherapy

    NARCIS (Netherlands)

    McLaughlin, PMJ; Kroesen, BJ; Dokter, WHA; van der Molen, H; de Groot, M; Brinker, MGL; Kok, K; Ruiters, MHJ; Buys, CHCM; de Leij, LFMH

    The human pancarcinoma-associated epithelial glycoprotein-2 (EGP-2), also known as 17-1A or EpCAM, is a 38-kDa transmembrane antigen, commonly used for targeted immunotherapy of carcinomas. Although strongly expressed by most carcinomas, EGP-2 is also expressed in most simple epithelia. To evaluate

  14. Oral Vaccination with the Porcine Rotavirus VP4 Outer Capsid Protein Expressed by Lactococcus lactis Induces Specific Antibody Production

    Directory of Open Access Journals (Sweden)

    Yi-jing Li

    2010-01-01

    Full Text Available The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

  15. A Live Vector Expressing HPV16 L1 Generates an Adjuvant-Induced Antibody Response In-vivo.

    Science.gov (United States)

    Shirbaghaee, Zeinab; Bolhassani, Azam; Mirshafiey, Abbas; Motevalli, Fatemeh; Zohrei, Negar

    2015-12-01

    The association between human papillomavirus (HPV) infections and cervical cancer has suggested the design of prophylactic and therapeutic vaccines against genital warts. The HPV capsid has made of two L1 and L2 coat proteins that have produced late in viral infections. Regarding to the recent studies, two commercial prophylactic vaccines have based on L1 viral like particles (VLPs) could strongly induce antibody responses, and protect human body from HPV infections. However, the use of these HPV vaccines has hindered due to their high cost and some limitations. Currently, among various vaccination strategies, live vector-based vaccines have attracted a great attention. Herein, a non-pathogenic strain of the protozoan organism known as Leishmania tarentolae has utilized to induce potent humoral immunity in mice model. At first, cloning of HPV16 L1 gene into Leishmania expression vector has performed and confirmed by PCR and digestion with restriction enzymes. The promastigotes of Leishmania tarentolae (L.tar) have transfected with linearized DNA construct by electroporation. Protein expression has analyzed by SDS-PAGE and western blotting. Then, the immunogenicity of leishmania expressing L1 protein (L.tar-L1) has assessed in mice model. Our data has indicated that subcutaneous immunization of mice with the recombinant L.tar-L1 has led to enhance the levels of IgG1 and lgG2a in comparison with control groups. Furthermore, there was no significant increase in antibody levels between two and three times of immunizations. The recombinant live vector was able to induce humoral immunity in mice without need of any adjuvant. However, further studies have required to increase its efficiency.

  16. Antibodies against outer-capsid proteins of grass carp reovirus expressed in E. coli are capable of neutralizing viral infectivity

    Directory of Open Access Journals (Sweden)

    Sun Xiaoyun

    2011-07-01

    Full Text Available Abstract Background Grass carp reovirus (GCRV, which causes severe infectious outbreaks of hemorrhagic disease in aquatic animals, is a highly pathogenic agent in the Aquareovirus genus of family Reoviridae. The outer capsid shell of GCRV, composed of the VP5-VP7 protein complex, is believed to be involved in cell entry. The objective of this study was to produce a major neutralization antibody for mitigating GCRV infection. Results Recombinant plasmids of GCRV outer capsid proteins VP5 and VP7 were constructed and expressed in prokaryotic cells in our previous work. In this study, we prepared GCRV Antibody (Ab, VP5Ab and VP7Ab generated from purified native GCRV, recombinant VP5 and VP7 respectively. Immunoblotting analysis showed that the prepared antibodies were specific to its antigens. In addition, combined plaque and cytopathic effect (CPE-based TCID50 (50% tissue culture infective dose assays showed that both VP5Ab and VP7Ab were capable of neutralizing viral infectivity. Particularly, the neutralizing activity of VP7Ab was 3 times higher than that of VP5Ab, suggesting that VP7 might be a dominating epitope. Moreover, the combination of VP5Ab and VP7Ab appeared to enhance GCRV neutralizing capacity. Conclusions The results presented in this study indicated that VP7 protein was the major epitope of GCRV. Furthermore, VP5Ab and VP7Ab in combination presented an enhanced capacity to neutralize the GCRV particle, suggesting that the VP5 and VP7 proteins may cooperate with each other during virus cell entry. The data can be used not only to further define the surface epitope domain of GCRV but may also be applicable in the designing of vaccines.

  17. Adenoviral gene transfer of PLD1-D4 enhances insulin sensitivity in mice by disrupting phospholipase D1 interaction with PED/PEA-15.

    Directory of Open Access Journals (Sweden)

    Angela Cassese

    Full Text Available Over-expression of phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA-15 causes insulin resistance by interacting with the D4 domain of phospholipase D1 (PLD1. Indeed, the disruption of this association restores insulin sensitivity in cultured cells over-expressing PED/PEA-15. Whether the displacement of PLD1 from PED/PEA-15 improves insulin sensitivity in vivo has not been explored yet. In this work we show that treatment with a recombinant adenoviral vector containing the human D4 cDNA (Ad-D4 restores normal glucose homeostasis in transgenic mice overexpressing PED/PEA-15 (Tg ped/pea-15 by improving both insulin sensitivity and secretion. In skeletal muscle of these mice, D4 over-expression inhibited PED/PEA-15-PLD1 interaction, decreased Protein Kinase C alpha activation and restored insulin induced Protein Kinase C zeta activation, leading to amelioration of insulin-dependent glucose uptake. Interestingly, Ad-D4 administration improved insulin sensitivity also in high-fat diet treated obese C57Bl/6 mice. We conclude that PED/PEA-15-PLD1 interaction may represent a novel target for interventions aiming at improving glucose tolerance.

  18. The Influence of Lead on Generation of Signalling Molecules and Accumulation of Flavonoids in Pea Seedlings in Response to Pea Aphid Infestation

    Directory of Open Access Journals (Sweden)

    Agnieszka Woźniak

    2017-08-01

    Full Text Available The aim of this study was to investigate the effect of an abiotic factor, i.e., lead at various concentrations (low causing a hormesis effect and causing high toxicity effects, on the generation of signalling molecules in pea (Pisum sativum L. cv. Cysterski seedlings and then during infestation by the pea aphid (Acyrthosiphon pisum Harris. The second objective was to verify whether the presence of lead in pea seedling organs and induction of signalling pathways dependent on the concentration of this metal trigger defense responses to A. pisum. Therefore, the profile of flavonoids and expression levels of genes encoding enzymes of the flavonoid biosynthesis pathway (phenylalanine ammonialyase and chalcone synthase were determined. A significant accumulation of total salicylic acid (TSA and abscisic acid (ABA was recorded in the roots and leaves of pea seedlings growing on lead-supplemented medium and next during infestation by aphids. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. This research provides insights into the cross-talk between the abiotic (lead and biotic factor (aphid infestation on the level of the generation of signalling molecules and their role in the induction of flavonoid biosynthesis.

  19. The Influence of Lead on Generation of Signalling Molecules and Accumulation of Flavonoids in Pea Seedlings in Response to Pea Aphid Infestation.

    Science.gov (United States)

    Woźniak, Agnieszka; Drzewiecka, Kinga; Kęsy, Jacek; Marczak, Łukasz; Narożna, Dorota; Grobela, Marcin; Motała, Rafał; Bocianowski, Jan; Morkunas, Iwona

    2017-08-24

    The aim of this study was to investigate the effect of an abiotic factor, i.e., lead at various concentrations (low causing a hormesis effect and causing high toxicity effects), on the generation of signalling molecules in pea ( Pisum sativum L. cv. Cysterski) seedlings and then during infestation by the pea aphid ( Acyrthosiphon pisum Harris). The second objective was to verify whether the presence of lead in pea seedling organs and induction of signalling pathways dependent on the concentration of this metal trigger defense responses to A. pisum . Therefore, the profile of flavonoids and expression levels of genes encoding enzymes of the flavonoid biosynthesis pathway (phenylalanine ammonialyase and chalcone synthase) were determined. A significant accumulation of total salicylic acid (TSA) and abscisic acid (ABA) was recorded in the roots and leaves of pea seedlings growing on lead-supplemented medium and next during infestation by aphids. Increased generation of these phytohormones strongly enhanced the biosynthesis of flavonoids, including a phytoalexin, pisatin. This research provides insights into the cross-talk between the abiotic (lead) and biotic factor (aphid infestation) on the level of the generation of signalling molecules and their role in the induction of flavonoid biosynthesis.

  20. Primary afferent and spinal cord expression of gastrin-releasing peptide: message, protein, and antibody concerns.

    Science.gov (United States)

    Solorzano, Carlos; Villafuerte, David; Meda, Karuna; Cevikbas, Ferda; Bráz, Joao; Sharif-Naeini, Reza; Juarez-Salinas, Dina; Llewellyn-Smith, Ida J; Guan, Zhonghui; Basbaum, Allan I

    2015-01-14

    There is continuing controversy relating to the primary afferent neurotransmitter that conveys itch signals to the spinal cord. Here, we investigated the DRG and spinal cord expression of the putative primary afferent-derived "itch" neurotransmitter, gastrin-releasing peptide (GRP). Using ISH, qPCR, and immunohistochemistry, we conclude that GRP is expressed abundantly in spinal cord, but not in DRG neurons. Titration of the most commonly used GRP antiserum in tissues from wild-type and GRP mutant mice indicates that the antiserum is only selective for GRP at high dilutions. Paralleling these observations, we found that a GRPeGFP transgenic reporter mouse has abundant expression in superficial dorsal horn neurons, but not in the DRG. In contrast to previous studies, neither dorsal rhizotomy nor an intrathecal injection of capsaicin, which completely eliminated spinal cord TRPV1-immunoreactive terminals, altered dorsal horn GRP immunoreactivity. Unexpectedly, however, peripheral nerve injury induced significant GRP expression in a heterogeneous population of DRG neurons. Finally, dual labeling and retrograde tracing studies showed that GRP-expressing neurons of the superficial dorsal horn are predominantly interneurons, that a small number coexpress protein kinase C gamma (PKCγ), but that none coexpress the GRP receptor (GRPR). Our studies support the view that pruritogens engage spinal cord "itch" circuits via excitatory superficial dorsal horn interneurons that express GRP and that likely target GRPR-expressing interneurons. The fact that peripheral nerve injury induced de novo GRP expression in DRG neurons points to a novel contribution of this peptide to pruritoceptive processing in neuropathic itch conditions. Copyright © 2015 Solorzano et al.

  1. Effects of Graded Levels of Pigeon Pea Meal on Growth ...

    African Journals Online (AJOL)

    The birds were fed on 0, 50, 75 and 100% dietary inclusions of toasted pigeon pea as replacement for soybean in a 28 day feeding trial. Significant interaction between dietary levels of pigeon pea and soybean were observed on feed efficiency and weight gain (p<0.05). Increase in dietary levels of pigeon pea reduced feed ...

  2. Possible causes of dry pea synergy to corn

    Science.gov (United States)

    Dry pea improves corn yield and tolerance to weed interference compared with soybean, spring wheat, or canola as preceding crops. To understand this synergy between dry pea and corn, we examined growth and nutrient concentration of corn following dry pea or soybean in sequence. Each corn plot was ...

  3. faba bean and field pea seed proportion for intercropping system

    African Journals Online (AJOL)

    Administrator

    Intercropping of Faba bean (Vicia fabae) and field pea (Pisum sativum) is an important cropping system in the. Horro highlands of ... productivity of the Faba bean/field pea was obtained from intercropping system. Growing Faba bean both as a .... as per the available research recommendation for. Faba bean and field pea ...

  4. Number and Effectiveness of Pea Rhizobia in Danish Soils

    DEFF Research Database (Denmark)

    Engvild, K.C.

    1989-01-01

    Most of 44 Danish soils tested contain between 1000 and 10 000 pea rhizobia (Rhizobium leguminosarum biovar viceae) per gram. Pea rhizobia were not detected in acid moor and forest soils. Only one case of failed nodulation in peas in the field has been noted, in spots in a reclaimed sandy heath...

  5. 7 CFR 457.140 - Dry pea crop insurance provisions.

    Science.gov (United States)

    2010-01-01

    ... varieties of contract seed peas, and deliver the production from those varieties to the processor/seed... contract seed pea variety by its respective production guarantee; (5) Multiplying each result of section 13... indemnity payment. (c) The value of contract seed pea production to count for each variety in the unit will...

  6. In vitro Regeneration of pigeon pea using leaf explants | Asande ...

    African Journals Online (AJOL)

    Pigeon pea production in countries like Kenya is faced with a number of challenges, particularly lack of high quality seeds. The objective of this study was to develop an in vitro regeneration system for pigeon pea varieties grown in Kenya, that is amenable to genetic transformation. In vitro regeneration of pigeon pea ...

  7. Experimental and in silico modelling analyses of the gene expression pathway for recombinant antibody and by-product production in NS0 cell lines.

    Directory of Open Access Journals (Sweden)

    Emma J Mead

    Full Text Available Monoclonal antibodies are commercially important, high value biotherapeutic drugs used in the treatment of a variety of diseases. These complex molecules consist of two heavy chain and two light chain polypeptides covalently linked by disulphide bonds. They are usually expressed as recombinant proteins from cultured mammalian cells, which are capable of correctly modifying, folding and assembling the polypeptide chains into the native quaternary structure. Such recombinant cell lines often vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based and engineering (nonlinear models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

  8. Recognition of phage-expressed peptides containing Asx-Pro sequences by monoclonal antibodies produced against Plasmodium falciparum circumsporozoite protein.

    Science.gov (United States)

    Wilson, D R; Wirtz, R A; Finlay, B B

    1997-05-01

    The immunodominant region of the Plasmodium falciparum circumsporozoite protein is comprised mainly of a series of tetrapeptide repeats that can, depending on the starting cadence chosen, be described as (NANP)n, (ANPN)n, (NPNA)n or (PNAN)n in one-letter amino acid code. Data from several studies suggest that the NPNA cadence alone is structurally correct, in that each NPNA tetrapeptide effectively forms a structural unit initiated by an Asx-Pro turn. To explore this idea further and to assess the immunological relevance of peptide conformation as it relates to the cadence of these tetrapeptide repeats, we used ELISA to compare the abilities of monoclonal antibodies (MAbs) produced against P. falciparum sporozoites to recognize repeat-related heptapeptides expressed on the surface of filamentous bacteriophage. Having included representatives of both NANP and NPNA cadences and other peptides in which the number and location of Asx-Pro sequences varied, we provide evidence that Asx-Pro sequences play an important role in peptide conformation and antibody recognition, that peptide conformation is influenced by the cadence of the tetrapeptide repeats and that peptide conformation is important to the abilities of these MAbs to recognize their epitopes.

  9. London's historic ''pea-soupers''

    International Nuclear Information System (INIS)

    Urbinato, D.

    1994-01-01

    Americans may think smog was invented in Los Angeles. Not so. In fact, a Londoner coined the term ''smog'' in 1905 to describe the city's insidious combination of natural fog and coal smoke. By then, the phenomenon was part of London history, and dirty, acrid smoke-filled ''pea-soupers'' were as familiar to Londoners as Big Ben and Westminster Abby. Smog in London predates Shakespeare by four centuries. Until the 12th century, most Londoners burned wood for fuel. But as the city grew and the forests shrank, wood became scarce and increasingly expensive. Large deposits of ''sea-coal'' off the northeast coast provided a cheap alternative. Soon, Londoners were burning the soft, bituminous coal to heat their homes and fuel their factories. Sea-coal was plentiful, but it didn't burn efficiently. A lot of its energy was spent making smoke, not heat. Coal smoke drifting through thousands of London chimneys combined with clean natural fog to make smog. If the weather conditions were right, it would last for days. Early on, no one had the scientific tools to correlate smog with adverse health effects, but complaints about the smoky air as an annoyance date back to at least 1272, when King Edward I, on the urging of important noblemen and clerics, banned the burning of sea-coal. Anyone caught burning or selling the stuff was to be tortured or executed. The first offender caught was summarily put to death. This deterred nobody. Of necessity, citizens continued to burn sea-coal in violation of the law, which required the burning of wood few could afford

  10. Expression and regulation of two idiotype families and subsets within an idiotype family among BALB/c antibodies against p-azophenylarsonate

    International Nuclear Information System (INIS)

    Brown, A.R.

    1984-01-01

    The expression and regulation of the two different iodiotype (id) families associated with the anti-p-azophenylarsonate (Ar) antibodies of BALB/c mice examined. Both families (5AF6 and 3C6) represented cross-reactive idiotypes (CRI) expressed in the anti-Ar of most individual BALB/c mice. In response to keyhole limpet hemocyanin-Ar, an average of about 28% of BALB/c anti-Ar had 5AF6 family idiotopes, while 3C6 family was expressed on about 16% of BALBc anti-Ar antibodies. Suppression induced by anti-idiotype treatment against one family did not suppress the expression of the other family suggesting that the two families were regulated independently. However, the relative expression of one family could influence the expression of the other, because depression of the 5AF6 family tended to increase the expression of the 3C6 family of anti-Ar. Analysis of the 5AF6 family showed that a majority of BALB/c mice produced antibodies heating most or all of the idiotopes associated with the family, but that a subset of about 35% of the antibodies synthesized lacked idiotopes associated with a monoclonal anti-Ar member of this family, 2.4. Treatment of mice with anti-idiotypes prepared against two different monoclonal anti-Ar of the 5AF6 family produced different effects: one enhanced while the other suppressed idiotype expression, suggesting that there are differences in the idiotopes associated with these two regulatory pathways. Additionally, results indicated that subsets of antibodies within the 5AF6 idiotype family could be regulated independently of each other

  11. Stamina pistilloida, the Pea ortholog of Fim and UFO, is required for normal development of flowers, inflorescences, and leaves.

    Science.gov (United States)

    Taylor, S; Hofer, J; Murfet, I

    2001-01-01

    Isolation and characterization of two severe alleles at the Stamina pistilloida (Stp) locus reveals that Stp is involved in a wide range of developmental processes in the garden pea. The most severe allele, stp-4, results in flowers consisting almost entirely of sepals and carpels. Production of ectopic secondary flowers in stp-4 plants suggests that Stp is involved in specifying floral meristem identity in pea. The stp mutations also reduce the complexity of the compound pea leaf, and primary inflorescences often terminate prematurely in an aberrant sepaloid flower. In addition, stp mutants were shorter than their wild-type siblings due to a reduction in cell number in their internodes. Fewer cells were also found in the epidermis of the leaf rachis of stp mutants. Examination of the effects of stp-4 in double mutant combinations with af, tl, det, and veg2-2-mutations known to influence leaf, inflorescence, and flower development in pea-suggests that Stp function is independent of these genes. A synergistic interaction between weak mutant alleles at Stp and Uni indicated that these two genes act together, possibly to regulate primordial growth. Molecular analysis revealed that Stp is the pea homolog of the Antirrhinum gene Fimbriata (Fim) and of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Differences between Fim/UFO and Stp mutant phenotypes and expression patterns suggest that expansion of Stp activity into the leaf was an important step during evolution of the compound leaf in the garden pea.

  12. Elucidating the Impact of CHO Cell Culture Media on Tryptophan Oxidation of a Monoclonal Antibody Through Gene Expression Analyses.

    Science.gov (United States)

    He, Luhong; Desai, Jairav Xolo; Gao, Jinxin; Hazeltine, Laurie B; Lian, Zhirui; Calley, John N; Frye, Christopher C

    2018-03-15

    Oxidation of monoclonal antibodies (mAb) is a common chemical modification with potential impact on a therapeutic protein's activity and immunogenicity. In a previous study, it was found that tryptophan oxidation (Trp-ox) levels of two mAb produced in Chinese hamster ovary (CHO) cells were significantly lowered by modifying cell culture medium/feed. In this study, transcriptome analysis by RNA-Seq was applied to further elucidate the underlying mechanism of those changes in lowering the Trp-ox levels. Cell samples from the 5L fed-batch conditions were harvested and subjected to RNA-Seq analysis. The results showed that the cell culture changes had little impact on neither the expression of the mAb transgenes nor genes related to glycosylation. However, those changes did significantly alter the expression of multiple genes (p-value ≤0.05 and absolute fold change ≥1.5 or adjusted p-value ≤0.1) involved in transport of copper, regulation of glutathione, iron storage, heme reduction, oxidative phosphorylation and Nrf2-mediated antioxidative response. These findings suggest a key underlying mechanism in lowering Trp-ox levels by CDM was likely to be collectively controlling ROS levels through regulation of those genes' expression. This is the first example, to our knowledge, applying transcriptomic analysis to mechanistically understand the impact of cell culture on mAb oxidation. This article is protected by copyright. All rights reserved.

  13. Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    P. Zhang

    2008-06-01

    Full Text Available Mouse PNAS-4 (mPNAS-4 has 96% identity with human PNAS-4 (hPNAS-4 in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data, it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2 and colon carcinoma cells (CT26 of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3. The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4 was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.

  14. Characterization of Niemann-Pick Type C2 protein expression in multiple cancers using a novel NPC2 monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Yi-Jen Liao

    Full Text Available Niemann-Pick Type C2 (NPC2 plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-, progesterone receptor (- and human epidermal growth factor receptor (+. On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor

  15. A Rare Cause of Abdominal Pain: Primary Epiploic Appendagitis (PEA

    Directory of Open Access Journals (Sweden)

    Gulbanu Erkan

    2016-07-01

    Full Text Available Primary epiploic appendagitis (PEA is a rare disease caused by torsion or spontaneous thrombosis of the central vein that drains epiploic appendages (EA. Primary Epiploic Appendagitis (PEA is an ischemic infarction. Although PEA is a self-limiting disease and does not require surgical intervention in most cases, it may mimic diseases that require surgical intervention or aggressive medical therapy, such as appendicitis, diverticulitis, or cholecystitis. In order to avoid unnecessary surgical intervention, PEA should be kept in mind when patients present with acute abdominal pain. In this report, we present a PEA case admitted with abdominal pain.

  16. A pea chloroplast translation elongation factor that is regulated by abiotic factors

    International Nuclear Information System (INIS)

    Singh, B.N.; Mishra, R.N.; Agarwal, Pradeep K.; Goswami, Mamta; Nair, Suresh; Sopory, S.K.; Reddy, M.K.

    2004-01-01

    We report the cloning and characterization of both the cDNA (tufA) and genomic clones encoding for a chloroplast translation elongation factor (EF-Tu) from pea. The analysis of the deduced amino acids of the cDNA clone reveals the presence of putative transit peptide sequence and four GTP binding domains and two EF-Tu signature motifs in the mature polypeptide region. Using in vivo immunostaining followed by confocal microscopy pea EF-Tu was localized to chloroplast. The steady state transcript level of pea tufA was high in leaves and not detectable in roots. The expression of this gene is stimulated by light. The differential expression of this gene in response to various abiotic stresses showed that it is down-regulated in response to salinity and ABA and up-regulated in response to low temperature and salicylic acid treatment. These results indicate that regulation of pea tufA may have an important role in plant adaptation to environmental stresses

  17. PeaMUST, a large multidisciplinary project dedicated to pea improvement

    OpenAIRE

    Burstin, Judith; Pilet Nayel, Marie-Laure; Rameau, Catherine; Thompson, Richard; Carrouée, Benoit; Rivière, N.; Brochot, Anne-Lise; Chaillet, Isabelle

    2014-01-01

    Background and Objectives: Legume use in arable rotations provides several significant ecological services that can help to meet the challenge of delivering high-quality food and feed in an economically and environmentally sustainable manner. Nevertheless, grain legumes are underused by European farmers mainly because of yield inconsistency. PeaMUST overall objective is to develop novel pea varieties and optimize plant-symbiotic interactions for seed yield and quality regularity, in the conte...

  18. Anticitrullinated Protein Antibodies Induce Inflammatory Gene Expression Profile in Peripheral Blood Cells from CCP-positive Patients with RA.

    Science.gov (United States)

    Gertel, Smadar; Karmon, Gidi; Szarka, Eszter; Shovman, Ora; Houri-Levi, Esther; Mozes, Edna; Shoenfeld, Yehuda; Amital, Howard

    2018-03-01

    Anticitrullinated protein antibodies (ACPA) have major diagnostic significance in rheumatoid arthritis (RA). ACPA are directed against different citrullinated antigens, including filaggrin, fibrinogen, vimentin, and collagen. The presence of ACPA is associated with joint damage and extraarticular manifestations, suggesting that ACPA may have a significant role in the pathogenesis of RA. To verify the effect of ACPA on RA-immune cells, peripheral blood mononuclear cells (PBMC) from cyclic citrullinated peptide (CCP)-positive patients with RA and healthy controls were cocultured in vitro with ACPA. ACPA-positive stained cells were analyzed by flow cytometry and the effect of ACPA on mRNA expression levels was evaluated by real-time PCR. We tested whether the stimulatory effects induced by ACPA could be inhibited by the addition of a new multiepitope citrullinated peptide (Cit-ME). We found that ACPA bind specifically to PBMC from CCP-positive patients with RA through the Fab portion. ACPA induce upregulation of pathogenic cytokine expression (4- to 13-fold increase) in PBMC derived from CCP-positive patients with RA. Moreover, ACPA upregulated IL-1β and IL-6 mRNA expression levels by 10- and 6-fold, respectively, compared to control IgG. Cit-ME, a genuine ligand of ACPA, inhibited the ACPA-induced upregulation of IL-1β and IL-6 by 30%. ACPA bind to a limited percentage of PBMC and upregulate inflammatory cytokine expression, suggesting that ACPA is involved in RA pathogenesis. Targeting ACPA to decrease their pathogenic effects might provide a novel direction in developing therapeutic strategies for RA.

  19. Yeast expressed recombinant Hemagglutinin protein of Novel H1N1 elicits neutralising antibodies in rabbits and mice

    Directory of Open Access Journals (Sweden)

    Athmaram TN

    2011-11-01

    Full Text Available Abstract Currently available vaccines for the pandemic Influenza A (H1N1 2009 produced in chicken eggs have serious impediments viz limited availability, risk of allergic reactions and the possible selection of sub-populations differing from the naturally occurring virus, whereas the cell culture derived vaccines are time consuming and may not meet the demands of rapid global vaccination required to combat the present/future pandemic. Hemagglutinin (HA based subunit vaccine for H1N1 requires the HA protein in glycosylated form, which is impossible with the commonly used bacterial expression platform. Additionally, bacterial derived protein requires extensive purification and refolding steps for vaccine applications. For these reasons an alternative heterologous system for rapid, easy and economical production of Hemagglutinin protein in its glycosylated form is required. The HA gene of novel H1N1 A/California/04/2009 was engineered for expression in Pichia pastoris as a soluble secreted protein. The full length HA- synthetic gene having α-secretory tag was integrated into P. pastoris genome through homologous recombination. The resultant Pichia clones having multiple copy integrants of the transgene expressed full length HA protein in the culture supernatant. The Recombinant yeast derived H1N1 HA protein elicited neutralising antibodies both in mice and rabbits. The sera from immunised animals also exhibited Hemagglutination Inhibition (HI activity. Considering the safety, reliability and also economic potential of Pichia expression platform, our preliminary data indicates the feasibility of using this system as an alternative for large-scale production of recombinant influenza HA protein in the face of influenza pandemic threat.

  20. Functional expression of single-chain variable fragment antibody against c-Met in the cytoplasm of Escherichia coli.

    Science.gov (United States)

    Heo, Mi-Ae; Kim, Su-Hyun; Kim, So-Yeon; Kim, Yu-Jin; Chung, Junho; Oh, Min-Kyu; Lee, Sun-Gu

    2006-05-01

    c-Met, a high affinity receptor for hepatocyte growth factor/scatter factor, shown to be overexpressed in a variety of malignant cells, is a potential biomarker as well as a therapeutic target. Thus, single-chain antibody fragment (scFv) specific for c-Met is expected to be efficiently employed in the clinical treatment or imaging of many cancer cells. Here, we constructed the expression system for anti-c-Met scFv fused with T7 tag at its N-terminus using pET vector and investigated the expression conditions to achieve a functional and soluble expression of the scFv in the cytoplasm of recombinant Escherichia coli. The redox potential of E. coli cytoplasm was the most critical factor for the functional expression of anti-c-Met scFv. The employment of a host with oxidizing cytoplasm, E. coli trxB/gor double mutant, improved the productivity of functional anti-c-Met scFv by approximately 10-fold compared to the production of anti-c-Met scFv in the reducing cytoplasm of wild type E. coli. Productivity of functional anti-c-Met scFv could be further enhanced by co-expressing molecular chaperones such as GroELS, trigger factor, and DsbC with the scFv. Coexpression of DsbC increased the yield of functional anti-c-Met scFv about 2.5-fold in the cytoplasm of E. coli trxB/gor mutant compared to the production of scFv without DsbC coexpression. Lowering the IPTG concentration from 1 to 0.05 mM led to the slight enhancement, approximately 1.6-fold, of productivity of functional scFv. Although the use of low temperature for anti-c-Met scFv expression increased the ratio of soluble scFv fraction to insoluble fraction, productivity of soluble scFv decreased owing to the significant reduction of expression rate. The addition of 0.5 M sucrose in the medium inhibited the formation of intracellular insoluble anti-c-Met scFv. To purify the anti-c-Met scFv simply, we fused hexahistidine at the C-terminus of scFv and purified the scFv showing 98% of purity through the interaction

  1. Pea Streak Virus Recorded in Europe

    Czech Academy of Sciences Publication Activity Database

    Sarkisova, Tatiana; Bečková, M.; Fránová, Jana; Petrzik, Karel

    2016-01-01

    Roč. 52, č. 3 (2016), s. 164-166 ISSN 1212-2580 R&D Projects: GA MZe QH71145 Institutional support: RVO:60077344 Keywords : Pea streak virus * alfalfa * carlavirus * partial sequence Subject RIV: EE - Microbiology, Virology Impact factor: 0.742, year: 2016

  2. Swimming of the pea crab (Pinnotheres pisum)

    NARCIS (Netherlands)

    Versteegh, C.P.C.; Muller, M.

    2014-01-01

    Aquatic organisms have to deal with different hydrodynamic regimes, depending on their size and speed during locomotion. The pea crab swims by beating the third and fourth pereiopod on opposite sides as pairs. Using particle tracking velocimetry and high-speed video recording, we quantify the

  3. Glatiramer acetate antibodies, gene expression and disease activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn Thorup; Hedegaard, Chris Juul; Krakauer, M

    2011-01-01

    Background: Glatiramer acetate (GA) treatment suppresses disease activity in multiple sclerosis (MS). The immunological response to treatment may differ in patients who are stable on GA therapy and patients with breakthrough disease activity, but the results of previous studies are inconsistent...... reaction (PCR) analysis in 39 untreated and 29 GA-treated relapsing-remitting MS patients. Definition of breakthrough disease was based on the occurrence of relapses, disability progression, or gadolinium (Gd)-enhanced MRI. Results: The expression of T helper type 1 (Th1) and Th17 cytokines...

  4. Chimeric bispecific OC/TR monoclonal antibody mediates lysis of tumor cells expressing the folate-binding protein (MOv18) and displays decreased immunogenicity in patients

    NARCIS (Netherlands)

    Luiten, R. M.; Warnaar, S. O.; Sanborn, D.; Lamers, C. H.; Bolhuis, R. L.; Litvinov, S. V.; Zurawski, V. R.; Coney, L. R.

    1997-01-01

    The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial

  5. Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

    Science.gov (United States)

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-07-05

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Decreased Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals

    Science.gov (United States)

    Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T.; Ackerman, Margaret E.; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

    2011-01-01

    In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. PMID:21565376

  7. Synthesis and Evaluation of Tc-99m-Labelled Monoclonal Antibody 1D09C3 for Molecular Imaging of Major Histocompatibility Complex Class II Protein Expression

    NARCIS (Netherlands)

    Malviya, Gaurav; de Vries, E. F. J.; Dierckx, Rudi A.; Signore, Alberto

    2011-01-01

    It is known that major histocompatibility complex class II protein HLA-DR is highly expressed in B-cell lymphomas and in a variety of autoimmune and inflammatory diseases. Therefore, a radiolabelled fully humanized IgG4 monoclonal antibody (mAb) can provide useful prognostic and diagnostic

  8. Sonic Hedgehog-signalling patterns the developing chicken comb as revealed by exploration of the pea-comb mutation.

    Directory of Open Access Journals (Sweden)

    Henrik Boije

    Full Text Available The genetic basis and mechanisms behind the morphological variation observed throughout the animal kingdom is still relatively unknown. In the present work we have focused on the establishment of the chicken comb-morphology by exploring the Pea-comb mutant. The wild-type single-comb is reduced in size and distorted in the Pea-comb mutant. Pea-comb is formed by a lateral expansion of the central comb anlage into three ridges and is caused by a mutation in SOX5, which induces ectopic expression of the SOX5 transcription factor in mesenchyme under the developing comb. Analysis of differential gene expression identified decreased Sonic hedgehog (SHH receptor expression in Pea-comb mesenchyme. By experimentally blocking SHH with cyclopamine, the wild-type single-comb was transformed into a Pea-comb-like phenotype. The results show that the patterning of the chicken comb is under the control of SHH and suggest that ectopic SOX5 expression in the Pea-comb change the response of mesenchyme to SHH signalling with altered comb morphogenesis as a result. A role for the mesenchyme during comb morphogenesis is further supported by the recent finding that another comb-mutant (Rose-comb, is caused by ectopic expression of a transcription factor in comb mesenchyme. The present study does not only give knowledge about how the chicken comb is formed, it also adds to our understanding how mutations or genetic polymorphisms may contribute to inherited variations in the human face.

  9. Expression and purification of a major allergen, Pla a 1, from Platanus acerifolia pollen and the preparation of its monoclonal antibody.

    Science.gov (United States)

    Ni, Wei-Wei; Huang, Wen; Wu, De-Qin; Zhou, Yan-Jun; Ji, Chun-Mei; Cao, Meng-Da; Guo, Miao; Sun, Jin-Lu; Wei, Ji-Fu

    2017-09-01

    Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1. Hybridoma cells were screened by indirect ELISA and limiting dilution. Positive cells were used to induce the formation of antibody‑containing ascites fluid, and the antibodies were purified using protein A‑agarose. The results of the present study demonstrated that recombinant Pla a 1 was successfully expressed and purified, and exhibited positive immunoglobulin E‑binding to serum from patients allergic to P. acerifolia. A total of 11 hybridomas that steadily secreted anti‑Pla a 1 antibody were obtained and an immunoblotting analysis indicated that all of these monoclonal antibodies specifically recognized the Pla a 1 protein. These results suggested that specific anti‑Pla a 1 antibodies may be obtained, which can be used for the rapid detection of Pla a 1 allergens and in the preparation of vaccines against P. acerifolia pollen.

  10. The Effects of Light and Temperature on Biotin Synthesis in Pea Sprouts.

    Science.gov (United States)

    Kamiyama, Shin; Ohnuki, Risa; Moriki, Aoi; Abe, Megumi; Ishiguro, Mariko; Sone, Hideyuki

    2016-01-01

    Biotin is an essential micronutrient, and is a cofactor for several carboxylases that are involved in the metabolism of glucose, fatty acids, and amino acids. Because plant cells can synthesize their own biotin, a wide variety of plant-based foods contains significant amounts of biotin; however, the influence of environmental conditions on the biotin content in plants remains largely unclear. In the present study, we investigated the effects of different cultivation conditions on the biotin content and biotin synthesis in pea sprouts (Pisum sativum). In the experiment, the pea sprouts were removed from their cotyledons and cultivated by hydroponics under five different lighting and temperature conditions (control [25ºC, 12-h light/12-h dark cycle], low light [25ºC, 4-h light/20-h dark cycle], dark [25ºC, 24 h dark], low temperature [12ºC, 12-h light/12-h dark cycle], and cold [6ºC, 12-h light/12-h dark cycle]) for 10 d. Compared to the biotin content of pea sprouts under the control conditions, the biotin contents of pea sprouts under the low-light, dark, and cold conditions had significantly decreased. The dark group showed the lowest biotin content among the groups. Expression of the biotin synthase gene (bio2) was also significantly decreased under the dark and cold conditions compared to the control condition, in a manner similar to that observed for the biotin content. No significant differences in the adenosine triphosphate content were observed among the groups. These results indicate that environmental conditions such as light and temperature modulate the biotin content of pea plant tissues by regulating the expression of biotin synthase.

  11. Profile and Functional Properties of Seed Proteins from Six Pea (Pisum sativum Genotypes

    Directory of Open Access Journals (Sweden)

    Nikola Ristic

    2010-12-01

    Full Text Available Extractability, extractable protein compositions, technological-functional properties of pea (Pisum sativum proteins from six genotypes grown in Serbia were investigated. Also, the relationship between these characteristics was presented. Investigated genotypes showed significant differences in storage protein content, composition and extractability. The ratio of vicilin:legumin concentrations, as well as the ratio of vicilin + convicilin: Legumin concentrations were positively correlated with extractability. Our data suggest that the higher level of vicilin and/or a lower level of legumin have a positive influence on protein extractability. The emulsion activity index (EAI was strongly and positively correlated with the solubility, while no significant correlation was found between emulsion stability (ESI and solubility, nor between foaming properties and solubility. No association was evident between ESI and EAI. A moderate positive correlation between emulsion stability and foam capacity was observed. Proteins from the investigated genotypes expressed significantly different emulsifying properties and foam capacity at different pH values, whereas low foam stability was detected. It appears that genotype has considerable influence on content, composition and technological-functional properties of pea bean proteins. This fact can be very useful for food scientists in efforts to improve the quality of peas and pea protein products.

  12. Developing new SSR markers from ESTs of pea (Pisum sativum L.).

    Science.gov (United States)

    Gong, Ya-ming; Xu, Sheng-chun; Mao, Wei-hua; Hu, Qi-zan; Zhang, Gu-wen; Ding, Ju; Li, Ya-dan

    2010-09-01

    The development of expressed sequence tags (ESTs) from pea has provided a useful source for mining novel simple sequence repeat (SSR) markers. In the present research, in order to find EST-derived SSR markers, 18 552 pea ESTs from the National Center for Biotechnology Information (NCBI) database were downloaded and assembled into 10 086 unigenes. A total of 586 microsatellites in 530 unigenes were identified, indicating that merely 5.25% of sequences contained SSRs. The most abundant SSRs within pea were tri-nucleotide repeat motifs, and among all the tri-nucleotide repeats, the motif GAA was the most abundant type. In total, 49 SSRs were used for primer design. EST-SSR loci were subsequently screened on 10 widely adapted varieties in China. Of these, nine loci showed polymorphic profiles that revealed two to three alleles per locus. The polymorphism information content value ranged from 0.18 to 0.58 with an average of 0.41. Furthermore, transferable analysis revealed that some of these loci showed transferability to faba bean. Because of their polymorphism and transferability, these nine novel EST-SSRs will be valuable tools for marker-assisted breeding and comparative mapping of pea in the future.

  13. Engineered resistance to Nosema bombycis by in vitro expression of a single-chain antibody in Sf9-III cells

    Science.gov (United States)

    Huang, Yukang; Chen, Jie; Sun, Bin; Zheng, Rong; Li, Boning; Li, Zeng; Tan, Yaoyao; Wei, Junhong; Pan, Guoqing; Li, Chunfeng

    2018-01-01

    Nosema bombycis is a destructive, obligate intracellular parasite of the Bombyx mori. In this study, a single-chain variable fragment (scFv) dependent technology is developed for the purpose of inhibiting parasite proliferation in insect cells. The scFv-G4, which we prepared from a mouse G4 monoclonal antibody, can target the N. bombycis spore wall protein 12 (NbSWP12). Indirect immunofluorescence assays showed that NbSWP12 located mainly on the outside of the N. bombycis cytoskeleton, although some of it co-localized with β-tubulin in the meront-stage of parasites. When meront division began, NbSWP12 became concentrated at both ends of each meront. Western blotting showed that scFv-G4 could express in Sf9-III cells and recognized native NbSWP12. The transgenic Sf9-III cell line showed better resistance than the controls when challenged with N. bombycis, indicating that NbSWP12 is a promising target in this parasite and this scFv dependent strategy could be a solution for construction of N. bombycis-resistant Bombyx mori. PMID:29447266

  14. Response of Pea Varieties to Damage Degree of Pea Weevil, Bruchus pisorum L.

    Science.gov (United States)

    Nikolova, Ivelina Mitkova

    2016-01-01

    A study was conducted to determine the response of five pea varieties (Pisum sativum L.) to damage degree of Bruchus pisorum: Glyans, Modus, Kamerton, and Svit (Ukrainian cultivars) and Pleven 4 (Bulgarian cultivar). The seeds were classified into three types: healthy seeds (type 1), damaged seeds with parasitoid emergence hole (type 2), and damaged seeds with bruchid emergence hole (type 3) and they were sown. It was found that the weight of 1000 seeds did not affect the field germination of the pea varieties. Healthy and damaged seeds with parasitoid emergence holes (first and second seed types) provide a very good opportunity for growth and development while plants from damaged seeds with bruchid emergence holes had poor germination and vigor and low productivity. These seeds cannot provide the creation of well-garnished seeding and stable crop yields. Among tested varieties, the Ukrainian variety Glyans had considerably higher seed weight, field germination, and index germination and weak egg-laying activity of B. pisorum compared to others. Use of spring pea cultivars that are weakly preferred by the pea weevil in breeding programs would reduce losses due to pea weevil and provide an environmentally safer option to its control.

  15. Response of Pea Varieties to Damage Degree of Pea Weevil, Bruchus pisorum L.

    Directory of Open Access Journals (Sweden)

    Ivelina Mitkova Nikolova

    2016-01-01

    Full Text Available A study was conducted to determine the response of five pea varieties (Pisum sativum L. to damage degree of Bruchus pisorum: Glyans, Modus, Kamerton, and Svit (Ukrainian cultivars and Pleven 4 (Bulgarian cultivar. The seeds were classified into three types: healthy seeds (type 1, damaged seeds with parasitoid emergence hole (type 2, and damaged seeds with bruchid emergence hole (type 3 and they were sown. It was found that the weight of 1000 seeds did not affect the field germination of the pea varieties. Healthy and damaged seeds with parasitoid emergence holes (first and second seed types provide a very good opportunity for growth and development while plants from damaged seeds with bruchid emergence holes had poor germination and vigor and low productivity. These seeds cannot provide the creation of well-garnished seeding and stable crop yields. Among tested varieties, the Ukrainian variety Glyans had considerably higher seed weight, field germination, and index germination and weak egg-laying activity of B. pisorum compared to others. Use of spring pea cultivars that are weakly preferred by the pea weevil in breeding programs would reduce losses due to pea weevil and provide an environmentally safer option to its control.

  16. IGHV1-69-Encoded Antibodies Expressed in Chronic Lymphocytic Leukemia React with Malondialdehyde-Acetaldehyde Adduct, an Immunodominant Oxidation-Specific Epitope

    DEFF Research Database (Denmark)

    Que, Xuchu; Widhopf Ii, George F; Amir, Shahzada

    2013-01-01

    The immunoglobulins expressed by chronic lymphocytic leukemia (CLL) B cells are highly restricted, suggesting they are selected for binding either self or foreign antigen. Of the immunoglobulin heavy-chain variable (IGHV) genes expressed in CLL, IGHV1-69 is the most common, and often is expressed...... are products of enhanced lipid peroxidation and a major target of innate natural antibodies. Specifically, CLL69C bound immunodominant OSE adducts termed MAA (malondialdehyde-acetaldehyde-adducts), which are found on apoptotic cells, inflammatory tissues, and atherosclerotic lesions. It also reacted...

  17. Effect of selection on green pea hybrid population structure

    Directory of Open Access Journals (Sweden)

    В. М. Стригун

    2013-05-01

    Full Text Available The article features the results of variability study for one of the productivity characteristics in green pea, namely «number of seeds in the seedpod» for hybrid  populations and for the selections made thereof. Statistical methods of analysis have established the levels of variability for the characteristics in F2 original population , the impact of the selection completed within F2 onto the variability of F3 hybrid population. This shows the efficiency of repeated selection from F3 population and its impact onto the level of expersiioon of the characteristic being studied in the resulting F6 population. As a conclusion, a good time is set for practical breeding with the characteristic of «number of seeds in a seedpod» for  the selection, as well as for the methods for achieving its stable expression, that is to facilitate the decrease of durations and means for the tests conduct.

  18. Species-Specific Monoclonal Antibodies to Escherichia coli-Expressed p36 Cytosolic Protein of Mycoplasma hyopneumoniae

    Science.gov (United States)

    Caron, J.; Sawyer, N.; Moumen, B. Ben Abdel; Bouh, K. Cheikh Saad; Dea, S.

    2000-01-01

    The p36 protein of Mycoplasma hyopneumoniae is a cytosolic protein carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the p36-encoding gene (948 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species and pathogenic bacteria that commonly colonize the porcine respiratory tract. The amplified p36 gene was subcloned into the pGEX-4T-1 vector to be expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The GST-p36 recombinant fusion protein was purified by affinity chromatography and cut by thrombin, and the enriched p36 protein was used to immunize female BALB/c mice for the production of anti-p36 monoclonal antibodies (MAbs). The polypeptide specificity of the nine MAbs obtained was confirmed by Western immunoblotting with cell lysates prepared from the homologous strain. Cross-reactivity studies of the anti-p36 MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture suggested that these anti-p36 MAbs were directed against a highly conserved epitope, or closely located epitopes, of the p36 protein. No reactivity was demonstrated against other mycoplasma species tested. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs infected experimentally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. The bacteria could be recovered from lung homogenates of pigs that were killed after the 3-week observation period by both PCR and cultivation procedures. Furthermore, the anti-p36 MAbs permitted effective detection by indirect immunofluorescence of M. hyopneumoniae in frozen lung sections from experimentally infected pigs. However, attempts to use the recombinant p36 protein as an antigen in an

  19. Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics.

    Science.gov (United States)

    Ramachandran, Vinoy K; East, Alison K; Karunakaran, Ramakrishnan; Downie, J Allan; Poole, Philip S

    2011-10-21

    The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.

  20. Anomalous expression of Thy1 (CD90) in B-cell lymphoma cells and proliferation inhibition by anti-Thy1 antibody treatment

    Energy Technology Data Exchange (ETDEWEB)

    Ishiura, Yoshihito [Department of Biochemistry, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kotani, Norihiro, E-mail: kotani@kochi-u.ac.jp [CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kochi System Glycobiology Center, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); Yamashita, Ryusuke [Department of Biochemistry, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Yamamoto, Harumi [Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Yoshida Shimo-Adachi, Sakyo, Kyoto 606-8501 (Japan); Kozutsumi, Yasunori [CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Yoshida Shimo-Adachi, Sakyo, Kyoto 606-8501 (Japan); Honke, Koichi [Department of Biochemistry, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan); CREST, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012 (Japan); Kochi System Glycobiology Center, Kochi University Medical School, Kohasu, Okocho, Nankoku, Kochi 783-8505 (Japan)

    2010-05-28

    The anti-CD20 monoclonal antibody (Ab) rituximab is accepted to be an effective therapeutic Ab for malignant B-cell lymphoma; however, discovery of other cell surface antigens is required for the option of antibody medicine. Considering that many tumor-associated antigens are glycans, we have searched glycoconjugates for the candidate antigens that therapeutic Abs target. To this end, we first focused on the difference in the glycogenes expression in terms of Epstein-Barr virus (EBV) infection of a Burkitt's lymphoma cell line, Akata. Using DNA array, flow cytometry and Western blotting, we found that Thy1 was highly expressed in EBV-positive Akata cells. Subsequently, Thy1 was found to be expressed in other B-cell lymphoma cell lines: BJAB, MutuI, and MutuIII, irrespective of EBV infection. Treatment of these cells with an anti-Thy1 monoclonal antibody inhibited proliferation more strongly than the therapeutic Ab rituximab. The B-cell lymphoma cell lines were classified based on the extent of the proliferation inhibition, which was not correlated with the expression level of Thy1. It is suggested that stable residence of receptor tyrosine kinases in lipid rafts sustains cell growth in B-cell lymphoma cells.

  1. Genomic tools in pea breeding programs: status and perspectives

    Directory of Open Access Journals (Sweden)

    Nadim eTAYEH

    2015-11-01

    Full Text Available Pea (Pisum sativum L. is an annual cool-season legume and one of the oldest domesticated crops. Dry pea seeds contain 22-25 percent protein, complex starch and fibre constituents and a rich array of vitamins, minerals, and phytochemicals which make them a valuable source for human consumption and livestock feed. Dry pea ranks third to common bean and chickpea as the most widely grown pulse in the world with more than 11 million tonnes produced in 2013. Pea breeding has achieved great success since the time of Mendel’s experiments in the mid-1800s. However, several traits still require significant improvement for better yield stability in a larger growing area. Key breeding objectives in pea include improving biotic and abiotic stress resistance and enhancing yield components and seed quality. Taking advantage of the diversity present in the pea genepool, many mapping populations have been constructed in the last decades and efforts have been deployed to identify loci involved in the control of target traits and further introgress them into elite breeding materials. Pea now benefits from next-generation sequencing and high-throughput genotyping technologies that are paving the way for genome-wide association studies and genomic selection approaches. This review covers the significant development and deployment of genomic tools for pea breeding in recent years. Future prospects are discussed especially in light of current progress towards deciphering the pea genome.

  2. Number and Effectiveness of Pea Rhizobia in Danish Soils

    DEFF Research Database (Denmark)

    Engvild, K.C.

    1989-01-01

    moor at pH 4.7. Soil suspensions of nine of the soils were tested as inoculum in large outdoor pot cultures of peas grown to maturity in nitrogen free vermiculture. One soil approached the effectiveness of commercial inoculants, and four soils were quite effective. Two reclaimed soils and two......Most of 44 Danish soils tested contain between 1000 and 10 000 pea rhizobia (Rhizobium leguminosarum biovar viceae) per gram. Pea rhizobia were not detected in acid moor and forest soils. Only one case of failed nodulation in peas in the field has been noted, in spots in a reclaimed sandy heath...... of the three noncultivated soils showed low effectiveness....

  3. Antitumour effects of single or combined monoclonal antibodies directed against membrane antigens expressed by human B cells leukaemia

    Directory of Open Access Journals (Sweden)

    Kosinski Marek

    2011-04-01

    Full Text Available Abstract Background The increasing availability of different monoclonal antibodies (mAbs opens the way to more specific biologic therapy of cancer patients. However, despite the significant success of therapy in breast and ovarian carcinomas with anti-HER2 mAbs as well as in non-Hodkin B cell lymphomas with anti-CD20 mAbs, certain B cell malignancies such as B chronic lymphocytic leukaemia (B-CLL respond poorly to anti-CD20 mAb, due to the low surface expression of this molecule. Thus, new mAbs adapted to each types of tumour will help to develop personalised mAb treatment. To this aim, we analyse the biological and therapeutic properties of three mAbs directed against the CD5, CD71 or HLA-DR molecules highly expressed on B-CLL cells. Results The three mAbs, after purification and radiolabelling demonstrated high and specific binding capacity to various human leukaemia target cells. Further in vitro analysis showed that mAb anti-CD5 induced neither growth inhibition nor apoptosis, mAb anti-CD71 induced proliferation inhibition with no early sign of cell death and mAb anti-HLA-DR induced specific cell aggregation, but without evidence of apoptosis. All three mAbs induced various degrees of ADCC by NK cells, as well as phagocytosis by macrophages. Only the anti-HLA-DR mAb induced complement mediated lysis. Coincubation of different pairs of mAbs did not significantly modify the in vitro results. In contrast with these discrete and heterogeneous in vitro effects, in vivo the three mAbs demonstrated marked anti-tumour efficacy and prolongation of mice survival in two models of SCID mice, grafted either intraperitoneally or intravenously with the CD5 transfected JOK1-5.3 cells. This cell line was derived from a human hairy cell leukaemia, a type of malignancy known to have very similar biological properties as the B-CLL, whose cells constitutively express CD5. Interestingly, the combined injection of anti-CD5 with anti-HLA-DR or with anti-CD71 led to

  4. Production process reproducibility and product quality consistency of transient gene expression in HEK293 cells with anti-PD1 antibody as the model protein.

    Science.gov (United States)

    Ding, Kai; Han, Lei; Zong, Huifang; Chen, Junsheng; Zhang, Baohong; Zhu, Jianwei

    2017-03-01

    Demonstration of reproducibility and consistency of process and product quality is one of the most crucial issues in using transient gene expression (TGE) technology for biopharmaceutical development. In this study, we challenged the production consistency of TGE by expressing nine batches of recombinant IgG antibody in human embryonic kidney 293 cells to evaluate reproducibility including viable cell density, viability, apoptotic status, and antibody yield in cell culture supernatant. Product quality including isoelectric point, binding affinity, secondary structure, and thermal stability was assessed as well. In addition, major glycan forms of antibody from different batches of production were compared to demonstrate glycosylation consistency. Glycan compositions of the antibody harvested at different time periods were also measured to illustrate N-glycan distribution over the culture time. From the results, it has been demonstrated that different TGE batches are reproducible from lot to lot in overall cell growth, product yield, and product qualities including isoelectric point, binding affinity, secondary structure, and thermal stability. Furthermore, major N-glycan compositions are consistent among different TGE batches and conserved during cell culture time.

  5. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Directory of Open Access Journals (Sweden)

    Imperiale Valentina

    2007-07-01

    Full Text Available Abstract Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc into an infectious disease-associated isoform, (PrPsc. Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP. Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

  6. Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

    Science.gov (United States)

    Flego, Michela; Ascione, Alessandro; Zamboni, Silvia; Dupuis, Maria L; Imperiale, Valentina; Cianfriglia, Maurizio

    2007-01-01

    Background A hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions. In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP). Results We adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv) phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells. Conclusion Sequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease. PMID:17605808

  7. Genetically Modified alpha-Amylase Inhibitor Peas Are Not Specifically Allergenic in Mice

    OpenAIRE

    Lee, Rui-Yun; Reiner, Daniela; Dekan, Gerhard; Moore, Andrew E.; Higgins, T. J. V.; Epstein, Michelle M.

    2013-01-01

    Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity ...

  8. Law enforcement fick and peleg model for study of water uptake in navel cargamanto yellow (phaseolus vulgaris l. and peas seca (pisan sativa during rehydration

    Directory of Open Access Journals (Sweden)

    John Fernando Agudelo Ospina

    2009-08-01

    Full Text Available This research validated Fick’s Law and Peleg’s model applicability for modeling water sorption of yellow navel cargamanto bean (Phaseolus Vulgaris L. and dry pea (Pisum sativum, using five different water temperatures 25, 35, 40, 47 y 55 ºC, to soak the analyzed materials; for bean an optimal mathematical expression was obtained to make estimations, opposite case was the observed thing for pea; the diffusion coefficient calculated were all of the order of 1.91 x 10-9 m2/s for bean and 3.51 x 10-10 for dry pea; the activation energies were 41.464 KJ/mol for bean and 56.246 KJ/mol for dry pea

  9. PeaMUST (2012-2019) – Pea Multi-Stress adaptation and biological regulations for yield improvement and stability

    OpenAIRE

    Burstin, Judith; Pilet-Nayel, Marie-Laure; Rameau, Catherine; Thompson, Richard; Labalette, Francoise; Leveugle, Magalie; Rémy, Amandine; Pinochet, Xavier

    2017-01-01

    PeaMUST (2012-2019) is a large French national project whose objective is to develop novel pea varieties and optimize plant-symbiotic interactions for stabilized seed yield and quality, in the context of climate change and pesticide reduction. With the involvement of 28 partners from the public and private sectors, it will take advantage of NGS sequencing, genotyping and phenotyping technologies, to tackle the challenge of multiple stresses that penalize the performance of the pea crop.

  10. Novel MeCP2 isoform-specific antibody reveals the endogenous MeCP2E1 expression in murine brain, primary neurons and astrocytes.

    Directory of Open Access Journals (Sweden)

    Robby M Zachariah

    Full Text Available Rett Syndrome (RTT is a severe neurological disorder in young females, and is caused by mutations in the X-linked MECP2 gene. MECP2/Mecp2 gene encodes for two protein isoforms; MeCP2E1 and MeCP2E2 that are identical except for the N-terminus region of the protein. In brain, MECP2E1 transcripts are 10X higher, and MeCP2E1 is suggested to be the relevant isoform for RTT. However, due to the unavailability of MeCP2 isoform-specific antibodies, the endogenous expression pattern of MeCP2E1 is unknown. To gain insight into the expression of MeCP2E1 in brain, we have developed an anti-MeCP2E1 antibody and validated its specificity in cells exogenously expressing individual MeCP2 isoforms. This antibody does not show any cross-reactivity with MeCP2E2 and detects endogenous MeCP2E1 in mice brain, with no signal in Mecp2(tm1.1Bird y/- null mice. Additionally, we show the endogenous MeCP2E1 expression throughout different brain regions in adult mice, and demonstrate its highest expression in the brain cortex. Our results also indicate that MeCP2E1 is highly expressed in primary neurons, as compared to primary astrocytes. This is the first report of the endogenous MeCP2E1 expression at the protein levels, providing novel avenues for understanding different aspects of MeCP2 function.

  11. Comparison of the α-amylase inhibitor-1 from common bean (Phaseolus vulgaris) varieties and transgenic expression in other legumes--post-translational modifications and immunogenicity.

    Science.gov (United States)

    Campbell, Peter M; Reiner, Daniela; Moore, Andrew E; Lee, Rui-Yun; Epstein, Michelle M; Higgins, T J V

    2011-06-08

    The seeds of peas (Pisum sativum) and chickpeas (Cicer arietinum) expressing a gene for α-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are protected from damage by old world bruchids (pea and cowpea weevils). Here, we used electrospray ionization time-of-flight mass spectrometry to compare the post-translational modifications of αAI from transgenic sources with the processed forms of the protein from several bean varieties. All sources showed microheterogeneity with differences in the relative abundance of particular variants due to differences in the frequency of addition of glycans, variable processing of glycans, and differences of C-terminal exopeptidase activity. The structural variation among the transgenics was generally within the range of the bean varieties. Previously, mice showed allergic reactions following ingestion of transgenic pea αAI but not bean αAI. Here, only minor differences were observed following intraperitoneal sensitization. Both of the transgenic pea and bean forms of αAI elicited Th1 and Th2 antibody isotype responses, suggesting that both proteins are immunogenic and could potentially be allergenic.

  12. Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody.

    Science.gov (United States)

    Piperno, G M; López-Requena, A; Predonzani, A; Dorvignit, D; Labrada, M; Zentilin, L; Burrone, O R; Cesco-Gaspere, M

    2015-12-01

    The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ∼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.

  13. EXPRESSION OF RECOMBINANT ANTIBODY FRAGMENT, ANTI BNP-SCFV ON THE PERIPLASM OF Escherichia coli FOR THE DETECTION OF HEART FAILURE

    Directory of Open Access Journals (Sweden)

    Shabarni Gaffar

    2017-05-01

    Full Text Available Basic natriuretic peptide (BNP is a polypeptide hormone consist of 32 amino acids that secreted by the heart ventricle to respond the excessive stretching of heart muscle cells. BNP can be used as prognostic marker for patients with heart failure. The presence of BNP in blood can be detected by BNP antibody, which is anti BNP-single chain variable fragment (Anti BNP-SCFV. The antibody is a combination of polypeptides between varying region on the heavy chain (VH and the light chain (VL of immunoglobulin. Anti BNP-SCFV will bind to BNP through the antigen-antibody interaction. Concentration of BNP in a patient’s blood can be detected through the interaction of BNP with Anti BNP-SCFV using immunosensor method. Production of recombinant Anti BNP-SCFV in Escherichia coli as host is reported in the present study. Anti BNP-SCFV was expressed in fusion form with OmpC signal peptide that direct the protein to a periplasmic space. Expression was performed under RhaBad promoter as control using L-rhamnose as inducer. SDS-PAGE characterization showed consistent band at 28 kDa, which was assumed as Anti BNP-SCFV. The optimum expression was found at four hours after induction with 4 mM inducer. Anti BNP-SCFV was secreted from the cell as characterized by the presence of the protein on periplasmic membrane and extracellular fraction.

  14. Expression of the rubber-like protein, resilin, in developing and functional insect cuticle determined using a Drosophila anti-Rec 1 resilin antibody.

    Science.gov (United States)

    Wong, Darren C C; Pearson, Roger D; Elvin, Christopher M; Merritt, David J

    2012-02-01

    The natural elastomeric protein, insect resilin, is the most efficient elastic material known, used to store energy for jumping and flight in a variety of insects. Here, an antibody to recombinant Drosophila melanogaster pro-resilin is used to examine resilin expression in Drosophila and a wider range of insects. Immunostaining of Drosophila embryos reveals anti-resilin reactivity in epidermal patches that exhibit a dynamic spatial and temporal expression through late embryogenesis. Resilin is also detected in stretch receptors in the embryo. In developing adult Drosophila, resilin pads are described at the base of wings and at the base of flexible sensory hairs in pupae. Resilin is also detected in embryos of the tephritid fruitfly, Bactrocera tryoni, and two well-known concentrations of insect resilin: the flight muscle tendon of the dragonfly and the pleural arch of the flea. The anti-Rec1 antibody antibody developed using Drosophila pro-resilin as antigen is cross-reactive and is useful for detection of resilin in diverse insects. For the first time, resilin expression has been detected during embryogenesis, revealing segmental patches of resilin in the developing epidermis of Drosophila. Copyright © 2011 Wiley Periodicals, Inc.

  15. Analysis of genetic diversity in pigeon pea germplasm using ...

    Indian Academy of Sciences (India)

    MANEESHA

    2017-08-16

    Aug 16, 2017 ... Pigeon pea (Cajanus cajan) is an important major pulse crop predominantly cultivated in semiarid tropical and subtropical regions of Asia and Africa. It is an impor- tant grain that ranks among the five edible legumes that is used in intercropping systems, for food, fodder and fire- wood. The pigeon pea ...

  16. Functional properties of processed pigeon pea ( Cajanus cajan ) flour

    African Journals Online (AJOL)

    Germination increased water absorption capacity, bulk density, oil absorption capacity, foaming capacity, foaming stability, emulsion activity, nitrogen solubility and decreased gelatin and wetability of the pigeon pea flour. Germinated pigeon pea flour has great potentials in food prperations that require hydration to improve ...

  17. Yield advantage and water saving in maize/pea intercrop

    NARCIS (Netherlands)

    Mao, L.; Zhang, L.; Li, W.; Werf, van der W.; Sun, J.; Spiertz, J.H.J.; Li, L.

    2012-01-01

    Intercropping is a well-established strategy for maximization of yield from limited land, but mixed results have been obtained as to its performance in terms of water use efficiency. Here, two maize/pea intercrop layouts were studied in comparison to sole maize and sole pea with and without plastic

  18. 78 FR 68410 - United States Standards for Whole Dry Peas

    Science.gov (United States)

    2013-11-14

    ... available for public inspection in the above office during regular business hours (7 CFR 1.27(b)). Please... the fundamental starting point to define commodity quality in the domestic and global marketplace... for whole dry peas, split peas, and lentils in today's marketing environment. According to information...

  19. Substitution Value of toasted Pigeon pea ( Cajanus cajan ) seed ...

    African Journals Online (AJOL)

    One hundred and eighty 7-day old broilers were used in a 28-day feeding trial to determine the substitution value of toasted pigeon pea seeds meal (TPSM) for soybean meal and maize in boiler starter diet. The brown coat coloured pigeon pea seeds were toasted at 100oC for 15minutes and milled. The inclusion levels of ...

  20. Revoluntionary Faith and Religious Disillusionment in Enrico Pea's ...

    African Journals Online (AJOL)

    Questa tragedia segna anche un momento cruciale di transizione nella produzione letteraria di Pea e, pur mostrando chiaramente l'influenza delle sue precedenti affinità con il Marxismo e con il movimento anarchico, guarda già in avanti con il profondo interesse e rispetto di Pea per le proprie radici cattoliche che si ...

  1. Symbiotic effectiveness of pea-rhizobia associations and the ...

    African Journals Online (AJOL)

    微软用户

    2011-05-02

    May 2, 2011 ... Interactions between pea (Pisum sativa L.) cultivars and Rhizobium strain affect the symbiotic relationship and ultimately both the nitrogen fixing capacity and the yield. Since Pisum sativum L. is poorly nodulated in the Loess Plateau of China where this crop is grown, the response of pea cultivars. Yannong ...

  2. Functional analysis of mildly refined fractions from yellow pea

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Boom, R.M.; Schutyser, M.A.I.

    2015-01-01

    Dry fractionation offers an attractive route to sustainably produce protein-enriched plant-based ingredients. For example, fine milling of peas followed by air classification separates starch granules from the protein matrix. Unlike conventional wet isolates, dry-enriched pea fractions consist of a

  3. Effective management of pigeon pea ( Cajanus cajan ) in a crop ...

    African Journals Online (AJOL)

    Effective management of pigeon pea ( Cajanus cajan ) in a crop/livestock integrated farming system in northern Ghana. ... Ghana Journal of Agricultural Science ... Pigeon pea plots with row lengths averaging 11 m and a planting geometry of 80 cm W 50 cm, were either pruned at 60 or 100 cm above ground level or not ...

  4. Genetic diversity in Ethiopian field pea ( Pisum sativum L ...

    African Journals Online (AJOL)

    Field pea is an ancient legume crop grown mainly for food in Ethiopia. Even though, there are over one thousand five hundred field pea collections, only a few studies has been conducted on the magnitude and pattern of genetic diversity at molecular level particularly with SSR markers. In this study, genetic diversity of 142 ...

  5. Dry fractionation for production of functional pea protein concentrates

    NARCIS (Netherlands)

    Pelgrom, P.J.M.; Vissers, A.M.; Boom, R.M.; Schutyser, M.A.I.

    2013-01-01

    Dry milling in combination with air classification was evaluated as an alternative to conventional wet extraction of protein from yellow field peas (Pisum sativum). Major advantages of dry fractionation are retention of native functionality of proteins and its lower energy and water use. Peas were

  6. VARIETIES AND BASIC TECHNIQUES OF PEA CULTIVATION IN KUBAN

    OpenAIRE

    A. G. Bessedin

    2013-01-01

    New varieties of pea of different time of ripening were developed in the Crimean experimental breeding station. These varieties were included in the State register of breeding achievements permitted for use in the areas of raw material for canning factories of Kuban. The cultivation technology of the pea varieties is described.

  7. VARIETIES AND BASIC TECHNIQUES OF PEA CULTIVATION IN KUBAN

    Directory of Open Access Journals (Sweden)

    A. G. Bessedin

    2013-01-01

    Full Text Available New varieties of pea of different time of ripening were developed in the Crimean experimental breeding station. These varieties were included in the State register of breeding achievements permitted for use in the areas of raw material for canning factories of Kuban. The cultivation technology of the pea varieties is described.

  8. The effect of Orobanche crenata infection severity in faba bean, field pea, and grass pea productivity.

    Directory of Open Access Journals (Sweden)

    Monica Fernandez-Aparicio

    2016-09-01

    Full Text Available Broomrape weeds (Orobanche and Phelipanche spp. are root holoparasites that feed off a wide range of important crops. Among them, Orobanche crenata attacks legumes complicating their inclusion in cropping systems along the Mediterranean area and West Asia. The detrimental effect of broomrape parasitism in crop yield can reach up to 100% depending on infection severity and the broomrape-crop association. This work provides field data of the consequences of O. crenata infection severity in three legume crops i.e. faba bean, field pea and grass pea. Regression functions modelled productivity losses and revealed trends in dry matter allocation in relation to infection severity. The host species differentially limits parasitic sink strength indicating different levels of broomrape tolerance at equivalent infection severities. Reductions in host aboveground biomass were observed starting at low infection severity and half maximal inhibitory performance was predicted as 4.5, 8.2 and 1.5 parasites per faba bean, field pea and grass pea plant, respectively. Reductions in host biomass occurred in both vegetative and reproductive organs, the latter resulting more affected. The proportion of resources allocated within the parasite was concomitant to reduction of host seed yield indicating that parasite growth and host reproduction compete directly for resources within a host plant. However, the parasitic sink activity does not fully explain the total host biomass reduction because combined biomass of host-parasite complex was lower than the biomass of uninfected plants. In grass pea, the seed yield was negligible at severities higher than 4 parasites per plant. In contrast, faba bean and field pea sustained low but significant seed production at the highest infection severity. Data on seed yield and seed number indicated that the sensitivity of field pea to O. crenata limited the production of grain yield by reducing seed number but maintaining seed size

  9. The Effect of Orobanche crenata Infection Severity in Faba Bean, Field Pea, and Grass Pea Productivity.

    Science.gov (United States)

    Fernández-Aparicio, Mónica; Flores, Fernando; Rubiales, Diego

    2016-01-01

    Broomrape weeds ( Orobanche and Phelipanche spp.) are root holoparasites that feed off a wide range of important crops. Among them, Orobanche crenata attacks legumes complicating their inclusion in cropping systems along the Mediterranean area and West Asia. The detrimental effect of broomrape parasitism in crop yield can reach up to 100% depending on infection severity and the broomrape-crop association. This work provides field data of the consequences of O. crenata infection severity in three legume crops, i.e., faba bean, field pea, and grass pea. Regression functions modeled productivity losses and revealed trends in dry matter allocation in relation to infection severity. The host species differentially limits parasitic sink strength indicating different levels of broomrape tolerance at equivalent infection severities. Reductions in host aboveground biomass were observed starting at low infection severity and half maximal inhibitory performance was predicted as 4.5, 8.2, and 1.5 parasites per faba bean, field pea, and grass pea plant, respectively. Reductions in host biomass occurred in both vegetative and reproductive organs, the latter resulting more affected. The increase of resources allocated within the parasite was concomitant to reduction of host seed yield indicating that parasite growth and host reproduction compete directly for resources within a host plant. However, the parasitic sink activity does not fully explain the total host biomass reduction because combined biomass of host-parasite complex was lower than the biomass of uninfected plants. In grass pea, the seed yield was negligible at severities higher than four parasites per plant. In contrast, faba bean and field pea sustained low but significant seed production at the highest infection severity. Data on seed yield and seed number indicated that the sensitivity of field pea to O. crenata limited the production of grain yield by reducing seed number but maintaining seed size. In contrast

  10. Overcoming maternal antibody interference by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of swine influenza virus.

    Science.gov (United States)

    Wesley, Ronald D; Lager, Kelly M

    2006-11-26

    Sows and gilts lack immunity to human adenovirus 5 (Ad-5) vectored vaccines so immunogens of swine pathogens can be expressed with these vaccines in order to immunize suckling piglets that have interfering, maternally derived antibodies. In this study 7-day-old piglets, that had suckled H3N2 infected gilts, were sham-inoculated with a non-expressing Ad-5 vector or given a primary vaccination with replication-defective Ad-5 viruses expressed the H3 hemagglutinin and the nucleoprotein of swine influenza virus (SIV) subtype H3N2. The hemagglutination inhibition (HI) titer of the sham-inoculated group (n = 12) showed continued antibody decay whereas piglets vaccinated with Ad-5 SIV (n = 23) developed an active immune response by the second week post-vaccination. At 4 weeks-of-age when the HI titer of the sham-inoculated group had decayed to 45, the sham-inoculated group and half of the Ad-5 SIV vaccinated pigs were boosted with a commercial inactivated SIV vaccine. The boosted pigs that had been primed in the presence of maternal interfering antibodies had a strong anamnestic response while sham-inoculated pigs did not respond to the commercial vaccine. Two weeks after the booster vaccination the pigs were challenged with a non-homologous H3N2 virulent SIV. The efficacy of the vaccination protocol was demonstrated by abrogation of clinical signs, by clearance of challenge virus from pulmonary lavage fluids, by markedly reduced virus shedding in nasal secretions, and by the absence of moderate or severe SIV-induced lung lesions. These recombinant Ad-5 SIV vaccines are useful for priming the immune system to override the effects of maternally derived antibodies which interfere with conventional SIV vaccines.

  11. Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo.

    Directory of Open Access Journals (Sweden)

    Stéphane Kerbrat

    Full Text Available TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4(+ T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15 kDa is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4(+ T cells. TCR-stimulated PEA-15-deficient CD4(+ T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4(+ T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4(+ CD62L(+ PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response.

  12. Absence of the Adaptor Protein PEA-15 Is Associated with Altered Pattern of Th Cytokines Production by Activated CD4+ T Lymphocytes In Vitro, and Defective Red Blood Cell Alloimmune Response In Vivo.

    Science.gov (United States)

    Kerbrat, Stéphane; Vingert, Benoit; Junier, Marie-Pierre; Castellano, Flavia; Renault-Mihara, François; Dos Reis Tavares, Silvina; Surenaud, Mathieu; Noizat-Pirenne, France; Boczkowski, Jorge; Guellaën, Georges; Chneiweiss, Hervé; Le Gouvello, Sabine

    2015-01-01

    TCR-dependent and costimulation signaling, cell division, and cytokine environment are major factors driving cytokines expression induced by CD4(+) T cell activation. PEA-15 15 (Protein Enriched in Astrocyte / 15 kDa) is an adaptor protein that regulates death receptor-induced apoptosis and proliferation signaling by binding to FADD and relocating ERK1/2 to the cytosol, respectively. By using PEA-15-deficient mice, we examined the role of PEA-15 in TCR-dependent cytokine production in CD4(+) T cells. TCR-stimulated PEA-15-deficient CD4(+) T cells exhibited defective progression through the cell cycle associated with impaired expression of cyclin E and phosphoRb, two ERK1/2-dependent proteins of the cell cycle. Accordingly, expression of the division cycle-dependent cytokines IL-2 and IFNγ, a Th1 cytokine, was reduced in stimulated PEA-15-deficient CD4(+) T cells. This was associated with abnormal subcellular compartmentalization of activated ERK1/2 in PEA-15-deficient T cells. Furthermore, in vitro TCR-dependent differentiation of naive CD4(+) CD62L(+) PEA-15-deficient T cells was associated with a lower production of the Th2 cytokine, IL-4, whereas expression of the Th17-associated molecule IL4I1 was enhanced. Finally, a defective humoral response was shown in PEA-15-deficient mice in a model of red blood cell alloimmunization performed with Poly IC, a classical adjuvant of Th1 response in vivo. Collectively, our data suggest that PEA-15 contributes to the specification of the cytokine pattern of activated Th cells, thus highlighting a potential new target to interfere with T cell functional polarization and subsequent immune response.

  13. Evaluation of HNA-expressing cell line-based antigen capture systems and a solid-phase system for detecting HNA-1a antibodies.

    Science.gov (United States)

    Amakishi, Etsuko; Irie, Yoriko; Nishizawa, Kanae; Kamada, Hiromi; Nakajima, Fumiaki; Matsuyama, Nobuki; Ishii, Hiroyuki; Matsukura, Harumichi; Yasui, Kazuta; Hirayama, Fumiya

    2015-04-01

    Granulocyte immunofluorescence and granulocyte agglutination tests are standard methods for detecting human neutrophil antigen (HNA) antibodies (Abs); however, these require a typed panel of neutrophils, which can be time-consuming to develop, and it remains difficult to determine antibody specificity in some cases. We established and evaluated four detection systems for HNA-1a Abs based on an HNA-1a-expressing cell line (KY cells) and antigen capture. We additionally evaluated a commercial solid-phase system. Eleven HNA-1a antibody-positive samples, including the World Health Organization Reference Reagent, and 40 serum samples derived from male blood donors were used as positive and negative control samples, respectively. Although specificity was >0.90 in all systems evaluated, the sensitivity varied among the systems. The KY cell-based monoclonal antibody specific immobilisation of granulocyte antigens (KY-MAIGA) system using certain, but not all, monoclonal Abs, and the solid-phase system revealed higher sensitivity than other systems. In conclusion, the KY-MAIGA and commercial solid-phase systems were superior in terms of specific and sensitive detection of HNA-1a Abs.

  14. The effect of salinity on seed germination and growth parameters of field pea (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    Jovičić Dušica

    2010-01-01

    Full Text Available Field pea (Pisum sativum L. seed contains a large amount of proteins, amino acids, sugars, carbohydrates, vitamins A and C, calcium and phosphorous, and hence it is widely used for many purposes. Although field pea has moderate requirements for its growth, it is sensitive to increased salt content in soil. This research included eight varieties (Javor, Jantar, Partner, Kristal, Pionir, Junior, Trezor, Dukat developed at Institute of Field and Vegetable Crops in Novi Sad. Sodium chloride solutions of various concentrations (0, 50, 100 and 150 mM were added in growing media to simulate saline conditions. The following were subsequently determined: seed germination, seedling length, fresh and dry weight of seedlings and 1000-seed weight. Among the analyzed varieties, variety Jantar expressed a high level of tolerance to increased salt content in growing media under laboratory conditions.

  15. Blood glucose response to pea fiber

    DEFF Research Database (Denmark)

    Hamberg, O; Rumessen, J J; Gudmand-Høyer, E

    1989-01-01

    Two new fiber types, pea fiber (PF) and sugar beet fiber (BF), were compared with wheat bran (WB) to investigate the effect on postprandial blood glucose and serum insulin responses in normal subjects. The control meal consisted of 150 g ground beef mixed with 50 g glucose and 20 g lactulose. Only...... addition of PF (15 g pure fiber) reduced the area under the incremental blood glucose curve significantly (by 65%, p less than 0.05). None of the fibers affected the area under the insulin-response curve significantly although it was reduced by all fibers. Mouth-to-cecum transit time, assessed...

  16. Molecular cloning of isoflavone reductase from pea (Pisum sativum L.): evidence for a 3R-isoflavanone intermediate in (+)-pisatin biosynthesis.

    Science.gov (United States)

    Paiva, N L; Sun, Y; Dixon, R A; VanEtten, H D; Hrazdina, G

    1994-08-01

    Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isoflavanones during the biosynthesis of chiral pterocarpan phytoalexins. A cDNA clone for IFR from pea (Pisum sativum) was isolated using the polymerase chain reaction and expressed in Escherichia coli. Analysis of circular dichroism (CD) spectra of the reduction product sophorol obtained using the recombinant enzyme indicated that the isoflavanone possessed the 3R stereochemistry, in contrast to previous reports indicating a 3S-isoflavanone as the product of the pea IFR. Analysis of CD spectra of sophorol produced using enzyme extracts of CuCl2-treated pea seedlings confirmed the 3R stereochemistry. Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (-)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry. At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively. IFR appears to be encoded by a single gene in pea. Its transcripts are highly induced in CuCl2-treated seedlings, consistent with the appearance of IFR enzyme activity and pisatin accumulation.

  17. Detection of NP, N3 and N7 antibodies to avian influenza virus by indirect ELISA using yeast-expressed antigens

    Directory of Open Access Journals (Sweden)

    Ammayappan Arun

    2009-10-01

    Full Text Available Abstract Background Avian influenza viruses, belonging to the family Orthomyxoviridae, possess distinct combinations of hemagglutinin (H and the neuraminidase (N surface glycoproteins. Typing of both H and N antigens is essential for the epidemiological and surveillance studies. Therefore, it is important to find a rapid, sensitive, and specific method for their assay, and ELISA can be useful for this purpose, by using recombinant proteins. Results The nucleoprotein (NP and truncated neuraminidase subtype 3 and 7 of avian influenza virus (AIV were expressed in Saccharomyces cerevisiae and used to develop an indirect enzyme-linked immunosorbent assay for antibody detection. The developed assays were evaluated with a panel of 64 chicken serum samples. The performance of NP-ELISA was compared with the commercially available ProFlok® AIV ELISA kit. The results showed comparable agreement and sensitivity between the two tests, indicating that NP-ELISA assay can be used for screening the influenza type A antibody in AIV infected birds. The N3 and N7- ELISAs also reacted specifically to their type specific sera and did not exhibit any cross-reaction with heterologous neuraminidase subtype specific sera. Conclusion The study demonstrates the expression of the NP, N3, and N7 proteins of AIV in yeast (S. cerevisiae and their application in developing an indirect ELISA for detecting NP, N3 and N7 antibodies from AIV-infected chicken sera. The described indirect ELISAs are rapid, sensitive, specific and can be used as promising tests during serological surveillance.

  18. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    Science.gov (United States)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  19. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    Directory of Open Access Journals (Sweden)

    Safar Farajnia

    2013-02-01

    Full Text Available Purpose: Recombinant tumor necrosis factor-alpha (TNF-α has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-scFv fragments for purification of TNF-α produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications.

  20. Intermediate monocytes in ANCA vasculitis: increased surface expression of ANCA autoantigens and IL-1β secretion in response to anti-MPO antibodies.

    LENUS (Irish Health Repository)

    O'Brien, Eóin C

    2015-01-01

    ANCA vasculitis encompasses several autoimmune conditions characterised by destruction of small vessels, inflammation of the respiratory tract and glomerulonephritis. Most patients harbour autoantibodies to myeloperoxidase (MPO) or proteinase 3 (PR3). Clinical and experimental data suggest that pathogenesis is driven by ANCA-mediated activation of neutrophils and monocytes. We investigated a potential role for distinct monocyte subsets. We found that the relative proportion of intermediate monocytes is increased in patients versus control individuals, and both MPO and PR3 are preferentially expressed on these cells. We demonstrate that MPO and PR3 are expressed independently of each other on monocytes and that PR3 is not associated with CD177. MPO expression correlates with that of Fc receptor CD16 on intermediate monocytes. Monocyte subsets respond differently to antibodies directed against MPO and PR3, with anti-MPO but not anti-PR3 leading to increased IL-1β, IL-6 and IL-8 production. In concordance with the observed higher surface expression of MPO on intermediate monocytes, this subset produces the highest quantity of IL-1β in response to anti-MPO stimulation. These data suggest that monocytes, specifically, the intermediate subset, may play a role in ANCA vasculitis, and also indicate that substantial differences exist between the effect of anti-MPO and anti-PR3 antibodies on these cells.

  1. Evaluation of the HerpeSelect Express rapid test in the detection of herpes simplex virus type 2 antibodies in patients with genital ulcer disease.

    Science.gov (United States)

    Al-Shobaili, Hani; Hassanein, Khaled M; Mostafa, Marwa Salah; Al Duways, Ali Saleh

    2015-01-01

    A rapid point-of-care test with high sensitivity and specificity is required in order to fulfill the need for early detection and screening of Herpes simplex virus type 2 (HSV-2) infection among patients with genital ulcer disease (GUD), for better management and control of virus transmission. The goal of this study is to evaluate the performance of the commercially available HerpeSelect Express rapid test in comparison with three ELISA assays: HerpeSelect ELISA, Kalon HSV-2 glycoprotein G2 assay, and monoclonal antibody blocking enzyme immunoassay, which was used as the gold standard for the detection of HSV-2 antibodies. This study showed high sensitivity (ranging from 82.6 to 100%) and specificity (100%) of the HerpeSelect Express rapid test when compared to the three ELISA assays. The agreement between the HerpeSelect Express rapid test with the three ELISAs ranged from 93.3 to 100%. The HerpeSelect Express rapid test has adequate sensitivity and specificity for confirming HSV-2 infection in patients with GUD, indicating its suitability for epidemiological studies. © 2014 Wiley Periodicals, Inc.

  2. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine.

    Directory of Open Access Journals (Sweden)

    Morten A Nielsen

    Full Text Available The disease caused by Plasmodium falciparum (Pf involves different clinical manifestations that, cumulatively, kill hundreds of thousands every year. Placental malaria (PM is one such manifestation in which Pf infected erythrocytes (IE bind to chondroitin sulphate A (CSA through expression of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally constrained receptor-binding domains may, theoretically, circumvent polymorphisms, reduce the risk of escape-mutants and induce cross-reactive antibodies. However, the sub-unit composition and small differences in the borders, may lead to exposure of novel immuno-dominant antibody epitopes that lead to non-functional antibodies, and furthermore influence the folding, stability and yield of expression. Candidate antigens from the pre-clinical development expressed in High-Five insect cells using the baculovirus expression vector system were transitioned into the Drosophila Schneider-2 cell (S2 expression-system compliant with clinical development. The functional capacity of antibodies against antigens expressed in High-Five cells or in S2 cells was equivalent. This enabled an extensive down-selection of S2 insect cell-expressed antigens primarily encompassing the minimal CSA-binding region of VAR2CSA. In general, we found differential potency of inhibitory antibodies against antigens with the same borders but of different var2csa sequences. Likewise, we found that subtle size differences in antigens of the same sequence gave varying levels of inhibitory antibodies. The study shows that induction of a functional response against recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens

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

    Science.gov (United States)

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

    2013-01-01

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

  4. Comparative assessment of the recognition of domain-specific CD163 monoclonal antibodies in human monocytes explains wide discrepancy in reported levels of cellular surface CD163 expression

    DEFF Research Database (Denmark)

    Maniecki, Maciej Bogdan; Etzerodt, Anders; Moestrup, Søren Kragh

    2011-01-01

    . Materials and Methods The cellular distribution of CD163 on human peripheral blood monocytes in freshly drawn blood and peripheral blood mononuclear cells isolated from buffy-coats was investigated by flow cytometry using CD163 monoclonal antibodies recognizing scavenger receptor cysteine-rich (SRCR) domain......Background CD163 is expressed exclusively on cells of the monocyte/macrophage lineage and is widely used as a marker of human macrophages. Further, it has been suggested as a diagnostic marker of monocyte/macrophage activity in inflammatory conditions and as a therapeutic target. However, studies...... 1 (MAC2-158), domain 4 (R-20), domain 7 (GHI/61), and domain 9 (RM3/1). The CD163 monoclonal antibodies were characterized in binding and endocytosis experiments in human macrophages and CD163-transfected Flp-In CHO cells. Calcium-dependent ligand binding was assessed using surface plasmon resonance...

  5. Comparison of internal ribosome entry site (IRES and Furin-2A (F2A for monoclonal antibody expression level and quality in CHO cells.

    Directory of Open Access Journals (Sweden)

    Steven C L Ho

    Full Text Available Four versions of tricistronic vectors expressing IgG1 light chain (LC, IgG1 heavy chain (HC, and dihydrofolate reductase (DHFR in one transcript were designed to compare internal ribosome entry site (IRES and furin-2A (F2A for their influence on monoclonal antibody (mAb expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

  6. Pea Marker Database (PMD) - A new online database combining known pea (Pisum sativum L.) gene-based markers.

    Science.gov (United States)

    Kulaeva, Olga A; Zhernakov, Aleksandr I; Afonin, Alexey M; Boikov, Sergei S; Sulima, Anton S; Tikhonovich, Igor A; Zhukov, Vladimir A

    2017-01-01

    Pea (Pisum sativum L.) is the oldest model object of plant genetics and one of the most agriculturally important legumes in the world. Since the pea genome has not been sequenced yet, identification of genes responsible for mutant phenotypes or desirable agricultural traits is usually performed via genetic mapping followed by candidate gene search. Such mapping is best carried out using gene-based molecular markers, as it opens the possibility for exploiting genome synteny between pea and its close relative Medicago truncatula Gaertn., possessing sequenced and annotated genome. In the last 5 years, a large number of pea gene-based molecular markers have been designed and mapped owing to the rapid evolution of "next-generation sequencing" technologies. However, the access to the complete set of markers designed worldwide is limited because the data are not uniformed and therefore hard to use. The Pea Marker Database was designed to combine the information about pea markers in a form of user-friendly and practical online tool. Version 1 (PMD1) comprises information about 2484 genic markers, including their locations in linkage groups, the sequences of corresponding pea transcripts and the names of related genes in M. truncatula. Version 2 (PMD2) is an updated version comprising 15944 pea markers in the same format with several advanced features. To test the performance of the PMD, fine mapping of pea symbiotic genes Sym13 and Sym27 in linkage groups VII and V, respectively, was carried out. The results of mapping allowed us to propose the Sen1 gene (a homologue of SEN1 gene of Lotus japonicus (Regel) K. Larsen) as the best candidate gene for Sym13, and to narrow the list of possible candidate genes for Sym27 to ten, thus proving PMD to be useful for pea gene mapping and cloning. All information contained in PMD1 and PMD2 is available at www.peamarker.arriam.ru.

  7. The prognostic value of oncogenic antigen 519 (OA-519) expression and proliferative activity detected by antibody MIB-1 in node-negative breast cancer

    DEFF Research Database (Denmark)

    Jensen, V; Ladekarl, M; Holm-Nielsen, P

    1995-01-01

    of invasion of skin or deep fascia (= T1N0M0 and T2N0M0). The median follow-up time was 104 months (range 5-143 months). Immunohistochemical analysis of OA-519 expression was performed on formalin-fixed, paraffin-embedded tissue. The proliferative activity was estimated using a Ki-67 equivalent monoclonal...... antibody (MIB-1), which is applicable on formalin-fixed, paraffin-embedded tissue after microwave pretreatment. OA-519 was expressed in about one-third of the tumours and the percentage of proliferating cells (the MIB-1 index) ranged between 1 and 72 per cent (median 17 per cent). Using multivariate Cox...... analysis, both the MIB-1 index and OA-519 expression were of independent prognostic value (2p

  8. Diversity of segetal weeds in pea (Pisum sativum L. depending on crops chosen for a crop rotation system

    Directory of Open Access Journals (Sweden)

    Marta K. Kostrzewska

    2014-04-01

    the basis of weed biomass was higher in the system with potato. The similarity indices, which express the convergence of floristic composition as well as of the density and biomass of weeds growing in pea fields in the two crop rotation systems compared, were within a broad range (42–86%. The biodiversity of weed communities was more closely correlated to total precipitation than to air temperature.

  9. Differential expression of IgE and IgG4 specific antibody responses in asymptomatic and chronic human filariasis

    NARCIS (Netherlands)

    Kurniawan, A.; Yazdanbakhsh, M.; van Ree, R.; Aalberse, R.; Selkirk, M. E.; Partono, F.; Maizels, R. M.

    1993-01-01

    A population of 164 adult individuals resident in an area endemic for Brugia malayi lymphatic filariasis has been studied for humoral immune responses to filarial parasites. Antibody levels to Ag extracted from adult worms were determined for each of the IgG subclasses, for IgM and for IgE. The

  10. Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

    NARCIS (Netherlands)

    Arafat, WO; Gomez-Navarro, J; Buchsbaum, DJ; Xiang, J; Casado, E; Barker, SD; Mahasreshti, PJ; Haisma, HJ; Barnes, MN; Siegal, GP; Alvarez, RD; Hemminki, A; Nettelbeck, DM; Curiel, DT

    Single chain antibodies (scFv) represent powerful interventional agents for the achievement of targeted therapeutics. The practical utility of these agents have been limited, however, by difficulties related to production of recombinant scFv and the achievement of effective and sustained levels of

  11. Tumor uptake of intravenously administered radiolabeled antibodies in ovarian carcinoma patients in relation to antigen expression and other tumor characteristics

    NARCIS (Netherlands)

    Buist, M. R.; Kenemans, P.; Molthoff, C. F.; Roos, J. C.; den Hollander, W.; Brinkhuis, M.; Baak, J. P.

    1995-01-01

    To study factors that possibly influence the heterogeneous tumor uptake of radiolabeled antibodies, tissues from 34 ovarian-carcinoma patients were obtained 2 to 8 days after i.v. injection with radiolabeled murine OV-TL3 or chimeric MOv18 (cMOv18). The tumor uptake and the ratio of tumor to normal

  12. Rheological and qualitative characteristics of pea flour incorporated cracker biscuits

    Directory of Open Access Journals (Sweden)

    Jolana Karovičová

    2013-01-01

    Full Text Available The suitability of pea flour for cracker biscuits production was investigated in this study. Pea flour was characterised by high protein (21.46 % and ash (3.11 % content and exhibited relatively high emulsifying (37.50 ml/100 ml and foaming (53.50 ml/100 ml capacity. The effect of pea flour incorporation to wheat dough (substitution levels 0, 10, 20 and 30 % on the rheological properties, physical characteristics and sensory parameters of cracker biscuits were also evaluated. Farinographic measurements showed that pea flour addition resulted in increasing of water absorption (from 58.90 to 61.80 % and dough development time (from 3.55 to 4.50 min, whereas dough stability was decreased (from 6.69 to 3.50 min. It was also found that incorporation of pea flour to cracker biscuits modified physical properties of final products by different ways (decreasing of volume index, width and spread ratio, increasing of thickness. From the sensory evaluation revealed that cracker biscuits prepared from blend flour contained 10 % pea flour showed no significant differences from wheat cracker biscuits. Higher levels of pea flour in the products adversely affected the odour, taste, firmness, colour and overall acceptance of final products.

  13. Symbiotic nitrogen fixation and nitrate uptake by the pea crop

    International Nuclear Information System (INIS)

    Jensen, E.S.

    1986-08-01

    Symbiotic nitrogen fixation and nitrate uptake by pea plants (Pisum sativum L.) were studied in field and pot experiments using the 15 N isotope dilution technique and spring barley as a non-fixing reference crop. Barley, although not ideal, seemed to be a suitable reference for pea in the 15 N-technique. Maximum N 2 fixation activity of 10 kg N fixed per ha per day was reached around the flat pod growth stage, and the activity decreased rapidly during pod-filling. The pea crop fixed between 100 and 250 kg N ha -1 , corresponding to from 45 to 80 per cent of total crop N. The amount of symbiotically fixed N 2 depended on the climatic conditions in the experimental year, the level of soil mineral N and the pea cultivar. Field-grown pea took up 60 to 70 per cent of the N-fertilizer supplied. The supply of 50 kg NO 3 -N ha -1 inhibited the N 2 fixation approximately 15 per cent. Small amounts of fertilizer N, supplied at sowing (starter-N), slightly stimulated the vegetative growth of pea, but the yields of seed dry matter and protein were not significantly influenced. In the present field experiments the environmental conditions, especially the distribution of rainfall during the growth season, seemed to be more important in determining the protein and dry matter yield of the dry pea crop, than the ability of pea to fix nitrogen symbiotically. However, fertilizer N supplied to pot-grown pea plants at the flat pod growth stage or as split applications significantly increased the yield of seed dry matter and protein. (author)

  14. An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8(+) T cells and antibody when expressed from modified vaccinia Ankara.

    Science.gov (United States)

    Quinan, Bárbara R; Flesch, Inge E A; Pinho, Tânia M G; Coelho, Fabiana M; Tscharke, David C; da Fonseca, Flávio G

    2014-05-23

    Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Prokaryotic expression of a truncated form of bovine herpesvirus 1 glycoprotein E (gE and its use in an ELISA for gE antibodies

    Directory of Open Access Journals (Sweden)

    Stephan A.M. Oliveira

    2013-01-01

    Full Text Available This article describes the expression of a truncated form of bovine herpesvirus 1 (BoHV-1 glycoprotein E (gE for use as immunodiagnostic reagent. A 651 nucleotide fragment corresponding to the amino-terminal third (217 amino acids of BoHV-1 gE - that shares a high identity with the homologous BoHV-5 counterpart - was cloned as a 6×His-tag fusion protein in an Escherichia coli expression vector. A soluble protein of approximately 25 kDa purified from lysates of transformed E. coli was recognized in Western blot (WB by anti-6xHis-tag and anti-BoHV-1 gE monoclonal antibodies. In addition, the recombinant protein was specifically recognized in WB by antibodies present in the sera of cattle seropositive to BoHV-1 and BoHV-5. An indirect ELISA using the expressed protein as coating antigen performed comparably to a commercial anti-gE ELISA and was able to differentiate serologically calves vaccinated with a gE-deleted BoHV-5 strain from calves infected with BoHV-1. Thus, the truncated gE may be useful for serological tests designed to differentiate BoHV-1/BoHV-5 infected animals from those vaccinated with gE-negative marker vaccines.

  16. Bgb expression in relation to the HLA-B17 antigen splits Bw57 and Bw58 and the cross-reactions of anti-Bgb antibodies.

    Science.gov (United States)

    Pollack, M S; Crawford, M N; Robinson, H M; Berger, R; Sabo, B; O'Neill, G J

    1982-07-01

    A substantial number of individuals typed as HLA-B17 do not have Bgb. To determine whether or not this discrepancy reflects genetic or ethnic group differences or the influence of B17 antigen subtypes, a large number of unrelated and related B17 individuals from the Caucasian, Hispanic, Black and Chinese ethnic groups were tested in parallel for Bgb and Bw57- or Bw58- B17 subtype. Higher Bgb expression was found in association with Bw57, but differences in expression of Bgb were also seen in different ethnic groups and in different related individuals carrying the same Bw57 or Bw58 haplotype. Genetic factors other than HLA thus influence the expression of HLa Antigens on red cells. Standard and antiglobulin lymphocytotoxicity tests indicate that all Bgb typing sera contain anti-Bw57 antilymphocyte antibodies and most also contain anti-Bw58 and cross-reacting anti-B12, B15 and/or Bw49 antibodies.

  17. Quality of peas modelled by a structural equation system

    DEFF Research Database (Denmark)

    Bech, Anne C.; Juhl, Hans Jørn; Martens, Magni

    2000-01-01

    The quality of peas has been studied in a joint project between a pea producing company in Denmark and several research institutions. The study included quality from a consumer point of view based on market research and quality from more internal company points of view based on measurement of phy...... by increasing the amount of sugars and more indirectly by improving the perception of colour through darker and less yellow peas. Perceived texture can be optimised by focusing on selected texture measurements. Udgivelsesdato: JUL...

  18. Response of Pea Varieties to Damage Degree of Pea Weevil, Bruchus pisorum L.

    OpenAIRE

    Nikolova, Ivelina Mitkova

    2016-01-01

    A study was conducted to determine the response of five pea varieties (Pisum sativum L.) to damage degree of Bruchus pisorum: Glyans, Modus, Kamerton, and Svit (Ukrainian cultivars) and Pleven 4 (Bulgarian cultivar). The seeds were classified into three types: healthy seeds (type 1), damaged seeds with parasitoid emergence hole (type 2), and damaged seeds with bruchid emergence hole (type 3) and they were sown. It was found that the weight of 1000 seeds did not affect the field germination of...

  19. Induction of neutralizing antibodies to Hendra and Nipah glycoproteins using a Venezuelan equine encephalitis virus in vivo expression system.

    Science.gov (United States)

    Defang, Gabriel N; Khetawat, Dimple; Broder, Christopher C; Quinnan, Gerald V

    2010-12-16

    The emergence of Hendra Virus (HeV) and Nipah Virus (NiV) which can cause fatal infections in both animals and humans has triggered a search for an effective vaccine. Here, we have explored the potential for generating an effective humoral immune response to these zoonotic pathogens using an alphavirus-based vaccine platform. Groups of mice were immunized with Venezuelan equine encephalitis virus replicon particles (VRPs) encoding the attachment or fusion glycoproteins of either HeV or NiV. We demonstrate the induction of highly potent cross-reactive neutralizing antibodies to both viruses using this approach. Preliminary study suggested early enhancement in the antibody response with use of a modified version of VRP. Overall, these data suggest that the use of an alphavirus-derived vaccine platform might serve as a viable approach for the development of an effective vaccine against the henipaviruses. Published by Elsevier Ltd.

  20. Modulation of immune response to Lol p I by pretreatment with anti-idiotypic antibody is not restricted to the idiotypic expression.

    Science.gov (United States)

    Boutin, Y; Hébert, J

    1994-05-01

    To study the role of anti-idiotypic antibodies in the regulation of the immune response to Lol p I (the major allergenic component of rye grass pollen), we have recently generated a panel of three MoAbs directed against distinct epitopes of Lolp I and an anti-idiotypic MoAb directed against the idiotype borne by one of the anti-Lol p I MoAbs (290A-167). The effects of pretreatment with this anti-idiotypic MoAb in BALB/c mice before immunization with the antigen have been examined. The anti-idiotypic MoAb or unrelated MoAb were given weekly for 8 weeks intraperitoneally. Mice then received the antigen (2 micrograms) adsorbed with alum (2 mg) at weeks 9, 11 and 13. Serum anti-Lol p I antibodies (IgG or IgE) and specific idiotypic responses were measured. Anti-Lol p I IgG antibodies could be detected before immunization with Lol p I only in mice pretreated with anti-idiotypic MoAb. Immunization with Lol p I induced an anti-Lol p I IgG response in both groups, but this response was higher in mice that received anti-idiotypic MoAb. Similar profiles were seen for specific IgE antibodies and idiotypic responses. Surprisingly, idiotypes borne by other anti-Lol p I MoAbs (539A-6 and 348A-6) had also been enhanced after pretreatment with the anti-290A-167 MoAb. These observations suggested that the pretreatment with this anti-idiotypic MoAb modulates not only the expression of the respective idiotype, but also affects other idiotype responses.

  1. Blood glucose response to pea fiber

    DEFF Research Database (Denmark)

    Hamberg, O; Rumessen, J J; Gudmand-Høyer, E

    1989-01-01

    Two new fiber types, pea fiber (PF) and sugar beet fiber (BF), were compared with wheat bran (WB) to investigate the effect on postprandial blood glucose and serum insulin responses in normal subjects. The control meal consisted of 150 g ground beef mixed with 50 g glucose and 20 g lactulose. Only...... addition of PF (15 g pure fiber) reduced the area under the incremental blood glucose curve significantly (by 65%, p less than 0.05). None of the fibers affected the area under the insulin-response curve significantly although it was reduced by all fibers. Mouth-to-cecum transit time, assessed...... by the hydrogen breath technique, was decreased by WB and BF, (p less than 0.05) but not by PF. PF is palatable and may prove beneficial as a fiber supplement for diabetics....

  2. A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

    Science.gov (United States)

    Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

    2017-05-01

    Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L. plants by agroinfection of germinated seeds

    Directory of Open Access Journals (Sweden)

    Yang Meiying

    2011-05-01

    Full Text Available Abstract Background For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation. Results By vacuum infiltration (0.08 Mpa, 1 min of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration. Conclusions Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product.

  4. Pea (Pisum sativum L.) in the Genomic Era

    Czech Academy of Sciences Publication Activity Database

    Smýkal, P.; Aubert, G.; Burstin, J.; Coyne, C.J.; Ellis, N.T.H.; Flavell, A.J.; Ford, R.; Hýbl, M.; Macas, Jiří; Neumann, Pavel; McPhee, K.E.; Redden, R.J.; Rubiales, D.; Weller, J.L.; Warkentin, T.D.

    2012-01-01

    Roč. 2, č. 2 (2012), s. 74-115 ISSN 2073-4395 Institutional research plan: CEZ:AV0Z50510513 Institutional support: RVO:60077344 Keywords : breeding * germplasm * genetic diversity * pea Subject RIV: EB - Genetics ; Molecular Biology

  5. Võimuliitlased ei pea Kiisleri kiirreformi võimalikuks / Urmas Seaver

    Index Scriptorium Estoniae

    Seaver, Urmas, 1973-

    2009-01-01

    Reformierakond ja Sotsiaaldemokraatlik Erakond tunnistavad haldusreformi vajalikkust, kuid ei pea võimalikuks regionaalminister Siim-Valmar Kiisleri plaani jätta juba sel sügisel Eestisse vaid 20 omavalitsust

  6. Effect of Pigeon Pea Hedgerow Alley Management on the Growth ...

    African Journals Online (AJOL)

    year (2010 and 2011 cropping seasons) field investigation conducted at the National Root Crops Research Institute, Umudike, South Eastern Nigeria. Treatments comprised three pigeon pea hedgerow alley populations of 20,000, 33,333 and ...

  7. Induced mutants in beans and peas resistant to rust

    International Nuclear Information System (INIS)

    Fadl, F.A.M.

    1983-01-01

    Beans (Phaseolus vulgaris) and peas (Pisum sativum) are important leguminous vegetable crops in Egypt. The area planted with beans is about 40,000 acres and peas 22,000 acres. These crops suffer from several diseases, particularly rusts, (Uromyces phaseoli/Uromyces pisi), which are mainly spread in northern Egypt. In our mutation induction programme we used 60 Co gamma rays and ethyl methane sulphonate (EMS). Bean and pea seeds were soaked in water for two hours before exposure to 8, 10 and 12 krad. For chemical treatments, bean and pea seeds were soaked in water for eight hours and then treated with 0.5 and 1.5% EMS for four hours. The M 1 was cultivated in 1978

  8. ASPECTS OF BREEDING IN VNIISSOK OF GREEN PEA FOR CANNING

    Directory of Open Access Journals (Sweden)

    E. P. Pronina

    2014-01-01

    Full Text Available The article presents the methods and results of the competitive breeding of promising high-quality varieties of vegetable green pea for canning, as well as the main achievements of FGBNU VNIISSOK.

  9. Characterization of the tumor marker muc16 (ca125 expressed by murine ovarian tumor cell lines and identification of a panel of cross-reactive monoclonal antibodies

    Directory of Open Access Journals (Sweden)

    Goodell Cara AR

    2009-06-01

    Full Text Available Abstract Objectives The ovarian tumor marker CA125 is expressed on human MUC16, a cell surface bound mucin that is also shed by proteolytic cleavage. Human MUC16 is overexpressed by ovarian cancer cells. MUC16 facilitates the binding of ovarian tumor cells to mesothelial cells lining the peritoneal cavity. Additionally, MUC16 also is a potent inhibitor of natural killer cell mediated anti-tumor cytotoxic responses. Extensive studies using human as well as murine ovarian tumor cell models are required to clearly define the function of MUC16 in the progression of ovarian tumors. The major objective of this study was to determine if the murine ovarian tumor cells, MOVCAR, express Muc16 and to characterize antibodies that recognize this mucin. Methods RT-PCR analysis was used for detecting the Muc16 message and size exclusion column chromatography for isolating Muc16 produced by MOVCAR cells. Soluble and cell-associated murine Muc16 were analyzed, respectively, by Western blotting and flow cytometry assays using a new panel of antibodies. The presence of N-linked oligosaccharides on murine Muc16 was determined by ConA chromatography. Results We demonstrate that murine Muc16 is expressed by mouse ovarian cancer cells as an ~250 kDa glycoprotein that carries both O-linked and N-linked oligosaccharides. In contrast to human MUC16, the murine ortholog is primarily released from the cells and cannot be detected on the cell surface. Since the released murine Muc16 is not detected by conventional anti-CA125 assays, we have for the first time identified a panel of anti-human MUC16 antibodies that also recognizes the murine counterpart. Conclusion The antibodies identified in this study can be used in future purification of murine Muc16 and exhaustive study of its properties. Furthermore, the initial identification and characterization of murine Muc16 is a vital preliminary step in the development of effective murine models of human ovarian cancer. These

  10. Pea (Pisum sativum L. in the Genomic Era

    Directory of Open Access Journals (Sweden)

    Robert J. Redden

    2012-04-01

    Full Text Available Pea (Pisum sativum L. was the original model organism used in Mendel’s discovery (1866 of the laws of inheritance, making it the foundation of modern plant genetics. However, subsequent progress in pea genomics has lagged behind many other plant species. Although the size and repetitive nature of the pea genome has so far restricted its sequencing, comprehensive genomic and post genomic resources already exist. These include BAC libraries, several types of molecular marker sets, both transcriptome and proteome datasets and mutant populations for reverse genetics. The availability of the full genome sequences of three legume species has offered significant opportunities for genome wide comparison revealing synteny and co-linearity to pea. A combination of a candidate gene and colinearity approach has successfully led to the identification of genes underlying agronomically important traits including virus resistances and plant architecture. Some of this knowledge has already been applied to marker assisted selection (MAS programs, increasing precision and shortening the breeding cycle. Yet, complete translation of marker discovery to pea breeding is still to be achieved. Molecular analysis of pea collections has shown that although substantial variation is present within the cultivated genepool, wild material offers the possibility to incorporate novel traits that may have been inadvertently eliminated. Association mapping analysis of diverse pea germplasm promises to identify genetic variation related to desirable agronomic traits, which are historically difficult to breed for in a traditional manner. The availability of high throughput ‘omics’ methodologies offers great promise for the development of novel, highly accurate selective breeding tools for improved pea genotypes that are sustainable under current and future climates and farming systems.

  11. Incidence of Lettuce mosaic virus in lettuce and its detection by polyclonal antibodies produced against recombinant coat protein expressed in Escherichia coli.

    Science.gov (United States)

    Sharma, Prachi; Sharma, Susheel; Singh, Jasvir; Saha, Swati; Baranwal, V K

    2016-04-01

    Lettuce mosaic virus (LMV), a member of the genus Potyvirus of family Potyviridae, causes mosaic disease in lettuce has recently been identified in India. The virus is seed borne and secondary infection occurs through aphids. To ensure virus freedom in seeds it is important to develop diagnostic tools, for serological methods the production of polyclonal antibodies is a prerequisite. The coat protein (CP) gene of LMV was amplified, cloned and expressed using pET-28a vector in Escherichia coli BL21DE3 competent cells. The LMV CP was expressed as a fusion protein containing a fragment of the E. coli His tag. The LMV CP/His protein reacted positively with a commercial antiserum against LMV in an immunoblot assay. Polyclonal antibodies purified from serum of rabbits immunized with the fusion protein gave positive results when LMV infected lettuce (Lactuca sativa) was tested at 1:1000 dilution in PTA-ELISA. These were used for specific detection of LMV in screening lettuce accessions. The efficacy of the raised polyclonal antiserum was high and it can be utilized in quarantine and clean seed production. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Monoclonal antibodies against muscle actin isoforms: epitope identification and analysis of isoform expression by immunoblot and immunostaining in normal and regenerating skeletal muscle [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Christine Chaponnier

    2016-06-01

    Full Text Available Higher vertebrates (mammals and birds express six different highly conserved actin isoforms that can be classified in three subgroups: 1 sarcomeric actins, α-skeletal (α-SKA and α-cardiac (α-CAA, 2 smooth muscle actins (SMAs, α-SMA and γ-SMA, and 3 cytoplasmic actins (CYAs, β-CYA and γ-CYA. The variations among isoactins, in each subgroup, are due to 3-4 amino acid differences located in their acetylated N-decapeptide sequence. The first monoclonal antibody (mAb against an actin isoform (α-SMA was produced and characterized in our laboratory in 1986 (Skalli  et al., 1986 . We have further obtained mAbs against the 5 other isoforms. In this report, we focus on the mAbs anti-α-SKA and anti-α-CAA obtained after immunization of mice with the respective acetylated N-terminal decapeptides using the Repetitive Immunizations at Multiple Sites Strategy (RIMMS. In addition to the identification of their epitope by immunoblotting, we describe the expression of the 2 sarcomeric actins in mature skeletal muscle and during muscle repair after micro-lesions. In particular, we analyze the expression of α-CAA, α-SKA and α-SMA by co-immunostaining in a time course frame during the muscle repair process. Our results indicate that a restricted myocyte population expresses α-CAA and suggest a high capacity of self-regeneration in muscle cells. These antibodies may represent a helpful tool for the follow-up of muscle regeneration and pathological changes.

  13. Expression of miR-142-5p in peripheral blood mononuclear cells from renal transplant patients with chronic antibody-mediated rejection.

    Directory of Open Access Journals (Sweden)

    Richard Danger

    Full Text Available In renal transplantation, the unresponsiveness of patients undergoing chronic antibody mediated rejection (CAMR to classical treatment stress on the need for accurate biomarkers to improve its diagnosis. We aim to determine whether microRNA expression patterns may be associated with a diagnosis of CAMR. We performed expression profiling of miRNAs in peripheral blood mononuclear cells (PBMC of kidney transplant recipients with CAMR or stable graft function. Among 257 expressed miRNAs, 10 miRNAs associated with CAMR were selected. Among them, miR-142-5p was increased in PBMC and biopsies of patients with CAMR as well as in a rodent model of CAMR. The lack of modulation of miR-142-5p in PBMC of patients with renal failure, suggests that its over-expression in CAMR was associated with immunological disorders rather than renal dysfunction. A ROC curve analysis performed on independent samples showed that miR-142-5p is a potential biomarker of CAMR allowing a very good discrimination of the patients with CAMR (AUC = 0.74; p = 0.0056. Moreover, its expression was decreased in PHA-activated blood cells and was not modulated in PBMC from patients with acute rejection, excluding a non-specific T cell activation expression. The absence of modulation of this miRNA in immunosuppressed patients suggests that its expression was not influenced by treatment. Finally, the analysis of miR-142-5p predicted targets under-expressed in CAMR PBMC in a published microarray dataset revealed an enrichment of immune-related genes. Altogether, these data suggest that miR-142-5p could be used as a biomarker in CAMR and these finding may improve our understanding of chronic rejection mechanisms.

  14. Induced mutations in beans and peas for resistance to rust

    International Nuclear Information System (INIS)

    Fadl, F.A.M.

    1983-01-01

    Gamma rays and ethyl methanesulphonate (EMS) were applied in a mutation-induction programme for rust resistance in bean and pea. Bean and pea seeds were pre-soaked 2 hours before irradiation with 9, 10 and 12 krad. For chemical mutagen treatments bean and pea seeds were pre-soaked for 8 hours and treated with 0.5 and 1.5% EMS for four hours. M 2 seeds of beans and peas were planted in 1979. Resistant M 2 plants were selected for their rust resistance and other morphological characters. M 3 seeds of selected plants were planted in 1980. In 1980 more seeds of the same varieties of beans and peas were treated with 0.1 and 0.3% EMS with the aim to produce rust-resistant mutants. Seed germination was reduced by gamma rays or EMS. Dwarf, malformed and abnormal plants were noticed. Some resistant M 2 plants selected gave high grain yields. Some were different in morphological characters. In the M 3 of selected plants various other mutant characters appeared, such as different height of plants, early and late flowering, resistance to powdery mildew in peas, altered grain yield, thickness of stem, pod shape and flower colour. (author)

  15. Protein quality traits of vegetable and field peas: varietal differences.

    Science.gov (United States)

    Saharan, K; Khetarpaul, N

    1994-01-01

    Four high yielding varieties of field (Rachna and RFP4) and vegetable peas (Bonnivielle and Arkal) were studied for their protein quality. Crude protein and true protein content of all four pea varieties varied from 19.5 to 20.6 percent and 18.7 to 19.8 percent, respectively. Non protein nitrogen formed only 3.94 to 4.84 percent of total nitrogen. Globulins were the major fractions followed by albumins and glutelins. All the four varieties of peas had similar methionine and tryptophan content. Lysine content of four pea varieties ranged from 7.56 to 9.65 g per 16 g of N. Cooking brought about an increase in in vitro protein digestibility of peas by 10 percent. The sodium dodecyl polyacrylamide gel electrophoresis of total protein revealed the presence of 22-25 bands with some difference in banding pattern of all four varieties. Some differences were observed in banding patterns of globulin and albumin of all four varieties, suggesting that composition of protein of pea varieties differed.

  16. Purification and immunolocalization of an annexin-like protein in pea seedlings

    Science.gov (United States)

    Clark, G. B.; Dauwalder, M.; Roux, S. J.

    1992-01-01

    As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

  17. Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus

    DEFF Research Database (Denmark)

    Sørensen, K.J.; Madsen, K.G.; Madsen, E.S.

    1998-01-01

    The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after...... experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins....... The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave...

  18. Evaluation of a novel type of imaging probe based on a recombinant bivalent mini-antibody construct for detection of CD44v6-expressing squamous cell carcinoma.

    Science.gov (United States)

    Haylock, Anna-Karin; Spiegelberg, Diana; Mortensen, Anja C; Selvaraju, Ram K; Nilvebrant, Johan; Eriksson, Olof; Tolmachev, Vladimir; Nestor, Marika V

    2016-02-01

    We have developed the CD44v6-targeting human bivalent antibody fragment AbD19384, an engineered recombinant human bivalent Fab antibody formed via dimerization of dHLX (synthetic double helix loop helix motif) domains, for potential use in antibody-based molecular imaging of squamous cell carcinoma in the head and neck region. This is a unique construct that has, to the best of our knowledge, never been assessed for molecular imaging in vivo before. The objective of the present study was to evaluate for the first time the in vitro and in vivo binding properties of radio-iodinated AbD19384, and to assess its utility as a targeting agent for molecular imaging of CD44v6-expressing tumors. Antigen specificity and binding properties were assessed in vitro. In vivo specificity and biodistribution of 125I-AbD19384 were next evaluated in tumor-bearing mice using a dual-tumor setup. Finally, AbD19384 was labeled with 124I, and its imaging properties were assessed by small animal PET/CT in tumor bearing mice, and compared with 2-deoxy-2-[18F]fluoro-D-glucose (18F-FDG). In vitro studies demonstrated CD44v6-specific binding with slow off-rate for AbD19384. A favorable biodistribution profile was seen in vivo, with tumor-specific uptake. Small animal PET/CT images of 124I-AbD19384 supported the results through clearly visible high CD44v6-expressing tumors and faintly visible low expressing tumors, with superior imaging properties compared to 18F-FDG. Tumor-to-blood ratios increased with time for the conjugate (assessed up to 72 h p.i.), although 48 h p.i. proved best for imaging. Biodistribution and small-animal PET studies demonstrated that the recombinant Fab-dHLX construct AbD19384 is a promising tracer for imaging of CD44v6 antigen expression in vivo, with the future aim to be used for individualized diagnosis and early detection of squamous cell carcinomas in the head and neck region. Furthermore, this proof-of-concept research established the feasibility of using

  19. The Influence of Sub-Unit Composition and Expression System on the Functional Antibody Response in the Development of a VAR2CSA Based Plasmodium falciparum Placental Malaria Vaccine

    DEFF Research Database (Denmark)

    Nielsen, Morten A; dos Santos Marques Resende, Mafalda; de Jongh, Willem A

    2015-01-01

    of VAR2CSA, a parasite-derived antigen. Protection against PM is mediated by antibodies that inhibit binding of IE in the placental intervillous space. VAR2CSA is a large antigen incompatible with large scale recombinant protein expression. Vaccines based on sub-units encompassing the functionally...... recombinant subunits of the VAR2CSA antigen is unpredictable, demonstrating the need for large-scale screening in order to identify antigens that induce a broadly strain-transcending antibody response....

  20. [Tendril inheritance in semi-leafless pea and its utilization in breeding].

    Science.gov (United States)

    Wang, Feng-Bao; Fu, Jin-Feng; Dong, Li-Feng; Zhu, Ying-Bo

    2003-03-01

    Using semi-leafless pea and common pea, the authors studied tendril character, its inheritance law and how to use it in pea breeding. The results were as follows: Semi-leafless pea had well developed tendrils; They twined with each other and formed an arbor structure. This ideal structure had greatly increased lodging resistance capability,improved canopy's air and light level, and remarkably increased new varieties yield. So, semi-leafless pea was one of the most important materials in lodging resistance breeding. Tendril gene, af, and seed color gene i, in semi-leafless pea was linked on chromosome I, and cross-over value was 5.72%.

  1. Expression patterns of bovine CD1 in vivo and assessment of the specificities of the anti-bovine CD1 antibodies.

    Directory of Open Access Journals (Sweden)

    Thi Kim Anh Nguyen

    Full Text Available Research addressing the in vivo effects of T cell activation by lipids, glycolipids, and lipopeptides is hampered by the absence of a suitable animal model. Mice and rats do not express CD1a, CD1b, and CD1c molecules that present pathogen-derived lipid antigens in humans. In cattle, two CD1A and three CD1B genes are transcribed. The proteins encoded by these genes differ in their antigen binding domains and in their cytoplasmic tails, suggesting that they may traffic differently in the cell and thus have access to different antigens. In the current study, we describe the genomic organization of the bovine CD1 locus and transcription of bovine CD1 genes in freshly isolated dendritic cells and B cells from different tissues. After determining the specificity of previously only partly characterized anti-CD1 antibodies by testing recombinant single chain bovine CD1 proteins and CD1-transfected cells, we were able to determine cell surface protein expression on freshly isolated cells. Our study suggests that CD1b1 and CD1b3 are more broadly expressed than CD1b5, and CD1a2 is more broadly expressed than CD1a1. Pseudoafferent lymph dendritic cells express CD1B genes, but no transcription is detected in lymph nodes. Even though B cells transcribe CD1B genes, there is no evidence of protein expression at the cell surface. Thus, patterns of CD1 protein expression are largely conserved among species.

  2. Expression patterns of bovine CD1 in vivo and assessment of the specificities of the anti-bovine CD1 antibodies.

    Science.gov (United States)

    Nguyen, Thi Kim Anh; Reinink, Peter; El Messlaki, Chema; Im, Jin S; Ercan, Altan; Porcelli, Steven A; Van Rhijn, Ildiko

    2015-01-01

    Research addressing the in vivo effects of T cell activation by lipids, glycolipids, and lipopeptides is hampered by the absence of a suitable animal model. Mice and rats do not express CD1a, CD1b, and CD1c molecules that present pathogen-derived lipid antigens in humans. In cattle, two CD1A and three CD1B genes are transcribed. The proteins encoded by these genes differ in their antigen binding domains and in their cytoplasmic tails, suggesting that they may traffic differently in the cell and thus have access to different antigens. In the current study, we describe the genomic organization of the bovine CD1 locus and transcription of bovine CD1 genes in freshly isolated dendritic cells and B cells from different tissues. After determining the specificity of previously only partly characterized anti-CD1 antibodies by testing recombinant single chain bovine CD1 proteins and CD1-transfected cells, we were able to determine cell surface protein expression on freshly isolated cells. Our study suggests that CD1b1 and CD1b3 are more broadly expressed than CD1b5, and CD1a2 is more broadly expressed than CD1a1. Pseudoafferent lymph dendritic cells express CD1B genes, but no transcription is detected in lymph nodes. Even though B cells transcribe CD1B genes, there is no evidence of protein expression at the cell surface. Thus, patterns of CD1 protein expression are largely conserved among species.

  3. Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice.

    Science.gov (United States)

    Moda, Fabio; Vimercati, Chiara; Campagnani, Ilaria; Ruggerone, Margherita; Giaccone, Giorgio; Morbin, Michela; Zentilin, Lorena; Giacca, Mauro; Zucca, Ileana; Legname, Giuseppe; Tagliavini, Fabrizio

    2012-01-01

    Prion diseases are caused by a conformational modification of the cellular prion protein (PrP (C)) into disease-specific forms, termed PrP (Sc), that have the ability to interact with PrP (C) promoting its conversion to PrP (Sc). In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP (C) region involved in the interaction with PrP (Sc) thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP (Sc) in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.

  4. Induction of feline immunodeficiency virus specific antibodies in cats with an attenuated Salmonella strain expressing the Gag protein.

    NARCIS (Netherlands)

    E.J. Tijhaar (Edwin); C.H.J. Siebelink (Kees); J.A. Karlas (Jos); M.C. Burger; F.R. Mooi (Frits); A.D.M.E. Osterhaus (Albert)

    1997-01-01

    textabstractSalmonella typhimurium aroA strains (SL3261), expressing high levels of the Gag protein of feline immunodeficiency virus (FIV) fused with maltose binding protein (SL3261-MFG), were constructed using an invertible promoter system that allows the stable expression of heterologous antigens

  5. Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

    Science.gov (United States)

    Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

    2014-02-01

    T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

  6. High expression of fusion proteins consisting of a single-chain variable fragment antibody against a tumor-associated antigen and interleukin-2 in Escherichia coli.

    Science.gov (United States)

    Napathorn, Suchada Chanprateep; Kuroki, Motomu; Kuroki, Masahide

    2014-08-01

    The aim of this study was to establish a strategy for high-level production of single-chain variable fragment (scFv) antibodies fused with interleukin-2 (IL-2) in Escherichia coli. We constructed two fusion sequences consisting of a scFv gene derived from a mouse monoclonal antibody against a tumor-associated antigen (MK-1) and human Interleukin-2(IL-2) gene, ligated the fusions into pET15b and transformed into three different E. coli strains. The effects of temperature, isopropyl-β-D-thiogalactopyranoside (IPTG) concentration and duration of IPTG induction were investigated. Employing E. coli strain Rosetta-gami B, which has an oxidizing cytoplasm that facilitates cytoplasmic disulfide bond formation, improved the level of soluble protein expression. Under optimal conditions, the highest levels of fusion protein expression and high percentages of the proteins were found in their soluble form. Specifically, 89.29% (0.28 g/l) of one fusion protein was soluble after a 10-h induction and 84.61% (0.26 g/l) of the other fusion protein was soluble after a separate 10-h induction. When analyzed by enzyme-linked immunosorbent assay, the partially-purified fusion proteins retained a specific binding activity to the cell lysate of Chinese hamster ovary (CHO) cells expressing MK-1. Taken together, the methods described herein permit the production of substantial amounts of the fusion proteins for conducting functional studies on the biological role of these fusion proteins. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  7. Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

    International Nuclear Information System (INIS)

    Chory, J.; Voytas, D.F.; Olszewski, N.E.; Ausubel, F.M.

    1987-01-01

    Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA 1 ) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [ 35 S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs

  8. Genetic diversity and population structure among pea (Pisum sativum L.) cultivars as revealed by simple sequence repeat and novel genic markers.

    Science.gov (United States)

    Jain, Shalu; Kumar, Ajay; Mamidi, Sujan; McPhee, Kevin

    2014-10-01

    Field pea (Pisum sativum L.) is an important cool season legume crop widely grown around the world. This research provides a basis for selection of pea germplasm across geographical regions in current and future breeding and genetic mapping efforts for pea improvement. Eleven novel genic markers were developed from pea expressed sequence tag (EST) sequences having significant similarity with gene calls from Medicago truncatula spanning at least one intron. In this study, 96 cultivars widely grown or used in breeding programs in the USA and Canada were analyzed for genetic diversity using 31 microsatellite or simple sequence repeat (SSR) and 11 novel EST-derived genic markers. The polymorphic information content varied from 0.01-0.56 among SSR markers and 0.04-0.43 among genic markers. The results showed that SSR and EST-derived genic markers displayed one or more highly reproducible, multi-allelic, and easy to score loci ranging from 200 to 700 bp in size. Genetic diversity was assessed through unweighted neighbor-joining method, and 96 varieties were grouped into three main clusters based on the dissimilarity matrix. Four subpopulations were determined through STRUCTURE analysis with no significant geographic separation of the subpopulations. The findings of the present study can be used to select diverse genotypes to be used as parents of crosses aimed for breeding improved pea cultivars.

  9. RESULTS AND PROSPECTS OF PEA BREEDING

    Directory of Open Access Journals (Sweden)

    I. P. Kotlyar

    2016-01-01

    Full Text Available In the laboratory of leguminous crops of the “All-Russian Scientific Research Institute of vegetable breeding and seed production”, more than 100 samples of pea are studied annually. As a result, pre-breeding collection of the sources of agronomical valuable traits was developed. It is utilized for development of new pea varieties resistant to lodging. In 2012- 2014, the crossing were conducted. As a result of selection in F2-F6, the lines of different groups of maturity with lodging resistance were selected. These transgressing forms are suitable for canning, deep freezing, and drying. The following samples are recommended for development of high yielded varieties: «102- 82А5Б4» is the middle-early variety, the progenitor of early ripening cv. «Korsar» applied for the state variety trial in 2015 and two promising samples «15 КСИ 15» and «9 КСИ». The «16-05» is the middle-early variety, the transgressive form for the trait «seed amount per nod» with high attachment of lower bean. The sample «K-8915» is the middle-early genotype, high productive, with high attachment of lower bean. The sample «Troyika» is recommended for development of high productive, late ripening initial breeding material. As a result of step crossing this sample is characterized by late maturity, by three bean per nod, and by high attaching of lower bean. The sample «CM-10 m» is distinct in the aphyllous type of leaf, resistance to powdery mildew, paired bean, suitable for canning and drying. This sample was selected as a transgressive form for stem anatomy structure (spongy cell tissue from sample developed by complex step hybridization. Owing the distinctive feature in the stem anatomy structure, this sample can be recommended as a main parental form for development of varieties resistant to lodging.

  10. A photosensitizer delivered by bispecific antibody redirected T lymphocytes enhances cytotoxicity against EpCAM-expressing carcinoma cells upon light irradiation.

    Science.gov (United States)

    Blaudszun, André-René; Moldenhauer, Gerhard; Schneider, Marc; Philippi, Anja

    2015-01-10

    Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy. Copyright © 2014

  11. Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies.

    Science.gov (United States)

    Morar, D; Tijhaar, E; Negrea, A; Hendriks, J; van Haarlem, D; Godfroid, J; Michel, A L; Rutten, V P M G

    2007-01-15

    Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.

  12. Gene-based SNP discovery and genetic mapping in pea.

    Science.gov (United States)

    Sindhu, Anoop; Ramsay, Larissa; Sanderson, Lacey-Anne; Stonehouse, Robert; Li, Rong; Condie, Janet; Shunmugam, Arun S K; Liu, Yong; Jha, Ambuj B; Diapari, Marwan; Burstin, Judith; Aubert, Gregoire; Tar'an, Bunyamin; Bett, Kirstin E; Warkentin, Thomas D; Sharpe, Andrew G

    2014-10-01

    Gene-based SNPs were identified and mapped in pea using five recombinant inbred line populations segregating for traits of agronomic importance. Pea (Pisum sativum L.) is one of the world's oldest domesticated crops and has been a model system in plant biology and genetics since the work of Gregor Mendel. Pea is the second most widely grown pulse crop in the world following common bean. The importance of pea as a food crop is growing due to its combination of moderate protein concentration, slowly digestible starch, high dietary fiber concentration, and its richness in micronutrients; however, pea has lagged behind other major crops in harnessing recent advances in molecular biology, genomics and bioinformatics, partly due to its large genome size with a large proportion of repetitive sequence, and to the relatively limited investment in research in this crop globally. The objective of this research was the development of a genome-wide transcriptome-based pea single-nucleotide polymorphism (SNP) marker platform using next-generation sequencing technology. A total of 1,536 polymorphic SNP loci selected from over 20,000 non-redundant SNPs identified using deep transcriptome sequencing of eight diverse Pisum accessions were used for genotyping in five RIL populations using an Illumina GoldenGate assay. The first high-density pea SNP map defining all seven linkage groups was generated by integrating with previously published anchor markers. Syntenic relationships of this map with the model legume Medicago truncatula and lentil (Lens culinaris Medik.) maps were established. The genic SNP map establishes a foundation for future molecular breeding efforts by enabling both the identification and tracking of introgression of genomic regions harbouring QTLs related to agronomic and seed quality traits.

  13. Restricted Cell Surface Expression of Receptor Tyrosine Kinase ROR1 in Pediatric B-Lineage Acute Lymphoblastic Leukemia Suggests Targetability with Therapeutic Monoclonal Antibodies

    Science.gov (United States)

    Dave, Hema; Anver, Miriam R.; Butcher, Donna O.; Brown, Patrick; Khan, Javed; Wayne, Alan S.; Baskar, Sivasubramanian; Rader, Christoph

    2012-01-01

    Background Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL), short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs) that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies. Methodology and Principal Findings Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues. Conclusions and Significance Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies. PMID:23285131

  14. Restricted cell surface expression of receptor tyrosine kinase ROR1 in pediatric B-lineage acute lymphoblastic leukemia suggests targetability with therapeutic monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Hema Dave

    Full Text Available Despite high cure rates for pediatric B-lineage acute lymphoblastic leukemia (B-ALL, short-term and long-term toxicities and chemoresistance are shortcomings of standard chemotherapy. Immunotherapy and chemoimmunotherapy based on monoclonal antibodies (mAbs that target cell surface antigens with restricted expression in pediatric B-ALL may offer the potential to reduce toxicities and prevent or overcome chemoresistance. The receptor tyrosine kinase ROR1 has emerged as a candidate for mAb targeting in select B-cell malignancies.Using flow cytometry, Western blotting, immunohistochemistry, and confocal immunofluorescence microscopy, we analyzed the cell surface expression of ROR1 across major pediatric ALL subtypes represented by 14 cell lines and 56 primary blasts at diagnosis or relapse as well as in normal adult and pediatric tissues. Cell surface ROR1 expression was found in 45% of pediatric ALL patients, all of which were B-ALL, and was not limited to any particular genotype. All cell lines and primary blasts with E2A-PBX1 translocation and a portion of patients with other high risk genotypes, such as MLL rearrangement, expressed cell surface ROR1. Importantly, cell surface ROR1 expression was found in many of the pediatric B-ALL patients with multiply relapsed and refractory disease and normal karyotype or low risk cytogenetics, such as hyperdiploidy. Notably, cell surface ROR1 was virtually absent in normal adult and pediatric tissues.Collectively, this study suggests that ROR1 merits preclinical and clinical investigations as a novel target for mAb-based therapies in pediatric B-ALL. We propose cell surface expression of ROR1 detected by flow cytometry as primary inclusion criterion for pediatric B-ALL patients in future clinical trials of ROR1-targeted therapies.

  15. Intragastric immunization with recombinant Lactobacillus casei expressing flagellar antigen confers antibody-independent protective immunity against Salmonella enterica serovar Enteritidis

    NARCIS (Netherlands)

    Kajikawa, A.; Satoh, E.; Leer, R.J.; Yamamoto, S.; Igimi, S.

    2007-01-01

    A recombinant Lactobacillus casei expressing a flagellar antigen from Salmonella enterica serovar Enteritidis was constructed and evaluated as a mucosal vaccine. Intragastric immunization of the recombinant strain conferred protective immunity against Salmonella infection in mice. This immunization

  16. Lactobacilli expressing variable domain of llama heavy-chain antibody fragments (lactobodies) confer protection against rotavirus-induced diarrhea

    NARCIS (Netherlands)

    Pant, N.; Hultberg, A.; Zhao, Y.; Svensson, L.; Pan-Hammarström, Q.; Johansen, K.; Pouwels, P.H.; Ruggeri, F.M.; Hermans, P.; Frenken, L.; Borén, T.; Marcotte, H.; Hammarström, L.

    2006-01-01

    Background. Rotavirus-induced diarrhea poses a worldwide medical problem in causing substantial morbidity and mortality among children in developing countries. We therefore developed a system for passive immunotherapy in which recombinant lactobacilli constitutively express neutralizing variable

  17. Stimulation of nodulation in field peas (Pisum sativum) by low concentrations of ammonium in hydroponic culture

    Science.gov (United States)

    Waterer, J. G.; Vessey, J. K.; Raper, C. D. Jr; Raper CD, J. r. (Principal Investigator)

    1992-01-01

    Although the inhibitory effects of high concentrations of mineral N (> 1.0 mM) on nodule development and function have often been studied, the effects of low, static concentrations of NH4+ (hydroponic culture on nodule establishment and nitrogenase activity in field peas [Pisum sativum L. cv. Express (Svalof AB)] for the initial 28 days after planting (DAP). Peas grown in the presence of low concentrations of NH4+ had significantly greater nodule numbers (up to 4-fold) than plants grown without NH4+. Nodule dry weight per plant was significantly higher at 14, 21 and 28 DAP in plants grown in the presence of NH4+, but individual nodule mass was lower than in plants grown without NH4+. The nodulation pattern of the plants supplied with NH4+ was similar to that often reported for supernodulating mutants, however the plants did not express other growth habits associated with supernodulation. Estimates of N2 fixation indicate that the plus-NH4+ peas fixed as much or more N2 than the plants supplied with minus-NH4+ nutrient solution. There were no significant differences in nodule numbers, nodule mass or NH4+ uptake between the plants grown at the two concentrations of NH4+. Nodulation appeared to autoregulate by 14 DAP in the minus-NH4+ treatment. Plant growth and N accumulation in the minus-NH4+ plants lagged behind those of the plus-NH4+ treatments prior to N2 fixation becoming well established in the final week of the experiment. The plus-NH4+ treatments appeared not to elicit autoregulation and plants continued to initiate nodules throughout the experiment.

  18. High-Throughput Sequencing of the Expressed Torafugu (Takifugu rubripes) Antibody Sequences Distinguishes IgM and IgT Repertoires and Reveals Evidence of Convergent Evolution.

    Science.gov (United States)

    Fu, Xi; Sun, Jianqiang; Tan, Engkong; Shimizu, Kentaro; Reza, Md Shaheed; Watabe, Shugo; Asakawa, Shuichi

    2018-01-01

    B-cell antigen receptor (BCR) or antibody diversity arises from somatic recombination of immunoglobulin (Ig) gene segments and is concentrated within the Ig heavy (H) chain complementarity-determining region 3 (CDR-H3). We performed high-throughput sequencing of the expressed antibody heavy-chain repertoire from adult torafugu. We found that torafugu use between 70 and 82% of all possible V (variable), D (diversity), and J (joining) gene segment combinations and that they share a similar frequency distribution of these VDJ combinations. The CDR-H3 sequence repertoire observed in individuals is biased with the preferential use of a small number of VDJ, dominated by sequences containing inserted nucleotides. We uncovered the common CDR-H3 amino-acid (aa) sequences shared by individuals. Common CDR-H3 sequences feature highly convergent nucleic-acid recombination compared with private ones. Finally, we observed differences in repertoires between IgM and IgT, including the unequal usage frequencies of V gene segment and the biased number of nucleotide insertion/deletion at VDJ junction regions that leads to distinct distributions of CDR-H3 lengths.

  19. High-Throughput Sequencing of the Expressed Torafugu (Takifugu rubripes Antibody Sequences Distinguishes IgM and IgT Repertoires and Reveals Evidence of Convergent Evolution

    Directory of Open Access Journals (Sweden)

    Xi Fu

    2018-02-01

    Full Text Available B-cell antigen receptor (BCR or antibody diversity arises from somatic recombination of immunoglobulin (Ig gene segments and is concentrated within the Ig heavy (H chain complementarity-determining region 3 (CDR-H3. We performed high-throughput sequencing of the expressed antibody heavy-chain repertoire from adult torafugu. We found that torafugu use between 70 and 82% of all possible V (variable, D (diversity, and J (joining gene segment combinations and that they share a similar frequency distribution of these VDJ combinations. The CDR-H3 sequence repertoire observed in individuals is biased with the preferential use of a small number of VDJ, dominated by sequences containing inserted nucleotides. We uncovered the common CDR-H3 amino-acid (aa sequences shared by individuals. Common CDR-H3 sequences feature highly convergent nucleic-acid recombination compared with private ones. Finally, we observed differences in repertoires between IgM and IgT, including the unequal usage frequencies of V gene segment and the biased number of nucleotide insertion/deletion at VDJ junction regions that leads to distinct distributions of CDR-H3 lengths.

  20. Fatty acid biosynthesis in pea root plastids

    International Nuclear Information System (INIS)

    Stahl, R.J.; Sparace, S.A.

    1989-01-01

    Fatty acid biosynthesis from [1- 14 C]acetate was optimized in plastids isolated from primary root tips of 7-day-old germinating pea seeds. Fatty acid synthesis was maximum at approximately 80 nmoles/hr/mg protein in the presence of 200 μM acetate, 0.5 mM each of NADH, NADPH and CoA, 6 mM each of ATP and MgCl 2 , 1 mM each of the MnCl 2 and glycerol-3-phosphate, 15 mM KHCO 3 , and 0.1M Bis-tris-propane, pH 8.0 incubated at 35C. At the standard incubation temperature of 25C, fatty acid synthesis was linear from up to 6 hours with 80 to 100 μg/mL plastid protein. ATP and CoA were absolute requirements, whereas KHCO 3 , divalent cations and reduced nucleotides all improved activity by 80 to 85%. Mg 2+ and NADH were the preferred cation and nucleotide, respectively. Dithiothreitol and detergents were generally inhibitory. The radioactive products of fatty acid biosynthesis were approximately 33% 16:0, 10% 18:0 and 56% 18:1 and generally did not vary with increasing concentrations of each cofactor

  1. A putative amino acid ABC transporter substrate-binding protein, NMB1612, from Neisseria meningitidis, induces murine bactericidal antibodies against meningococci expressing heterologous NMB1612 proteins.

    Science.gov (United States)

    Hung, Miao-Chiu; Humbert, María Victoria; Laver, Jay R; Phillips, Renee; Heckels, John E; Christodoulides, Myron

    2015-08-26

    The nmb1612 (NEIS1533) gene encoding the ~27-kDa putative amino acid ATP-binding cassette (ABC) transporter, periplasmic substrate-binding protein from Neisseria meningitidis serogroup B (MenB) strain MC58 was cloned and expressed in Escherichia coli, and the purified recombinant (r)NMB1612 was used for animal immunization studies. Immunization of mice with rNMB1612 adsorbed to Al(OH)3 and in liposomes with and without MPLA, induced antiserum with bactericidal activity in an assay using baby rabbit complement, against the homologous strain MC58 (encoding protein representative of Allele 62) and killed heterologous strains encoding proteins of three other alleles (representative of Alleles 1, 64 and 68), with similar SBA titres. However, strain MC58 was not killed (titre bactericidal assay (hSBA) using anti-rNMB1612 sera, although another strain (MC168) expressing the same protein was killed (median titres of 16-64 in the hSBA). Analysis of the NMB1612 amino acid sequences from 4351 meningococcal strains in the pubmlst.org/Neisseria database and a collection of 13 isolates from colonized individuals and from patients, showed that antibodies raised against rNMB1612 could potentially kill at least 72% of the MenB strains in the complete sequence database. For MenB disease occurring specifically in the UK from 2013 to 2015, >91% of the isolates causing disease in this recent period expressed NMB1612 protein encoded by Allele 1 and could be potentially killed by sera raised to the recombinant antigen in the current study. The NMB1612 protein was surface-accessible and expressed by different meningococcal strains. In summary, the properties of (i) NMB1612 protein conservation and expression, (ii) limited amino acid sequence variation between proteins encoded by different alleles, and (iii) the ability of a recombinant protein to induce cross-strain bactericidal antibodies, would all suggest a promising antigen for consideration for inclusion in new meningococcal vaccines

  2. A specific anti-Aire antibody reveals aire expression is restricted to medullary thymic epithelial cells and not expressed in periphery.

    Science.gov (United States)

    Hubert, François-Xavier; Kinkel, Sarah A; Webster, Kylie E; Cannon, Ping; Crewther, Pauline E; Proeitto, Anna I; Wu, Li; Heath, William R; Scott, Hamish S

    2008-03-15

    Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is an autoimmune disorder caused by mutations in the autoimmune regulator gene AIRE. We examined the expression of Aire in different organs (thymus, spleen, and lymph nodes) in C57BL/6 mice, using a novel rat mAb, specific for murine Aire. Using flow cytometry, directly fluorochrome-labeled mAb revealed Aire expression in a rare thymic cellular subset that was CD45(-), expressed low levels of Ly51, and was high for MHC-II and EpCam. This subset also expressed a specific pattern of costimulatory molecules, including CD40, CD80, and PD-L1. Immunohistochemical analysis revealed that Aire(+) cells were specifically localized to the thymus or, more precisely, to the cortico-medulla junction and medulla, correlating with the site of negative selection. Although in agreement with previous studies, low levels of Aire mRNA was detected in all dendritic cell subtypes however lacZ staining, immunohistochemistry and flow cytometry failed to detect Aire protein. At a cellular level, Aire was expressed in perinuclear speckles within the nucleus. This report provides the first detailed analysis of Aire protein expression, highlighting the precise location at both the tissue and cellular level.

  3. POSSIBLE WAYS TO SAVE SEED PRODUCTION OF PEA DOMESTIC VARIETIES IN CURRENT CONDITIONS

    Directory of Open Access Journals (Sweden)

    N. A. Samarin

    2013-01-01

    Full Text Available The possible ways of development of seed production of pea in Russia and increasing of competitive growth of domestic pea varieties suitable for processing industry are represented in the article.

  4. Pea varieties resources in Russia in context to the situation in Western Europe.

    Science.gov (United States)

    Gorpintchenko, T; Anikanova, Z

    2001-10-01

    The largest share of legumes grain production in Russia falls at peas and amounts to 75%. The pea is grown practically in all agricultural regions of Russia. The most favourable pea-growing regions are the forest-steppe zone of European part of Russia and Volga region. 84 varieties of pea are being included in the official "National List of the Breeding Achievements Permitted to Use in the Russian Federation" in 2001. 24 of these varieties correspond to the requirements for the most valuable food pea ones. The pea varieties especially suitable for food are identified by a special classification. 32.5% percent of a total area sown with the peas in Europe falls to Russia. Total peas production in Russia is 10.6% of a total European production.

  5. Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2011-07-01

    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

  6. Antibody affinity maturation

    DEFF Research Database (Denmark)

    Skjødt, Mette Louise

    surface expression of various antibody formats in the generated knockout strain. Functional scFv and scFab fragments were efficiently displayed on yeast whereas impaired chain assembly and heavy chain degradation was observed for display of full-length IgG molecules. To identify the optimal polypeptide......-antibody interface and the antibody intraface.the microenvironment and ecology of Acaryochloris and Prochloron, and in this thesis we attempted to further describe the distribution, growth characteristics and adaptive/regulatory mechanisms of these two cyanobacteria, both in their natural habitat and under defined...

  7. Virotherapy of canine tumors with oncolytic vaccinia virus GLV-1h109 expressing an anti-VEGF single-chain antibody.

    Directory of Open Access Journals (Sweden)

    Sandeep S Patil

    Full Text Available Virotherapy using oncolytic vaccinia virus (VACV strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor single-chain antibody (scAb GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients.

  8. Radioimmunotherapy with an antibody to HPV16 E6 oncoprotein is effective in experimental cervical tumor expressing low levels of E6

    Science.gov (United States)

    Jiang, Zewei; Wang, Xing Guo; Einstein, Mark H; Goldberg, Gary L; Casadevall, Arturo

    2010-01-01

    Purpose HPV16 is associated with ∼50% of all cervical cancers worldwide. The E6 and E7 genes of oncogenic HPV types, such as HPV16, are necessary for the HPV transforming function and tumorogenesis making them ideal targets for novel treatments. Radioimmunotherapy employs systemically administered radiolabeled monoclonal antibodies (mAbs) that bind to tumor-associated antigens. Previously we demonstrated in mice that radioimmunotherapy targeting viral antigens with mAb to HPV16 E6 suppressed CasKi cervical tumors expressing high levels of E6 (∼600 copies of HPV per cell). However, that study opened the question whether radioimmunotherapy can suppress the growth of cervical tumors with low E6 and E7 expression, such as may be seen in patients. Experimental Design We evaluated the expression of E6 in patients' tumors and in the SiHa cell line expressing low levels of E6 and E7 (1–2 copies of HPV per cell) and found them comparable. We initiated SiHa tumors in nude mice, radiolabeled C1P5 mAb to E6 with a beta-emitter 188-Rhenium (188Re) and treated tumor-bearing mice with: (1) 200 µCi 188Re-C1P5 alone; (2) proteasome inhibitor MG132 alone; (3) MG132 followed by 200 µCi 188Re-C1P5; (4) unlabeled C1P5; (5) 200 µCi 188Re-18B7 (isotype-matching control mAb); (6) no treatment. 188Re-C1P5 alone and in combination with MG-132 significantly retarded tumor growth compared to all control groups. Conclusions Our data demonstrate the possibility to suppress tumor growth by targeting viral antigens even in cervical tumors with low E6 expression and provide additional evidence for the potential usefulness of radioimmunotherapy targeting HPV-related antigens in the clinic. PMID:20861673

  9. Interleukin-17 expression positively correlates with disease severity of lupus nephritis by increasing anti-double-stranded DNA antibody production in a lupus model induced by activated lymphocyte derived DNA.

    Directory of Open Access Journals (Sweden)

    Zhenke Wen

    Full Text Available Lupus nephritis is one of the most serious manifestations and one of the strongest predictors of a poor outcome in systemic lupus erythematosus (SLE. Recent evidence implicated a potential role of interlukin-17 (IL-17 in the pathogenesis of lupus nephritis. However, the correlation between IL-17 expression level and the severity of lupus nephritis still remains incompletely understood. In this study, we found that serum IL-17 expression level was associated with the severity of lupus nephritis, which was evaluated by histopathology of kidney sections and urine protein. Of note, we showed that enforced expression of IL-17 using adenovirus construct that expresses IL-17 could enhance the severity of lupus nephritis, while blockade of IL-17 using neutralizing antibody resulted in decreased severity of lupus nephritis. Consistently, we observed an impaired induction of lupus nephritis in IL-17-deficient mice. Further, we revealed that IL-17 expression level was associated with immune complex deposition and complement activation in kidney. Of interest, we found that IL-17 was crucial for increasing anti-double-stranded DNA (dsDNA antibody production in SLE. Our results suggested that IL-17 expression level positively correlated with the severity of lupus nephritis, at least in part, because of its contribution to anti-dsDNA antibody production. These findings provided a novel mechanism for how IL-17 expression level correlated with disease pathogenesis and suggested that management of IL-17 expression level was a potential and promising approach for treatment of lupus nephritis.

  10. Codon modification for the DNA sequence of a single-chain Fv antibody against clenbuterol and expression in Pichia pastoris

    Science.gov (United States)

    To improve expression efficiency of the recombinant single-chain variable fragment (scFv) against clenbuterol (CBL) obtained from mouse in the methylotrophic yeast Pichia pastoris (P. pastoris) GS115, the DNA sequence encoding for CBL-scFv was designed and synthesized based on the codon bias of P. p...

  11. 7 CFR 319.56-45 - Shelled garden peas from Kenya.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 5 2010-01-01 2010-01-01 false Shelled garden peas from Kenya. 319.56-45 Section 319... Shelled garden peas from Kenya. Garden peas (Pisum sativum) may be imported into the continental United States from Kenya only under the following conditions and in accordance with all other applicable...

  12. Significance of seed-borne Ascochyta on pea and test of management strategies

    OpenAIRE

    Wolffhechel, H.; Bødker, L.; Nielsen, G.C.

    2005-01-01

    Large amounts of Danish pea seeds are rejected each year due to high levels of Ascochyta infections. Selection of less susceptible pea varieties may reduce seed infections, while intercropping with barley only gives a small reduction. Increasing seeding rates has no effect. Short pea varieties generally seem to be more susceptible to seed infection by Ascochyta than taller varieties.

  13. Evidence that the "waxy" protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase.

    Science.gov (United States)

    Smith, A M

    1990-11-01

    The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between "waxy" proteins and starch-granule-bound starch synthases generally.

  14. Neuregulin 1 expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models.

    Science.gov (United States)

    Meetze, Kristan; Vincent, Sylvie; Tyler, Steven; Mazsa, Elizabeth K; Delpero, Andrea R; Bottega, Steve; McIntosh, Donna; Nicoletti, Richard; Winston, William M; Weiler, Solly; Feng, Bin; Gyuris, Jeno; Weng, Zhigang

    2015-03-01

    ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203. ©2014 American Association for Cancer Research.

  15. Changes in some constituents of pea seed during commercial canning.

    Science.gov (United States)

    Ros, G; Rincon, F

    1993-05-01

    Changes in crude protein (CP), non protein nitrogen (NPN), total sulphur (TS) contents, carbon/nitrogen (C/N) and nitrogen/sulphur (N/S) ratios and albumin protein (AP) were studied in four sizes of peas at various stages of the commercial canning process. Of all the parameters considered, NPN, AP, and TS showed the most regular trend to decrease during the commercial canning process. In this paper the use of these three parameters is proposed to obtain a Heat Treatment Index (HTI) applicable to raw, blanched and canned peas, according to the equation: HTI = (NPN x AP)/TS.

  16. Model of high-productive varieties in forage pea

    OpenAIRE

    KOSEV, VALENTIN

    2015-01-01

    A linear equation of regression was used for establishment of the influence of quantitative characteristics on the grain productivity in forage pea and for development of a model for breeding work. The model for pea plant with high productivity was characterized by average height of 60–70 cm, 8–10 formed pods, 30–40 seeds per plant and 160–260 g in regard to 1000-seed weight. The obtained results showed that the greatest effect on grain productivity had the seed number per plant, first pod he...

  17. STUDY ON QUANTITATIVE CHARACTERS OF ORGANIC SEEDS IN GARDEN PEA

    Directory of Open Access Journals (Sweden)

    S. Kalapchieva

    2013-01-01

    Full Text Available The experiment was conducted in the Maritsa Vegetable Crops Research Institute, Plovdiv during the period 2008-2009. Four garden pea varieties were grown in the conditions of organic and conventional production. The purpose of the experiment was to study the quantitative characters of seeds obtained in organic production. It was established that the organic garden pea seeds differ with lower absolute weight compared to the seeds obtained in conventional system but they kept high germination (89,5-96,5 close to the control variant. The late variety Vyatovo grown with organic fertilizers and biopesticides are with the lowest percentage of injuries by Bruchus pisi l.

  18. Interaction of pea (Pisum sativum L.) lectins with rhizobial strains.

    Science.gov (United States)

    Bajaj, M; Soni, G; Singh, C K

    2001-01-01

    Lectins from two varieties (PG-3 and LFP-48) of pea have been purified by affinity chromatography on Sephadex G-50. The specific activity increased by 23 and 25 folds, respectively. These lectins from both the varieties were found to be specific for mannose. The purified fluorescein isothiocyanate (FITC)-labelled lectins showed binding reaction with homologous as well as heterologous strains of Rhizobium spp. The results revealed that pea lectins are not highly specific to their respective rhizobia. Moreover, these lectins showed a greater stimulatory effect on homologous Rhizobium leguminosarum strains.

  19. [Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25-35)].

    Science.gov (United States)

    Kolobov, V V; Davydova, T V; Fomina, V G

    2014-01-01

    Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp 1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ25-35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp 1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.

  20. Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities.

    Science.gov (United States)

    Gorgoglione, Bartolomeo; Wang, Tiehui; Secombes, Christopher J; Holland, Jason W

    2013-07-16

    The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate/inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell/antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease.

  1. Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities

    Science.gov (United States)

    2013-01-01

    The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate / inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell / antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease. PMID:23865616

  2. Effect of domestic processing and cooking methods on phytic acid and polyphenol contents of pea cultivars (Pisum sativum).

    Science.gov (United States)

    Bishnoi, S; Khetarpaul, N; Yadav, R K

    1994-06-01

    All the pea varieties differed significantly (p pea cultivars had significantly (p pea varieties. All the domestic processing and cooking methods could reduce the contents of phytic acid and polyphenols but germination for 48 hours seemed to have a marked lowering effect on the levels of these antinutrients in peas.

  3. Physicochemical and sensory characteristics of soy sauce substituted with pigeon pea (Cajanus cajan (Linn.))

    Science.gov (United States)

    Retnaningsih, C.; Sumardi; Meiliana; Surya, A.

    2018-01-01

    The objective of this study wasto investigate the physicochemical and sensory properties of the soy sauce substituted with pigeon pea. Soybean was substituted by 20%, 50%, 75%, and 100% of pigeon pea. The observation included viscosity, total solids, protein levels, antioxidant activity, and sensory characteristics. The results showed that the more substitution of pigeon pea, the less the protein content of soy sauce and the more the antioxidant activity as well as total solids. The most favored group was 25% pigeon pea substitution. It is suggested that soy sauce could be prepared using 25% to 75% pigeon pea substitution.

  4. Anti-K1 (Kell Antibody Expressed in Maternal Breastmilk: A Case Report of a Neonate with Multiple Intrauterine Transfusions and Postnatal Exposure to Kell Antibody in Maternal Breastmilk

    Directory of Open Access Journals (Sweden)

    Patrick DeMoss

    2017-01-01

    Full Text Available Hemolytic disease of the fetus and newborn is a common consideration in newborn medicine, especially among the jaundiced. Maternal breastmilk provides numerous benefits to the infant, including nutrition and immunologic factors. Here, we present an infant who received three intrauterine transfusions for anemia secondary to anti-K1 (Kell, anti-C, and anti-e antibodies and whose maternal breastmilk tested positive for anti-Kell antibodies. The infant required another transfusion at 4 weeks of life for anemia. We review the pathophysiology of anti-Kell antibodies, the immunology of breast milk, and the intersection of these two topics.

  5. ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Sixiang [University of Wisconsin, Materials Science Program, Madison, WI (United States); Hong, Hao; Orbay, Hakan; Yang, Yunan; Ohman, Jakob D. [University of Wisconsin, Department of Radiology, Madison, WI (United States); Graves, Stephen A.; Nickles, Robert J. [University of Wisconsin, Department of Medical Physics, Madison, WI (United States); Liu, Bai; Wong, Hing C. [Altor BioScience, Miramar, FL (United States); Cai, Weibo [University of Wisconsin, Materials Science Program, Madison, WI (United States); University of Wisconsin, Department of Radiology, Madison, WI (United States); University of Wisconsin, Department of Medical Physics, Madison, WI (United States); University of Wisconsin Carbone Cancer Center, Madison, WI (United States); University of Wisconsin, Departments of Radiology and Medical Physics, Madison, WI (United States)

    2015-07-15

    To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and {sup 64}Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of {sup 64}Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of {sup 64}Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. {sup 64}Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management. (orig.)

  6. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

    Science.gov (United States)

    Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; von Bergwelt-Baildon, M.; Lardinois, Didier; Müller, Philipp; Karanikas, Vaios; Zippelius, Alfred

    2016-01-01

    ABSTRACT T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting folate receptor 1 and CD3 (FolR1-TCB). We observed a considerable heterogeneity in T-cell activation, cytokine production and tumor cell killing upon exposure to FolR1-TCB among different FolR1-expressing tumors. Of note, tumors presenting with a high frequency of PD-1hi TILs displayed significantly impaired tumor cell killing and T-cell function. Further characterization of additional T-cell inhibitory receptors revealed that PD-1hi TILs defined a T-cell subset with particularly high levels of multiple inhibitory receptors compared with PD-1int and PD-1neg T-cells. PD-1 blockade could restore cytokine secretion but not cytotoxicity of TILs in a subset of patients with scarce PD-1hi expressing cells; in contrast, patients with abundance of PD-1hi expressing T-cells did not benefit from PD-1 blockade. Our data highlight that FolR1-TCB is a promising novel immunotherapeutic treatment option which is capable of activating intratumoral T-cells in different carcinomas. However, its therapeutic efficacy may be substantially hampered by a pre-existing dysfunctional state of T-cells, reflected by abundance of intratumoral PD-1hi T-cells. These findings present a rationale for combinatorial approaches of TCBs with other therapeutic strategies targeting T-cell dysfunction. PMID:27057429

  7. Comparison of P-glycoprotein expression in cell lines and xenogragraft sections using I-125 MRK-16 monoclonal antibody (MAB)

    Energy Technology Data Exchange (ETDEWEB)

    Mehta, B.M.; Kostakoglu, L.; Levchenko, A. [Kettering Cancer, New York, NY (United States)] [and others

    1994-05-01

    P-glycoprotein (Pgp) is known to be associated with multidrug resistance (MDR). Quantitation of P-glycoprotein expression may permit appropriate therapy depending on Pgp expression in tumors. The present study was undertaken to evaluate the utility of quantitative autoradiography (QAR) in the quantification of MDR using MRK-16, a murine IgG mAb reactive against Pgp. Balb/c mice were xenografted with colchicine resistant BE(2)C/CHC cells. Animals with established tumors were sacrificed, and 8 {mu}m tumor sections were prepared. Mab MRK-16 was labeled with I-125 (150 {mu}Ci/0.625 nmole) by the iodogen method and subsequently purified by size exclusion chromatography. Consecutive tumor sections were incubated overnight at 4{degrees}C with serial dilutions of I-125 MRK-16. Similarly cell suspensions containing 1 X 10{sup 7} cells per ml were also incubated with serial dilutions. QAR analysis of tissue sections of BE(2)C/CHC tumors growing as xenografts in nude mice, determined the binding affinity (K{sub a}) for MRK-16 to be 1 x 10{sup 9} L/M and the number of binding sites (B{sub max}) to be 137, 700 per cell (222 picomols/g); it compared very well with the K{sub a} value of 5 x 10{sup 8} L/M and the B{sub max} value of 130,000 per cell (217 picomols/g) obtained from binding analysis with cell suspensions.

  8. A brief review on the early distribution of pea (Pisum sativum L. in Europe

    Directory of Open Access Journals (Sweden)

    Ljuština Marija

    2010-01-01

    Full Text Available Pea was a part of the everyday diet of the European hunter-gatherers at the end of the last Ice Age. The major criteria to determine the domestication in pea are non-dehiscent pods, larger seed size and smooth seed testa. Pea seeds were found among the earliest findings of cultivated crops at the site of Tell El-Kerkh, Syria, from 10th millennium BP. Along with cereals and lentil, pea has definitely become associated with the start of the 'agricultural revolution' in the Old World. Pea entered Europe in its southeast regions and progressed into its interior via Danube. Its distribution was rapid, since the available evidence reveals its presence in remote places at similar periods. The linguistic evidence supports the fact that pea had been present in nearly all regions of Europe. Most of European peoples have their own words denoting pea, meaning that it preceded the diversification of their own proto-languages.

  9. In vivo imaging and specific targeting of P-glycoprotein expression in multidrug resistant nude mice xenografts with [{sup 125}I]MRK-16 monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Scott, Andrew M.; Rosa, Eddie; Mehta, Bippin M.; Divgi, Chaitanya R.; Finn, Ronald D.; Biedler, June L.; Tsuruo, Takashi; Kalaigian, Hovannes; Larson, Steven M

    1995-05-01

    Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression. In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning. The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P-glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model. MRK-16 was labeled with {sup 125}I by the iodogen method, with subsequent purification by size exclusion chromatography. Groups of 10 Balb/c mice were each xenografted with colchicine-resistant or -sensitive neuroblastoma cell lines, respectively. Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with [{sup 125}]MRK-16 and an isotype matched control antibody, A33. Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targeting of [{sup 125}I]MRK-16. Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g {+-} SD) (18.76 {+-} 2.94 vs 10.93 {+-} 0.96; p < 0.05). Quantitative autoradiography verified these findings (19.13 {+-} 0.622 vs 12.08 {+-} 0.38, p < 0.05). Specific binding of [{sup 125}I]MRK-16 was confirmed by comparison to [{sup 131}I]A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors. Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure. We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with [{sup 125}I]MRK-16. These findings suggest a potential clinical application for radiolabeled MRK-16 in the in vivo evaluation of multidrug resistance in tumors.

  10. Photosynthetic responses of pea plants (Pisum sativum L. cv. Little ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-08-04

    Aug 4, 2008 ... meteorological parameters, levels of solar radiation as influenced .... three times during vegetative, flowering and reproductive growth stages of pea plants on expanded leaves of the upper canopy under direct sun light. The Pn rates ..... Global Change Environment: The Importance of Different Plant Traits.

  11. Relationships between yield and some yield components in Pea ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-03

    Dec 3, 2008 ... end of the study, positive and significant relationship were found among seed yield and pods per plant and biological yield in both years. The strongest and direct positive effects were the biological yield (p = 0.6500), numbers of ... Pea is an important plant in human and animal nutrition because of its high ...

  12. Maanteeamet ei pea uue maantee ehitamist õigustatuks / Gerli Romanovitsh

    Index Scriptorium Estoniae

    Romanovitš, Gerli, 1977-

    2004-01-01

    Ilmunud ka: Severnoje Poberezhje, 8. dets. 2004, lk. 1, 4. Maanteeamet ei pea Kukruse-Jõhvi teelõigu asemele uue maantee ehitamist asulast põhja poole rahaliselt õigustatuks, kuna transiitliikluse Kukruselt väljasuunamine ei vähendaks kahe linna ühendava tee turvalisust ning investeeringud tuleks õnnetuste vältimiseks teha topelt

  13. Development of Pea (Pisum sativum L.) and Chickpea (Cicer ...

    African Journals Online (AJOL)

    2012r

    snack was found to have much higher fat content than the non-fried snacks: fried snack (27.94 % w/w, wet weight ..... have contributed to lower perceived intensity for “moist” texture among assessors, in contrast with the ... processing of green peas due to loss of chlorophylls is well established in the literature (Coultate, 2002 ...

  14. Sustainable protein production and consumption : pigs of peas?

    NARCIS (Netherlands)

    Aiking, H.; Boer, de J.; Vereijken, J.M.

    2006-01-01

    Sustainable Protein Production and Consumption: Pigs or Peas? is a book that presents and explores the PROFETAS programme for development of a more sustainable food system by studying the feasibility of substituting meat with plant based alternatives. The emphasis is on improving the food system by

  15. Symbiotic effectiveness of pea-rhizobia associations and the ...

    African Journals Online (AJOL)

    Selected strains were isolated from the root nodules of pea (Pisum sativum L.), broad bean (Vicia faba L.) and lentil (Lens culinaris L.) plants in the Loess Plateau of China. Analyses focused on the nodule number, nodule dry weight, plant dry weight, nitrogenase activity, total N accumulation of per plant and seed yield.

  16. Determination of mycoflora of pea (Pisum sativum) seeds and the ...

    African Journals Online (AJOL)

    Jane

    2011-07-06

    Jul 6, 2011 ... Mycoflora of pea seeds and the effectiveness of Rhizobium leguminosarum on important seed-borne pathogens were determined. Mycoflora were changed according to seed groups with or without surface sterilization. The most common isolated fungi were Fusarium spp., Alternaria spp., Macrophomina.

  17. Allelic reůationships of Pea Nodulation Mutants

    Czech Academy of Sciences Publication Activity Database

    Novák, Karel

    2003-01-01

    Roč. 94, č. 2 (2003), s. 191-193 ISSN 0022-1503 R&D Projects: GA ČR GA521/00/0937 Institutional research plan: CEZ:AV0Z5020903 Keywords : allelic * relationships * pea Subject RIV: EE - Microbiology, Virology Impact factor: 1.707, year: 2003

  18. Development of Pea (Pisum sativum L.) and Chickpea (Cicer ...

    African Journals Online (AJOL)

    Moisture content was determined by the air-oven drying method and fat content was quantified by the soxhlet extraction method. Quantitative Descriptive Analysis was performed by 7 experienced assessors of CFTRI using 15 cm line scales. The fried green pea snack obtained highest score for “crispy” and lowest score for ...

  19. Performance of active Photosystem II centers in photoinhibited pea leaves

    NARCIS (Netherlands)

    Schansker, G.; Rensen, van J.J.S.

    1999-01-01

    Effects of photoinhibition on photosynthesis in pea (Pisum sativum L.) leaves were investigated by studying the relationship between the severity of a photoinhibitory treatment (measured as F_v/F_m) and several photoacoustic and chlorophyll a fluorescence parameters. Because of the observed linear

  20. Determination of mycoflora of pea ( Pisum sativum ) seeds and the ...

    African Journals Online (AJOL)

    Mycoflora of pea seeds and the effectiveness of Rhizobium leguminosarum on important seed-borne pathogens were determined. Mycoflora were changed according to seed groups with or without surface sterilization. The most common isolated fungi were Fusarium spp., Alternaria spp., Macrophomina phaseolina, ...

  1. Functional Properties of Processed Pigeon Pea ( Cajanus Cajan ...

    African Journals Online (AJOL)

    Pigeon pea (Cajanus cajan) seeds were carefully sorted cleaned and divided into two lots for flour production. One portion was germinated following soaking in water for 36 hours, with water being changed every 6 hours, at a ratio of 1:10, including an hour air rest before re-soaking. The seeds were germinated to obtain ...

  2. Short Communication: Occurrence of the pea aphid, Acyrthosiphon ...

    African Journals Online (AJOL)

    The occurrence of pea aphid, Acyrthosiphon pisum (Harris) (Homoptera: Aphididae), on wild annual and perennial leguminous plants was studied at two locations (Adet and Wondata) in West Gojam, Ethiopia in 1999/2000 seasons. Annual and perennial leguminous wild or volunteer plants encountered in the study areas ...

  3. Nutritional evaluation of cookies produced from pigeon pea ...

    African Journals Online (AJOL)

    Cookies were produced from pigeon pea (Cajanus cajan), sorghum (Sorghum bicolor) and cocoyam (Xanthosoma sagittifolium) flour blends. Ten different cookie formulations were produced. The cookies were evaluated for proximate composition, antinutritional factors and protein quality. The protein content ranged from ...

  4. occurrence of the pea aphid, acyrthosiphon pisum (harris)

    African Journals Online (AJOL)

    Preferred Customer

    alfalfa (Medicago sativa), clover (Trifolium sp.) and other leguminous crops ... holocyclic life cycle (i.e., it reproduces parthenogenetically ... Table 1. Plant species infested by pea aphid at Adet and. Wondata, Ethiopia, in 1999/2000. Host plant species. Total. Number of plants. Vetch Clover Lupine. Alfalfa. Infested plants.

  5. Analysis of genetic diversity in pigeon pea germplasm using ...

    Indian Academy of Sciences (India)

    Pigeon pea (Cajanus cajan), an important legume crop is predominantly cultivated in tropical and subtropical regions of Asia and Africa. It is normally considered to have a low degree of genetic diversity, an impediment in undertaking crop improvement programmes.We have analysed genetic polymorphism of domesticated ...

  6. Pigeon pea seed production and delivery system: Experience from ...

    African Journals Online (AJOL)

    Mo

    Non-availability of seed is one of the main constraints to adoption of improved crop varieties. The cause of this is mainly due to lack of efficient and sustainable seed delivery system and seed production skills in Uganda. The private seed companies are not interested in pigeon pea seed production due to low profit margins ...

  7. Models and tools for studying drought stress responses in peas.

    Science.gov (United States)

    Magyar-Tábori, Katalin; Mendler-Drienyovszki, Nóra; Dobránszki, Judit

    2011-12-01

    The pea (Pisum sativum L.) is an important pulse crop but the growing area is limited because of its relatively low yield stability. In many parts of the world the most important abiotic factor limiting the survival and yield of plants is the restricted water supply, and the crop productivity can only be increased by improving drought tolerance. Development of pea cultivars well adapted to dry conditions has been one of the major tasks in breeding programs. Conventional breeding of new cultivars for dry conditions required extensive selection and testing for yield performance over diverse environments using various biometrical approaches. Several morphological and biochemical traits have been proven to be related to drought resistance, and methods based on physiological attributes can also be used in development of better varieties. Osmoregulation plays a role in the maintenance of turgor pressure under water stress conditions, and information on the behaviour of genotypes under osmotic stress can help selection for drought resistance. Biotechnological approaches including in vitro test, genetic transformation, and the use of molecular markers and mutants could be useful tools in breeding of pea. In this minireview we summarized the present status of different approaches related to drought stress improvement in the pea.

  8. PRIMA IS A NEW CULTIVAR OF VEGETABLE PEA

    Directory of Open Access Journals (Sweden)

    A. G. Besedin

    2015-01-01

    Full Text Available In 2013, the new vegetable pea cv.Prima was applied for the State Variety Trial. This variety is characterized by very yearly-ripeness, that allows to increase the raw material processing in the canning factories of the Kuban.

  9. The hebridological analysis of productivity traits in pea hybrids

    Directory of Open Access Journals (Sweden)

    Світлана Володимирівна Коблай

    2015-09-01

    Full Text Available To study the nature of inheritance of quantitative traits that influence productivity of pea hybrids were obtained first and second generations for which sample and varieties with different leaf morphotype are served as parental form. As the results of hybridological analysis it is determined the degree of domination and revealed the heterosis combinations

  10. Bitterness of saponins and their content in dry peas

    NARCIS (Netherlands)

    Heng, L.; Vincken, J.P.; Koningsveld, van G.A.; Legger, A.; Gruppen, H.; Boekel, van M.A.J.S.; Roozen, J.; Voragen, A.G.J.

    2006-01-01

    The bitterness of a saponin mixture (containing saponin B and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) saponin in a ratio of 1:4) and saponin B obtained from dry peas were established by a trained panel using line scaling. Both saponins were found to be bitter. However, the saponin

  11. Genotype x Environment Interaction for Yield in Field Pea ( Pisum ...

    African Journals Online (AJOL)

    In this study, sixteen genotypes of field pea (Pisum sativum L.) were tested over locations (Shambu and Arjo) for three years (2000-2002) and at Gedo (2002) with the objectives of identifying high yielding, stable and adaptable varieties for the Western highlands of Ethiopia. Regression and additive main and multiplication ...

  12. Response of pigeon pea genotypes to different races of Fusarium ...

    African Journals Online (AJOL)

    To develop high yielding Fusarium wilt resistant varieties, it is essential to identify new sources of resistance that can withstand multiple races of the pathogen. The aim of this study was to identify [Fusarium udum (L.) Millsp.] races occurring in the study fields, determine reaction of pigeon pea hybrids to the different isolates ...

  13. CHARACTERISTICS OF CHAIN OF PEA VARIETIES FOR VEGQ ETABLE CANNING

    Directory of Open Access Journals (Sweden)

    N. A. Samarin

    2013-01-01

    Full Text Available The characteristics of seven pea varieties of different groups of ripening are described. These cultivars provide the sustainable row material input in canning factories of Krasnodar region. In bogharic agriculture it takes 24,26 days. When using the irrigation system and different time of sowing it takes 35,40 days.

  14. Diversity of Rhizobium leguminosarum from pea fields in Washington State

    Science.gov (United States)

    Rhizobia-mediated biological nitrogen (N) fixation in legumes contributes to yield potential in these crops and also provides residual fertilizer to subsequent cereals. Our objectives were to collect isolates of Rhizobium leguminosarum from several pea fields in Washington, examine genetic diversity...

  15. EMERGE: A Randomized Phase II Study of the Antibody-Drug Conjugate Glembatumumab Vedotin in Advanced Glycoprotein NMB-Expressing Breast Cancer.

    Science.gov (United States)

    Yardley, Denise A; Weaver, Robert; Melisko, Michelle E; Saleh, Mansoor N; Arena, Francis P; Forero, Andres; Cigler, Tessa; Stopeck, Alison; Citrin, Dennis; Oliff, Ira; Bechhold, Rebecca; Loutfi, Randa; Garcia, Agustin A; Cruickshank, Scott; Crowley, Elizabeth; Green, Jennifer; Hawthorne, Thomas; Yellin, Michael J; Davis, Thomas A; Vahdat, Linda T

    2015-05-10

    Glycoprotein NMB (gpNMB), a negative prognostic marker, is overexpressed in multiple tumor types. Glembatumumab vedotin is a gpNMB-specific monoclonal antibody conjugated to the potent cytotoxin monomethyl auristatin E. This phase II study investigated the activity of glembatumumab vedotin in advanced breast cancer by gpNMB expression. Patients (n = 124) with refractory breast cancer that expressed gpNMB in ≥ 5% of epithelial or stromal cells by central immunohistochemistry were stratified by gpNMB expression (tumor, low stromal intensity, high stromal intensity) and were randomly assigned 2:1 to glembatumumab vedotin (n = 83) or investigator's choice (IC) chemotherapy (n = 41). The study was powered to detect overall objective response rate (ORR) in the glembatumumab vedotin arm between 10% (null) and 22.5% (alternative hypothesis) with preplanned investigation of activity by gpNMB distribution and/or intensity (Stratum 1 to Stratum 3). Glembatumumab vedotin was well tolerated as compared with IC chemotherapy (less hematologic toxicity; more rash, pruritus, neuropathy, and alopecia). ORR was 6% (five of 83) for glembatumumab vedotin versus 7% (three of 41) for IC, without significant intertreatment differences for predefined strata. Secondary end point revealed ORR of 12% (10 of 83) versus 12% (five of 41) overall, and 30% (seven of 23) versus 9% (one of 11) for gpNMB overexpression (≥ 25% of tumor cells). Unplanned analysis showed ORR of 18% (five of 28) versus 0% (0 of 11) in patients with triple-negative breast cancer (TNBC), and 40% (four of 10) versus 0% (zero of six) in gpNMB-overexpressing TNBC. Glembatumumab vedotin is well tolerated in heavily pretreated patients with breast cancer. Although the primary end point in advanced gpNMB-expressing breast cancer was not met for all enrolled patients (median tumor gpNMB expression, 5%), activity may be enhanced in patients with gpNMB-overexpressing tumors and/or TNBC. A pivotal phase II trial (METRIC

  16. A combination of p53-activating APR-246 and phosphatidylserine-targeting antibody potently inhibits tumor development in hormone-dependent mutant p53-expressing breast cancer xenografts

    Directory of Open Access Journals (Sweden)

    Liang Y

    2018-03-01

    Full Text Available Yayun Liang,1 Benford Mafuvadze,1 Cynthia Besch-Williford,2 Salman M Hyder1 1Deparment of Biomedical Sciences and Dalton Cardiovascular Research Center, Columbia, MO, USA; 2IDEXX BioResearch, Columbia, MO, USA Background: Between 30 and 40% of human breast cancers express a defective tumor suppressor p53 gene. Wild-type p53 tumor suppressor protein promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor–dependent angiogenesis, whereas mutant p53 protein (mtp53 lacks these functions, resulting in tumor cell survival and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for combating mtp53-expressing breast cancer. Methods: In this study, we sought to determine whether administration of APR-246, a small-molecule drug that restores p53 function, in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits advanced hormone-dependent breast cancer tumor growth. Results: APR-246 reduced cell viability in mtp53-expressing BT-474 and T47-D human breast cancer cells in vitro, and significantly induced apoptosis in a dose-dependent manner. However, APR-246 did not reduce cell viability in MCF-7 breast cancer cells, which express wild-type p53. We next examined APR-246’s anti-tumor effects in vivo using BT-474 and T47-D tumor xenografts established in female nude mice. Tumor-bearing mice were treated with APR-246 and/or 2aG4 and tumor volume followed over time. Tumor growth was more effectively suppressed by combination treatment than by either agent alone, and combination therapy completely eradicated some tumors. Immunohistochemistry analysis of tumor tissue sections demonstrated that combination therapy more effectively induced apoptosis and reduced cell proliferation in tumor xenografts than either agent alone. Importantly, combination therapy dramatically reduced the density of blood

  17. Performance of fourteen improved pea lines (Pisum sativum L. in Challapata zone, Oruro

    Directory of Open Access Journals (Sweden)

    Maiza Benedicto

    2015-02-01

    Full Text Available In Challapata zone, cultivated pea varieties are low yielding and long cycle. The research objective was to determine the performance of fourteen pea lines developed by “Pairumani Fitoecogenetics Investigation Center” (CIFP in Challapata zone (Oruro. The 14 pea lines with local pea variety, were planted in row and column generalized experimental design with four replications in tree location randomly selection in Challapata zone (Oruro, between October 2011 and April 2012. The results indicate, that, in general, all the improved lines were superior in green pod yield to the local pea variety (3.69 t.ha-1, between 6.13 and 16.58 t.ha-1, (65.9 and 349.3% respectively. among the improved lines, Pea5_102-1, Pea5_102-6, Pea5_102-5, Pea5_102-2, Pea5_102-3 and Pea5_102-4, with high green pod yield (13.05 and 16.58 t.ha-1, large pod (8.49 to 9.25 cm, mayor number of grains for pod (5.27 to 7.20 grains and intermediate cycle (85 days to the floración, are the superior performance. The lines Pea5_102-14, Pea5_102-10 (Pairumani 3 and Pea5_102-13, because of their characteristics of high green pod yield, the longest pod, the mayor number of grains for pod, early maturity, preference and wide adaptability, and according to the farmer’s criteria, are the most recommend for their use in Challapata zone (Oruro.

  18. Organic meat quality of dual purpose young bulls supplemented with pea (Pisum sativum L.) or soybean.

    Science.gov (United States)

    Corazzin, Mirco; Piasentier, Edi; Saccà, Elena; Bazzoli, Ilario; Bovolenta, Stefano

    2018-02-01

    One of the main constraints established by organic legislation that limits the development of the rearing of young bulls is the ban on the use of genetically modified organisms (GMO). Most of the worldwide cultivated soybean is GMO, therefore the use of alternative protein sources should be evaluated. In this study, the effect of dietary substitution of soybean with pea (Pisum sativum L.) on carcass characteristics and meat quality of dual purpose young bulls reared following the organic method was investigated. Twenty-four young bulls of Rendena breed were randomly assigned to two diet treatments differing in protein supplement (soybean (SB) or field pea (FP)). Carcass characteristics and meat chemical composition, colour, cooking loss and Warner-Bratzler shear force did not differ between groups. Regarding meat fatty acid composition, SB showed higher concentrations of C18:0 and C18:1 t and lower C16:1n-9c, C14:0, C17:1n-9c and C18:1n-9c than FP. In descriptive sensory analysis, trained judges were not able to differentiate meats from SB and FP, which also had similar overall liking expressed by consumers. The results of this study indicate that FP can replace SB in the diet of dual purpose young bulls with only a minor influence on fatty acid composition and no effect on carcass characteristics and meat quality. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  19. Antiprothrombin Antibodies

    Directory of Open Access Journals (Sweden)

    Polona Žigon

    2015-05-01

    Full Text Available In patients with the antiphospholipid syndrome (APS, the presence of a group of pathogenic autoantibodies called antiphospholipid antibodies causes thrombosis and pregnancy complications. The most frequent antigenic target of antiphospholipid antibodies are phospholipid bound β2-glycoprotein 1 (β2GPI and prothrombin. The international classification criteria for APS connect the occurrence of thrombosis and/or obstetric complications together with the persistence of lupus anticoagulant, anti-cardiolipin antibodies (aCL and antibodies against β2GPI (anti-β2GPI into APS. Current trends for the diagnostic evaluation of APS patients propose determination of multiple antiphospholipid antibodies, among them also anti-prothrombin antibodies, to gain a common score which estimates the risk for thrombosis in APS patients. Antiprothrombin antibodies are common in APS patients and are sometimes the only antiphospholipid antibodies being elevated. Methods for their determination differ and have not yet been standardized. Many novel studies confirmed method using phosphatidylserine/prothrombin (aPS/PT ELISA as an antigen on solid phase encompass higher diagnostic accuracy compared to method using prothrombin alone (aPT ELISA. Our research group developed an in-house aPS/PT ELISA with increased analytical sensitivity which enables the determination of all clinically relevant antiprothrombin antibodies. aPS/PT exhibited the highest percentage of lupus anticoagulant activity compared to aCL and anti-β2GPI. aPS/PT antibodies measured with the in-house method associated with venous thrombosis and presented the strongest independent risk factor for the presence of obstetric complications among all tested antiphospholipid antibodies

  20. Differentiation of foot-and-mouth disease virus infected animals from vaccinated animals using a blocking ELISA based on baculovirus expressed FMDV 3ABC antigen and a 3ABC monoclonal antibody

    DEFF Research Database (Denmark)

    Sørensen, K.J.; de Stricker, K.; Dyrting, K.C.

    2005-01-01

    A blocking ELISA that differentiated foot-and-mouth disease virus (FMDV) infected animals from vaccinated animals was developed which uses baculovirus expressed FMDV 3ABC non-structural protein as antigen and monoclonal antibody against FMDV 3ABC non-structural protein as capture and detector...... and sheep collected after experimental or natural infection. The blocking ELISA based on recombinant FMDV 3ABC antigen and a monoclonal antibody to 3ABC is a promising tool for FMD control and eradication campaigns, where vaccination has been carried out....

  1. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  2. ATR-FTIR spectroscopy reveals involvement of lipids and proteins of intact pea pollen grains to heat stress tolerance

    Directory of Open Access Journals (Sweden)

    Rachid eLahlali

    2014-12-01

    Full Text Available With climate change, pea will be more frequently subjected to heat stress in semi-arid regions like Saskatchewan during flowering. The pollen germination percentage of two pea cultivars was reduced by heat stress (36°C with an important decrease in cultivar ‘CDC Golden’ compared to ‘CDC Sage’. Lipids, protein and other pollen coat compositions of whole intact pollen grains of both pea cultivars were investigated using mid infrared (mid-IR Attenuated Total Reflectance (ATR-Fourier Transform Infrared (FTIR spectroscopy. Curve-fitting of ATR absorbance spectra in the protein region enabled estimation and comparison of different protein secondary structures between the two cultivars. CDC Sage had relatively greater amounts of α-helical structures (48.6-43.6%; band at 1654 cm-1 and smaller amounts of β-sheets (41.3-46% than CDC Golden. The CDC Golden had higher amounts of β-sheets (46.3-51.7% compared to α-helical structures (35.3-36.2%. Further, heat stress resulted in prominent changes in the symmetrical and asymmetrical CH2 bands from lipid acyl chain, ester carbonyl band, and carbohydrate region. The intensity of asymmetric and symmetric CH2 vibration of heat stressed CDC Golden was reduced considerably in comparison to the control and the decrease was higher compared to CDC sage. In addition, CDC Golden showed an increase in intensity at the oxidative band of 3015 cm-1. These results reveal that the whole pollen grains of both pea cultivars responded differently to heat stress. The tolerance of CDC Sage to heat stress (expressed as pollen germination percentage may be due to its protein richness with α-helical structures which would protect against the destructive effects of dehydration due to heat stress. The low pollen germination percentage of CDC Golden after heat stress may be also due to its sensitivity to lipid changes due to heat stress.

  3. Estimation of pea (Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses.

    Science.gov (United States)

    Cieslarová, Jaroslava; Hanáček, Pavel; Fialová, Eva; Hýbl, Miroslav; Smýkal, Petr

    2011-11-01

    Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5'untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10(-3) per locus per generation, with a 95% confidence interval of 1.2 × 10(-4) to 2.7 × 10(-2). The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.

  4. ATR-FTIR spectroscopy reveals involvement of lipids and proteins of intact pea pollen grains to heat stress tolerance.

    Science.gov (United States)

    Lahlali, Rachid; Jiang, Yunfei; Kumar, Saroj; Karunakaran, Chithra; Liu, Xia; Borondics, Ferenc; Hallin, Emil; Bueckert, Rosalind

    2014-01-01

    With climate change, pea will be more frequently subjected to heat stress in semi-arid regions like Saskatchewan during flowering. The pollen germination percentage of two pea cultivars was reduced by heat stress (36°C) with an important decrease in cultivar 'CDC Golden' compared to 'CDC Sage.' Lipids, protein and other pollen coat compositions of whole intact pollen grains of both pea cultivars were investigated using mid infrared (mid-IR) attenuated total reflectance (ATR)-Fourier transform infrared (FTIR) spectroscopy. Curve fitting of ATR absorbance spectra in the protein region enabled estimation and comparison of different protein secondary structures between the two cultivars. CDC Sage had relatively greater amounts of α-helical structures (48.6-43.6%; band at 1654 cm(-1)) and smaller amounts of β-sheets (41.3-46%) than CDC Golden. The CDC Golden had higher amounts of β-sheets (46.3-51.7%) compared to α-helical structures (35.3-36.2%). Further, heat stress resulted in prominent changes in the symmetrical and asymmetrical CH2 bands from lipid acyl chain, ester carbonyl band, and carbohydrate region. The intensity of asymmetric and symmetric CH2 vibration of heat stressed CDC Golden was reduced considerably in comparison to the control and the decrease was higher compared to CDC Sage. In addition, CDC Golden showed an increase in intensity at the oxidative band of 3015 cm(-1). These results reveal that the whole pollen grains of both pea cultivars responded differently to heat stress. The tolerance of CDC Sage to heat stress (expressed as pollen germination percentage) may be due to its protein richness with α-helical structures which would protect against the destructive effects of dehydration due to heat stress. The low pollen germination percentage of CDC Golden after heat stress may be also due to its sensitivity to lipid changes due to heat stress.

  5. Cloning and expression of canine CD25 for validation of an anti-human CD25 antibody to compare T regulatory lymphocytes in healthy dogs and dogs with osteosarcoma.

    Science.gov (United States)

    Rissetto, K C; Rindt, H; Selting, K A; Villamil, J A; Henry, C J; Reinero, C R

    2010-05-15

    T regulatory cells (Tregs) are a unique subset of T helper cells that serve to modify/inhibit effector cells of the immune system and thus are essential to prevent autoimmunity. Overzealous Treg activity may contribute to impaired immune responses to cancer. Tregs can be phenotypically identified by proteins expressed on the cell surface (CD4 and CD25) and inside the cell (forkhead box3 (FoxP3)), although in dogs, no anti-canine CD25 antibody exists. We hypothesized that a mouse anti-human CD25 antibody definitively recognizes the canine protein and can be used to identify Tregs in dogs. We describe cloning and transfection of the canine CD25 gene into human HeLa cells with subsequent expression of the canine protein on the cell surface detected using an anti-human CD25 antibody in a flow cytometric assay. Validation of this antibody was used to identify CD4+CD25+FoxP3+ Tregs in 39 healthy dogs and 16 dogs with osteosarcoma (OSA). Results were expressed in five different ways and showed significantly fewer %CD4+CD25+ T lymphocytes expressing FoxP3 in blood of older dogs (>/=7 years) compared with the other two age groups (dogs and those with OSA. While the CD25 antibody can be successfully used in a flow cytometric assay to identify Tregs, this study does not support clinical utility of phenotypic recognition of Tregs in dogs with OSA. Copyright 2010 Elsevier B.V. All rights reserved.

  6. Effect of pea, pea hulls, faba beans and faba bean hulls on the ileal microbial composition in weaned piglets

    NARCIS (Netherlands)

    Meulen, van der J.; Panneman, H.; Jansman, A.J.M.

    2010-01-01

    Grain legumes produced in Europe such as pea, faba beans and lupins are alternative vegetable protein sources for imported soy protein in animal feeds. These legume seeds contain constituents that are not digested and may act as a substrate for microbial fermentation in the gastrointestinal tract,

  7. Nasal immunization of mice with Lactobacillus casei expressing the Pneumococcal Surface Protein A: induction of antibodies, complement deposition and partial protection against Streptococcus pneumoniae challenge.

    Science.gov (United States)

    Campos, Ivana B; Darrieux, Michelle; Ferreira, Daniela M; Miyaji, Eliane N; Silva, Débora A; Arêas, Ana Paula M; Aires, Karina A; Leite, Luciana C C; Ho, Paulo L; Oliveira, Maria Leonor S

    2008-04-01

    Strategies for the development of new vaccines against Streptococcus pneumoniae infections try to overcome problems such as serotype coverage and high costs, present in currently available vaccines. Formulations based on protein candidates that can induce protection in animal models have been pointed as good alternatives. Among them, the Pneumococcal Surface Protein A (PspA) plays an important role during systemic infection at least in part through the inhibition of complement deposition on the pneumococcal surface, a mechanism of evasion from the immune system. Antigen delivery systems based on live recombinant lactic acid bacteria (LAB) represents a promising strategy for mucosal vaccination, since they are generally regarded as safe bacteria able to elicit both systemic and mucosal immune responses. In this work, the N-terminal region of clade 1 PspA was constitutively expressed in Lactobacillus casei and the recombinant bacteria was tested as a mucosal vaccine in mice. Nasal immunization with L. casei-PspA 1 induced anti-PspA antibodies that were able to bind to pneumococcal strains carrying both clade 1 and clade 2 PspAs and to induce complement deposition on the surface of the bacteria. In addition, an increase in survival of immunized mice after a systemic challenge with a virulent pneumococcal strain was observed.

  8. Expression analysis and specific blockade of the receptor for human thymic stromal lymphopoietin (TSLP) by novel antibodies to the human TSLPRα receptor chain.

    Science.gov (United States)

    Borowski, Andreas; Vetter, Tina; Kuepper, Michael; Wohlmann, Andreas; Krause, Sebastian; Lorenzen, Thomas; Virchow, Johann Christian; Luttmann, Werner; Friedrich, Karlheinz

    2013-02-01

    Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7)-like cytokine with a pivotal role in development and maintenance of atopic diseases such as allergic asthma and atopic dermatitis. Moreover, recent studies show an involvement of TSLP in the progression of various cancers. TSLP signaling is mediated by the TSLP receptor (TSLPR), a heterodimeric type I cytokine receptor. It consists of the IL-7 receptor alpha chain (IL-7Rα), which is shared with the IL-7 receptor, and the TSLPRα chain as a specific subunit. Blocking signal release by TSLP without affecting IL-7 function is a potentially interesting option for the treatment of atopic diseases or certain tumors. By employing the extracellular domain of human TSLPRα chain (hTSLPRα(ex)) as an antigen, we generated a set of monoclonal antibodies. Several binders to native and/or denatured receptor protein were identified and characterized by cytometry and Western blot analysis. A screen based on a STAT3-driven reporter gene assay in murine pro-B cells expressing a functional hTSLPR yielded two hybridoma clones with specific antagonistic properties towards hTSLP, but not IL-7. Kinetic studies measuring blockade of hTSLP-dependent STAT phosphorylation in a TSLP-responsive cell line revealed an inhibitory constant in the nanomolar range. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Adjuvant-enhanced antibody and cellular responses to inclusion bodies expressing FhSAP2 correlates with protection of mice to Fasciola hepatica.

    Science.gov (United States)

    Rivera, Francheska; Espino, Ana M

    2016-01-01

    Fasciola hepatica saposin-like protein-2 (FhSAP2) is a protein differentially expressed in various developmental stages of F. hepatica. Recombinant FhSAP2 has demonstrated the induction of partial protection in mice and rabbits when it is administered subcutaneously (SC) in Freund's adjuvant. Because FhSAP2 is overexpressed in bacteria in the form of inclusion bodies (IBs), we isolated IBs expressing FhSAP2 and tested their immunogenicity when administered SC in mice emulsified in two different adjuvants: QS-21 and Montanide TM ISA720. Animals received three injections containing 20 μg of protein two weeks apart and 4 weeks after the third injection, mice were infected with 10 F. hepatica metacercariae by oral route. The percentages of protection induced by FhSAP2-IBs were estimated to be between 60.0 and 62.5% when compared with adjuvant-vaccinated, infected controls. By determining the levels of IgG1 and IgG2a antibodies and IL-4 and IFNγ cytokines in the serum of experimental animals, it was found that both Th1 and Th2 immune responses were significantly increased in the FhSAP2-IBs vaccinated groups compared with the adjuvant-vaccinated, infected control groups. The adjuvant-vaccinated groups had significantly lower IgG1 to IgG2a ratios and lower IL-4 to IFNγ ratios than the FhSAP2-IBs vaccinated animals, which is indicative of higher levels of Th2 immune responses. Irrespective to the adjuvant used, animals vaccinated with FhSAP2-IBs exhibited significantly higher survival percentage and less liver damage than the adjuvant-control groups. This study suggests that FhSAP2 has potential as vaccine against F. hepatica and that the protection elicited by this molecule could be linked to a mechanism driven by the CD4-Th1 cells. Published by Elsevier Inc.

  10. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Science.gov (United States)

    Mani, Shailendra; Tripathi, Lav; Raut, Rajendra; Tyagi, Poornima; Arora, Upasana; Barman, Tarani; Sood, Ruchi; Galav, Alka; Wahala, Wahala; de Silva, Aravinda; Swaminathan, Sathyamangalam; Khanna, Navin

    2013-01-01

    Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4). A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs) which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E) protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+)-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (pdengue vaccine.

  11. Influence of partial replacement of soya bean meal by faba beans or peas in heavy pigs diet on meat quality, residual anti-nutritional factors and phytoestrogen content.

    Science.gov (United States)

    Gatta, Domenico; Russo, Claudia; Giuliotti, Lorella; Mannari, Claudio; Picciarelli, Piero; Lombardi, Lara; Giovannini, Luca; Ceccarelli, Nello; Mariotti, Lorenzo

    2013-06-01

    The study evaluated the partial substitution of soybean meal by faba beans (18%) or peas (20%) as additional protein sources in diets destined for typical Italian heavy pig production. It compared animal performances, meat quality, the presence of residual anti-nutritional factors (ANF) and phytoestrogens in plasma and meat and the possible effects on pig health, by evaluating oxidative, inflammatory and pro-atherogenic markers. The results showed that the productive performances, expressed as body weight and feed conversion ratio, of pigs fed with faba bean and pea diets were similar to those of pigs fed only the soybean meal. Meat quality of pigs fed with the three diets was similar in colour, water-holding capacity, tenderness and chemical composition. Despite the higher levels of phytoestrogen in the plasma of pigs fed only the soybean meal, phytoestrogen concentration in the muscle was equivalent to that of animals fed diets with faba beans, whereas pigs fed a diet with peas showed a lower concentration. Inflammation and pro-atherogenic parameters did not show significant differences among the three diets. Overall, the partial substitution of soybean meal by faba beans appears more interesting than with peas, particularly in relation to the higher amount of polyphenols in the diet and the highest concentration of phytoestrogens found in the plasma and muscle of animals, while the pyrimidine anti-nutritional compounds present in the diet did not appear to accumulate and had no effect on the growth performance of animals.

  12. Apple Latent Spherical Virus Vector as Vaccine for the Prevention and Treatment of Mosaic Diseases in Pea, Broad Bean, and Eustoma Plants by Bean Yellow Mosaic Virus

    Directory of Open Access Journals (Sweden)

    Nozomi Satoh

    2014-11-01

    Full Text Available We investigated the protective effects of a viral vector based on an Apple latent spherical virus (ALSV harboring a segment of the Bean yellow mosaic virus (BYMV genome against mosaic diseases in pea, broad bean, and eustoma plants caused by BYMV infection. In pea plants pre-inoculated with the ALSV vaccine and challenge inoculated with BYMV expressing green fluorescence protein, BYMV multiplication occurred in inoculated leaves, but was markedly inhibited in the upper leaves. No mosaic symptoms due to BYMV infection were observed in the challenged plants pre-inoculated with the ALSV vaccine. Simultaneous inoculation with the ALSV vaccine and BYMV also prevented mosaic symptoms in broad bean and eustoma plants, and BYMV accumulation was strongly inhibited in the upper leaves of plants treated with the ALSV vaccine. Pea and eustoma plants were pre-inoculated with BYMV followed by inoculation with the ALSV vaccine to investigate the curative effects of the ALSV vaccine. In both plant species, recovery from mosaic symptoms was observed in upper leaves and BYMV accumulation was inhibited in leaves developing post-ALSV vaccination. These results show that ALSV vaccination not only prevents mosaic diseases in pea, broad bean, and eustoma, but that it is also effective in curing these diseases.

  13. Salicylic acid negatively affects the response to salt stress in pea plants.

    Science.gov (United States)

    Barba-Espín, G; Clemente-Moreno, M J; Alvarez, S; García-Legaz, M F; Hernández, J A; Díaz-Vivancos, P

    2011-11-01

    We studied the effect of salicylic acid (SA) treatment on the response of pea plants to salinity. Sodium chloride (NaCl)-induced damage to leaves was increased by SA, which was correlated with a reduction in plant growth. The content of reduced ascorbate and glutathione in leaves of salt-treated plants increased in response to SA, although accumulation of the respective oxidised forms occurred. An increase in hydrogen peroxide also occurred in leaves of salt-exposed plants treated with SA. In the absence of NaCl, SA increased ascorbate peroxidase (APX; 100 μm) and glutathione-S transferase (GST; 50 μm) activities and increased catalase (CAT) activity in a concentration-dependent manner. Salinity decreased glutathione reductase (GR) activity, but increased GST and CAT activity. In salt-stressed plants, SA also produced changes in antioxidative enzymes: 100 μm SA decreased APX but increased GST. Finally, a concentration-dependent increase in superoxide dismutase (SOD) activity was induced by SA treatment in salt-stressed plants. Induction of PR-1b was observed in NaCl-stressed plants treated with SA. The treatment with SA, as well as the interaction between salinity and SA treatment, had a significant effect on PsMAPK3 expression. The expression of PsMAPK3 was not altered by 70 mm NaCl, but was statistically higher in the absence than in the presence of SA. Overall, the results show that SA treatment negatively affected the response of pea plants to NaCl, and this response correlated with an imbalance in antioxidant metabolism. The data also show that SA treatment could enhance the resistance of salt-stressed plants to possible opportunistic pathogen attack, as suggested by increased PR-1b gene expression. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

  14. Effect of hot aqueous ethanol treatment on anti-nutritional factors, protein denaturation and functional properties in raw pea and pea protein isolate

    NARCIS (Netherlands)

    Tolman, G.H.

    1995-01-01

    The effect of hot aqueous ethanol treatment on several nutritionally relevant mainly protein-related parameters in raw peas (var. Solara) and ultra-filtrated pea protein isolate was examined. Of all test samples, water absorptive capacity (WAC), weight loss and protein loss owing to the processing

  15. Modified cytokeratins expressed on the surface of carcinoma cells undergo endocytosis upon binding of human monoclonal antibody and its recombinant Fab fragment

    DEFF Research Database (Denmark)

    Ditzel, H J; Garrigues, U; Andersen, C B

    1997-01-01

    Previously, we have reported on successful imaging of colon, rectal, and pancreatic carcinomas in patients by using a radiolabeled all-human monoclonal antibody, COU-1, directed against modified cytokeratin. To further develop this antibody for use as an immunoconjugate, COU-1 was cloned by phage...

  16. [The significances of peripheral neutrophils CD(55) and myeloperoxidase expression in patients with myeloperoxidase-specific anti-neutrophil cytoplasmic antibody associated vasculitis].

    Science.gov (United States)

    Zhou, X L; Zheng, M J; Shuai, Z W; Zhang, L; Zhang, M M; Chen, S Y

    2017-06-01

    Objective: To investigate the expression of CD(55) and myeloperoxidase (MPO) on neutrophils in patients with MPO-specific anti-neutrophil cytoplasmic antibody associated vasculitis(MPO-AAV), and analyze the relationship between the expression and clinical manifestation. Methods: Forty untreated patients with active MPO-AAV (patient group) and 30 healthy volunteers (control group) were enrolled in this study. The CD(55) on neutrophils and both membrane and cytoplasmic MPO were detected by flow cytometry. Serum fragment-from the activated complement factor B(Ba) and MPO were measured by ELISA. The clinical activity of vasculitis was valued by Birmingham vasculitis activity score-version 3(BVAS-V3). The significance of laboratory data was evaluated by Spearman correlation test and multivariate linear regression analysis. Results: (1)The mean fluorescence intensity(MFI) of CD(55) expressed on neutrophils was significantly higher than that in control group[4 068.6±2 306.0 vs 2 999.5±1 504.9, P =0.033]. Similar results of serum MPO and Ba in patient group were found compared to controls [500.0(381.0, 612.7) IU/L vs 286.9(225.5, 329.1) IU/L, P <0.001; 35.2(25.2, 79.5) ng/L vs 18.0(15.0, 28.0) ng/L, P <0.001], respectively. However, MIF of cytoplasmic MPO in patients was significantly lower than that of control group(1 577.1±1 175.9 vs 3 105.3±2 323.0, P =0.003) . (2) In patient group, cytoplasmic intensity of MPO was negatively associated with the serum levels of MPO( r =-0.710, P <0.001) and Ba ( r =-0.589, P =0.001). Moreover, serum MPO was positively associated with serum Ba( r =0.691, P <0.001). Membrane intensity of CD(55) on neutrophils was positively correlated with patient age ( r =0.514, P =0.001), C reactive protein ( r =0.376, P =0.018), peripheral neutrophils count ( r =0.485, P =0.001) and BVAS-V3 ( r =0.484, P =0.002), whereas negative correlation between membrane CD(55) and disease duration was seen ( r =-0.403, P =0.01). (3) The result of multiple

  17. Inheritance of resistance to powdery mildew in pea and pathogenesis-related aspects

    Directory of Open Access Journals (Sweden)

    Ricardo Lima dos Santos

    2012-06-01

    Full Text Available The inheritance of resistance to powdery mildew in the pea cultivar MK-10 and some histological aspects of infection were assessed. For the inheritance study, F1, F2, backcrosses and F3 generations of MK-10 crossed with two susceptible populations were evaluated. Histological evaluations included percentage of germinated conidia, percentage of conidia that formed appresoria, percentage of conidia that established colonies, and number of haustoria per colony. Segregation ratios obtained in the resistance inheritance study were compared by Chi-square (ײ test and the histological data were analyzed by Tukey's test at 5% probability. It was concluded that resistance of MK-10 to powdery mildew is due to a pair of recessive alleles since it is expressed in the pre-penetration stage and completed by post-penetration localized cellular death, characteristic of the presence of the pair of recessive alleles er1er1.

  18. PIGEON PEA (Cajanus cajan AN ALTERNATIVE IN THE FOOD INDUSTRY

    Directory of Open Access Journals (Sweden)

    Carmen Lucia Navarro V

    2014-12-01

    Full Text Available Due to the current situation of inadequate nutrition in the population of many countries, including Colombia. Search sources rich in proteins and low-cost alternatives. The pigeon pea (Cajanus cajan is an important legume that contain a mo derate amount of protein, calories, vitamins and minerals, its use in foods is limited by the presence of anti-nutritional factors, which can be reduced or eliminated through the use of treatments. The proteins have functional properties that can be take advantage in meat, dairy and bakery products. The purpose of this review is to present an overview of the skills nutritional and functional properties of pigeon pea application opportunities in various applications in the food industry.

  19. Evaluation of Pigeon Pea Lines for Biological Soil Decompaction

    Directory of Open Access Journals (Sweden)

    Rodolfo Godoy

    2009-01-01

    Full Text Available Soil decompaction is generally achieved through mechanical cultivation practices; however biological processes can significantly add to this process through root growth, development, and later senescence. This study was carried out in Piracicaba, SP, Brazil and had the purpose of selecting, among forty one pure pigeon pea lines, the most efficient genotypes that promote soil decompaction by roots penetrating compacted soil layers. Utilizing artificially compacted 30 mm high soil blocks, in a series of experiments, these lines were compared to the cultivar Fava Larga taken as a standard. Three lines were preliminarily selected out of the initial group, and afterwards, in more detailed screenings by monitoring soil resistance to penetration and also evaluating the behavior of Tanzania grass plants seeded after pigeon pea, two of them, g5-94 and g8-95, were selected as possessing the most fit root system to penetrate compacted soil layers.

  20. Sample size for estimating average productive traits of pigeon pea

    Directory of Open Access Journals (Sweden)

    Giovani Facco

    2016-04-01

    Full Text Available ABSTRACT: The objectives of this study were to determine the sample size, in terms of number of plants, needed to estimate the average values of productive traits of the pigeon pea and to determine whether the sample size needed varies between traits and between crop years. Separate uniformity trials were conducted in 2011/2012 and 2012/2013. In each trial, 360 plants were demarcated, and the fresh and dry masses of roots, stems, and leaves and of shoots and the total plant were evaluated during blossoming for 10 productive traits. Descriptive statistics were calculated, normality and randomness were checked, and the sample size was calculated. There was variability in the sample size between the productive traits and crop years of the pigeon pea culture. To estimate the averages of the productive traits with a 20% maximum estimation error and 95% confidence level, 70 plants are sufficient.

  1. Uptake and distribution of 232U in peas and barley

    International Nuclear Information System (INIS)

    Schreckhise, R.G.; Cline, J.F.

    1980-01-01

    The uptake of 232 U from soil and its distribution in peas and barley were examined under conditions which isolated root uptake from deposition on aboveground plant parts. Aboveground plant parts were harvested at maturity and analyzed for 232 U content by alpha-energy-analysis. The ratio of concentration (CR) of 232 U in the dry barley seeds to dry soil was 1.6 x 10 -4 while the CR values of the stem/leaf to dry soil fraction was 3.6 x 10 -3 . The Cr values for the pea seed, stem/pod and leaf components were 5.4 x 10 -4 , 3.3 x 10 -3 and 1.7 x 10 -2 , respectively. This indicates that the CR values used in certain radiological dose-assessment models may be high by about a factor of 100 when evaluating the consumption of seeds of legumes or cereal grains by man. (author)

  2. Model of high-productive varieties in forage pea

    Directory of Open Access Journals (Sweden)

    Valentin Kosev

    2015-06-01

    Full Text Available A linear equation of regression was used for establishment of the influence of quantitative characteristics on the grain productivity in forage pea and for development of a model for breeding work. The model for pea plant with high productivity was characterized by average height of 60–70 cm, 8–10 formed pods, 30–40 seeds per plant and 160–260 g in regard to 1000-seed weight. The obtained results showed that the greatest effect on grain productivity had the seed number per plant, first pod height and 1000-seed weight. Kristal variety had high ecological plasticity and could be considered as close to an ideal type, suitable for growing under wide range of environments. Pleven 4 and Rezonator were determined as high-productive varieties and with low stability, Kerpo and Pikardi - as low-productive but stable varieties. Druzba was identified as unstable and low-productive variety.

  3. Genetic improvement of field pea (Pisum sativum L. in Bulgaria

    Directory of Open Access Journals (Sweden)

    Kosev Valentin

    2010-01-01

    Full Text Available Field pea attained greater importance as a cultivated plant in Bulgaria at the beginning of the 20th century. Until 1964, only breeding for forage was in use, with developed winter varieties №5 and Pleven 2. Recently, field pea achieved the greatest increase (283.3% in the sown areas, since the variety structure was updated annually. There are 10 registered varieties in total, with 7 spring and 3 winter ones. The composition of the Bulgarian Pisum collections is highly variable, with accessions of diverse status. The greatest efficiency is obtained in a combination of bulk method in the early generations and certain features of pedigree, single seed method, with possible modifications and inclusion of the mutational variability.

  4. Intercropping of wheat and pea as influenced by nitrogen fertilization

    DEFF Research Database (Denmark)

    Ghaley, B.B.; Hauggaard-Nielsen, Henrik; Jensen, Henning Høgh

    2005-01-01

    The effect of sole and intercropping of field pea (Pisum sativum L.) and spring wheat (Triticum aestivum L.) on crop yield, fertilizer and soil nitrogen (N) use was tested on a sandy loam soil at three levels of urea fertilizer N (0, 4 and 8 g N m−2) applied at sowing. The 15N enrichment and natu...... with lower soil N levels, and vice versa for wheat, paving way for future option to reduce N inputs and negative environmental impacts of agricultural crop production......., grain N concentration, the proportion of N derived from symbiotic N2 fixation, and soil N accumulation. With increasing fertilizer N supply, intercropped and sole cropped wheat responded with increased yield, grain N yield and soil N accumulation, whereas the opposite was the case for pea. Fertilizer N...

  5. Characterization of DNA-binding proteins from pea mitochondria

    DEFF Research Database (Denmark)

    Hatzack, F.A.; Dombrowski, S.; Brennicke, A.

    1998-01-01

    in competition experiments. Purification by hydroxyapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography separated two polypeptides with apparent molecular masses of 32 and 44 kD. Both proteins bound to conserved structures of the pea atp9 and the heterologous Oenothera berteriana atp......1 promoters and to sequences just upstream. Possible functions of these proteins in mitochondrial promoter recognition are discussed....

  6. Eesti ei pea ümberasujatele midagi tagastama / Helle Kalda

    Index Scriptorium Estoniae

    Kalda, Helle, 1950-

    2006-01-01

    Omandireformi aluste seaduse 7 paragrahvi lõikest 3 ja varade tagastamisest nn. järelümberasunutele. Sama ka Meie Maa 12. jaan. 2006, lk. 2 ; Vooremaa 17. jaan. 2006, lk. 2 ; Virumaa Teataja 2. veeb. 2006, lk. 11 ; Pärnu Postimees 9. veeb. 2006, lk. 15 ; Pärnu Postimees 9. veeb. 2006, lk. 15, pealkiri kujul : Ümberasujatele ei pea midagi tagastama

  7. Sort sourceof pea in Ukraine and in the world

    Directory of Open Access Journals (Sweden)

    А. М. Шевченко

    2006-03-01

    Full Text Available The sorts different by economic-biologic character of unshed seed, breeded in Ukraine and abroad obtained wide spreading in production. Expediency using lugansky or samarsky type of determinant stability to healthy growth. Breeding sorts with tendril type of leaf give the positive results in increase stability of the plants to damping-off. The sorts combining named recessive character with another aconomic-value traits of grain pea made and suggested to production.

  8. Quality Inspection and Grading of Canned Green Peas using Computer Vision

    OpenAIRE

    Jinesh V N

    2015-01-01

    Canned Green Peas are widely used vegetable and is the preferred food during emergency food supply in natural disaster for victims. It is highly nutritive and is rich in protein. The quality of these Canned Green Peas is determined by its color, smell and shape. A computer vision system is used to inspect the quality of peas. The sample for the experiment was acquired from proposed image acquisition system with image resolution 400X300. The proposed system facilitates the color and dimension ...

  9. Genetic diversity and trait genomic prediction in a pea diversity panel

    OpenAIRE

    Burstin, Judith; Salloignon, Pauline; Chabert Martinello, Marianne; Magnin Robert, Jean-Bernard; Siol, Mathieu; Jacquin, Françoise; Chauveau, Aurelie; Pont, Caroline; Aubert, Gregoire; Delaitre, Catherine; Truntzer, Caroline; Duc, Gérard

    2015-01-01

    Background Pea (Pisum sativum L.), a major pulse crop grown for its protein-rich seeds, is an important component of agroecological cropping systems in diverse regions of the world. New breeding challenges imposed by global climate change and new regulations urge pea breeders to undertake more efficient methods of selection and better take advantage of the large genetic diversity present in the Pisum sativum genepool. Diversity studies conducted so far in pea used Simple Sequence Repeat (SSR)...

  10. The influence of feeding GMO-peas on growth of animal models

    Directory of Open Access Journals (Sweden)

    Petr Mares

    2014-02-01

    Full Text Available Introduction of genetically modified (GM food or feed into the commercial sale represents a very complicated process. One of the most important steps in approval process is the evaluation of all risks on the health status of people and animal models. Within our project the genetically modified peas was breeded that showed significant resistance against Pea seed-borne mosaic virus and Pea enation mosaic virus. Preclinical studies have been conducted to found out the effect of GMO peas on animals - rats of outbreeding line Wistar. In a total, 24 male, specific pathogen free Wistar rats were used in the experiment. At the beginning of the experiment, the animals were 28 days old. The three experimental groups with 8 individuals were created. The first group of rats was fed with GMO peas, the second group of rats consumed mix of pea cultivar Raman and the third group was control without pea addition (wheat and soya were used instead of pea. In the present study we focused our attention on health, growth and utility features of rats fed with GM pea. All characteristic were observed during the experiment lasting 35 days. Consumed feed was weighted daily and the weight of the animals was measured every seven days. The average values were compared within the groups. The aim of the experiment was to verify if resistant lines of pea influence the weight growth of animal models. The results of our experiment showed that even a high concentration (30% of GM pea did not influence growth rate of rats to compare with both rats fed with pea of Raman cultivar and control group. We did not observe any health problems of animal models during the experiment.

  11. Impact of climate and diseases on pea yields: what perspectives with climate change?

    OpenAIRE

    Bénézit Maud; Biarnès Véronique; Jeuffroy Marie-Hélène

    2017-01-01

    For farmers, pea crop is characterized by a large yield variability between years, between areas, and even between fields in a same small area in a given year. In dry year, spring pea crops are mostly affected by water stress and high temperature but significant yield losses can also be caused by a root disease, Aphanomyces euteiches, particularly during wet years. Winter pea can escape partially from drought, high temperature, and root disease during the reproductive phase of the crop cycle....

  12. Selenium and phosphorus interaction in pea (pisum sativum L.)

    International Nuclear Information System (INIS)

    Singh, Mahendra; Bhandari, D.K.

    1975-01-01

    The interaction of selenium and phosphorus on the dry matter yield and concentration and uptake of phosphorus, sulfur and selenium was studied in pea (Pisum sativnum) var. T 163. The fertilizer was tagged with P 32 . It was observed that increased concentration of applied selenium in soil decreased the dry matter yield and increased the concentration and uptake of total P, soil P and selenium in pea plants. Increased concentration of P alone increased dry matter yield, concentration and uptake of total, soil and fertilizer P and selenium which was beyond safe limits, and decreased concentration and uptake of sulphur. Selenium and phosphorus showed strong synergetic relationship by increasing the concentration of each other in plants while both showed antagonistic effect on the concentration of sulphur. Phosphorus compensated the toxic effect of selenium and improved the growth and dry matter yield of pea plants. The highest selenium concentration of 22.4 ppm was observed in 100 ppm phosphorus with 5 ppm selenium treated pots while lowest (0.10 ppm) in control. (author)

  13. OPPORTUNITIES TO USE PEA - WHEAT MIXES IN ORGANIC FARMING

    Directory of Open Access Journals (Sweden)

    Grigori Ivanov

    2015-12-01

    Full Text Available This article presented the results of productivity and quality of the green mass of pea-wheat mixes grown in conditions of organic farming. Are explored 5 wheat varieties - Sadovo 1, Geia 1, Guinness, Farmer, Liusil and 4 varieties of winter peas -Mir, Vesela, №11, L12AB, at different ratio between them - 50:50 and 30:70%. The selection of varieties is made based on previous studies of their complex characteristics – ripening, yield, chemistry (Angelova S., T.Georgieva, M.Sabeva, 2011. Setting up and raising the experimental mixture of seeds has been made in a medium free of organic and mineral fertilizers. We have studied the changes in green mass yield and the biochemistry of surface biomass. The cultivation of pea–wheat mixtures under conditions of organic farming leads to increased yields of green mass in comparison with the self-seeding of wheat and peas. According to the results obtained at early ripening and the highest crude protein content average of three years is the mixture Sadovo1–Mir 30:70%. The most productive is the mixture Sadovo1-Mir 50-50%.

  14. Structural and functional properties of starches from field peas.

    Science.gov (United States)

    Wang, Shujun; Sharp, Peter; Copeland, Les

    2011-06-15

    Starch was isolated from seven varieties of field peas (Pisumsativum L.) and characterised using a combination of physical, chemical and functional tests. The total starch content of the peas ranged between 34% and 42.7% of dry matter, and the amylose content of the starch was between 35% and 38%. Average particle diameter of the seven starches varied between 21.4 and 26.1μm. All of the pea starches gave a typical C-type X-ray diffraction pattern, with relative crystallinity ranging between 36% and 55% and the proportion of B-type crystallites between 3.8% and 30.4%. Although there were only small differences between the starches in amylose content, they displayed significant variability in functional properties, including swelling power, pasting characteristics, thermal transition temperatures in the differential scanning calorimeter, and in susceptibility to invitro attack by α-amylase. The results indicate the importance of structural characteristics of starch molecules, particularly amylopectin, as determinants of the properties of native starch granules. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Shailendra Mani

    Full Text Available Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4. A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (p<0.05. The formation of immunogenic DENV-2 E

  16. Relating physico-chemical properties of frozen green peas (Pisum sativum L.) to sensory quality.

    Science.gov (United States)

    Nleya, Kathleen M; Minnaar, Amanda; de Kock, Henriëtte L

    2014-03-30

    The acceptability of frozen green peas depends on their sensory quality. There is a need to relate physico-chemical parameters to sensory quality. In this research, six brands of frozen green peas representing product sold for retail and caterer's markets were purchased and subjected to descriptive sensory evaluation and physico-chemical analyses (including dry matter content, alcohol insoluble solids content, starch content, °Brix, residual peroxidase activity, size sorting, hardness using texture analysis and colour measurements) to assess and explain product quality. The sensory quality of frozen green peas, particularly texture properties, were well explained using physico-chemical methods of analysis notably alcohol insoluble solids, starch content, hardness and °Brix. Generally, retail class peas were of superior sensory quality to caterer's class peas although one caterer's brand was comparable to the retail brands. Retail class peas were sweeter, smaller, greener, more moist and more tender than the caterer's peas. Retail class peas also had higher °Brix, a(*) , hue and chroma values; lower starch, alcohol insoluble solids, dry matter content and hardness measured. The sensory quality of frozen green peas can be partially predicted by measuring physico-chemical parameters particularly °Brix and to a lesser extent hardness by texture analyser, alcohol insoluble solids, dry matter and starch content. © 2013 Society of Chemical Industry.

  17. Effect of gamma irradiation on physicochemical properties of stored pigeon pea (Cajanus cajan) flour.

    Science.gov (United States)

    Bamidele, Oluwaseun P; Akanbi, Charles T

    2013-09-01

    The effect of gamma irradiation at various doses (5, 10, 15, 20 kGy) was observed on pigeon pea flour stored for 3 months on proximate composition, functional properties, and peroxide value. Sensory evaluation was also carried out on bean cake (moinmoin) made from nonirradiated and irradiated pigeon pea flour. The results showed that stored gamma-irradiated samples had significantly lower (P pigeon pea flour showed no significant difference from the moinmoin sample prepared from nonirradiated flour. It can be concluded that gamma irradiation can extend the shelf life of pigeon pea flour.

  18. Engineered Lactobacillus rhamnosus GG expressing IgG-binding domains of protein G: Capture of hyperimmune bovine colostrum antibodies and protection against diarrhea in a mouse pup rotavirus infection model.

    Science.gov (United States)

    Günaydın, Gökçe; Zhang, Ran; Hammarström, Lennart; Marcotte, Harold

    2014-01-16

    Rotavirus-induced diarrhea causes more than 500,000 deaths annually in the world, and although vaccines are being made available, new effective treatment strategies should still be considered. Purified antibodies derived from hyperimmune bovine colostrum (HBC), from cows immunized with rotavirus, were previously used for treatment of rotavirus diarrhea in children. A combination of HBC antibodies and a probiotic strain of Lactobacillus (L. rhamnosus GG) was also found to be more effective than HBC alone in reducing diarrhea in a mouse model of rotavirus infection. In order to further improve this form of treatment, L. rhamnosus GG was engineered to display surface expressed IgG-binding domains of protein G (GB1, GB2, and GB3) which capture HBC-derived IgG antibodies (HBC-IgG) and thus target rotavirus. The expression of IgG-binding domains on the surface of the bacteria as well as their binding to HBC-IgG and to rotavirus (simian strain RRV) was demonstrated by Western blot, flow cytometry, and electron microscopy. The prophylactic effect of engineered L. rhamnosus GG and anti-rotaviral activity of HBC antibodies was evaluated in a mouse pup model of RRV infection. The combination therapy with engineered L. rhamnosus GG (PG3) and HBC was significantly more effective in reducing the prevalence, severity, and duration of diarrhea in comparison to HBC alone or a combination of wild-type L. rhamnosus GG and HBC. The new therapy reduces the effective dose of HBC between 10 to 100-fold and may thus decrease treatment costs. This antibody capturing platform, tested here for the first time in vivo, could potentially be used to target additional gastrointestinal pathogens. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Insulin-related peptide 5 is involved in regulating embryo development and biochemical composition in pea aphid with wing polyphenism

    Directory of Open Access Journals (Sweden)

    Shan-Shan eGuo

    2016-02-01

    Full Text Available In aphids there is a fecundity-dispersal trade-off between wingless and winged morphs. Recent research on the molecular mechanism of wing morphs associated with dispersal reveals that insulin receptors in the insulin signaling (IS pathway regulate alteration of wing morphs in planthoppers. However, little is known about whether genes in the IS pathway are involved in developmental regulation in aphid nymphs with different wing morphs. In this study, we show that expression of the insulin-related peptide 5 gene (Apirp5 affects biochemical composition and embryo development of wingless pea aphids, Acyrthosiphon pisum. After comparing expression levels of major genes in the IS pathway between third instar winged and wingless nymphs, we found that Apirp5 showed higher expression in head and thorax of the wingless nymphs than in the winged nymphs. Although microinjection treatment affects physical performance in aphids, nymphs with RNA interference of Apirp5 had less weight, smaller embryo size and higher carbohydrate and protein contents compared to control group. Comparison between winged and wingless nymphs showed a similar trend. These results indicate that Apirp5 is involved in embryo development and metabolic regulation in wing dimorphic pea aphid.

  20. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Miyamura, Tatsuo; Saito, Izumu (National Institute of Health, Tokyo (Japan)); Katayama, Tohru (Tokyo National Chest Hospital (Japan)); Kikuchi, Shu; Tateda, Akira (Sendai National Hospital (Japan)); Houghton, M.; Choo, Quilim; Kuo, G. (Chiron Corporation, Emeryville, CA (USA))

    1990-02-01

    A cDNA clone has been derived from the plasma of a chimpanzee with chronic non-A, non-B viral hepatits (NANBH). The authors have assayed for antibodies reacting with the encoded antigen in sera from posttransfusion hepatitis patients (643 samples from 23 patients) and their corresponding donors collected during the past 10 years in Japan. The antibody was detected in 15 out of 17 (88.2%) posttransfusion NANBH (PT-NANBH) patients whose sera over time displayed multiple alanine aminotransferase (ALT) peaks. In general, the antibody was detected after several peaks of serum ALT elevations and, once detected, it persisted for years. Of the 15 well-defined cases of PT-NANBH that showed multiple ALT peaks and hepatitis C virus seroconversions, 11 (73.3%) were shown to be transfused with at least one unit of blood positive for the antibody. The retrospective analysis showed that all tested donor blood found to be positive for the antibody had been transfused to recipients who afterwards developed NANBH. These data strongly suggest that the cloned cDNA originated from an etiological agent of NANBH termed the hepatitis C virus. Furthermore, the present study demonstrates that had the screening been done with the anti-hepatitis C virus assay, 11 out of 17 (64.7%) cases of chronic PT-NANBH and 1 out of 6 (16.6%) acute PT-NANBH would have been prevented.

  1. Root biomass accumulation in some varieties and hybrids of pea (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    VILIANA VASILEVA

    2015-04-01

    Full Text Available Root biomass accumulation in spring and winter varieties and hybrids pea was recorded in field experiment in the Institute of Forage Crops, Pleven, Bulgaria (2011-2013. Spring (Shtambovyi and Pleven 4 and winter (Rosacrono and Pleven 10 varieties and their hybrids (Shtambovyi x Pleven 10 and Rosacrono х Pleven 4 (in first and second generation, F1 and F2 were investigated for fresh and dry root biomass accumulation at three phenological stages of plant development (budding, beginning of flowering and maturity. It was found that the tested varieties and hybrids pea accumulated different amount root biomass at the different stages of plant development. Pleven 10 (67.5 kg/da fresh and 11.28 kg/da dry root biomass and Rosacrono х Pleven 4 (F1 (68.7 kg/da fresh and 14.8 kg/da dry root biomass accumulated the greatest amount of root biomass at the budding stage. Winter variety Rosocrono accumulated the greatest amount of root biomass at the beginning of flowering (59.04 kg/da fresh and 16.20 kg/da dry root biomass. In this stage, hybrids Rosacrono х Pleven 4 formed significantly more root biomass in both generations, in F1 reached to 108.0 kg/da fresh and to 26.10 kg/da dry root biomass. At the maturity Pleven 10 formed 20.88 kg/da dry root biomass and the hybrid Shtambovyi х Pleven 10 (F1 - 24.96 kg/da, respectively. The genetical part in phenotypic expression for investigated traits in Pleven 10 х Shtambovyi was relative high and existed probability for selection of homozygous genotypes in F3 and F4 hybrid generations. The data for root biomass accumulation at different phenological stages could be used as parameters for characterization and differentiations of samples, hybrids and lines in this crop.

  2. Heat stress differentially modifies ethylene biosynthesis and signaling in pea floral and fruit tissues.

    Science.gov (United States)

    Savada, Raghavendra P; Ozga, Jocelyn A; Jayasinghege, Charitha P A; Waduthanthri, Kosala D; Reinecke, Dennis M

    2017-10-01

    Ethylene biosynthesis is regulated in reproductive tissues in response to heat stress in a manner to optimize resource allocation to pollinated fruits with developing seeds. High temperatures during reproductive development are particularly detrimental to crop fruit/seed production. Ethylene plays vital roles in plant development and abiotic stress responses; however, little is known about ethylene's role in reproductive tissues during development under heat stress. We assessed ethylene biosynthesis and signaling regulation within the reproductive and associated tissues of pea during the developmental phase that sets the stage for fruit-set and seed development under normal and heat-stress conditions. The transcript abundance profiles of PsACS [encode enzymes that convert S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid (ACC)] and PsACO (encode enzymes that convert ACC to ethylene), and ethylene evolution were developmentally, environmentally, and tissue-specifically regulated in the floral/fruit/pedicel tissues of pea. Higher transcript abundance of PsACS and PsACO in the ovaries, and PsACO in the pedicels was correlated with higher ethylene evolution and ovary senescence and pedicel abscission in fruits that were not pollinated under control temperature conditions. Under heat-stress conditions, up-regulation of ethylene biosynthesis gene expression in pre-pollinated ovaries was also associated with higher ethylene evolution and lower retention of these fruits. Following successful pollination and ovule fertilization, heat-stress modified PsACS and PsACO transcript profiles in a manner that suppressed ovary ethylene evolution. The normal ethylene burst in the stigma/style and petals following pollination was also suppressed by heat-stress. Transcript abundance profiles of ethylene receptor and signaling-related genes acted as qualitative markers of tissue ethylene signaling events. These data support the hypothesis that ethylene biosynthesis is

  3. Antibody biotechnology

    African Journals Online (AJOL)

    STORAGESEVER

    2009-07-06

    Jul 6, 2009 ... and automated, the hybrid cells can be stored for many years in liquid nitrogen and antibodies production is homogeneous. The hybridoma method .... they may be modified to vehicle active molecules such as radio-isotopes, toxins, cytokines, enzyme etc. In these cases, the therapeutic effect is due to ...

  4. Catalytic Antibodies

    Indian Academy of Sciences (India)

    The ability of the highly evolved machinery of immune system to produce structurally and functionally complex ... to Pauling, if the structure of the antigen binding site of antibodies were to be produced in a random ..... where the immune system of the body is destructive, as in autoimmune disorders or after organ transplant.

  5. Catalytic Antibodies

    Indian Academy of Sciences (India)

    While chemistry provides the framework for understanding the structure and function of biomolecules, the immune sys- tem provides a highly evolved natural process to generate one class of complex biomolecules – the antibodies. A combination of the two could be exploited to generate new classes of molecules with novel ...

  6. In situ localization of chalcone synthase mRNA in pea root nodule development.

    NARCIS (Netherlands)

    Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.

    1992-01-01

    In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it

  7. Effect of processing on the nutritive value of pigeon pea ( Cajanus ...

    African Journals Online (AJOL)

    Effect of processing on the nutritive value of pigeon pea (Cajanus cajan) seeds for finisher broilers. PN Onu, PE Nwakpu, SN Okongwu. Abstract. A 28 – day feeding trial was conducted to evaluate the effect of processing of pigeon pea seeds on the performance and nutrient digestibility of finisher broilers. Four experimental ...

  8. Danish Rhizobium leguminosarum strains nodulating ‘Afghanistan’ pea (Pisum sativum)

    DEFF Research Database (Denmark)

    Jensen, Erik Steen; Sørensen, Lasse Holst; Engvild, Kjeld Christensen

    1986-01-01

    A wild pea (Pisum sativum L.) native to Afghanistan normally known to be resisant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection...... pattern with Rhizobium leguminosarum strains isolated from a modern pea variety cultivated in the same field....

  9. Exploring variation in pea protein composition by natural selection and genetic transformation

    NARCIS (Netherlands)

    Tzitzikas, E.

    2005-01-01

    Pea (Pisumsativum L.) seeds are a rich and valuable source of proteins, which can have potential for food industrial applications. Pea storage proteins are classified into two major classes: the salt-soluble globulins, and the water-soluble

  10. Simple Identification of the Neutral Chlorinated Auxin in Pea by Thin Layer Chromatography

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1980-01-01

    One of the neutral chlorinated auxins of immature pea seeds was readily identified by thin layer procedures simple enough to serve in student's laboratory courses. 4-Chloroindole-3-acetic acid methyl ester was extracted from 50 g of commercial, frozen peas by either water or acetone, concentrated...

  11. Vooruit met de geit. Marktkansen voor Geitenvlees! Een duik in de keten van The Green Peas

    NARCIS (Netherlands)

    Livestock Research,

    2012-01-01

    De geitensector loopt tegen verschillende problemen aan. The Green Peas is gevraagd door Wageningen UR Livestock Research (WUR) om onderzoek te doen naar het verwaarden van duurzaam, Nederlands geitenvlees. The Green Peas is gevraagd vanwege haar expertise op het gebied van duurzaam voedselonderzoek

  12. Effect of enzyme treatment on pea starch physicomechanical properties of biofilms

    OpenAIRE

    A. Sh. Zakirova; T. N. Manahova; A. V. Kanarskiy; Z. A. Kanarskaya

    2013-01-01

    The regularities of change in physical and mechanical properties of biofilms based on pea starch treated with pullulanase enzyme preparation were obtained. The possibility of formation of linear pea starch amylopectin polymers, which contribute to improvement of the mechanical and rheological properties of biofilms was identified.

  13. Effect of enzyme treatment on pea starch physicomechanical properties of biofilms

    Directory of Open Access Journals (Sweden)

    A. Sh. Zakirova

    2013-01-01

    Full Text Available The regularities of change in physical and mechanical properties of biofilms based on pea starch treated with pullulanase enzyme preparation were obtained. The possibility of formation of linear pea starch amylopectin polymers, which contribute to improvement of the mechanical and rheological properties of biofilms was identified.

  14. Effect of gamma irradiation on pollen and seed fertility in pigeon pea

    International Nuclear Information System (INIS)

    Ahmed John, S.

    1997-01-01

    A study was undertaken in pigeon pea parents and their F 1 hybrid to analyse the pollen and seed fertility following gamma irradiation. It is found that the reduction of pollen and seed fertility in pigeon pea was lesser over those of black gram and cowpea. 5 refs., 1 tab

  15. Use of Cowpea and Pigeon pea as Nutritional Ingredients in Culture ...

    African Journals Online (AJOL)

    Use of Cowpea and Pigeon pea as Nutritional Ingredients in Culture Media. ... Sudan Journal of Medical Sciences ... Cheap, locally available plant seeds such as cowpea (Vigna unguiculata) and pigeon pea (Cajanus cajan) could be used in the design and formulation of microbial culture media in order to reduce the cost.

  16. Sustainable control of pea bacterial blight : approaches for durable genetic resistance and biocontrol by endophytic bacteria

    NARCIS (Netherlands)

    Elvira-Recuenco, M.

    2000-01-01

    Key-words: bacterial blight, biological control, biodiversity, endophytic bacteria, L-form, pea, PDRl retrotransposon, Pisum sativum, Pisum abyssinicum, Pseudomonas syringae pv. pisi, race specific resistance, race non-specific resistance, Spanish landraces.

    Pea bacterial blight

  17. Barley uptake of N deposited in the rhizosphere of associated field pea

    DEFF Research Database (Denmark)

    Jensen, E.S.

    1996-01-01

    vulgare L.) was determined in three pot experiments using a direct split-root N-15-labelling technique. The donor (pea) and receiver (barley) plants were grown with their root systems either mixed within the same soil compartment or separated by a 20 mu m mesh bag. Pea-derived N was detected in barley...

  18. OLIGOSACCHARIDE LEVELS IN IMMATURE AND NATURE SEEDS FROM SEVERAL VARIETIES OF PIGEON PEAS (Cajanus cajan

    Directory of Open Access Journals (Sweden)

    JAIRO OSVALDO CAZETTA

    2009-07-01

    Full Text Available

    ABSTRACT: Oligosaccharide level were evaluated in immature and nature seeds from different varieties of pigeon peas (Cajanus cajan and compared to those of common beans (Phaseolus vulgaris and peas (Pisum sativum: The effect of seed processing by soaking and cooking was also determined. Immature pigeon peas seeds presented low oligosaccharide levels when compared to mature pigeon pea seeds or to pea seeds, thus representing a good alternative for human consumption. The mature pigeon pea seeds of the “Kaki” variety had oligosaccharide levels closest to those of common beans, whereas seeds of the “Paraíba” and “Fava-larga” varieties had higher levels. Soaking and cooking promoted a reduction of the oligosaccharide levels present in unprocessed mature seeds, with large amounts of these sugars being detected in the cooking water. This reduction was much more pronounced in immature seeds but the oligosaccharide levels present in their cooking water. This reduction was much more pronounced in immature seeds but the oligosaccharide levels present in their cooking water were also low, indicating that processing induced oligosaccharide degradation in immature seeds. KEYWORDS: Oligosaccharides & cooking; pigeon peas; common beans; peas.

  19. Grass pea and neurolathyrism: farmers' perception on its consumption and protective measure in North Shewa, Ethiopia.

    Science.gov (United States)

    Girma, Anteneh; Tefera, Beneberu; Dadi, Legesse

    2011-03-01

    Neurolathyrism in Ethiopia is caused by food dependency on grass pea (Lathyrus sativus L.). In the study area, a large proportion of the farmers are growing grass pea since it can withstand harsh environments. Socio-economic factors (poverty; lack of money to buy other food legumes) and environmental problems (such as water logging and frost hazards) influence consumption of grass pea. Most of the respondents have the idea that some chemical contained in grass pea causes a health problem. Different processing and preparation methods are used to prepare grass pea into different food forms. The major processing methods include washing and soaking, as the farmers apply these methods mainly because they assume that the chemical that causes lathyrism, scientifically known as β-ODAP (β-N-oxalyl-L-α,β-diaminopropionic acid) is reduced through washing and soaking. The farmers adopt different strategies to avoid the problem of lathyrism such as avoiding consumption of grass pea in the form that they suspect to cause the problem, blending/mixing with other crops, applying different processing/detoxification methods. Since grass pea is consumed with a fear of lathyrism, future research should concentrate either on developing grass pea varieties with safe level of β-ODAP content or improving the traditional/indigenous processing methods. Copyright © 2010 Elsevier Ltd. All rights reserved.

  20. The forage utilization of winter pea-cereal mixture in agriculture low-input system

    Directory of Open Access Journals (Sweden)

    Josef Hakl

    2011-01-01

    Full Text Available The aim of this paper is to evaluate the forage utilization of winter catch crop in relation to yield and mixture composition. In 2006–2009, the plot experiment with winter pea in mixtures with rye and triticale was conducted under completely randomized design with four replicates. The productivity of mixture was above 10 t.ha−1 whilst the significantly lowest value was observed for pea monoculture. The rye represented the most productive component in mixture but it achieved lower forage quality in comparison with triticale. The ratio of pea varied from 7 to 38 % in dependence on year and companion cereal. The triticale mixture provided higher ratio in comparison with rye and significant differences between rye varieties were also detected. The quality of mixture was depended mainly on ratio of crops in the mixture. The ratio of pea significantly increased crude protein content in mixture according to linear regression where crude protein = 9.56 + 0.11* weight percentage pea ratio (P < 0.000, R2 = 0.89. Quality of pea forage was also influenced by companion crop where pea in mixture with rye achieved significantly lower quality. The amount of weeds was highest in the pea monoculture and lowest in mixture with rye.

  1. Doing the Basics Better in Africa: How School Support, Autonomy, and Accountability Improved Outcomes for Girls in PEAS Schools

    Science.gov (United States)

    Hills, Libby

    2017-01-01

    Promoting Equality in African Schools (PEAS) seeks to expand access to sustainably delivered, quality secondary education in Africa. PEAS builds and runs chains of not-for-profit, low-cost private schools in public-private partnership with governments. External evaluation data show that PEAS schools in Uganda are delivering higher quality…

  2. Identification of LATE BLOOMER2 as a CYCLING DOF FACTOR Homolog Reveals Conserved and Divergent Features of the Flowering Response to Photoperiod in Pea.

    Science.gov (United States)

    Ridge, Stephen; Sussmilch, Frances C; Hecht, Valérie; Vander Schoor, Jacqueline K; Lee, Robyn; Aubert, Gregoire; Burstin, Judith; Macknight, Richard C; Weller, James L

    2016-10-01

    The molecular pathways responsible for the flowering response to photoperiod have been extensively studied in Arabidopsis thaliana and cereals but remain poorly understood in other major plant groups. Here, we describe a dominant mutant at the LATE BLOOMER2 (LATE2) locus in pea (Pisum sativum) that is late-flowering with a reduced response to photoperiod. LATE2 acts downstream of light signaling and the circadian clock to control expression of the main photoperiod-regulated FT gene, FTb2, implying that it plays a primary role in photoperiod measurement. Mapping identified the CYCLING DOF FACTOR gene CDFc1 as a strong candidate for LATE2, and the late2-1D mutant was found to carry a missense mutation in CDFc1 that impairs its capacity to bind to the blue-light photoreceptor FKF1 in yeast two-hybrid assays and delays flowering in Arabidopsis when overexpressed. Arabidopsis CDF genes are important negative regulators of CONSTANS (CO) transcription, but we found no effect of LATE2 on the transcription of pea CO-LIKE genes, nor on genes in any other families previously implicated in the activation of FT in Arabidopsis. Our results reveal an important component of the pea photoperiod response pathway and support the view that regulation of FTb2 expression by photoperiod occurs via a CO-independent mechanism. © 2016 American Society of Plant Biologists. All rights reserved.

  3. Influence of gibberellic acid on the growth and flowering initiation of two types of peas (Pisum sativum L. differing in photoperiod response

    Directory of Open Access Journals (Sweden)

    S. Łukasik

    2015-06-01

    Full Text Available It was found that GA3 (0.03 mg per one plant caused significant delay of the flowering of two different genotypes of peas under conditions of an increasing natural day length (March - May. It was expressed both in a greater number of vegetative nodes and in a greater number of days to the first flower. Under conditions of a decreasing day length (August - November most of G type plants treated with GA3 reacted with complete inhibition of the flowering. In K type pea, GA3 treatment in the discussed conditions affected only the number of days from the sowing time to the appearence of the first flower. This stage was greater in treated plants in comparison with the control ones.

  4. GREEN PEA GALAXIES REVEAL SECRETS OF Lyα ESCAPE

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Huan; Wang, Junxian [CAS Key Laboratory for Research in Galaxies and Cosmology, Department of Astronomy, University of Science and Technology of China (China); Malhotra, Sangeeta; Rhoads, James E. [Arizona State University, School of Earth and Space Exploration (United States); Gronke, Max; Dijkstra, Mark [Institute of Theoretical Astrophysics, University of Oslo (Norway); Jaskot, Anne [Smith College, Northampton, MA (United States); Zheng, Zhenya, E-mail: yanghuan@mail.ustc.edu.cn, E-mail: huan.y@asu.edu, E-mail: Sangeeta.Malhotra@asu.edu, E-mail: James.Rhoads@asu.edu [Pontificia Universidad Católica de Chile, Santiago (Chile)

    2016-04-01

    We analyze archival Lyα spectra of 12 “Green Pea” galaxies observed with the Hubble Space Telescope, model their Lyα profiles with radiative transfer models, and explore the dependence of the Lyα escape fraction on various properties. Green Pea galaxies are nearby compact starburst galaxies with [O iii] λ5007 equivalent widths (EWs) of hundreds of Å. All 12 Green Pea galaxies in our sample show Lyα lines in emission, with an Lyα EW distribution similar to high-redshift Lyα emitters. Combining the optical and UV spectra of Green Pea galaxies, we estimate their Lyα escape fractions and find correlations between Lyα escape fraction and kinematic features of Lyα profiles. The escape fraction of Lyα in these galaxies ranges from 1.4% to 67%. We also find that the Lyα escape fraction depends strongly on metallicity and moderately on dust extinction. We compare their high-quality Lyα profiles with single H i shell radiative transfer models and find that the Lyα escape fraction anticorrelates with the derived H i column densities. Single-shell models fit most Lyα profiles well, but not the ones with the highest escape fractions of Lyα. Our results suggest that low H i column density and low metallicity are essential for Lyα escape and make a galaxy an Lyα emitter.

  5. Antibody pretargeting advances cancer radioimmunodetection and radioimmunotherapy.

    Science.gov (United States)

    Goldenberg, David M; Sharkey, Robert M; Paganelli, Giovanni; Barbet, Jacques; Chatal, Jean-François

    2006-02-10

    This article reviews the methods of pretargeting, which involve separating the targeting antibody from the subsequent delivery of an imaging or therapeutic agent that binds to the tumor-localized antibody. This provides enhanced tumor:background ratios and the delivery of a higher therapeutic dose than when antibodies are directly conjugated with radionuclides, as currently practiced in cancer radioimmunotherapy. We describe initial promising clinical results using streptavidin-antibody constructs with biotin-radionuclide conjugates in the treatment of patients with malignant gliomas, and of bispecific antibodies with hapten-radionuclides in the therapy of tumors expressing carcinoembryonic antigen, such as medullary thyroid and small-cell lung cancers.

  6. Effect of pre-emergence herbicides on growth parameters of green pea.

    Science.gov (United States)

    Wágner, G; Nádasy, E

    2006-01-01

    Green pea (Pisum sativum L.), is one of the important vegetable crop in Hungary. Chemical weed control has an important role in pea growing. Pre-emergence herbicides are used most frequently in a green pea culture because they eliminate competition between crop plant and weeds even at the critical early growth stage. Preemergence treatment combined with mechanical methods makes unnecessary the post-emergence protection. Herbicides with different mode of action can influence the growth of pea and cause phytotoxic symptoms. Sensitivity of plant varieties against herbicides is different in the first place due to the thickness ofleave's wax layer. The aim of our experience was to study the effect of five pre-emergence herbicides with different mode of action on the fresh and dry matter production and growing of a green pea variety (Pisum sativum cv. Petit Provencal). The examined herbicides were Pivot (imazethapyr), Proponit 840 EC (propisochlor), Sencor 70 WG (metribuzin), Stomp 330 (pendimethalin), and Afalon Dispersion (linuron). Pot experiments were carried out under greenhouse conditions in four replications. Herbicides were applied in the suggested and double doses. Four weeks after sowing the length, fresh- and dry weight of the shoots and the roots of pea were determined. It was an interesting observation that Pivot raised fresh weight significantly. Sencor 70 WG caused the most dramatic fresh mass reduction. We have got similar tendencies in dry matter production of green pea samples. Double rate of Stomp 330 and Sencor 70 WG decreased length of shoots and roots significantly. We established that examined pre-emergence herbicides could influence growth parameters to a different extent. Sencor 70 WG strongly inhibited growth of pea and caused severe phytotoxic symptoms. Double dose of Proponit 840 EC and Stomp 330 also damaged the pea but to a lesser extent. Afalon Dispersion and Pivot proved to be the most suitable herbicides; pea wasn't sensitive to these

  7. In vitro functional test of two subclasses of an anti-RhD antibody produced by transient expression in COS cells

    DEFF Research Database (Denmark)

    Nielsen, Leif Kofoed; Norderhaug, Lars; Sandlie, Inger

    2006-01-01

    For over 35 years hemolytic disease of the fetus and newborn (HDFN) due to RhD has been effectively prevented by anti-RhD antibodies obtained from alloimmunized women or deliberately immunized men. However, due to the reduced number of immunized women and for ethical reasons it is foreseen...

  8. Characterization of monoclonal antibodies recognizing HLA-G or HLA-E:new tools to analyze the expression of nonclassical HLA calss I molecules

    Czech Academy of Sciences Publication Activity Database

    Menier, C.; Saez, B.; Hořejší, Václav; Martinozzi, S.; Krawice-Radanne, I.; Bruel, S.; Le Danff, C.; Reboul, M.; Hilgert, Ivan; Rabreau, M.; Larrad, M. L.; Pla, M.; Carosella, E. D.; Rouas-Freiss, N.

    2002-01-01

    Roč. 64, č. 3 (2002), s. 315-326 ISSN 0198-8859 R&D Projects: GA MŠk LN00A026 Keywords : Characterization * monoclonal antibody * molecules Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.573, year: 2002

  9. Antibody response to chicken parvovirus following inoculation with inactivated virus and recombinant viruses expressing chicken parvovirus viral protein 2(VP2).

    Science.gov (United States)

    We reported earlier that day-old broiler chickens showed typical runting-stunting syndrome (RSS) post infection with chicken parvovirus (ChPV). There was also evidence that ChPV-specific maternal antibodies could provide significant protection against parvovirus induced enteric disease. Here, we st...

  10. Characterization of monoclonal antibodies recognizing HLA-G or HLA-E: new tools to analyze the expression of nonclassical HLA class I molecules

    Czech Academy of Sciences Publication Activity Database

    Menier, C.; Saez, B.; Hořejší, Václav; Martinozzi, S.; Krawice-Radanne, I.; Bruel, S.; Le Danff, C.; Reboul, M.; Hilgert, Ivan; Rabreau, M.; Larrad, M. L.; Pla, M.; Carosella, E. D.; Rouas-Freiss, N.

    2003-01-01

    Roč. 64, č. 3 (2003), s. 315-326 ISSN 0198-8859 R&D Projects: GA MŠk LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : HLA-G * HLA-E * monoclonal antibody Subject RIV: EC - Immunology Impact factor: 2.619, year: 2003

  11. Competitive nodulation blocking of cv. Afghanistan pea is related to high levels of nodulation factors made by some strains of Rhizobium leguminosarum bv. viciae.

    Science.gov (United States)

    Hogg, Bridget; Davies, Andrea E; Wilson, Karen E; Bisseling, Ton; Downie, J Allan

    2002-01-01

    Cultivar Afghanistan peas are resistant to nodulation by many strains of Rhizobium leguminosarum bv. viciae but are nodulated by strain TOM, which carries the host specificity gene nodX. Some strains that lack nodX can inhibit nodulation of cv. Afghanistan by strain TOM. We present evidence that this "competitive nodulation-blocking" (Cnb) phenotype may result from high levels of Nod factors inhibiting nodulation of cv. Afghanistan peas. The TOM nod gene region (including nodX) is cloned on pIJ1095, and strains (including TOM itself) carrying pIJ1095 nodulate cv. Afghanistan peas very poorly but can nodulate other varieties normally. The presence of pIJ1095, which causes increased levels of Nod factor production, correlates with Cnb. Nodulation of cv. Afghanistan by TOM is also inhibited by a cloned nodD gene that increases nod gene expression and Nod factor production. Nodulation of cv. Afghanistan can be stimulated if nodD on pIJ1095 is mutated, thus severely reducing the level of Nod factor produced. Repression of nod gene expression by nolR eliminates the Cnb phenotype and can stimulate nodulation of cv. Afghanistan. Addition of Nod factors to cv. Afghanistan roots strongly inhibits nodulation. The Cnb+ strains and added Nod factors inhibit infection thread initiation by strain TOM. The sym2A allele determines resistance of cv. Afghanistan to nodulation by strains of R. leguminosarum bv. viciae lacking nodX. We tested whether sym2A is involved in Cnb by using a pea line carrying the sym2A region introgressed from cv. Afghanistan; nodulation in the introgressed line was inhibited by Cnb+ strains. Therefore, the sym2A region has an effect on Cnb, although another locus (or loci) may contribute to the stronger Cnb seen in cv. Afghanistan.

  12. [Demonstration of β-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies].

    Science.gov (United States)

    Aydın, Cevahir; Ataoğlu, Haluk

    2015-01-01

    Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method

  13. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  14. Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

    Science.gov (United States)

    Choi, Sun Hee; Hagiwara-Komoda, Yuka; Atsumi, Go; Shimada, Ryoko; Hisa, Yusuke; Naito, Satoshi

    2013-01-01

    In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus. PMID:23616656

  15. Strigolactones positively regulate chilling tolerance in pea and in Arabidopsis.

    Science.gov (United States)

    Cooper, James W; Hu, Yan; Beyyoudh, Leila; Yildiz Dasgan, H; Kunert, Karl; Beveridge, Christine A; Foyer, Christine H

    2018-01-17

    Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here we characterised the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. However, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling-induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in max3-9, max4-1, max2-1 mutants that are defective in SL synthesis or signalling. When grown on agar plates the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24 decreased leaf area in the wild type, max3-9 and max4-1 mutants but not in max2-1 in the absence of stress. Moreover, a chilling-induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance. This article is protected by copyright. All rights reserved.

  16. Analysis of nodule senescence in pea (Pisum sativum L.) using laser microdissection, real-time PCR, and ACC immunolocalization.

    Science.gov (United States)

    Serova, Tatiana A; Tikhonovich, Igor A; Tsyganov, Viktor E

    2017-05-01

    A delay in the senescence of symbiotic nodules could prolong active nitrogen fixation, resulting in improved crop yield and a reduced need for chemical fertilizers. The molecular genetic mechanisms underlying nodule senescence have not been extensively studied with a view to breeding varieties with delayed nodule senescence. In such studies, plant mutants with the phenotype of premature degradation of symbiotic structures are useful models to elucidate the genetic basis of nodule senescence. Using a dataset from transcriptome analysis of Medicago truncatula Gaertn. nodules and previous studies on pea (Pisum sativum L.) nodules, we developed a set of molecular markers based on genes that are known to be activated during nodule senescence. These genes encode cysteine proteases, a thiol protease, a bZIP transcription factor, enzymes involved in the biosynthesis of ethylene (ACS2 for ACC synthase and ACO1 for ACC oxidase) and ABA (AO3 for aldehyde oxidase), and an enzyme involved in catabolism of gibberellins (GA 2-oxidase). We analyzed the transcript levels of these genes in the nodules of two pea wild-types (cv. Sparkle and line Sprint-2) and two mutant lines, one showing premature nodule senescence (E135F (sym13)) and one showing no morphological signs of symbiotic structure degradation (Sprint-2Fix - (sym31)). Real-time PCR analyses revealed that all of the selected genes showed increased transcript levels during nodule aging in all phenotypes. Remarkably, at 4 weeks after inoculation (WAI), the transcript levels of all analyzed genes were significantly higher in the early senescent nodules of the mutant line E135F (sym13) and in nodules of the mutant Sprint-2Fix - (sym31) than in the active nitrogen-fixing nodules of wild-types. In contrast, the transcript levels of the same genes of both wild-types were significantly increased only at 6 WAI. We evaluated the expression of selected markers in the different histological nodule zones of pea cv. Sparkle and its

  17. Antibody-IL2 Fusion Protein Delivery by Gene Transfer

    National Research Council Canada - National Science Library

    Nicolet, Charles

    1997-01-01

    The purpose of the work described is to assess the feasibility of a gene therapy approach to deliver a specific antibody cytokine fusion protein called CC49-1L2 to a tumor expressing antigen reactive with the antibody...

  18. Mechanisms of protection of pea plants by polysaccharides extracted from a strain of Rhizobium against Orobanche crenata

    International Nuclear Information System (INIS)

    Khairi, Hanene; Temani, Randa

    2009-01-01

    The Broomrape causes notable damage on the leguminous crops and became major factor limiting production of pea in the Mediterranean region. The effect of the polysaccharides extracted from P.SOM Rhizobium strain on the development of Orobanche crenata on pea was studied. The results showed that the lipopolysaccharides significantly reduce the infestation of pea by O. crenata. This limitation of infestation results from the reduction of seeds germination rates of the parasite resulting in reduction of the tubercles number on pea roots. Moreover, necrosis of orobanche before or after attachment on pea roots treated by LPS can explain this reduction of parasitism. A correlation was observed between the reduction of pea infection by the broomrape and the activation phenolic compounds pathway. This activation resulted to increase of two enzymes (peroxidase and polyphenoloxidase) activities these enzymes are implicated in plant defense. The results of our study showed that the LPS seem implied in the induction of pea resistance against the broomrape.

  19. Nitrogen acquisition by pea and barley and the effect of their crop residues on available nitrogen for subsequent crops

    DEFF Research Database (Denmark)

    Jensen, E.S.

    1996-01-01

    Nitrogen acquisition by field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) grown on a sandy loam soil and availability of N in three subsequent sequences of a cropping system were studied in an outdoor pot experiment. The effect of crop residues on the N availability was evaluated....... The uptake of soil-derived N by a test crop (N catch crop) of white mustard (Sinapis alba L.) grown in the autumn was higher after pea than after barley. The N uptake in the test crop was reduced by 27% and 34% after pea and barley residue incorporation, respectively, probably due to N immobilization....... The dry matter production and total N uptake of a spring barley crop following pea or barley, with a period of unplanted soil in the autumn/winter, were significantly higher after pea than after barley. The barley crop following pea and barley recovered 11% of the pea and 8% of the barley residue N...

  20. Expression of inhibitory receptors on intratumoral T cells modulates the activity of a T cell-bispecific antibody targeting folate receptor

    OpenAIRE

    Schreiner, Jens; Thommen, Daniela S.; Herzig, Petra; Bacac, Marina; Klein, Christian; Roller, Andreas; Belousov, Anton; Levitsky, Victor; Savic, Spasenija; Moersig, Wolfgang; Uhlenbrock, Franziska; Heinzelmann-Schwarz, Viola A.; Umana, Pablo; Pisa, Pavel; Lardinois, Didier

    2015-01-01

    T-cell bispecific antibodies (TCBs) are a novel therapeutic tool designed to selectively recruit T-cells to tumor cells and simultaneously activate them. However, it is currently unknown whether the dysfunctional state of T-cells, embedded into the tumor microenvironment, imprints on the therapeutic activity of TCBs. We performed a comprehensive analysis of activation and effector functions of tumor-infiltrating T-cells (TILs) in different tumor types, upon stimulation by a TCB targeting fola...

  1. Germinated Pigeon Pea (Cajanus cajan): a novel diet for lowering oxidative stress and hyperglycemia.

    Science.gov (United States)

    Uchegbu, Nneka N; Ishiwu, Charles N

    2016-09-01

    This work studied the antioxidant activity of extract of germinated pigeon pea (Cajanus cajan) in alloxan-induced diabetic rats. Germination was carried out in a dark chamber under room temperature (28°C). The total phenolic, 1,1,diphenyl-2-picrylhy-drazyl free radical (DPPH) scavenging, the inhibition of α-amylase and α-glucosidase were done in vitro and blood glucose levels of the animal were investigated. Lipid peroxidation (LPO) and reduced glutathione (GSH) were analyzed spectrophotometrically. The total phenolic and DPPH scavenging activity increased by 30% and 63%, respectively, after germinating pigeon pea. Also after germination there was an increase in the inhibitory potential of pigeon pea extract against α-glucosidase compared with the nongerminated pigeon pea extract. There was a significant increase (P pigeon pea extract gave rise to a reduced fasting blood glucose level in diabetic rats. On administration of germinated pigeon pea extract, LPO reduced drastically but there was an increase in the level of GSH. This study concluded that intake of germinated pigeon pea is a good dietary supplement for controlling hyperglycemia and LPO.

  2. Protein cross-linking, peroxidase and beta-1,3-endoglucanase involved in resistance of pea against Orobanche crenata.

    Science.gov (United States)

    Pérez-de-Luque, Alejandro; González-Verdejo, Clara I; Lozano, M Dolores; Dita, Miguel A; Cubero, José I; González-Melendi, Pablo; Risueño, María C; Rubiales, Diego

    2006-01-01

    Root holoparasitic angiosperms, like Orobanche spp, completely lack chlorophyll and totally depend on their host for their supply of nutrients. O. crenata is a severe constraint to the cultivation of legumes and breeding for resistance remains the most economical, feasible, and environmentally friendly method of control. Due to the lack of resistance in commercial pea cultivars, the use of wild relatives for breeding is necessary, and an understanding of the mechanisms underlying host resistance is needed in order to improve screening for resistance in breeding programmes. Compatible and incompatible interactions between O. crenata and pea have been studied using cytochemical procedures. The parasite was stopped in the host cortex before reaching the central cylinder, and accumulation of H2O2, peroxidases, and callose were detected in neighbouring cells. Protein cross-linking in the host cell walls appears as the mechanism of defence, halting penetration of the parasite. In situ hybridization studies have also shown that a peroxidase and a beta-glucanase are differently expressed in cells of the resistant host (Pf651) near the penetration point. The role of these proteins in the resistance to O. crenata is discussed.

  3. Importance of new winter pea genotyp in production of the milk on family farms

    Directory of Open Access Journals (Sweden)

    Gordana Županac

    2009-12-01

    Full Text Available Forage pea (Pisum sativum L. is becoming more represented gorage leguminoza on the fields Republic of Croatia. Three year field trials (2003-2005 were carried out to determine the effect of seed winter pea inoculation and nitrogen top-dressing on productivity of new winter pea genotype G3 in production of milk on family farms. Just before sowing the inoculation of pea seed was performed by the variety of Rhizobium leguminosarum bv. viciae 1001 which is part of the microbiological collection of the Department of Microbiology at the Faculty of Agriculture University of Zagreb. The results of the research showed that the highest total nodule number on pea root (39.7 nodule/plant as well as nodule dry matter weight (0.203 g/plant was determined on the inoculated variant. Average highest yield of winter pea dry matter was, once more, determined on the inoculated variant (4.33 t ha-1. Total dry matter yield of winter pea and wheat mixture were ranging from 8.92 t ha-1 (control up to 10.64 t ha-1 (nitrogen top-dressing. Average highest yield of winter pea crude protein was, once more, determined on the inoculated variant (266 kg ha-1 in 2003, (672 kg ha-1 in 2004 and (853 kg ha-1 in 2005. The conclusion of this research is that the highest dry matter yield (4.33 t ha-1 and crude protein yield was obtained with the inoculation of new genotype winter pea G3.

  4. Impact of climate and diseases on pea yields: what perspectives with climate change?

    Directory of Open Access Journals (Sweden)

    Bénézit Maud

    2017-01-01

    Full Text Available For farmers, pea crop is characterized by a large yield variability between years, between areas, and even between fields in a same small area in a given year. In dry year, spring pea crops are mostly affected by water stress and high temperature but significant yield losses can also be caused by a root disease, Aphanomyces euteiches, particularly during wet years. Winter pea can escape partially from drought, high temperature, and root disease during the reproductive phase of the crop cycle. However, when winters are mild, without progressive negative temperatures, which provide frost acclimation, available cultivars are not resistant enough to frost and are susceptible to aerial diseases such as ascochyta blight and bacterial blight (Pseudomonas syringae pv. pisi, thus leading to yield losses. A better adaptation of sowing dates, an improvement of lodging resistance and a limitation of the sowing density can limit the development of ascochyta blight for winter pea crops. For spring pea, an increased use of Aphanomyces soil test could avoid to sow the crop in infested fields. Current spring pea varieties are the result of changes in plant architecture including the reduction of 1000-seed weight that have led to yield losses by increasing the fragility of variety facing these stresses. The development of a pea crop model, simulating the effect of various stress encountered on winter and spring pea crops, can help to better define the regions adapted for the production of these two types of cultivars, and also help the breeders to better define and choose which trait to improve in order to increase the pea productivity and yield stability. National and European projects are in course to breed new varieties more adapted to different stresses.

  5. [Meristematic characteristics of tumors initiated by Agrobacterium tumefaciens in pea plants].

    Science.gov (United States)

    Vinogradova, A P; Lebedeva, M A; Lutova, L A

    2015-01-01

    It is known that two key groups of plant hormones--auxins and cytokinins--play an important role in plant tumor development. The formation of Agrobacterium-induced tumors results from the horizontal transfer of bacterial oncogenes involved in the biosynthesis of these hormones in the plant genome. The role of transcriptional factors in plant tumor development is poorly investigated. It can be assumed that tumor development associated with abnormal cell proliferation can be controlled by the same set of transcription factors that control normal cell proliferation and, in particular, transcription factors that regulate meristem activity. In the present study, we analyzed the histological organization and distribution of proliferating cells in tumors induced by Agrobacterium tumefaciens on pea hypocotyls. In addition, the expression of a set of meristem-specific genes with Agrobacterium tumefaciens-induced tumor development was analyzed. In general, our results indicate that meristematic structures are present in A. tumefaciens-induced tumors and that the development of such tumors is associated with increased expression of a key gene regulating the root apical meristem--the WOX5 gene.

  6. Systemic Induction of the Defensin and Phytoalexin Pisatin Pathways in Pea (Pisum sativum against Aphanomyces euteiches by Acetylated and Nonacetylated Oligogalacturonides

    Directory of Open Access Journals (Sweden)

    Sameh Selim

    2017-06-01

    Full Text Available Oligogalacturonides (OGs are known for their powerful ability to stimulate the plant immune system but little is known about their mode of action in pea (Pisum sativum. In the present study, we investigated the elicitor activity of two fractions of OGs, with polymerization degrees (DPs of 2–25, in pea against Aphanomyces euteiches. One fraction was nonacetylated (OGs − Ac whereas the second one was 30% acetylated (OGs + Ac. OGs were applied by injecting the upper two rachises of the plants at three- and/or four-weeks-old. Five-week-old roots were inoculated with 105 zoospores of A. euteiches. The root infection level was determined at 7, 10 and 14 days after inoculation using the quantitative real-time polymerase chain reaction (qPCR. Results showed significant root infection reductions namely 58, 45 and 48% in the plants treated with 80 µg OGs + Ac and 59, 56 and 65% with 200 µg of OGs − Ac. Gene expression results showed the upregulation of genes involved in the antifungal defensins, lignans and the phytoalexin pisatin pathways and a priming effect in the basal defense, SA and ROS gene markers as a response to OGs. The reduction of the efficient dose in OGs + Ac is suggesting that acetylation is necessary for some specific responses. Our work provides the first evidence for the potential of OGs in the defense induction in pea against Aphanomyces root rot.

  7. Characterization of FRO1, a Pea Ferric-Chelate Reductase Involved in Root Iron Acquisition1

    Science.gov (United States)

    Waters, Brian M.; Blevins, Dale G.; Eide, David J.

    2002-01-01

    To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1 expression in roots, leaves, and nodules were determined. FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants. PMID:12011340

  8. Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition.

    Science.gov (United States)

    Waters, Brian M; Blevins, Dale G; Eide, David J

    2002-05-01

    To acquire iron, many plant species reduce soil Fe(III) to Fe(II) by Fe(III)-chelate reductases embedded in the plasma membrane of root epidermal cells. The reduced product is then taken up by Fe(II) transporter proteins. These activities are induced under Fe deficiency. We describe here the FRO1 gene from pea (Pisum sativum), which encodes an Fe(III)-chelate reductase. Consistent with this proposed role, FRO1 shows similarity to other oxidoreductase proteins, and expression of FRO1 in yeast conferred increased Fe(III)-chelate reductase activity. Furthermore, FRO1 mRNA levels in plants correlated with Fe(III)-chelate reductase activity. Sites of FRO1 expression in roots, leaves, and nodules were determined. FRO1 mRNA was detected throughout the root, but was most abundant in the outer epidermal cells. Expression was detected in mesophyll cells in leaves. In root nodules, mRNA was detected in the infection zone and nitrogen-fixing region. These results indicate that FRO1 acts in root Fe uptake and they suggest a role in Fe distribution throughout the plant. Characterization of FRO1 has also provided new insights into the regulation of Fe uptake. FRO1 expression and reductase activity was detected only in Fe-deficient roots of Sparkle, whereas both were constitutive in brz and dgl, two mutants with incorrectly regulated Fe accumulation. In contrast, FRO1 expression was responsive to Fe status in shoots of all three plant lines. These results indicate differential regulation of FRO1 in roots and shoots, and improper FRO1 regulation in response to a shoot-derived signal of iron status in the roots of the brz and dgl mutants.

  9. Ileal digestibility of sunfl ower meal, pea, rapeseed cake, and lupine in pigs

    DEFF Research Database (Denmark)

    Nørgaard, Jan Værum; Fernández, José Adalberto; Jørgensen, Henry

    2012-01-01

    .05) for soybean meal and pea compared to sunfl ower meal, rapeseed cake, and lupine. The SID of Lys and His were lowest (P soybean meal and pea to be a high-digestible protein source relative to sunfl ower......The standardized ileal digestibility (SID) of CP and AA was evaluated in soybean (Glycine max) meal, sunfl ower (Helianthus annuus) meal, rapeseed cake, and fi eld pea (Pisum sativum) using 10 pigs and in lupine (Lupinus angustifolius) using 7 pigs. Pigs were fi tted with either a T...

  10. Rheological and textural properties of cracker dough with addition of pea dietary fiber

    Directory of Open Access Journals (Sweden)

    Dokić Ljubica

    2015-01-01

    Full Text Available Consumers are becoming aware of health benefits of dietary fiber intake. In past years, the development of products containing different fiber is increasing. The aim of this research was to look into the effect of addition pea fiber to dough for crackers. Rheological and textural properties were evaluated. In order to develop dough in which wheat flour was partly substituted with pea fiber, it was necessary to increase water content. The addition of 5% and 10% of pea fiber affected intramolecular linkages of gluten and lowered elastic properties of the dough. Dough with addition of 30% of fiber was brittle, grainy and impossible to process.

  11. Resistance to rusts (uromyces pisi and u. viciae-fabae) in pea

    OpenAIRE

    Barilli, Eleonora; Sillero, Josefina C.; Prats, Elena; Rubiales, Diego

    2014-01-01

    Pea is the second most important food legume crop in the world. Rust is a pea disease widely distributed, particularly in regions with warm, humid weather. Pea rust can be incited by Uromyces viciae-fabae and by U. pisi. U. viciae-fabae prevails in tropical and subtropical regions such as India and China, while U. pisi prevails in temperate regions. Chemical control of rust is possible, but the use of host plant resistance is the most desired means of rust control. In this paper we revise and...

  12. Danish Rhizobium leguminosarum strains nodulating ‘Afghanistan’ pea (Pisum sativum)

    DEFF Research Database (Denmark)

    Jensen, Erik Steen; Sørensen, Lasse Holst; Engvild, Kjeld Christensen

    1986-01-01

    A wild pea (Pisum sativum L.) native to Afghanistan normally known to be resisant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection ...... pattern with Rhizobium leguminosarum strains isolated from a modern pea variety cultivated in the same field.......A wild pea (Pisum sativum L.) native to Afghanistan normally known to be resisant to nodulation with European strains of Rhizobium leguminosarum was nodulated early and effectively in field soil in Denmark. Isolates from nodules formed effective nodules abundantly on 'Afghanistan' on reinfection...

  13. Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector.

    Science.gov (United States)

    Liang, Bo; Ngwuta, Joan O; Herbert, Richard; Swerczek, Joanna; Dorward, David W; Amaro-Carambot, Emerito; Mackow, Natalie; Kabatova, Barbora; Lingemann, Matthias; Surman, Sonja; Yang, Lijuan; Chen, Man; Moin, Syed M; Kumar, Azad; McLellan, Jason S; Kwong, Peter D; Graham, Barney S; Schaap-Nutt, Anne; Collins, Peter L; Munir, Shirin

    2016-11-01

    Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high-quality" RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499-9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. Human respiratory syncytial virus (RSV) and

  14. Development of pea protein-based bioplastics with antimicrobial properties.

    Science.gov (United States)

    Perez-Puyana, Víctor; Felix, Manuel; Romero, Alberto; Guerrero, Antonio

    2017-06-01

    In the present work, bioplastics from renewable polymers were studied in order to reduce the huge generation of plastic wastes, causing an environmental problem that continues owing to the increasing demand for plastic products. Bioplastics with much better antimicrobial properties, in particular against Gram-positive bacteria, were obtained with the addition of nisin to the initial protein/plasticizer mixture. However, the addition of nisin produces more rigid but less deformable bioplastics (higher Young's modulus but lower strain at break). The results obtained are useful to demonstrate the antimicrobial properties of pea protein-based bioplastics by adding nisin and make them suitable as potential candidates to replace conventional plastics in food packaging. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  15. Induction of mutation in peas (Pisum sativum) in Peru

    International Nuclear Information System (INIS)

    Gomez Pando, L.; Torres Aranda, M.; Romero Loli, M.

    1984-01-01

    The production of peas, a staple food in Peru, can be increased by crop rotation with cereals in high lands (3000 m and above). Cultivation in high lands not only gives cultivar of higher proteic content but also improves the fertility of the soils. However, the low temperature (in the freezing region) in the high lands and the associated plant diseases are the major problems for this kind of cultivation. The present report describes the development of freezing and disease resistant mutants through mutagenesis with gamma radiation. Two varieties, Alderman and Amarilla, which had been adopted to high lands are selected for the present study. Two doses were used, 14 and 18 Krad, employing 4600 seeds/dose for the Alderman variety and 3600 seeds/dose for Amarilla. Preliminary results are presented

  16. Macromolecular organization of xyloglucan and cellulose in pea epicotyls

    International Nuclear Information System (INIS)

    Hayashi, T.; Maclachlan, G.

    1984-01-01

    Xyloglucan is known to occur widely in the primary cell walls of higher plants. This polysaccharide in most dicots possesses a cellulose-like main chain with three of every four consecutive residues substituted with xylose and minor addition of other sugars. Xyloglucan and cellulose metabolism is regulated by different processes; since different enzyme systems are probably required for the synthesis of their 1,4-β-linkages. A macromolecular complex composed of xyloglucan and cellulose only was obtained from elongating regions of etiolated pea stems. It was examined by light microscopy using iodine staining, by radioautography after labeling with [ 3 H]fructose, by fluorescence microscopy using a fluorescein-lectin (fructose-binding) as probe, and by electron microscopy after shadowing. The techniques all demonstrated that the macromolecule was present in files of cell shapes, referred to here as cell-wall ghosts, in which xyloglucan was localized both on and between the cellulose microfibrils

  17. Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs.

    Science.gov (United States)

    Sestak, K; Zhou, Z; Shoup, D I; Saif, L J

    1999-05-01

    The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV- from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n = 52) and conventional young pigs (n = 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative = 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to

  18. Monoclonal antibodies against rat leukocyte surface antigens

    NARCIS (Netherlands)

    van den Berg, T. K.; Puklavec, M. J.; Barclay, A. N.; Dijkstra, C. D.

    2001-01-01

    Monoclonal antibodies have proven to be powerful tools for studying the properties of leukocyte surface antigens and the cells that express them. In the past decades many monoclonal antibodies (mAb) for identifying the different rat leukocyte surface antigens have been described. A list of mAb is

  19. A single, plastic population of Mycosphaerella pinodes causes ascochyta blight on winter and spring peas (Pisum sativum) in France.

    Science.gov (United States)

    Le May, Christophe; Guibert, Michèle; Leclerc, Aurélie; Andrivon, Didier; Tivoli, Bernard

    2012-12-01

    Plant diseases are caused by pathogen populations continuously subjected to evolutionary forces (genetic flow, selection, and recombination). Ascochyta blight, caused by Mycosphaerella pinodes, is one of the most damaging necrotrophic pathogens of field peas worldwide. In France, both winter and spring peas are cultivated. Although these crops overlap by about 4 months (March to June), primary Ascochyta blight infections are not synchronous on the two crops. This suggests that the disease could be due to two different M. pinodes populations, specialized on either winter or spring pea. To test this hypothesis, 144 pathogen isolates were collected in the field during the winter and spring growing seasons in Rennes (western France), and all the isolates were genotyped using amplified fragment length polymorphism (AFLP) markers. Furthermore, the pathogenicities of 33 isolates randomly chosen within the collection were tested on four pea genotypes (2 winter and 2 spring types) grown under three climatic regimes, simulating winter, late winter, and spring conditions. M. pinodes isolates from winter and spring peas were genetically polymorphic but not differentiated according to the type of cultivars. Isolates from winter pea were more pathogenic than isolates from spring pea on hosts raised under winter conditions, while isolates from spring pea were more pathogenic than those from winter pea on plants raised under spring conditions. These results show that disease developed on winter and spring peas was initiated by a single population of M. pinodes whose pathogenicity is a plastic trait modulated by the physiological status of the host plant.

  20. Thiamine treatments alleviate aphid infestations in barley and pea.

    Science.gov (United States)

    Hamada, Afaf M; Jonsson, Lisbeth M V

    2013-10-01

    Treatment of plants with thiamine (Vitamin B1) has before been shown to activate plant defence against microorganisms. Here, we have studied the effects of thiamine treatments of plants on aphid reproduction and behaviour. The work was mainly carried out with bird cherry-oat aphid (Rhopalosiphum padi L.) on barley (Hordeum vulgare L.). Aphid population growth and aphid acceptance on plants grown from seeds soaked in a 150μM thiamine solution were reduced to ca. 60% of that on control plants. R. padi life span and the total number of offspring were reduced on barley plants treated with thiamine. Healthy aphids and aphids infected with the R. padi virus were similarly affected. Spraying or addition of thiamine at 150μM to nutrient solutions likewise resulted in reduced aphid population growth to ca. 60%, as did plant exposure to thiamine odour at 4mM. Thiamine treatments resulted in reduced aphid population growth also when tested with grain aphid (Sitobion avenae F.) on barley and pea aphid (Acyrthosiphon pisum H.) on pea (Pisum sativum L.). There was no direct effect of thiamine on aphid reproduction or thiamine odour on aphid behaviour, as evaluated using artificial diets and by olfactometer tests, respectively. Two gene sequences regulated by salicylic acid showed higher transcript abundance and one gene sequence regulated by methyl jasmonate showed lower transcript abundance in thiamine-treated plants but not in control plants after aphid infestation. These results suggest that the aphid antibiosis and antixenosis effects may be related to priming of defence, but more studies are needed to explain the effects against aphids. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Xyloglucan galactosyl- and fucosyltransferase activity from pea epicotyl microsomes

    International Nuclear Information System (INIS)

    Faik, A.; Chileshe, C.; Sterling, J.; Maclachlan, G.

    1997-01-01

    Microsomal membranes from growing tissue of pea (Pisum sativum L.) epicotyls were incubated with the substrate UDP-[14C]galactose (Gal) with or without tamarind seed xyloglucan (XG) as a potential galactosyl acceptor. Added tamarind seed XG enhanced incorporation of [14C]Gal into high-molecular-weight products (eluted from columns of Sepharose CL-6B in the void volume) that were trichloroacetic acid-soluble but insoluble in 67% ethanol. These products were hydrolyzed by cellulase to fragments comparable in size to XG subunit oligosaccharides. XG-dependent galactosyltransferase activity could be solubilized, along with XG fucosyltransferase, by the detergent 3-[(3-cholamidopropyl)-dimethylammonio]-1 propanesulfonate. When this enzyme was incubated with tamarind (Tamarindus indica L.) seed XG or nasturtium (Tropaeolum majus L.) seed XG that had been partially degalactosylated with an XG-specific beta-galactosidase, the rates of Gal transfer increased and fucose transfer decreased compared with controls with native XG. The reaction products were hydrolyzed by cellulase to 14C fragments that were analyzed by gel-filtration and high-performance liquid chromatography fractionation with pulsed amperometric detection. The major components were XG subunits, namely one of the two possible monogalactosyl octasaccharides (-XXLG-) and digalactosyl nonasaccharide (-XLLG-), whether the predominant octasaccharide in the acceptor was XXLG (as in tamarind seed XG) or XLXG (as in nasturtium seed XG). It is concluded that the first xylosylglucose from the reducing end of the subunits was the Gal acceptor locus preferred by the solubilized pea transferase. These observations are incorporated into a model for the biosynthesis of cell wall XGs

  2. Natural acquired inhibitory antibodies to Plasmodium vivax Duffy binding protein (PvDBP-II) equally block erythrocyte binding of homologous and heterologous expressed PvDBP-II on the surface of COS-7 cells.

    Science.gov (United States)

    Valizadeh, Vahideh; Zakeri, Sedigheh; Mehrizi, Akram A; Mirkazemi, Sedigheh; Djadid, Navid D

    2016-02-01

    The binding domain of Plasmodium vivax Duffy binding protein (PvDBP-II) is a promising blood-stage vaccine candidate for vivax malaria. For the development of a successful vivax malaria vaccine based on DBP-II, the antigenic diversity and also naturally occurring functional antibodies to different PvDBP-II variant types in the various populations must be determined. However, similar to other blood-stage antigens, allelic variation within the PvDBP-II is a fundamental challenge for the development of a broadly efficient vaccine. The present study was performed to define whether the polymorphisms in PvDBP-II influence the nature of functional inhibitory activity of naturally acquired or induced anti-DBP-II antibodies in mice. In this investigation, five genetically distinct variants of PvDBP-II were transiently expressed on the COS-7 cell surface. Erythrocyte-binding inhibition assay (EBIA) was performed using human sera infected with corresponding and non-corresponding P. vivax variants as well as by the use of mice sera immunized with different expressed recombinant PvDBP-IIs. EBIA results showed that the inhibitory percentage varied between 50 and 63 % by using sera from infected individuals, and in case of mouse antisera, inhibition was in the range of 76-86 %. Interestingly, no significant difference was detected in red blood cell binding inhibition when different PvDBP-II variants on the COS-7 cell surfaces were incubated with heterologous and homologous sera infected with PvDBP-II variants. This suggests that the detected polymorphisms in all five forms of PvDBP-II may not affect functional activity of anti-DBP-II antibodies. In conclusion, our results revealed that there are functional cross-reactive antibody responses to heterologous PvDBP-II variants that might provide a broader inhibitory response against all, or at least the majority of strains compared to single allele of this protein that should be considered in development of PvDBP-II-based vaccine.

  3. Dietary Green Pea Protects against DSS-Induced Colitis in Mice Challenged with High-Fat Diet.

    Science.gov (United States)

    Bibi, Shima; de Sousa Moraes, Luís Fernando; Lebow, Noelle; Zhu, Mei-Jun

    2017-05-18

    Obesity is a risk factor for developing inflammatory bowel disease. Pea is unique with its high content of dietary fiber, polyphenolics, and glycoproteins, all of which are known to be health beneficial. We aimed to investigate the impact of green pea (GP) supplementation on the susceptibility of high-fat diet (HFD)-fed mice to dextran sulfate sodium (DSS)-induced colitis. Six-week-old C57BL/6J female mice were fed a 45% HFD or HFD supplemented with 10% GP. After 7-week dietary supplementation, colitis was induced by adding 2.5% DSS in drinking water for 7 days followed by a 7-day recovery period. GP supplementation ameliorated the disease activity index score in HFD-fed mice during the recovery stage, and reduced neutrophil infiltration, mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and inflammatory markers interleukin (IL)-6, cyclooxygenase-2 (COX-2), IL-17, interferon-γ (IFN-γ), and inducible nitric oxide synthase (iNOS) in HFD-fed mice. Further, GP supplementation increased mucin 2 content and mRNA expression of goblet cell differentiation markers including Trefoil factor 3 (Tff3), Krüppel-like factor 4 (Klf4), and SAM pointed domain ETS factor 1 (Spdef1) in HFD-fed mice. In addition, GP ameliorated endoplasmic reticulum (ER) stress as indicated by the reduced expression of Activating transcription factor-6 (ATF-6) protein and its target genes chaperone protein glucose-regulated protein 78 (Grp78), the CCAAT-enhancer-binding protein homologous protein (CHOP), the ER degradation-enhancing α-mannosidase-like 1 protein (Edem1), and the X-box binding protein 1 (Xbp1) in HFD-fed mice. In conclusion, GP supplementation ameliorated the severity of DSS-induced colitis in HFD-fed mice, which was associated with the suppression of inflammation, mucin depletion, and ER stress in the colon.

  4. The effect of Normast (PEA) in neuropathic pain in spinal cord injury

    DEFF Research Database (Denmark)

    Andresen, Sven Robert; Bing, Jette; Hansen, Rikke Bod Middelhede

    2015-01-01

    and psychological functioning in patients with spinal cord injury. Methods: A randomized, double-blind, placebo-controlled parallel multicenter study. We have included 66 patients with neuropathic pain due to spinal cord injury. Questionnaires regarding neuropathic pain, spasticity, insomnia, anxiety and depression......Introduction: Neuropathic pain and spasticity after spinal cord injury represent significant problems. Palmitoylethanolamide (PEA) is a fatty acid that is produced in many cells in the body, and it is thought to potentiate the body's own cannabis-like substances (endocannabinoids). PEA is suggested...... to reduce pain and inflammation but randomized controlled trials are lacking. Normast is a medical supplement which contains (PEA) approved for use in Denmark. The primary aim is to investigate the effect of Normast (PEA) on neuropathic pain, and secondary to study the effect of Normast on spasticity...

  5. Wrinkled Peas and White-Eyed Fruit Flies: The Molecular Basis of Two Classical Genetic Traits.

    Science.gov (United States)

    Guilfoile, Patrick

    1997-01-01

    Focuses on bridging the gap between classical and molecular genetics for two traits: wrinkled seeds in garden peas and white eye color in fruit flies. Discusses the molecular details of the underlying basis of these traits. Contains 15 references. (JRH)

  6. Physiological responses of PEA (Pisum sativum cv. meteor) to irrigation salinity

    International Nuclear Information System (INIS)

    Shahid, M.A.; Pervez, M.A.; Balal, R.M.; Azhar, N.; Shahzad, J.; Ubaidullah

    2008-01-01

    The effects of irrigation water or soil salinity on physiological aspects of pea (Pisum sativum cv.Meteor) were contrived. Ten weeks old pea plants were treated with NaCl at 0, 40, 90 and 140 mM in nutrient solution Plants were grown in controlled environment and harvested at each 3 days interval for decisiveness 0 physiological parameters. Photosynthetic rate, relative water content, stomatal conductance and chlorophyll contents reduced by increasing the NaCI concentration while CO/sub 2/ concentration and free proline content intensified. By experiment it was adumbrated that high salinity level along with prolonged accentuate duration is more drastic to pea plants physiology. Results also exhibited that pea plants could indulge 40 and 90 mM NaCl but are sensitive to 140 mM. (author)

  7. [Labelling of nif-plasmid pEA9 from Enterobacter agglomerans 339].

    Science.gov (United States)

    Liu, Cheng-jun; Klingmüller, Walter

    2002-07-01

    The authors describe the in vivo labelling of the plasmid pEA9 in Enterobacter agglomerans 339 with a kanamycin resistance gene. For labelling purposes the donor plasmid pST5 was constructed. This plasmid contains the nif ENX region from pEA9,in which a kanamycin resistance gene is cloned.pST5 was transformed into E.a.339 and subsequently cured from the host. Curing was achieved with AP medium. Fourty strains that had lost pST5,but retained the kanamycin resistance, could be isolated. It showed that none of these clones contained co-integrates of pST5 and pEA9. This is evident that in all clones the kanamycin resistance gene was integrated into pEA9 by homologous recombination.

  8. Nitrogen immobilization and mineralization during initial decomposition of 15N-labelled pea and barley residues

    DEFF Research Database (Denmark)

    Jensen, E.S.

    1997-01-01

    The immobilization and mineralization of N following plant residue incorporation were studied in a sandy loam soil using N-15-labelled field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) straw. Both crop residues caused a net immobilization of soil-derived inorganic N during...... the complete incubation period of 84 days. The maximum rate of N immobilization was found to 12 and 18 mg soil-derived N g(-1) added C after incorporation of pea and barley residues, respectively. After 7 days of incubation, 21% of the pea and 17% of the barley residue N were assimilated by the soil microbial...... the decomposition of the barley residue. The net mineralization of residue-derived N was 2% in the barley and 22% in the pea residue treatment after 84 days of incubation. The results demonstrated that even if crop residues have a relative low C/N ratio (15), transient immobilization of soil N in the microbial...

  9. Impact of the semi-leafless field pea on dry matter yield

    Directory of Open Access Journals (Sweden)

    Tihomir CUPIC

    2013-03-01

    Full Text Available The field pea breeding at the Agricultural Institute Osijek has primarily been aimed at continued development of new cultivar types with high yield, potential early group maturity and resistance to lodging. Letin is a new semi-leafless -winter field pea (Pisum sativum with purple flowers and a mottled light brown seed coat. Semi- leafless and leafed genotypes were evaluated for plant height, lodging, maturity, forage yield and quality in two locations. The forage yield of pea genotypes has an average of 32.21 t*ha-1. Higher yield was obtained from semi-leafless genotype (34.48 t*ha-1 . The leafed genotype has achieved better nutritive characteristics. The obtained results point to a need for new tests and the possible introduction of semi- leafless cultivars in the production as a pea-cereal mixture or as pure crops to be utilized by cutting.

  10. Breeding high yielding varieties of pigeon pea, mungbean and black gram using induced mutations

    International Nuclear Information System (INIS)

    Pawar, S.E.; Wanjari, K.B.

    1994-01-01

    The present communication emphasis the developing of high yielding varieties of pigeon pea, mungbean and black gram using induced mutation with disease resistance in these crops. This would help in stabilisation of the higher yield potential

  11. Genetic background and agronomic value of leaf types in pea (Pisum sativum

    Directory of Open Access Journals (Sweden)

    Mikić Aleksandar

    2011-01-01

    Full Text Available Pea (Pisum sativum L. has a compound leaf like many other legume species. The 'semi-leafless' pea (afaf tltl, with all leaflets transformed into tendrils, is considered one of the most important achievements in pea breeding, due to a significantly enhanced standing ability and equally efficient dry matter production in comparison to normal-leafed genotypes (AFAF TLTL. 'Semi-leafless' cultivars provide high and stable grain yield and are dominant in the modern dry pea production worldwide. There are also 'semi-leafless' cultivars that are autumn-sown and those for forage production. The genotypes with all tendrils transformed into leaflets (afaf tltl, called 'acacia' or 'tendril-less', are extremely prone to lodging and may have importance in breeding for forage production. Little is known about the potential agronomic value of 'acacia-tendril-less' (afaf tltl genotypes.

  12. SSR genetic linkage map construction of pea (Pisum sativum L. based on Chinese native varieties

    Directory of Open Access Journals (Sweden)

    Xuelian Sun

    2014-04-01

    Full Text Available Simple sequence repeat (SSR markers have previously been applied to linkage mapping of the pea (Pisum sativum L. genome. However, the transferability of existing loci to the molecularly distinct Chinese winter pea gene pool was limited. A novel set of pea SSR markers was accordingly developed. Together with existing SSR sequences, the genome of the G0003973 (winter hardy × G0005527 (cold sensitive cross was mapped using 190 F2 individuals. In total, 157 SSR markers were placed in 11 linkage groups with an average interval of 9.7 cM and total coverage of 1518 cM. The novel markers and genetic linkage map will be useful for marker-assisted pea breeding.

  13. Inhibition of protein kinase C in PHA-activated PBMC treated with anti-HLA class I monoclonal antibody is associated to decreased proliferation and expression of cell cycle related genes.

    Science.gov (United States)

    Rita, S; Michele, R; Patrizia, O; Alessansdra, B; Sara, B; Enrico, G

    1994-01-01

    Treatment of PHA-activated PBMC with anti-HLA class I monoclonal antibody (mAb 01.65) shows: 1) depletion of particulate protein Kinase C (PKC) and partial reduction of cytosolic PKC after only 10 min.; 2) inhibition of tritiated thymidine (3H-Td) incorporation; 3) slowing down of cell cycle; 4) reduced expression of four cell cycle related genes. These findings suggest that the depletion of PKC is reflected on the cell cycle progression and expression of cell cycle related genes. We studied, in PHA-activated PBMC cultures, the effect of N-N-Staurosporine (StSp) acting as PKC inhibitor at nanomolar concentrations, alone and combined with mAb 01.65. StSp, inhibits the proliferative response of PHA stimulated PBMC in a competitive fashion with mAb 01.65. We report here that StSp alone and combined with mAb 01.65 affects also the expression of c-myc and cdc2 and the membrane expression of two proliferation markers: IL-2R (CD25) and TfR (CD71).

  14. The role of sulfur nutrition in the pea response to water deficit

    OpenAIRE

    Henriet, Charlotte; Zivy, Michel; Vernoud, Vanessa; Gallardo, Karine

    2016-01-01

    Pea (Pisum sativum L.) produces seeds rich in proteins for human and animal nutrition and its cultivation enriches the soils in nitrogen, thus decreasing the need for nitrogen fertilization. Increasing pea cultivation and productivity is an agroecological challenge which requires to improve its tolerance to environmental stresses. Water deficit and the lack of sulfur in soils are two abiotic stresses that interact in the current context of climate change and low-input practices. Sulfur metabo...

  15. Model system evaluation of the effects of pea and pH on the ...

    African Journals Online (AJOL)

    Model system evaluation of the effects of pea and pH on the emulsion properties of beef. Ş Kurt, H.G. Ceylan. Abstract. The effects of dried ground pea (0 - 1%) and pH (4.80 - 7.20) on the emulsion properties of beef were investigated using the model system. The study was designed according to the central composite ...

  16. Shelf Life Extension of Maple pea (Pisum sativum var. arvense L. Spread Using Sous Vide

    Directory of Open Access Journals (Sweden)

    Ķirse Asnate

    2016-12-01

    Full Text Available The aim of this study was to determine the effect of sous vide packaging on the shelf life of maple pea (Pisum sativum var. arvense L. spread. Pea spreads were made of ground re-hydrated cooked maple peas ‘Bruno’ (Pisum sativum var. arvense L., to which salt, citric acid, oil, and spices were added. Pea spread was stored in polyamide/polyethylene (PA/PE film pouches, packaged in vacuum and hermetically sealed. Pea spread pouches were heat treated in a water bath, then rapidly cooled in ice-water and stored at 4.0 ± 0.5 °C. Sous vide was applied in three different heat regimens +(65.0; 80.0 and 100.0 ± 0.5 °C with cooking times 5, 10, 15, 20, 25, and 30 min at a constant temperature. Total plate count was determined according to ISO 4833-1:2014 on Plate Count Agar and Enterobacteriaceae determination was performed in accordance with ISO 21528-2:2004 on Violet Red Bile Glucose Agar. Total plate count in pea spread without thermal treatment was 3.41 log10 CFU g−1, in all sous vide packaged pea spread samples microbial contamination was significantly lower (p < 0.05. Enterobacteriaceae were not detected in any samples. It is possible to extend the shelf life of sous vide maple pea spread up to 14 weeks when stored at 4.0 ± 0.5 °C.

  17. Selenium bioavailability from naturally produced high-selenium peas and oats in selenium-deficient rats.

    Science.gov (United States)

    Yan, Lin; Johnson, LuAnn K

    2011-06-08

    This study determined the bioavailability of selenium (Se) from yellow peas and oats harvested from the high-Se soil of South Dakota, United States. The Se concentrations were 13.5 ± 0.2 and 2.5 ± 0.1 mg/kg (dry weight) for peas and oats, respectively. Male weanling Sprague-Dawley rats were depleted of Se by feeding them a 30% Torula yeast-based diet (4.1 μg Se/kg) for 56 days, and then they were replenished with Se for an additional 50 days by feeding them the same diet supplemented with 20, 30, or 40 μg Se/kg from peas or oats, respectively. Selenium bioavailability was determined on the basis of the restoration of Se-dependent enzyme activities and tissue Se concentrations in Se-depleted rats, comparing those responses for yellow peas and oats to those for l-selenomethionine (SeMet; used as a reference) by using a slope-ratio method. Dietary supplementation with peas or oats resulted in linear or log-linear, dose-dependent increases in glutathione peroxidase activities in blood and liver and in thioredoxin reductase activity in liver. Supplementation with peas or oats resulted in linear or log-linear, dose-dependent increases in Se concentrations of plasma, liver, gastrocnemius muscle, and kidneys. The overall bioavailability was approximately 88% for Se from yellow peas and 92% from oats, compared to SeMet. It was concluded that Se from naturally produced high-Se yellow peas or oats is highly bioavailable in this model and that these high-Se foods may be a good dietary source of Se.

  18. Importance of winter pea cv. Maksimirski rani in milk production on family farms

    Directory of Open Access Journals (Sweden)

    Darko Uher

    2010-03-01

    Full Text Available Forage pea (Pisum sativum L. is gaining importance as a forage legume in the Republic of Croatia. Pea seed contains 20-30 percent of protein, it is utilized without thermal treatment in feeding different types and categories of livestock, and with stable yield it provides an appreciable income per hectare. Two-year field trials (2005-2006 were carried out to determine the effect of winter pea seed inoculation and nitrogen top-dressing on the number and mass (g/plant-1 of root nodules and also on the yield and quality of winter pea cv. Maksimirski rani in a mixture with wheat cv. Sana. Just before sowing, pea seeds were inoculated with the strain Rhizobium leguminosarum bv. viciae 1001 from the microbial collection of the Department of Microbiology, Faculty of Agriculture, University of Zagreb. The highest number of root nodules (43 nodules/plant, as well as the highest nodule mass (0.219 g/plant-1 were determined in the inoculated variant. The highest number of pods (19.0 and seeds per plant (60 were determined in the inoculated variant as well. The highest 1000-seed mass (132 g and seed mass per plant (7.93 g were also determined in the inoculated variant. Average pea seed yield ranged from 2949 kg ha-1 (control up to 3353 kg ha-1 (inoculation. The conclusion of this research is that the highest seed (3353 kg ha-1 and crude protein yields (833 kg ha-1 were obtained with inoculated forage winter pea cv. Maksimirski rani. Seed inoculation of the studied pea cultivar Maksimirski rani with the strain Rhizobium leguminosarum bv. viciae 1001 influenced also higher milk production per hectare compared to the control and the nitrogen top-dressed variant.

  19. Accession to Ukraine’s market of pea (Pisum sativum L. sensu lato

    Directory of Open Access Journals (Sweden)

    О. І. Безручко

    2012-07-01

    Full Text Available The article details economical value, directions of use for valuable legume crop, pea for grain production purposes, growth rates of its production during recent years worldwide and in our country, development of its varietal resources in Ukraine. A complete characterization has been provided for new pea varieties meant for grain production listed in the State Register of Varieties Suitable for Dissemination in Ukraine in 2012, for which descriptions are available.

  20. Exceptional Antibodies Produced by Successive Immunizations.

    Directory of Open Access Journals (Sweden)

    Patricia J Gearhart

    2015-12-01

    Full Text Available Antibodies stand between us and pathogens. Viruses mutate quickly to avoid detection, and antibodies mutate at similar rates to hunt them down. This death spiral is fueled by specialized proteins and error-prone polymerases that change DNA sequences. Here, we explore how B lymphocytes stay in the race by expressing activation-induced deaminase, which unleashes a tsunami of mutations in the immunoglobulin loci. This produces random DNA substitutions, followed by selection for the highest affinity antibodies. We may be able to manipulate the process to produce better antibodies by expanding the repertoire of specific B cells through successive vaccinations.

  1. Evaluation of Semiochemical-Baited Traps for Monitoring the Pea Leaf Weevil, Sitona lineatus (Coleoptera: Curculionidae) in Field Pea Crops.

    Science.gov (United States)

    St Onge, Amanda; Cárcamo, Héctor A; Evenden, Maya L

    2018-02-08

    The pea leaf weevil (PLW), Sitona lineatus L., is a pest of field pea (Pisum sativum L.) and faba bean (Vicia faba L.) that recently invaded the Canadian Prairie Provinces. Although most damage is done by larvae that feed on root nodules, adults are easier to monitor than larvae. Both male and female weevils respond to a male-produced aggregation pheromone and to volatiles released by host plants. The current study tests the attractiveness of synthetic aggregation pheromone, 4-methyl-3,5-heptanedione, and host plant volatiles linalool, (Z)-3-hexenol, and (Z)-3-hexenyl acetate to PLWs in spring when weevils are reproductively active and in fall when weevils seek overwintering sites. Different combinations of semiochemical lures at various doses, released from a variety of devices were tested in pitfall traps. Semiochemical-baited traps captured both male and female weevils in both seasons but the sex ratio varied with season. Weevils did not respond in a dose-dependent manner to pheromone, as all pheromone lures were equally attractive. Pheromone release rate was determined by the release device and not the pheromone dose in the lure. The addition of plant volatiles sometimes increased weevil captures but plant volatiles alone were not attractive to PLW adults. An additional study tested the effect of trap type on weevil capture. Of the 12 different trap types tested, pheromone-baited pitfall traps were most successful in attracting and retaining weevils. Bycatch of other Sitona species was limited to a few specimens of the sweet clover weevil, Sitona cylindricollis Fahraeus. © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Use of semi-leafless peas (Pisum sativum L with enzyme addition in diets for broilers

    Directory of Open Access Journals (Sweden)

    Erja Koivunen

    2016-08-01

    Full Text Available The aim was to study the effects of dietary pea inclusion and the addition of Avizyme 1200 -enzyme cocktail on broiler performance, intestinal viscosity and organoleptic quality of meat. The experimental design was a 4 × 2 factorial, the factors being dietary pea inclusion (0, 150, 300 and 450 g kg-1 in the diets fed from day 9 to day 38 and the addition of Avizyme 1200 enzyme cocktail including amylase, protease and xylanase during the entire experiment. The growth of birds improved (p ≤ 0.018 with pea inclusion in a cubic manner. Feed conversion ratio (FCR of the birds fed on unsupplemented diets improved along pea inclusion, while pea inclusion impaired the FCR on the birds on enzyme cocktail supplemented diets (p ≤ 0.006. The use of the enzyme cocktail improved the body weight, body weight gain and FCR of the broilers and decreased intestinal viscosity (p 0.05. In conclusion, 450 g kg-1 peas can be used in the broiler grower diets without negative effects on the bird performance. The use of enzyme cocktail improves bird performance.

  3. Saponin content and trypsin inhibitor activity in processed and cooked pigeon pea cultivars.

    Science.gov (United States)

    Duhan, A; Khetarpaul, N; Bishnoi, S

    2001-01-01

    Four high-yielding varieties of pigeon pea namely UPAS-120, Manak, JCPL-151. ICPL-87 had considerable amounts of antinutrients i.e. saponins and trypsin inhibitors. Saponin content of these unprocessed cultivars ranged from 2164 to 3494 mg/100 g. There were significant varietal variations in trypsin inhibitor activity (1007-1082 TIU/g) of these pigeon pea cultivars. Some simple, inexpensive and easy-to-use domestic processing and cooking methods, namely, soaking (6, 12, 18 h), soaking (12 h)-dehulling, ordinary cooking, pressure cooking and germination (24, 36, 48 h) were found to be quite effective in lowering the level of saponins and trypsin inhibitors in all the pigeon pea cultivars. Pressure cooking of soaked and dehulled seeds lowered the content of saponins to a maximum extent (28 to 38%) followed by ordinary cooking of soaked and dehulled seeds (28 to 35%), soaked dehulled raw seeds (22 to 27%) and 48 h germinated seeds (15 to 19%). Loss of TIA was marginal due to soaking but ordinary as well as pressure cooking of unsoaked and soaked-dehulled pigeon pea seeds reduced the TIA drastically. Pressure cooking of pigeon pea seeds completely destroyed the TIA while it was reduced to the extent of 86-88% against the control in 48 h pigeon pea sprouts.

  4. Antibodies from plants for bionanomaterials.

    Science.gov (United States)

    Edgue, Gueven; Twyman, Richard M; Beiss, Veronique; Fischer, Rainer; Sack, Markus

    2017-11-01

    Antibodies are produced as part of the vertebrate adaptive immune response and are not naturally made by plants. However, antibody DNA sequences can be introduced into plants, and together with laboratory technologies that allow the design of antibodies recognizing any conceivable molecular structure, plants can be used as 'green factories' to produce any antibody at all. The advent of plant-based transient expression systems in particular allows the rapid, convenient, and safe production of antibodies, ranging from laboratory-scale expression to industrial-scale manufacturing. The key features of plant-based production include safety, speed, low cost, and convenience, allowing newcomers to rapidly master the technology and use it to its full advantage. Manufacturing in plants has recently achieved significant milestones and offers more than just an alternative to established microbial and mammalian cell platforms. The use of plants for product development in particular offers the power and flexibility to easily coexpress many different genes, allowing the plug-and-play construction of novel bionanomaterials, perfectly complementing existing approaches based on plant virus-like particles. As well as producing single antibodies for applications in medicine, agriculture, and industry, plants can be used to produce antibody-based supramolecular structures and scaffolds as a new generation of green bionanomaterials that promise a bright future based on clean and renewable nanotechnology applications. WIREs Nanomed Nanobiotechnol 2017, 9:e1462. doi: 10.1002/wnan.1462 For further resources related to this article, please visit the WIREs website. © 2017 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.

  5. Use of a fragment of glycoprotein G-2 produced in the baculovirus expression system for detecting herpes simplex virus type 2-specific antibodies

    NARCIS (Netherlands)

    Ikoma, M; Liljeqvist, JA; Glazenburg, KL; The, TH; Welling-Wester, S; Groen, J.

    Fragments of glycoprotein G (gG-2(281-594His)), comprising residues 281 to 594 of herpes simplex virus type 2 (HSV-2), glycoprotein G of HSV-1 (gG-1(t26-189His)), and glycoprotein D of HSV-1 (gD-1(1-313)), were expressed in the baculovirus expression system to develop an assay for the detection of

  6. Intracellular sucrose communicates metabolic demand to sucrose transporters in developing pea cotyledons.

    Science.gov (United States)

    Zhou, Yuchan; Chan, Katie; Wang, Trevor L; Hedley, Cliff L; Offler, Christina E; Patrick, John W

    2009-01-01

    Mechanistic inter-relationships in sinks between sucrose compartmentation/metabolism and phloem unloading/translocation are poorly understood. Developing grain legume seeds provide tractable experimental systems to explore this question. Metabolic demand by cotyledons is communicated to phloem unloading and ultimately import by sucrose withdrawal from the seed apoplasmic space via a turgor-homeostat mechanism. What is unknown is how metabolic demand is communicated to cotyledon sucrose transporters responsible for withdrawing sucrose from the apoplasmic space. This question was explored here using a pea rugosus mutant (rrRbRb) compromised in starch biosynthesis compared with its wild-type counterpart (RRRbRb). Sucrose influx into cotyledons was found to account for 90% of developmental variations in their absolute growth and hence starch biosynthetic rates. Furthermore, rr and RR cotyledons shared identical response surfaces, indicating that control of transporter activity was likely to be similar for both lines. In this context, sucrose influx was correlated positively with expression of a sucrose/H(+) symporter (PsSUT1) and negatively with two sucrose facilitators (PsSUF1 and PsSUF4). Sucrose influx exhibited a negative curvilinear relationship with cotyledon concentrations of sucrose and hexoses. In contrast, the impact of intracellular sugars on transporter expression was transporter dependent, with expression of PsSUT1 inhibited, PsSUF1 unaffected, and PsSUF4 enhanced by sugars. Sugar supply to, and sugar concentrations of, RR cotyledons were manipulated using in vitro pod and cotyledon culture. Collectively the results obtained showed that intracellular sucrose was the physiologically active sugar signal that communicated metabolic demand to sucrose influx and this transport function was primarily determined by PsSUT1 regulated at the transcriptional level.

  7. Applications of recombinant antibodies in plant pathology.

    Science.gov (United States)

    Ziegler, Angelika; Torrance, Lesley

    2002-09-01

    Summary Advances in molecular biology have made it possible to produce antibody fragments comprising the binding domains of antibody molecules in diverse heterologous systems, such as Escherichia coli, insect cells, or plants. Antibody fragments specific for a wide range of antigens, including plant pathogens, have been obtained by cloning V-genes from lymphoid tissue, or by selection from large naive phage display libraries, thus avoiding the need for immunization. The antibody fragments have been expressed as fusion proteins to create different functional molecules, and fully recombinant assays have been devised to detect plant viruses. The defined binding properties and unlimited cheap supply of antibody fusion proteins make them useful components of standardized immunoassays. The expression of antibody fragments in plants was shown to confer resistance to several plant pathogens. However, the antibodies usually only slowed the progress of infection and durable 'plantibody' resistance has yet to be demonstrated. In future, it is anticipated that antibody fragments from large libraries will be essential tools in high-throughput approaches to post-genomics research, such as the assignment of gene function, characterization of spatio-temporal patterns of protein expression, and elucidation of protein-protein interactions.

  8. Myosins 1 and 6, myosin light chain kinase, actin and microtubules cooperate during antibody-mediated internalisation and trafficking of membrane-expressed viral antigens in feline infectious peritonitis virus infected monocytes.

    Science.gov (United States)

    Dewerchin, Hannah L; Desmarets, Lowiese M; Noppe, Ytse; Nauwynck, Hans J

    2014-02-12

    Monocytes infected with feline infectious peritonitis virus, a coronavirus, express viral proteins in their plasma membranes. Upon binding of antibodies, these proteins are quickly internalised through a new clathrin- and caveolae-independent internalisation pathway. By doing so, the infected monocytes can escape antibody-dependent cell lysis. In the present study, we investigated which kinases and cytoskeletal proteins are of importance during internalisation and subsequent intracellular transport. The experiments showed that myosin light chain kinase (MLCK) and myosin 1 are crucial for the initiation of the internalisation. With co-localisation stainings, it was found that MLCK and myosin 1 co-localise with antigens even before internalisation started. Myosin 6 co-localised with the internalising complexes during passage through the cortical actin, were it might play a role in moving or disintegrating actin filaments, to overcome the actin barrier. One minute after internalisation started, vesicles had passed the cortical actin, co-localised with microtubules and association with myosin 6 was lost. The vesicles were further transported over the microtubules and accumulated at the microtubule organising centre after 10 to 30 min. Intracellular trafficking over microtubules was mediated by MLCK, myosin 1 and a small actin tail. Since inhibiting MLCK with ML-7 was so efficient in blocking the internalisation pathway, this target can be used for the development of a new treatment for FIPV.

  9. Mass spectral identification and gas-liquid chromatographic determination of methyl 4-chloroindolyl-3-acetate in canned and frozen peas.

    Science.gov (United States)

    Heikes, D L

    1980-11-01

    Methyl 4-chloroindolyl-3-acetate (MCIA), a naturally occurring auxin, has been identified in canned and frozen peas by gas-liquid chromatography (GLC)/mass spectrometry. Eight samples were analyzed for MCIA using slightly modified official AOAC multiple pesticide residue procedures employing GLC with microcoulometric detection. Levels of MCIA averaged 0.20 ppm for canned peas and 0.16 ppm for frozen peas. Recovery of MCIA from a fortified sample was 40%.

  10. Quality evaluation of stiff porridges prepared from Irish potato (Solanum tuberosum) and pigeon pea (Cajanus cajan) starch blends

    OpenAIRE

    Abu, Joseph Oneh; Enyinnaya, Chinma Chiemela; James, Samaila; Okeleke, Ezinne

    2011-01-01

    Quality attributes of stiff porridges prepared from Irish potato and pigeon pea starch blends were studied. Starches were extracted from Irish potato and pigeon pea using a wet extraction method. Various ratios of the starches were mixed and analyzed for chemical, functional and pasting properties. The starch blends were then prepared into stiff porridges for sensory evaluation using a 20-man sensory panel. Substitution of Irish potato starch with pigeon pea starch led to increases in protein...

  11. Raw, extruded and expanded pea (Pisum sativum in dairy cows diets

    Directory of Open Access Journals (Sweden)

    Gianfranco Piva

    2010-01-01

    Full Text Available The objective of the study was to evaluate the nutritive value of raw, extruded or expanded peas relative to soybean meal in lactating dairy cows feeding. Twenty four Italian Holstein cows (8 primiparous and 16 pluriparous, 604 ± 109 kg body weight, 34.5 ± 2.5 kg/d milk yield, were randomly assigned to four dietary treatments in a 4x4 Latin square arrangement with periods of four weeks and washout period of seven days. Diets were fed ad libitum(5% orts. The bulk of the base diet on a dry matter basis was corn silage (31.2%, alfalfa hay (16.7%, grass hay (4.1%, protein supplement (10.3%, whole cotton seed (8.5%, corn and barley mix (24.9%, soybean meal (3.4% and calcium soap (0.9%. The pea (2.5 kg/cow/day partially replaced the soybean meal and totally replaced the barley meal of the base diet. The unprocessed or differently processed pea did not affect the dry matter intake. The extruded pea group had a 3.2% increase (P< 0.05 of the milk yield compared to the control group. When estimated as contrast analysis, the technological treatment (extruded or expanded on peas did not modify the milk yield and composition. Among pea diets, animal fed the extruded pea had the higher (P< 0.05 milk protein content, although not different than that of the control group. The rumen acetate was reduced (P< 0.05 and the butyrate and valerate were increased (P< 0.05 in animals fed extruded pea compared to the control. No differences were observed among feeding groups on blood parameters except for the cholesterol level higher (P< 0.05 in animals fed the expanded pea diet. There were no effects of diets on milk rennet coagulation characteris- tics. Results support the partial substitution of soybean meal and the total substitution of barley meal with peas in diets for lactating cows with no negative effects on milk yield and composition.

  12. Effect of Processing on the in Vitro and in Vivo Protein Quality of Yellow and Green Split Peas (Pisum sativum).

    Science.gov (United States)

    Nosworthy, Matthew G; Franczyk, Adam J; Medina, Gerardo; Neufeld, Jason; Appah, Paulyn; Utioh, Alphonsus; Frohlich, Peter; House, James D

    2017-09-06

    In order to determine the effect of extrusion, baking, and cooking on the protein quality of yellow and green split peas, a rodent bioassay was conducted and compared to an in vitro method of protein quality determination. The Protein Digestibility-Corrected Amino Acid Score (PDCAAS) of green split peas (71.4%) was higher than that of yellow split peas (67.8%), on average. Similarly, the average Digestible Indispensable Amino Acid Score (DIAAS) of green split peas (69%) was higher than that of yellow split peas (67%). Cooked green pea flour had lower PDCAAS and DIAAS values (69.19% and 67%) than either extruded (73.61%, 70%) or baked (75.22%, 70%). Conversely, cooked yellow split peas had the highest PDCCAS value (69.19%), while extruded yellow split peas had the highest DIAAS value (67%). Interestingly, a strong correlation was found between in vivo and in vitro analysis of protein quality (R 2 = 0.9745). This work highlights the differences between processing methods on pea protein quality and suggests that in vitro measurements of protein digestibility could be used as a surrogate for in vivo analysis.

  13. NUTRALYS® pea protein: characterization of in vitro gastric digestion and in vivo gastrointestinal peptide responses relevant to satiety

    Directory of Open Access Journals (Sweden)

    Joost Overduin

    2015-04-01

    Design: Under in vitro simulated gastric conditions, the digestion of NUTRALYS® pea protein was compared to that of two dairy proteins, slow-digestible casein and fast-digestible whey. In vivo, blood glucose and gastrointestinal hormonal (insulin, ghrelin, cholecystokinin [CCK], glucagon-like peptide 1 [GLP-1], and peptide YY [PYY] responses were monitored in nine male Wistar rats following isocaloric (11 kcal meals containing 35 energy% of either NUTRALYS® pea protein, whey protein, or carbohydrate (non-protein. Results: In vitro, pea protein transiently aggregated into particles, whereas casein formed a more enduring protein network and whey protein remained dissolved. Pea-protein particle size ranged from 50 to 500 µm, well below the 2 mm threshold for gastric retention in humans. In vivo, pea-protein and whey-protein meals induced comparable responses for CCK, GLP-1, and PYY, that is, the anorexigenic hormones. Pea protein induced weaker initial, but equal 3-h integrated ghrelin and insulin responses than whey protein, possibly due to the slower gastric breakdown of pea protein observed in vitro. Two hours after meals, CCK levels were more elevated in the case of protein meals compared to that of non-protein meals. Conclusions: These results indicate that 1 pea protein transiently aggregates in the stomach and has an intermediately fast intestinal bioavailability in between that of whey and casein; 2 pea-protein- and dairy-protein-containing meals were comparably efficacious in triggering gastrointestinal satiety signals.

  14. A monoclonal antibody raised against a thermo-stabilised β1-adrenoceptor interacts with extracellular loop 2 and acts as a negative allosteric modulator of a sub-set of β1-adrenoceptors expressed in stable cell lines.

    Science.gov (United States)

    Soave, Mark; Cseke, Gabriella; Hutchings, Catherine J; Brown, Alastair J H; Woolard, Jeanette; Hill, Stephen J

    2018-01-01

    Recent interest has focused on antibodies that can discriminate between different receptor conformations. Here we have characterised the effect of a monoclonal antibody (mAb3), raised against a purified thermo-stabilised turkey β 1 -adrenoceptor (β 1 AR-m23 StaR), on β 1 -ARs expressed in CHO-K1 or HEK 293 cells. Immunohistochemical and radioligand-binding studies demonstrated that mAb3 was able to bind to ECL2 of the tβ 1 -AR, but not its human homologue. Specific binding of mAb3 to tβ 1 -AR was inhibited by a peptide based on the turkey, but not the human, ECL2 sequence. Studies with [ 3 H]-CGP 12177 demonstrated that mAb3 prevented the binding of orthosteric ligands to a subset (circa 40%) of turkey β 1 -receptors expressed in both CHO K1 and HEK 293 cells. MAb3 significantly reduced the maximum specific binding capacity of [ 3 H]-CGP-12177 without influencing its binding affinity. Substitution of ECL2 of tβ 1 -AR with its human equivalent, or mutation of residues D186S, P187D, Q188E prevented the inhibition of [ 3 H]-CGP 12177 binding by mAb3. MAb3 also elicited a negative allosteric effect on agonist-stimulated cAMP responses. The identity of the subset of turkey β 1 -adrenoceptors influenced by mAb3 remains to be established but mAb3 should become an important tool to investigate the nature of β 1 -AR conformational states and oligomeric complexes. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Development of an Immunoperoxidase Monolayer Assay for the Detection of Antibodies against Peste des Petits Ruminants Virus Based on BHK-21 Cell Line Stably Expressing the Goat Signaling Lymphocyte Activation Molecule.

    Directory of Open Access Journals (Sweden)

    Jialin Zhang

    Full Text Available From 2013 to 2015, peste des petits ruminants (PPR broke out in more than half of the provinces of China; thus, the application and development of diagnostic methods are very important for the control of PPR. Here, an immunoperoxidase monolayer assay (IPMA was developed to detect antibodies against PPR. However, during IPMA development, we found that Vero cells were not the appropriate choice because staining results were not easily observed. Therefore, we first established a baby hamster kidney-goat signaling lymphocyte activation molecule (BHK-SLAM cell line that could stably express goat SLAM for at least 20 generations. Compared with Vero cells, the PPR-mediated cytopathic effect occurred earlier in BHK-SLAM cells, and large syncytia appeared after virus infection. Based on this cell line and recombinant PPR virus expressing the green fluorescent protein (GFP (rPPRV-GFP, an IPMA for PPR diagnosis was developed. One hundred and ninety-eight PPR serum samples from goats or sheep were tested by the IPMA and virus neutralization test (VNT. Compared with the VNT, the sensitivity and specificity of the IPMA were 91% and 100%, respectively, and the coincidence rate of the two methods was 95.5%. The IPMA assay could be completed in 4 h, compared with more than 6 d for the VNT using rPPRV-GFP, and it is easily performed, as the staining results can be observed under a microscope. Additionally, unlike the VNT, the IPMA does not require antigen purification, which will reduce its cost. In conclusion, the established IPMA will be an alternative method that replaces the VNT for detecting antibodies against PPRV in the field.

  16. Bglbrick strategy for the construction of single domain antibody fusions

    Directory of Open Access Journals (Sweden)

    Ellen R. Goldman

    2017-12-01

    Full Text Available Single domain antibodies, recombinantly expressed variable domains derived from camelid heavy chain antibodies, are often expressed as multimers for detection and therapeutic applications. Constructs in which several single domain antibodies are genetically fused serially, as well as those in which single domain antibodies are genetically linked with domains that naturally form multimers, yield improvement in apparent binding affinity due to avidity. Here, using a single domain antibody that binds envelope protein from the Dengue virus, we demonstrated the construction of single domain antibody dimers using the Bglbrick cloning strategy. Constructing single domain antibodies and multimerization domains as Bglbrick parts enables the easy mixing and matching of parts. The dimeric constructs provided enhanced fluorescent signal in assays for detection of Dengue virus like particles over the monomeric single domain antibody.

  17. Immunohistochemical Expression of CD-10, BCL-6 and MUM-1 Antibodies and Immediate Clinical Response in Patients of Diffuse Large B-Cell Lymphomas after Six Cycles of Chemotherapy

    International Nuclear Information System (INIS)

    Hassan, U.; Ishtiaq, S.; Hussain, M.

    2014-01-01

    Objective: To determine the expression of CD-10, BCL-6 and MUM-1 in patients with diffuse large B-cell lymphoma (DLBCL) and its association with immediate clinical response after six cycles of CHOP chemotherapy. Study Design: Analytical study. Place and Duration of Study: Armed Forces Institute of Pathology (AFIP), Rawalpindi in collaboration with Nuclear medicine, Oncology and Radiotherapy Institute (NORI), Islamabad from September 2010 to September 2011. Methodology: CD-10, BCL-6 and MUM-1 antibodies were applied on cases diagnosed as DLBCL. Immediate clinical response was noted after 6 cycles of chemotherapy with the help of oncologist and divided into complete response, partial response, stable disease and relapse/ progression. Patient's age, results of expression of CD-10, BCL-6 and MUM-1 and results of immediate clinical response to chemotherapy were noted. Regarding analysis of prognostic markers (CD-10, BCL-6 and MUM-1), chi-square test was used for immediate clinical response to chemotherapy in DLBCL. Results: CD-10 was positive in 40% cases, BCL-6 in 58.7% cases and MUM-1 was positive in 46.7% cases. About 41.3% of patients showed complete response, 10.6% partial response, 17.3% stable disease and 30.8% showed relapse/progression. CD-10 expression in DLBCL was associated with better immediate clinical response (p = 0.011) whereas MUM-1 expression in DLBCL was associated with poor immediate clinical response (p < 0.0001). However, there was no statistically significant association of BCL-6 with immediate clinical response (p = 0.22). Conclusion: DLBCL shows expression of CD-10, BCL-6 and MUM-1 in nearly fifty percent of the cases. CD-10 is associated with good whereas MUM is associated with poor response. However, there was no association of BCL-6 with immediate clinical response. (author)

  18. Mitochondrial ultrastructure and tissue respiration of pea leaves under clinorotation

    Science.gov (United States)

    Brykov, Vasyl

    2016-07-01

    Respiration is essential for growth, maintenance, and carbon balance of all plant cells. Mitochondrial respiration in plants provides energy for biosynthesis, and its balance with photosynthesis determines the rate of plant biomass accumulation (production). Mitochondria are not only the energetic organelles in a cell but they play an essential regulatory role in many basic cellular processes. As plants adapt to real and simulated microgravity, it is very important to understand the state of mitochondria in these conditions. Disturbance of respiratory metabolism can significantly affect the productivity of plants in long-term space flights. We have established earlier that the rate of respiration in root apices of pea etiolated seedlings rose after 7 days of clinorotation. These data indicate the oxygen increased requirement by root apices under clinorotation, that confirms the necessity of sufficient substrate aeration in space greenhouses to provide normal respiratory metabolism and supply of energy for root growth. In etiolated seedlings, substrate supply of mitochondria occurs at the expense of the mobilization of cotyledon nutrients. A goal of our work was to study the ultrastructure and respiration of mitochondria in pea leaves after 12 days of clinorotation during (2 rpm/min). Plants grew at a light level of 180 μµmol m ^{-2} s ^{-1} PAR and a photoperiod of 16 h light/4 h dark. It was showed an essential increase in the mitochondrion area on 53% in palisade parenchyma cells at the sections. Such phenomenon can not be described as swelling of mitochondria, since enlarged mitochondria contained a more quantity of crista 1.76 times. In addition, the cristae total area per organelle also increased in comparison with that in control. An increase in a size of mitochondria in the experimental conditions is supposed to occur by a partial alteration of the chondriom. Thus, a size of 49% mitochondria in control was 0.1 - 0.3 μµm ^{2}, whereas only 26

  19. Dengue-1 Virus Envelope Glycoprotein Gene Expressed in Recombinant Baculovirus Elicits Virus-Neutralizing Antibody in Mice and Protects them from Virus Challenge

    Science.gov (United States)

    1991-01-01

    8217 terminus of E. When the recombinant virus was grown in Spodoptera frugiperda cells. about I mg of E antigen was made per 10’ cells. Recombinant E antigen...assay with DEN-I virus coprotein gene and its expression in Spodoptera hyperimmune mouse ascitic fluid. This heat-in- frugiperda cells activated...part by gene expression and evolution . Science 229: DOD grants DAMD17-C-2237, DAMD17-C-6156, 726-733. and DAMDI 7-C-6612. I1. Rice CM. Strauss EG

  20. Genetic diversity and trait genomic prediction in a pea diversity panel.

    Science.gov (United States)

    Burstin, Judith; Salloignon, Pauline; Chabert-Martinello, Marianne; Magnin-Robert, Jean-Bernard; Siol, Mathieu; Jacquin, Françoise; Chauveau, Aurélie; Pont, Caroline; Aubert, Grégoire; Delaitre, Catherine; Truntzer, Caroline; Duc, Gérard

    2015-02-21

    Pea (Pisum sativum L.), a major pulse crop grown for its protein-rich seeds, is an important component of agroecological cropping systems in diverse regions of the world. New breeding challenges imposed by global climate change and new regulations urge pea breeders to undertake more efficient methods of selection and better take advantage of the large genetic diversity present in the Pisum sativum genepool. Diversity studies conducted so far in pea used Simple Sequence Repeat (SSR) and Retrotransposon Based Insertion Polymorphism (RBIP) markers. Recently, SNP marker panels have been developed that will be useful for genetic diversity assessment and marker-assisted selection. A collection of diverse pea accessions, including landraces and cultivars of garden, field or fodder peas as well as wild peas was characterised at the molecular level using newly developed SNP markers, as well as SSR markers and RBIP markers. The three types of markers were used to describe the structure of the collection and revealed different pictures of the genetic diversity among the collection. SSR showed the fastest rate of evolution and RBIP the slowest rate of evolution, pointing to their contrasted mode of evolution. SNP markers were then used to predict phenotypes -the date of flowering (BegFlo), the number of seeds per plant (Nseed) and thousand seed weight (TSW)- that were recorded for the collection. Different statistical methods were tested including the LASSO (Least Absolute Shrinkage ans Selection Operator), PLS (Partial Least Squares), SPLS (Sparse Partial Least Squares), Bayes A, Bayes B and GBLUP (Genomic Best Linear Unbiased Prediction) methods and the structure of the collection was taken into account in the prediction. Despite a limited number of 331 markers used for prediction, TSW was reliably predicted. The development of marker assisted selection has not reached its full potential in pea until now. This paper shows that the high-throughput SNP arrays that are being