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Sample records for antibody expressing pea

  1. Analysis of pea HMG-I/Y expression suggests a role in defence gene regulation.

    Science.gov (United States)

    Klosterman, Steven J; Choi, Jane J; Hadwiger, Lee A

    2003-07-01

    SUMMARY HMG-I/Y proteins are characterized by the presence of AT-hook motifs, DNA binding domains that recognize AT-rich tracts of DNA. By facilitating protein:protein and protein:DNA interactions in the vicinity of these AT-rich binding sites, HMG-I/Y positively or negatively regulates gene expression. Several pea defence gene promoters have AT-rich tracts of DNA that are potential targets for modulation via HMG-I/Y. In this study, a comparison of the expression of a pea defence gene (DRR206) mRNA relative to the expression of HMG-I/Y mRNA was monitored by Northern analysis following the inoculation of a fungal pathogen, Fusarium solani or treatment with chitosan and a F. solani DNase (Fsph DNase). In pea pod endocarp tissue, HMG-I/Y expression was observed at high levels in untreated tissue and at lower levels 6 h following inoculation or wounding of the tissue. Western blots with an antipea HMG-I/Y polyclonal antibody also revealed that pea HMG-I/Y is expressed at decreased levels 6 h following inoculation or elicitor treatment. HMG-I/Y extracted from pea caused alterations in the gel migration of radio-labelled AT-rich sequences from the pea DRR206 promoter, suggesting that similar interactions could exist in vivo. Agroinfiltration was utilized to express the pea HMG-I/Y gene in tobacco containing a chimeric gene fusion of a promoter from the PR gene, DRR206, and the beta-glucuronidase (GUS) reporter gene. Transient expression of pea HMG-I/Y led to a decrease in GUS reporter gene activity in the heterologous tobacco system. These data implicate pea HMG-I/Y abundance in the down-regulation of DRR206 gene expression, and possibly HMG-I/Y depletion in the expression of defence genes in pea.

  2. Effect of pectin methylesterase gene expression on pea root development.

    Science.gov (United States)

    Wen, F; Zhu, Y; Hawes, M C

    1999-06-01

    Expression of an inducible gene with sequences common to genes encoding pectin methylesterase (PME) was found to be tightly correlated, both spatially and temporally, with border cell separation in pea root caps. Partial inhibition of the gene's expression by antisense mRNA in transgenic pea hairy roots prevented the normal separation of root border cells from the root tip into the external environment. This phenotype was correlated with an increase in extracellular pH, reduced root elongation, and altered cellular morphology. The translation product of the gene exhibited PME activity in vitro. These results are consistent with the long-standing hypothesis that the demethylation of pectin by PME plays a key role in cell wall metabolism.

  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. PMID:19156736

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

    Science.gov (United States)

    Teshome, Abel; Bryngelsson, Tomas; Mendesil, Esayas; Marttila, Salla; Geleta, Mulatu

    2016-01-01

    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 [neoplastic (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 Np 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 Np pods dropped significantly (7%). In order to enhance Np expression at the field level, intercropping of Np genotypes with sorghum was investigated. As result, the percentage of Np pods was threefold in intercropped Np genotypes as compared to those without intercropping. Therefore, intercropping Np 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 Np genotypes is lower in comparison to wild type genotypes. Therefore, promoting Np formation under field conditions via intercropping can serve as part of an integrated pea weevil management strategy especially for small scale farming systems. PMID:27242855

  5. Expression of Recombinant Antibodies

    OpenAIRE

    Frenzel, André; Hust, Michael; Schirrmann, Thomas

    2013-01-01

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

  6. PEA-15真核表达载体的构建及表达%Construction and expression of eukaryotic expression vector of PEA-15

    Institute of Scientific and Technical Information of China (English)

    张弘广; 庄晓飞; 王春利; 郭石平; 马炎炎

    2014-01-01

    目的 构建PEA-15真核表达载体pcDNA3.1-PEA-15,并在人类食管癌细胞(EC-109)中表达.探讨PEA-15对EC-109细胞的影响.方法 采用反转录聚合酶链反应(RT-PCR)技术检测EC-109细胞中的PEA-15基因表达.构建重组真核表达载体pcDNA3.1-PEA-15.酶切及测序鉴定重组质粒正确后,脂质体介导转染至EC-109细胞中.采用RT-PCR、Western blot法检测基因及蛋白表达.四甲基偶氮唑蓝(MTT)法检测细胞生长抑制率.结果 RT-PCR显示PEA-15在EC-109细胞中表达.PCR扩增片段与预期片段大小相符,测序结果表明真核表达载体pcDNA3.1-PEA-15构建成功.转染后的细胞中PEA-15表达均增加(t值分别为4.078、5.269,均P<0.05).转染质粒72h后,细胞的生长抑制率较转染空质粒组降低[(7.12±0.86)%、(12.55±1.78)%,t=6.163,P<0.05].结论 成功构建重组真核表达载体pcDN A3.1-PEA-15,并在EC-109细胞中进行了表达;转染后对食管癌细胞生长有促进作用,其表达可能促进食管癌的发生.%Objective To construct the eukaryotic expression vector of pcDNA3.1-PEA-15 and express it in the human esophageal cancer (EC-109) cells,and to explore the effect of PEA-15 on EC-109 cells.Methods The PEA-15 gene was amplified from EC-109 by reverse transcriptase polymerase chain reaction (RT-PCR) and ligated to eukaryotic expression vector pcDNA3.1.After confirmation of recombinant plasmid was correctly by endonuc]eases digestion and DNA sequencing,the construct was transfected it into EC-109 through liposome inducing.The expression of PEA-15 in transfected EC-109 was detected by RT-PCR and Western blot.The cell growth inhibition ratio was evaluated by MTT assay.Results RT-PCR indicated that PEA-15 was highly expressed in EC-109 cells.The amplified fragment by RT-PCR was coincident with hypothesis enzyme digestion analysis and DNA sequencing confirmed that the pcDNA3.1-PEA-15 was constructed successfully.The expression of PEA-15 gene was increased obviously

  7. ERβ and PEA3 co-activate IL-8 expression and promote the invasion of breast cancer cells.

    Science.gov (United States)

    Chen, Ying; Chen, Li; Li, Ji-Yu; Mukaida, Naofumi; Wang, Qiaoqiao; Yang, Chen; Yin, Wen-Jin; Zeng, Xiao-Hua; Jin, Wei; Shao, Zhi-ming

    2011-03-01

    Metastasis represents the major remaining cause of mortality in human breast cancer. Interleukin-8 (IL-8), a proinflammatory chemokine, plays an important role during tumor angiogenesis and metastasis. In this study, we found that IL-8 and ERβ showed positive association. Overexpression of ERβ or PEA3 could up-regulate IL-8 promoter activity, mRNA and secretion; silencing of ERβ or PEA3 decreased IL-8 mRNA and secretion. ERβ and PEA3 increased IL-8 expression through binding to the IL-8 promoter and increased cell invasion. HER2 could increase ERβ and PEA3 expression and their binding to the IL-8 promoter. We conclude that ERβ and PEA3 play important roles in tumor invasion by regulating IL-8 expression, and HER2 maybe the upstream of ERβ and PEA3 - IL-8 pathway.

  8. Inducible expression of Pisum sativum xyloglucan fucosyltransferase in the pea root cap meristem, and effects of antisense mRNA expression on root cap cell wall structural integrity.

    Science.gov (United States)

    Wen, Fushi; Celoy, Rhodesia M; Nguyen, Trang; Zeng, Weiqing; Keegstra, Kenneth; Immerzeel, Peter; Pauly, Markus; Hawes, Martha C

    2008-07-01

    Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.

  9. Differential gene expression according to race and host plant in the pea aphid.

    Science.gov (United States)

    Eyres, Isobel; Jaquiéry, Julie; Sugio, Akiko; Duvaux, Ludovic; Gharbi, Karim; Zhou, Jing-Jiang; Legeai, Fabrice; Nelson, Michaela; Simon, Jean-Christophe; Smadja, Carole M; Butlin, Roger; Ferrari, Julia

    2016-09-01

    Host-race formation in phytophagous insects is thought to provide the opportunity for local adaptation and subsequent ecological speciation. Studying gene expression differences amongst host races may help to identify phenotypes under (or resulting from) divergent selection and their genetic, molecular and physiological bases. The pea aphid (Acyrthosiphon pisum) comprises host races specializing on numerous plants in the Fabaceae and provides a unique system for examining the early stages of diversification along a gradient of genetic and associated adaptive divergence. In this study, we examine transcriptome-wide gene expression both in response to environment and across pea aphid races selected to cover the range of genetic divergence reported in this species complex. We identify changes in expression in response to host plant, indicating the importance of gene expression in aphid-plant interactions. Races can be distinguished on the basis of gene expression, and higher numbers of differentially expressed genes are apparent between more divergent races; these expression differences between host races may result from genetic drift and reproductive isolation and possibly divergent selection. Expression differences related to plant adaptation include a subset of chemosensory and salivary genes. Genes showing expression changes in response to host plant do not make up a large portion of between-race expression differences, providing confirmation of previous studies' findings that genes involved in expression differences between diverging populations or species are not necessarily those showing initial plasticity in the face of environmental change. PMID:27474484

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

    Science.gov (United States)

    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. PMID:26798990

  11. Vector-Mediated In Vivo Antibody Expression.

    Science.gov (United States)

    Schnepp, Bruce C; Johnson, Philip R

    2014-08-01

    This article focuses on a novel vaccine strategy known as vector-mediated antibody gene transfer, with a particular focus on human immunodeficiency virus (HIV). This strategy provides a solution to the problem of current vaccines that fail to generate neutralizing antibodies to prevent HIV-1 infection and AIDS. Antibody gene transfer allows for predetermination of antibody affinity and specificity prior to "immunization" and avoids the need for an active humoral immune response against the HIV envelope protein. This approach uses recombinant adeno-associated viral (rAAV) vectors, which have been shown to transduce muscle with high efficiency and direct the long-term expression of a variety of transgenes, to deliver the gene encoding a broadly neutralizing antibody into the muscle. Following rAAV vector gene delivery, the broadly neutralizing antibodies are endogenously synthesized in myofibers and passively distributed to the circulatory system. This is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. Vector-mediated gene transfer studies in mice and monkeys with anti-HIV and simian immunodeficiency virus (SIV)-neutralizing antibodies demonstrated long-lasting neutralizing activity in serum with complete protection against intravenous challenge with virulent HIV and SIV. These results indicate that existing potent anti-HIV antibodies can be rapidly moved into the clinic. However, this methodology need not be confined to HIV. The general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets such as hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. PMID:26104192

  12. Evaluation of expression stability of candidate references genes among green and yellow pea cultivars (Pisum sativum L.) subjected to abiotic and biotic stress

    Science.gov (United States)

    Dry pea (Pisum sativum) is grown as human and animal feed throughout the world. Large yield losses in pea due to biotic and abiotic stresses compel an improved understanding of mechanisms of stress tolerance and genetic determinants conditioning these tolerances. The availability of stably expressed...

  13. Expression of PED/PEA-15 mRNA and protein in esophageal carcinoma%食管癌中PED/PEA-15mRNA及蛋白表达的临床意义

    Institute of Scientific and Technical Information of China (English)

    庄晓飞; 张弘广; 王春利; 郭石平; 马炎炎; 解立武

    2013-01-01

    Objective To investigate the expression of PED/PEA-15 mRNA and protein in esophageal carcinoma tissue and their clinical significances.Methods The expression of PED/PEA-15 mRNA was detected by RT-PCR,and the expression of PED/PEA-15 protein was measured by immunohistochemistry in 50 cases of esophageal carcinoma,50 cases of corresponding paracarcinoma tissue,and 50 cases of corresponding normal esophageal tissue.Results The expression of PED/PEA-15 mRNA was 1.14±0.49 in esophageal carcinoma,which was significantly higher than that in para-carcinoma tissue (0.59±0.31) and normal esophageal carcinoma tissue (0.53±0.22) (F =44.085,P < 0.001).The immunohistochemistry results showed that PED/PEA-15 protein expression was significantly higher than that in para-carcinoma tissue and normal esophageal tissue (x2 =36.967,P < 0.001; x2 =26.272,P < 0.001).The expression of PED/PEA-15 mRNA and protein were significantly associated with pathological grade,clinical stage of esophageal carcinoma (P < 0.05),but were not significantly correlated to the age of onset,gender,pathological types (P > 0.05).Conclusion The expression of PED/PEA-15 mRNA and protein are increased in esophageal carcinoma,which may play an important role in the occurrence and development of esophageal carcinoma.%目的 探讨PED/PEA-15 mRNA及蛋白在食管癌组织中的表达情况及其临床意义.方法 应用半定量反转录-聚合酶链反应(RT-PCR)检测50例食管癌、相应癌旁组织和正常食管组织中PED/PEA-15基因的相对表达量,并通过免疫组织化学检测PED/PEA-15蛋白的表达.结果 半定量RT-PCR显示,PED/PEA-15基因在食管癌组织中均呈高表达,癌旁组织及正常食管组织呈低表达或不表达,相对表达量分别为1.14±0.49、0.59±0.31和0.53±0.22,PED/PEA-15 mRNA在食管癌组织中表达较癌旁组织、正常食管组织升高(F=44.085,P<0.01).免疫组织化学结果显示,食管癌组织中PED/PEA-15蛋白阳性表达率

  14. 胃癌组织中磷酸化 PEA -15蛋白的表达及临床意义%Study on the expression of phosphorylated PEA - 15 and its clinical significance in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    李莹; 麻日虎; 王玉旋; 刘荣火

    2015-01-01

    Objective The physiological activity of PEA - 15 in regulating cell proliferation is dependent on the phosphorylation status. However,little is known about the impact of phosphorylated PEA - 15 on the progression of tumor. The aim of this study is to explore the expres-sion of phosphorylated PEA - 15 protein in gastric cancer and its clinical significance. Methods Immunohistochemical method was used to detect the expression of phosphorylated PEA - 15 protein in 50 patients with gastric cancer and 50 tissue specimens from patients with chronic gastritis as controls. Results The expression of phosphorylated PEA - 15 protein was significantly increased in patients with gastric cancer detected by immu-nohistochemical method,while its expression in patients with chronic gastritis was undetectable( P < 0. 05). The expression level of phosphoryla-ted PEA - 15 protein was significantly associated with the differentiated degree and clinical stage of gastric cancer( P < 0. 05),but it was not sig-nificantly correlated to age of onset and gender of patients( P ﹥ 0. 05). Conclusion The results suggest that phosphorylated PEA - 15 may play an important role in the development and aggression of gastric cancer. The regulation of phosphorylation status of PEA - 15 can be used as an effec-tive therapeutic marker in the treatment of patients with gastric cancer.%目的:探讨磷酸化 PEA -15蛋白在胃癌组织中的表达情况及其临床意义。方法取新鲜胃癌标本50例,并以50例慢性胃炎组织作对照。运用免疫组织化学的方法检测磷酸化 PEA -15蛋白的表达,并观察磷酸化 PEA -15蛋白的表达与患者性别、年龄、肿瘤分化程度和临床分期的关系。结果胃癌组织中磷酸化 PEA -15蛋白阳性表达增高,而慢性胃炎组织标本未测到( P <0.05)。且磷酸化 PEA -15蛋白的表达随着胃癌恶性程度增高及临床分期发展而增高( P <0.05),而与发病年龄及性别无关( P ﹥0

  15. Soluble expression and characterization of a GFP-fused pea actin isoform(PEAc1)

    Institute of Scientific and Technical Information of China (English)

    Ai Xiao LIU; Shao Bin ZHANG; Xiao Jing XU; Dong Tao REN; Guo Qin LIU

    2004-01-01

    A pea actin isoform PEAc1 with green fluorescent protein (GFP) fusion to its C-terminus and His-tag to its Nterminus, was expressed in prokaryotic cells in soluble form, and highly purified with Ni-Chelating SepharoseTM Fast Flow column. The purified fusion protein (PEAc1-GFP) efficiently inhibited DNase I activities before polymerization,and activated the myosin Mg-ATPase activities after polymerization. The PEAc 1-GFP also polymerized into green fluorescent filamentous structures with a critical concentration of 0.75 μM. These filamentous structures were labeled by TRITC-phalloidin, a specific agent for staining actin microfilaments, and identified as having 9 nm diameters by negative staining. These results indicated that PEAc1 preserved the essential characteristics of actin even with His-tag and GFP fusion, suggesting a promising potential to use GFP fusion protein in obtainning soluble plant actin isoform to analyze its physical and biochemical properties in vitro. The PEAc 1-GFP was also expressed in tobacco BY2 cells,which offers a new pathway for further studying its distribution and function in vivo.

  16. Dehydration induces expression of GALACTINOL SYNTHASE and RAFFINOSE SYNTHASE in seedlings of pea (Pisum sativum L.).

    Science.gov (United States)

    Lahuta, Lesław B; Pluskota, Wioletta E; Stelmaszewska, Joanna; Szablińska, Joanna

    2014-09-01

    The exposition of 7-day-old pea seedlings to dehydration induced sudden changes in the concentration of monosaccharides and sucrose in epicotyl and roots tissues. During 24h of dehydration, the concentration of glucose and, to a lesser extent, fructose in seedling tissues decreased. The accumulation of sucrose was observed in roots after 4h and in epicotyls after 8h of stress. Epicotyls and roots also began to accumulate galactinol and raffinose after 8h of stress, when small changes in the water content of tissues occurred. The accumulation of galactinol and raffinose progressed parallel to water withdrawal from tissues, but after seedling rehydration both galactosides disappeared. The synthesis of galactinol and raffinose by an early induction (during the first hour of treatment) of galactinol synthase (PsGolS) and raffinose synthase (PsRS) gene expression as well as a later increase in the activity of both enzymes was noted. Signals possibly triggering the induction of PsGolS and PsRS gene expression and accumulation of galactinol and raffinose in seedlings are discussed.

  17. Evaluation and Application of Two High-Iron Transgenic Rice Lines Expressing a Pea Ferritin Gene

    Institute of Scientific and Technical Information of China (English)

    YE Hong-xai; LI Mei; Guo Ze-jian; Shu Qing-yao; xu Xiao-hui; BAO Jin-song; SHEN Sheng-quan

    2008-01-01

    A totaI of 105 transgenic rice lines independently transformed with a pea ferritin gene (Fer)were previously obtained.After seven generations of selfing and β-glucuronidase(GUS)assisted selection,82 transgenic lines with stable agronomic traits were got.Among the 82 transgenic lines,two high-iron transgenic rice lines Fer34 and Fer65,with the iron contents in the milled rice being 4.82 and 3.46 times of that of the wild type Xiushui 11,respectively were identified.In the two transgenic lines,the exogenous Fer gene was highly expressed,and inherited as a single locus.The transgene had no negative effect on the agronomic traits of rice plant,other mineral nutritional components,appearance quailty and eating quailty of the milled rice,indicating that these two lines were elite high-iron breeding lines.Furthermore,the practical application and further studies facilitating utilization of the two elite breeding lines were discussed.

  18. Prokaryotic expression and characterization of a pea actin isoform (PEAcl) fused to GFP

    Institute of Scientific and Technical Information of China (English)

    ZHANG Shaobin; REN Dongtao; XU Xiaojing; LIU Guoqin

    2004-01-01

    Actins widely exist in eukaryotic cells and play important roles in many living activities. As there are many kinds of actin isoforms in plant cells, it is difficult to purify each actin isoform in sufficient quantities for analyzing its physicochemical properties. In the present study, a pea(Pisum Sativum L.) actin isoform (PEAc1) fused to His-tag at its amino terminus and GFP (green fluorescent protein) at its Carboxyl terminus were expressed in E. Coli in inclusion bodies. The fusion protein (PEAc1-GFP) was highly purified with the yield of above 2 mg/L culture by dissolving inclusions in 8 mol/L urea, renaturing by dialysis in a gradient of urea, and affinity binding to Ni-resin. The purified mono meric PEAc1-GFP could efficiently bind on Dnase Ⅰ and inhibit the latter's enzyme activity. PEAc1-GFP could polymerize into green fluorescent filamentous structures (F-PEAc1-GFP), which could be labeled by TRITC-phalloidin, a specific agent for observing microfilaments. The PEAc1-GFP polymerization curve was identical with that of chicken skeletal muscle actin. The critical concentration for PEAc1-GFP to polymerize into filaments is 0.24 μmol/L. The F-PEAc1-GFP could stimulate myosin Mg-ATPase activity in a protein concentration dependant manner (about 4 folds at1 mg/mL F-PEAc1-GFP). The results above show that the PEAcl fused to GFP retained the assembly characteristic of actin, indicating that gene fusion, prokaryotic expression,denaturation and renaturation, and affinity chromatography is a useful strategy for obtaining plant actin isoform proteins in a large amount.

  19. Occurrence of the Transition of Apical Architecture and Expression Patterns of Related Genes during Conversion of Apical Meristem Identity in G2 Pea

    Institute of Scientific and Technical Information of China (English)

    Da-Yong Wang; Qing Li; Ke-Ming Cui; Yu-Xian Zhu

    2009-01-01

    G2 pea exhibits an apical senescence delaying phenotype under short-day (SD) conditions; however, the structural basis for its apical development is still largely unknown. In the present study, the apical meristem of SD-grown G2 pea plants underwent a transition from vegetative to indeterminate inflorescence meristem, but the apical meristem of long-day (LD)-grown G2 pea plants would be further converted to determinate floral meristem. Both SD signal and GA3 treatment enhanced expression of the putative calcium transporter PPF1, and pea homologs of TFL1 (LF and DET), whereas LD signal suppressed their expression at 60 d post-flowering compared with those at 40 d post-flowering. Both PPF1 and LF expressed at the vegetative and reproductive phases in SD-grown apical buds, but floral initiation obviously increased the expression level of PPF1 compared with the unchanged expression level of LF from 40 to 60 d post-flowering. In addition, although the floral initiation significantly enhanced the expression levels of PPF1 and DET, DET was mainly expressed after floral initiation in SD-grown apical buds. Therefore, the main structural difference between LD- and SD-grown apical meristem in G2 pea lies in whether their apical indeterminate inflorescence medstem could be converted to the determinate structure.

  20. 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. PMID:12972656

  1. 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

    ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression. The ENOD12 gene family in pea (Pisum sativum) has two members. A cDNA clone representing PsENOD12A [26] and a PsENOD12B genomic clone [7] have been...

  2. The PsENOD12 gene is expressed at two different sites in Afghanistan Pea Pseudonodules induced by Auxin transport inhibitors

    NARCIS (Netherlands)

    Scheres, B.J.G.; McKhann, H.I.; Zalensky, A.; Löbler, M.; Bisseling, T.; Hirsch, A.M.

    1992-01-01

    A number of early nodulin genes are expressed in specific cell types as pea (Pisum sativum) root nodules develop. The Pisum sativum early nodulin PsENOD2 is detected only in the uninfected cells of the nodule parenchyma, whereas PsENOD12 is expressed at two spatially removed sites: in root hairs and

  3. The expression of PED/PEA-15 in gastric cancer and its clinical significance%PED/PEA-15在胃癌中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    高磊; 何若冲

    2013-01-01

    Objective To detect the expression of inhibitor of apoptosis protein PED/PEA-15 in gastric cancer, and to investigate the relationship between ped/pea-15 and gastric cancer and its clinical significance. Methods Operation specimens of gastric cancer between January and December in 2012 were collected from the first affiliated hospital of Shan xi Medical University. All specimens had been proved by pathology. Immunohistochemical method was used to detect the expression of PED/PEA-15 in 40 cases of gastric cancer tissues, 40 cases of corresponding para carcinoma tissues and 40 cases of corresponding normal tissues adjacent to the tumor.The experimental result were evaluated according to the percentage of positive cells and the staining intensity. Row×columns unordered contingency table chi-square test was used in comparison of sample rate among three groups(test levelα=0.05).Chi-square test of independent samples was used in comparison of sample rate between the two groups(test level α=0.0167).The fourfold table chi-square test was used for analysis of relationship between expression of PED/PEA-15 and clinical pathology of gastric cancer(significance level α=0.05). Results The positive rate of the inhibitor of apoptosis protein PED/PEA-15 in gastric carcinoma, the tissue adjacent to carcinoma and normal tissues adjacent to the tumor was 70% (28/40), 25%(10/40) and 13%(5/40) respectively, The expression of PED/PEA-15 in gastric significantly increased compared with the adjacent cancer tissues and normal tissues adjacent to the tumor(P0.05). Conclusion The inhibitor of apoptosis protein PED/PEA-15 showed high expression in gastric carcinoma, which may play an important role in the occurrence and development of gastric carcinoma. Thus it may serve as a reference indicator for biological behaviors of gastric carcinoma and a new target for gene therapy.%目的:检测凋亡抑制蛋白PED/PEA-15在胃癌中的表达,以探讨其与胃癌发生的联系及临床意

  4. Stable isotope labelled mass spectrometry for quantification of the relative abundances for expressed proteins induced by PeaT1

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The protein elicitor from the mycelium of Alternaria tenuissima has been isolated.The elicitor triggered resistance to the tobacco mosaic virus in tobacco by inducing relative oxygen species,but without causing hypersensitive necrosis.The elicitor is reported to impart resistance against Verticillium dahliae and to increase yield in cotton,but its mechanism is not yet clear.In this study,the stable isotope labelled mass spectrometry method was used to quantify the relative abundances of protein expression induced by PeaT1 in Arabidopsis.A significant difference in the relative abundances for the expression of different proteins related to metabolism,modification,regulatory,defense,stress and antioxidation was found in Arabidopsis.

  5. Expression and assembly of a fully active antibody in algae

    OpenAIRE

    Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

    2003-01-01

    Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene...

  6. Expression and assembly of a fully active antibody in algae

    Science.gov (United States)

    Mayfield, Stephen P.; Franklin, Scott E.; Lerner, Richard A.

    2003-01-01

    Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem. Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii. We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C. reinhardtii chloroplast promoters and 5' and 3' RNA elements. This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo. Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification. We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene. These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

  7. Prokaryotic Expression and Polyclonal Antibody Preparation of PRL3

    Institute of Scientific and Technical Information of China (English)

    SHEN Xing-gui; LI Wan-nan; WANG Jing; JIANG Yi-qun; LI Qing-shan; FU Xue-qi

    2007-01-01

    Phosphatase of regenerating liver 3(PRL3), which belongs to the superfamily of protein tyrosine phosphatases (PTPs), represents a group of low molecular weight PTPs that participate in tumorigenesis and metastasis processes.Presented here are the results of cloning, prokaryotic expression, purification, and polyclonal antibody preparation of PRL3. To obtain a specific polyclonal antibody against PRL3, the authors have prepared GST-PRL3 to immunize rabbits and purify an anti-PRL3 polyclonal antibody by negative selection affinity columns. Western blot analysis shows that the anti-PRL3 polyclonal antibody has a specific binding ability with PRL3 protein. The anti-PRL3 polyclonal antibody provides a good tool to further study the function of PRL3.

  8. Anti-DNA antibodies: Sequencing, cloning, and expression

    Energy Technology Data Exchange (ETDEWEB)

    Barry, M.M.

    1992-01-01

    To gain some insight into the mechanism of systemic lupus erythematosus, and the interactions involved in proteins binding to DNA four anti-DNA antibodies have been investigated. Two of the antibodies, Hed 10 and Jel 242, have previously been prepared from female NZB/NZW mice which develop an autoimmune disease resembling human SLE. The remaining two antibodies, Jel 72 and Jel 318, have previously been produced via immunization of C57BL/6 mice. The isotypes of the four antibodies investigated in this thesis were determined by an enzyme-linked-immunosorbent assay. All four antibodies contained [kappa] light chains and [gamma]2a heavy chains except Jel 318 which contains a [gamma]2b heavy chain. The complete variable regions of the heavy and light chains of these four antibodies were sequenced from their respective mRNAs. The gene segments and variable gene families expressed in each antibody were identified. Analysis of the genes used in the autoimmune anti-DNA antibodies and those produced by immunization indicated no obvious differences to account for their different origins. Examination of the amino acid residues present in the complementary-determining regions of these four antibodies indicates a preference for aromatic amino acids. Jel 72 and Jel 242 contain three arginine residues in the third complementary-determining region. A single-chain Fv and the variable region of the heavy chain of Hed 10 were expressed in Escherichia coli. Expression resulted in the production of a 26,000 M[sub r] protein and a 15,000 M[sub r] protein. An immunoblot indicated that the 26,000 M[sub r] protein was the Fv for Hed 10, while the 15,000 M[sub r] protein was shown to bind poly (dT). The contribution of the heavy chain to DNA binding was assessed.

  9. Pollination-, development-, and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds.

    Science.gov (United States)

    Ozga, Jocelyn A; Yu, Jody; Reinecke, Dennis M

    2003-03-01

    To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA(20) to biologically active GA(1) using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (-2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6-10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarp PsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA(3) were the same or lower than deseeded controls. These data show that PsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarp PsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA(20) to GA(1) occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth.

  10. Photoregulated expression of the PsPK3 and PsPK5 genes in pea seedlings.

    Science.gov (United States)

    Khanna, R; Lin, X; Watson, J C

    1999-01-01

    The PsPK3 and PsPK5 genes of the garden pea encode protein-serine/threonine kinases whose catalytic domains are closely related to known signal transducing kinases from animals and fungi. The PsPK3 polypeptide is predicted to be located in the nucleus, whereas PsPK5 is a homologue of NPH1, the probable blue light receptor for phototropism from Arabidopsis. We found previously that when etiolated pea seedlings are illuminated with continuous white light, PsPK3 and PsPK5 transcript levels within apical buds decline substantially, reaching their minimum levels within one day of exposure to light. The role of light in regulating the expression of the PsPK3 and PsPK5 genes was investigated further. To gain insight into the rapidity with which expression changes, 6-day old, dark-grown pea seedlings were transferred to continuous white light, and PsPK3 and PsPK5 RNA levels monitored over the ensuing 24 h. While transcripts from the RbcS gene family increase, the PsPK3 and PsPK5 mRNAs decline rapidly to their minimum levels. PsPK5 mRNA declines 10-fold in ca. 2 h, whereas PsPK3 mRNA declines 4-fold in ca. 8 h. We used single pulses of light to elucidate which photoreceptor triggers the negative regulation of PsPK3 and PsPK5 gene expression. To assess phytochrome involvement, etiolated seedlings were treated with single pulses of red light, red followed by far-red light, or far-red light alone. RbcS induction by a red light pulse is reversible with a subsequent far-red light pulse, clearly showing that phytochrome mediates its induction. Likewise, RbcS expression is induced with a single pulse of blue light or a dichromatic pulse of red+blue light. However, none of these pulses trigger the PsPK3 and PsPK5 mRNA levels to decline. Given the lack of effectiveness of light pulses, etiolated seedlings were transferred to continuous light of three different qualities to determine the spectral sensitivity of PsPK3 and PsPK5 gene expression. Exposure to continuous red, continuous

  11. [Features of Expression of the PsSst] and PsIgn1 Genes in Nodules of Pea (Pisum sativum L.) Symbiotic Mutants].

    Science.gov (United States)

    Zhukova, V A; Rychagova, T S; Fedorina, Ya V; Pinaeva, A G; Andronova, E E; Borisova, A Yu; Tikhonovich, I A

    2016-04-01

    The sequences of the PsSst1 and PsIgn1 genes of pea (Pisum sativum L.) homologous to the symbiotic LjSST1 and LjIGN1 genes of Lotusjaponicus (Regel.) K. Larsen are determined. The expression level of PsSst1 and PsIgn1 genes is determined by real-time PCR in nodules of several symbiotic mutants and original lines of pea. Lines with increased (Sprint-2Fix⁻ (Pssym31)) and decreased (P61 (Pssym25)) expression level of both genes are revealed along with the lines characterized by changes in the expression level of only one of these genes. The revealed features of the PsSst1 and PsIgn1 expression allow us to expand the phenotypic characterization of pea symbiotic mutants. In addition, PsSst1 and PsIgn1 cDNA is sequenced in selected mutant lines, characterized by a decreased expression level of these genes in nodules, but no mutations are found.

  12. High efficient expression of Lhcb2 gene from pea in E. coli and reconstitution of its expressed product with pigment in vitro

    Institute of Scientific and Technical Information of China (English)

    孙钦秒; 李良璧; 毛大璋; 匡廷云

    2000-01-01

    Lhcb2 gene from pea (Pisum sativum L.) was subcloned into bacterial expression vector pET-3d, and its protein overexpressed was obtained from E. coli (BL21) containing PetpLhcb2 by site-directed mutagenesis method. Bacteria transformed with this construct yielded up to 40 percent of total protein of E. coli. Using the modified method with three subsequent cycles of freezing (1 min, -196℃) and thawing (15 min, 25℃), Lhcb2 protein purified was highly reconstituted with pigments to yield pigment-protein complexes. The reconstituted LHCB2 monomers were very similar to native LHCll monomers from spinach in partially denaturing polyacrylamide gel electrophoresis, fluorescence and absorbance spectroscopy. These results showed that Lhcb2 proteins overexpressed were reconstituted successfully with pigments and had similar organization and structure to the native LHCII monomers.

  13. High efficient expression of Lhcb2 gene from pea in E. coli and reconstitution of its expressed product with pigment in vitro

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Lhcb2 gene from pea (Pisum sativum L.) was subcloned into bacterial expression vector pET-3d, and its protein overexpressed was obtained from E. coli (BL21) containing PetpLhcb2 by site-directed mutagenesis method. Bacteria transformed with this construct yielded up to 40 percent of total protein of E. coli. Using the modified method with three subsequent cycles of freezing (1 min, -196℃) and thawing (15 min, 25℃), Lhcb2 protein purified was highly reconstituted with pigments to yield pigment-protein complexes. The reconstituted LHCB2 monomers were very similar to native LHCII monomers from spinach in partially denaturing polyacrylamide gel electrophoresis, fluorescence and absorbance spectroscopy. These results showed that Lhcb2 proteins overexpressed were reconstituted successfully with pigments and had similar organization and structure to the native LHCII monomers.

  14. Hormone interactions and regulation of Unifoliata, PsPK2, PsPIN1 and LE gene expression in pea (Pisum sativum) shoot tips.

    Science.gov (United States)

    Bai, Fang; DeMason, Darleen A

    2006-07-01

    The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development. PMID:16760220

  15. Pea (Pisum sativum) genes involved in symbiosis with nitrogen-fixing bacteria.1.Analysis of the expression of the early nodulin gene ENOD12 using the polymerase chain-reaction

    OpenAIRE

    Zalenskii, A.O.; Kozik, A.V.; Scheres, B.J.G.; Bisseling, A.; Tikhonovich, I.A.

    1991-01-01

    The polymerase chain reaction (PCR) was used to detect the transcription products of the early nodulin gene in the pea. Single-stranded DNA copies were prepared using a primer corresponding to the terminal part of a previously sequenced cDNA clone and a total RNA isolate. The presence of amplification products was detected using Southern hybridization. Expression of the ENOD12 gene was found to occur at the earliest developmental stages of the symbiosis between the pea and nitrogenfixing bact...

  16. The expression and significance of PED/PEA-15 and it's phosphorylation state in epithe-lial ovarian tumors%PED/PEA-15及其磷酸化状态在卵巢上皮肿瘤中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    刘记; 金兰; 罗永红

    2016-01-01

    目的::探讨PED/PEA-15( phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes)蛋白及其磷酸化状态在卵巢上皮性肿瘤中的表达及临床意义。方法:免疫组化法检测40例卵巢上皮良性肿瘤(良性组)、40例卵巢交界性肿瘤(交界性组)、50例卵巢上皮癌(恶性组)中PED/PEA-15及其磷酸化状态的阳性表达水平。结果:PEA-15蛋白在良性组中表达高于交界性组及恶性组( P0.05)。卵巢癌中PEA-15磷酸化状态pPEA-15-Ser104蛋白表达高于交界性及良性上皮肿瘤(P0.05);另一位点磷酸化状态pPEA-15-Ser116在不同性质卵巢组织、不同年龄、组织学分级、临床分期、淋巴结转移分组中表达无差异( P>0.05)。结论:PEA-15及其磷酸化状态在卵巢癌的发生发展中发挥重要作用,可否通过调控PED/PEA-15的磷酸化状态从而使之成为卵巢癌治疗的重要靶点仍需进一步研究。%Objective:To investigate the expression and clinical significance of PED/PEA-15 protein and its phosphorylation state in epithelial ovarian tumors. Methods:The posi-tive expression level of PED/PEA-15 and it's phosphorylation state in 40 cases of epithelial be-nign ovarian tumor,40 cases of epithelial ovarian borderline tumors and 50 cases of epithelial o-varian cancer was analyzed by immunohistochemical method. Results:The expression of PEA-15 protein in the benign group was higher than that in the borderline group and the malignant group (P0 . 05 ) . There were no significant differences in the expression of pPEA-15-Ser116 in different ovarian tissues,in differ-ent age,histological grade,clinical stage and lymph node metastasis (P>0. 05). Conclusion:PEA-15 and it's phosphorylation state play an important role in the occurrence and development of ovarian cancer. Whether the regulation of phosphorylation status of PED/PEA-15 can be used as an effective therapeutic marker in ovarian epithelial malignant tumor is still need to be

  17. Heterologous expression of viral suppressors of RNA silencing complements virulence of the HC-Pro mutant of clover yellow vein virus in pea.

    Science.gov (United States)

    Atsumi, Go; Nakahara, Kenji S; Wada, Tomoko Sugikawa; Choi, Sun Hee; Masuta, Chikara; Uyeda, Ichiro

    2012-06-01

    Many plant viruses encode suppressors of RNA silencing, including the helper component-proteinase (HC-Pro) of potyviruses. Our previous studies showed that a D-to-Y mutation at amino acid position 193 in HC-Pro (HC-Pro-D193Y) drastically attenuated the virulence of clover yellow vein virus (ClYVV) in legume plants. Furthermore, RNA-silencing suppression (RSS) activity of HC-Pro-D193Y was significantly reduced in Nicotiana benthamiana. Here, we examine the effect of expression of heterologous suppressors of RNA silencing, i.e., tomato bushy stunt virus p19, cucumber mosaic virus 2b, and their mutants, on the virulence of the ClYVV point mutant with D193Y (Cl-D193Y) in pea. P19 and 2b fully and partially complemented Cl-D193Y multiplication and virulence, including lethal systemic HR in pea, respectively, but the P19 and 2b mutants with defects in their RSS activity did not. Our findings strongly suggest that the D193Y mutation exclusively affects RSS activity of HC-Pro and that RSS activity is necessary for ClYVV multiplication and virulence in pea. PMID:22398917

  18. CONSTRUCTION AND EXPRESSION OF A HUMAN-MOUSE CHIMERIC ANTIBODY AGAINST HUMAN BLADDER CANCER

    Institute of Scientific and Technical Information of China (English)

    白银; 王琰; 周丽君; 俞莉章

    2001-01-01

    To construct and express a human-mouse chimeric antibody against human bladder cancer. Method: The variable region genes of anti-human bladder cancer monoclonal antibody BDI-1 were cloned by RT-PCR. A human-mouse chimeric antibody expression vector was constructed and transfected into CHO cells. The chimeric antibody against bladder cancer was expressed and characterized. Result: Eukaryotic expression vector of the chimeric antibody against human bladder carcinoma was successfully constructed, and was expressed in eukaryotic cells; the expressed chimeric antibody ch-BDI showed same specificity as its parent McAb against human bladder cancer cells. Conclusion: The constructed chimeric antibody was expressed successfully in eukaryotic cells, and the chimeric antibody had desired affinity against human bladder cancer cells.

  19. Differential Expression of Proteins and mRNAs from Border Cells and Root Tips of Pea.

    Science.gov (United States)

    Brigham, L. A.; Woo, H. H.; Nicoll, S. M.; Hawes, M. C.

    1995-10-01

    Many plants release large numbers of metabolically active root border cells into the rhizosphere. We have proposed that border cells, cells produced by the root cap meristem that separate from the rest of the root upon reaching the periphery of the cap, are a singularly differentiated part of the root system that modulates the environment of the plant root by producing specific substances to be released into the rhizosphere. Proteins synthesized in border cells exhibit profiles that are very distinct from those of the root tip (root cap, root meristem, and adjacent cells). In vivo-labeling experiments demonstrate that 13% of the proteins that are abundant in preparations from border cells are undetectable in root tip preparations. Twenty-five percent of the proteins synthesized by border cells in a 1-h period are rapidly excreted into the incubation medium. Quantitative variation in levels of specific marker proteins, including glutamine synthetase, heat-shock protein 70, and isoflavone reductase, also occurs between border cells and cells in the root tip. mRNA differential-display assays demonstrate that these large qualitative and quantitative differences in protein expression are correlated with similarly distinct patterns of gene expression. These observations are consistent with the hypothesis that a major switch in gene expression accompanies differentiation into root border cells, as expected for cells with specialized functions in plant development.

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

    Directory of Open Access Journals (Sweden)

    Dayalan G Srinivasan

    Full Text Available 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. Full-length de novo assembly of RNA-seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species.

    Science.gov (United States)

    Alves-Carvalho, Susete; Aubert, Grégoire; Carrère, Sébastien; Cruaud, Corinne; Brochot, Anne-Lise; Jacquin, Françoise; Klein, Anthony; Martin, Chantal; Boucherot, Karen; Kreplak, Jonathan; da Silva, Corinne; Moreau, Sandra; Gamas, Pascal; Wincker, Patrick; Gouzy, Jérôme; Burstin, Judith

    2015-10-01

    Next-generation sequencing technologies allow an almost exhaustive survey of the transcriptome, even in species with no available genome sequence. To produce a Unigene set representing most of the expressed genes of pea, 20 cDNA libraries produced from various plant tissues harvested at various developmental stages from plants grown under contrasting nitrogen conditions were sequenced. Around one billion reads and 100 Gb of sequence were de novo assembled. Following several steps of redundancy reduction, 46 099 contigs with N50 length of 1667 nt were identified. These constitute the 'Caméor' Unigene set. The high depth of sequencing allowed identification of rare transcripts and detected expression for approximately 80% of contigs in each library. The Unigene set is now available online (http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi), allowing (i) searches for pea orthologs of candidate genes based on gene sequences from other species, or based on annotation, (ii) determination of transcript expression patterns using various metrics, (iii) identification of uncharacterized genes with interesting patterns of expression, and (iv) comparison of gene ontology pathways between tissues. This resource has allowed identification of the pea orthologs of major nodulation genes characterized in recent years in model species, as a major step towards deciphering unresolved pea nodulation phenotypes. In addition to a remarkable conservation of the early transcriptome nodulation apparatus between pea and Medicago truncatula, some specific features were highlighted. The resource provides a reference for the pea exome, and will facilitate transcriptome and proteome approaches as well as SNP discovery in pea.

  2. Selection of reference genes for expression analysis using quantitative real-time PCR in the pea aphid, Acyrthosiphon pisum (Harris (Hemiptera, Aphidiae.

    Directory of Open Access Journals (Sweden)

    Chunxiao Yang

    Full Text Available To facilitate gene expression study and obtain accurate qRT-PCR analysis, normalization relative to stable expressed housekeeping genes is required. In this study, expression profiles of 11 candidate reference genes, including actin (Actin, elongation factor 1 α (EF1A, TATA-box-binding protein (TATA, ribosomal protein L12 (RPL12, β-tubulin (Tubulin, NADH dehydrogenase (NADH, vacuolar-type H+-ATPase (v-ATPase, succinate dehydrogenase B (SDHB, 28S ribosomal RNA (28S, 16S ribosomal RNA (16S, and 18S ribosomal RNA (18S from the pea aphid Acyrthosiphon pisum, under different developmental stages and temperature conditions, were investigated. A total of four analytical tools, geNorm, Normfinder, BestKeeper, and the ΔCt method, were used to evaluate the suitability of these genes as endogenous controls. According to RefFinder, a web-based software tool which integrates all four above-mentioned algorithms to compare and rank the reference genes, SDHB, 16S, and NADH were the three most stable house-keeping genes under different developmental stages and temperatures. This work is intended to establish a standardized qRT-PCR protocol in pea aphid and serves as a starting point for the genomics and functional genomics research in this emerging insect model.

  3. Occurrence of viruses infecting pea in Iran.

    Science.gov (United States)

    Esfandiari, N; Kohi-Habibi, M; Mosahebi, Gh

    2006-01-01

    A survey was conducted to determine the incidence of Alfalfa mosaic virus (AMV), Bean yellow mosaic virus (BYMV), Broad bean wilt virus-1 (BBWV), Pea leafroll virus (PLRV), Pea enation mosaic virus (PEMV), Pea seed borne mosaic virus (PSbMV), Potato virus x(PVX), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) on pea (Pisum sativum) in Iran. A Total of 1276 random and 684 symptomatic pea samples were collected during the spring and summer of 2002-2004 in Tehran province of Iran, where pea is grown, and tested by enzyme-linked immunosorbent assay (ELISA) using specific polyclonal antibodies. Serological diagnoses were confirmed by electron microscopy and host range studies. Incidence of viruses in decreasing order was PVX (69%), ToMV (59%), PSbMV (36.6%), BBWV-1 (26.1%), BYMV (20.3%), AMV (17.77%), TSWV (12.6%), PEMV (10.9%), PLRV (6.78%). In this survey, natural occurrence of AMV, BBWV-1, PSbMV, TSWV, PVX and ToMV was reported for the first time on the pea in Iran. PMID:17390891

  4. Improved expression of single-chain antibodies in Ustilago maydis.

    Science.gov (United States)

    Sarkari, Parveen; Reindl, Michèle; Stock, Janpeter; Müller, Olaf; Kahmann, Regine; Feldbrügge, Michael; Schipper, Kerstin

    2014-12-10

    To produce the full repertoire of biopharmaceutical proteins, alternative expression platforms are required. Systems that enable secretion of the target protein are favored because this facilitates downstream processing. Ustilago maydis is a promising fungal model organism for future applications in protein expression. Recently, we described the exploitation of a novel unconventional secretion mechanism for the export of heterologous proteins. In this mode of secretion, the endochitinase Cts1 functions as a carrier for export with the main advantage of avoiding potentially harmful N-glycosylation. The major limitation until now was a low yield of secreted full-length protein. For optimization, we identified two bottlenecks: mRNA amount and extracellular proteolytic activity. By generating novel expression vectors harboring a strong constitutive promoter as well as eliminating harmful proteases, yields were increased significantly. A scFv antibody fragment against the cMyc epitope served as proof-of-principle and could be purified in its active, full-length form from the culture supernatant. Thus, we improved the novel expression system in U. maydis such that it can now be investigated with respect to other targets with potential applications for instance in diagnostics and medicine.

  5. The antibody preparation and expression of human Pescadillo

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene- sis, the cDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated. Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells, and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer, colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a po- tential target in cancer diagnosis and therapy.

  6. The antibody preparation and expression of human Pescadillo

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hao; NING Kang; ZHU JianHua; LI JieZhi; HUANG CuiFen; LIU AiJun; YE QiNong; LI JiePing; WANG XiaoHui; SUN Yan; YUAN Bin; YANG ZhiHong; JIANG YanChao; ZENG Min; DING LiHua

    2007-01-01

    To explore the biological roles of human Pescadillo and investigate its potential effect on tumorigene sis, the eDNA of Pescadillo was fused with that of GST. After purification and elution, the purified GST-Pescadillo fusion protein was obtained, and the antibody against the fusion protein was generated.Endogenous Pescadillo protein was observed to be remarkably induced by estrogen. It was mainly distributed in the tissues such as breast, ovary and intestine, all of which contain proliferating cells,and was also detected in many cell lines of human cancer: renal carcinoma, hepatoma, ovarian cancer,colon carcinoma, and breast cancer. The expression level of Pescadillo was increased significantly in breast cancer tissues compared with their paired margin tissues. Taken together, these data suggest that Pescadillo may play important roles in the initiation and development of cancer and may be a potential target in cancer diagnosis and therapy.

  7. Expression and clinical significance of anti-apoptotic factors PED/PEA-15 and XIAP in hepatocellular carcinoma%凋亡抑制蛋白PED/PEA-15和XIAP在肝细胞肝癌中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    孙德光; 吴益峰; 高振明; 梁锐; 王立明

    2010-01-01

    Objective To investigate the expression of apoptosis arrestin PED/PEA-15 and XIAP in hepatocellular carcinoma ( HCC) and explore their relationship with clinicopathological factors of HCC.Methods The mRNA expressions of PED/PEA-15 and XIAP genes were detected by RT-PCR ( reverse transcription-polymerase chain reaction) in resected liver tumor tissues and corresponding adjacent noncancerous tissues from 40 HCC patients and normal liver tissues from 12 patients with benign lesions.Their protein levels were detected by Western blot. Results The mRNA expressions of PED/PEA-15 and XIAP genes and their proteins were significantly higher than those in adjacent tissues and normal tissues (0.636±0.061±0.352±0.068±0.179 ±0. 036;0. 579 ±0.090±0.344 ±0.084±0.184±0.038) (P <0. 01). The mRNA levels of PED/PEA-15 and XIAP genes and their protein levels were significantly associated with the pathological grade and clinical stage of HCC (P < 0. 05). However there was not a significant correlation with such clinicopathological features as age, gender, size of tumor, tumor load,metastasis and recurrence (P > 0. 05). Conclusion The expressions of apoptosis arrestin PED/PEA-15 and XIAP are elevated in HCC as compared with adjacent tissues and normal tissues. Thus it may serve as both a reference indicator for biological behaviors of HCC and a new target for gene therapy.%目的 探讨凋亡抑制蛋白PED/PEA-15和XIAP在肝细胞肝癌中的表达及临床病理因素的关系.方法 我们应用RT-PCR和Western印迹检测了40例肝细胞肝癌和相应癌旁组织及12例正常肝组织中PED/PEA-15和XIAP mRNA及蛋白的表达,并结合临床病理特征进行分析.结果 肝细胞肝癌组织中PED/PEA-15和XIAP mRNA蛋白的表达均明显高于癌旁组织和正常肝组织(相对吸光度比值为0.636±0.061、0.352±0.068、0.179±0.036与0.579±0.090、0.344±0.084、0.184±0.038)(P均<0.01),PED/PEA-15和XIAP mRNA和蛋白的表达在不同年龄、性

  8. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants

    OpenAIRE

    Hehle, Verena K; Paul, Matthew J.; Roberts, Victoria A.; van Dolleweerd, Craig J.; Ma, Julian K.-C.

    2015-01-01

    This study examined the degradation pattern of a murine IgG1κ monoclonal antibody expressed in and extracted from transformed Nicotiana tabacum. Gel electrophoresis of leaf extracts revealed a consistent pattern of recombinant immunoglobulin bands, including intact and full-length antibody, as well as smaller antibody fragments. N-terminal sequencing revealed these smaller fragments to be proteolytic cleavage products and identified a limited number of protease-sensitive sites in the antibody...

  9. 肝细胞肝癌中PED/PEA-15 mRNA及蛋白表达的临床意义%Expression and significance of PED/PEA-15 mRNA in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    孙德光; 吴益峰; 高振明; 梁锐; 庄成君; 王立明

    2010-01-01

    目的:探讨PED/PEA-15 mRNA及蛋白在肝癌组织、癌旁组织及正常肝组织中的表达情况及其临床意义.方法:应用半定量逆转录.聚合酶链反应检测40例肝细胞肝癌、相应癌旁组织和12例正常肝组织中PED/PEA.15基因的相对表达量.并通过免疫组化检测PED/PEA-15蛋白的表达.结果:半定量RT-PCR显示,PED/PEA-15基因在肝癌组织中均呈高表达,癌旁组织及正常肝组织呈低表达或不表达,相对表达量分别为1.201±0.301、0.64±0.101和0.565±0.077,PED/PEA-15 mRNA在肝癌组织中表达较癌旁组织、正常肝组织显著升高(P0.05).结论:PED/PEA-15 mRNA及蛋白在肝癌组织中表达水平明显升高,可能在肝癌的发生及发展过程中发挥重要作用.

  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.

    2016-01-01

    Abstract 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-1BaL 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. PMID:26950606

  11. Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: a case study.

    Science.gov (United States)

    Dorvignit, Denise; Palacios, Julio L; Merino, Maylin; Hernández, Tays; Sosa, Katya; Casaco, Angel; López-Requena, Alejandro; Mateo de Acosta, Cristina

    2012-01-01

    The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials. PMID:22647435

  12. Expression and biological characterization of an anti-CD20 biosimilar candidate antibody

    Science.gov (United States)

    Dorvignit, Denise; Palacios, Julio L.; Merino, Maylin; Hernández, Tays; Sosa, Katya; Casacó, Angel; López-Requena, Alejandro; Mateo de Acosta, Cristina

    2012-01-01

    The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials. PMID:22647435

  13. Monoclonal Antibody Expression and Novel Purification in Nicotiana benthamiana

    OpenAIRE

    Fulton, Andrew Dale

    2011-01-01

    Over the past few decades researchers and industrial professionals alike have realized the vast potential of monoclonal antibodies to treat diseases ranging from arthritis, immune and infectious diseases to cancer. There are a number of antibodies on the market that constitute a large portion of the biopharmaceutical niche in the drug industry. Blockbuster drugs (selling greater than $1 billion/year), include antibodies such as Avastin (bevacizumab), Herceptin (trastuzumab), Rituxan (rituxi...

  14. Protein Kinase B/Akt Binds and Phosphorylates PED/PEA-15, Stabilizing Its Antiapoptotic Action

    OpenAIRE

    Trencia, Alessandra; Perfetti, Anna; Cassese, Angela; Vigliotta, Giovanni; Miele, Claudia; Oriente, Francesco; Santopietro, Stefania; Giacco, Ferdinando; Condorelli, Gerolama; Formisano, Pietro; Beguinot, Francesco

    2003-01-01

    The antiapoptotic protein PED/PEA-15 features an Akt phosphorylation motif upstream from Ser116. In vitro, recombinant PED/PEA-15 was phosphorylated by Akt with a stoichiometry close to 1. Based on Western blotting with specific phospho-Ser116 PED/PEA-15 antibodies, Akt phosphorylation of PED/PEA-15 occurred mainly at Ser116. In addition, a mutant of PED/PEA-15 featuring the substitution of Ser116→Gly (PEDS116→G) showed 10-fold-decreased phosphorylation by Akt. In intact 293 cells, Akt also i...

  15. Expression of anti-SRP19 antibody in muscle tissues from patients with autoimmune necrotizing myopathy.

    Science.gov (United States)

    Wang, Q; Duan, F; Liu, P; Wang, P F; Wang, M X

    2016-01-01

    This study aimed to investigate the role of anti-SRP19 antibody in muscle tissues of patients with autoimmune necrotizing myopathy. Immunohistochemistry staining was used to determine the expression of anti-SRP19 antibodies in muscle tissues of autoimmune necrotizing myopathy patients. Results demonstrated that anti-SRP19 antibody was expressed in 71.4% (20/28) of muscle tissue specimens from patients with autoimmune necrotizing myopathy. Anti-SRP19 antibody expression was mainly localized in cytoplasm of necrotic muscle fibers surrounding the small blood vessels and interstitial cells. There were no significant differences in the age, course of disease, muscle, and creatine kinase levels between patients with positive or negative expression of anti-SRP19 antibodies. The expression levels of anti-SRP19, serum anti-nuclear antibodies, as well as anti-Ro-52, anti- SSA, anti-Sm, and anti-Jo-1 antibodies were not significantly different among groups. This study demonstrates that anti-SRP19 antibody is highly expressed in muscle tissues of patients with autoimmune necrotizing myopathy, and suggests that this protein may be involved in the origin and progression of the disease. PMID:27525944

  16. Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots

    Science.gov (United States)

    Wu, L. L.; Mitchell, J. P.; Cohn, N. S.; Kaufman, P. B.

    1993-01-01

    The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

  17. Enhanced Survival and Nodule Occupancy of Pigeon pea Nodulating Rhizobium sp. ST1 expressing fegA Gene of Bradyrhizobium japonicum 61A152

    Directory of Open Access Journals (Sweden)

    G. Archana

    2009-01-01

    Full Text Available Problem statement: Rhizobial isolates belonging to genera (Rhizobium sp. and Mesorhizobium sp. in our laboratory produced only catecholate type of siderophores. Although FhuA and FegA (ferrichrome receptors homologs were found to be present in the sequenced genomes of few rhizobia (e.g., 1 in R. etli and 2 in Mesorhizobium sp. BNC1, laboratory isolates of the corresponding genera failed to utilize ferrichrome, a siderophore which is present in nanomolar concentrations in the soil. This inability was considered as a negative fitness factor with respect to rhizospheric colonization by these rhizobia. Approach: The 2.4 kb fegA gene (encoding ferrichrome receptor was amplified along with its native promoter from Bradyrhizobium japonicum 61A152 and cloned in a broad host range plasmid vector pUCPM18. The plasmid construct pFJ was transferred by conjugation into Rhizobium sp. ST1 to give transconjugant ST1pFJ12. The consequence of FegA expression on the transconjugant was tested under lab and soil conditions, using physiological experiments. Results: Ability of the transconjugant ST1pFJ12 to utilize ferrichrome and expression of a 79 kD protein band on the outer membrane of the transconjugant confirmed FegA expression. Transconjugant ST1pFJ12 exhibited increased growth rate as compared to the parent strain ST1, in minimal media containing ferrichrome as the sole iron source, confirming the positive effect of FegA expression. Inoculation of pigeon pea seedlings with transconjugant ST1pFJ12 led to a marked increase in plant growth parameters as compared to plants inoculated with the parent strain ST1, the effect being more pronounced when Ustilago maydis, a ferrichrome producer was co-inoculated in the systems. Nodule occupancy on pigeon pea plant when inoculated with the transconjugant ST1pFJ12 alone was 57% which increased to 66% when co-inoculated with U. maydis as compared with 37 and 30

  18. Monoclonal antibodies produced to bean yellow mosaic virus, clover yellow vein virus, and pea mosaic virus which cross-react among the three viruses.

    Science.gov (United States)

    Scott, S W; McLaughlin, M R; Ainsworth, A J

    1989-01-01

    Monoclonal antibodies prepared against individual potyviruses that infect forage legumes cross-reacted among the viruses. The reaction occurs between capsid subunits and presumably involves epitopes located in the trypsin-resistant core of the coat protein. PMID:2480762

  19. Expression of PIN and AUX1 genes encoding putative carrier proteins for auxin polar transport in etiolated pea epicotyls [correction of epicotyles] under simulated microgravity conditions on a three-dimensional clinostat.

    Science.gov (United States)

    Hoshino, Tomoki; Hitotsubashi, Reiko; Miyamoto, Kensuke; Tanimoto, Eiichi; Ueda, Junichi

    2003-10-01

    Etiolated pea (Pisum sativum L. cv. Alaska) seedlings grown under simulated microgravity conditions on a 3-dimensional clinostat showed automorphosis-like growth and development similar to that observed in true microgravity conditions in space. Application of inhibitors of auxin polar transport phenocopied automorphosis-like growth on 1 g conditions, suggesting that automorophosis is closely related to auxin polar transport. Strenuous efforts to know the relationships between automorphosis and auxin polar transport in pea seedlings at molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin efflux and influx carrier protein, respectively. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 and AtPINs, and between PsAUX1 and AtAUX1. Expression of PsPIN1 and PsAUX1 genes in etiolated pea seedlings grown on the clinostat were substantially affected, but that of PsPIN2 was not. Roles of these genes in auxin polar transport and automorphosis of etiolated pea seedlings are also described. PMID:14676360

  20. Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: A case study

    OpenAIRE

    Dorvignit, Denise; Palacios, Julio L.; Merino, Maylin; Hernández, Tays; Sosa, Katya; Casacó, Angel; López-Requena, Alejandro; Mateo de Acosta, Cristina

    2012-01-01

    The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the ...

  1. Rescue and expression of human immunoglobulin genes to generate functional human monoclonal antibodies.

    Science.gov (United States)

    Lewis, A P; Parry, N; Peakman, T C; Crowe, J S

    1992-07-01

    Human monoclonal antibody production has been hampered for many years by the instability of cell lines and low levels of expression of the antibodies. We describe here the rescue of human immunoglobulin genes utilizing micro-mRNA preparation from a small number of human hybridoma cells and conventional cDNA cloning. This allows cloning and immediate high-level expression from full-length human heavy and light chain cDNA molecules and provides a mechanism to rescue whole human monoclonal antibodies of proven efficacy.

  2. 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.

  3. Quantitating Antibody Uptake In Vivo: Conditional Dependence on Antigen Expression Levels

    Science.gov (United States)

    Thurber, Greg M.; Weissleder, Ralph

    2010-01-01

    Purpose Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation. Procedures Using a cell line with high EpCAM expression and moderate EGFR expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high affinity antibodies. Results As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors. Conclusions These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes. PMID:20809210

  4. Immunodiagnosis of Citrus leprosis virus C using a polyclonal antibody to an expressed putative coat protein.

    Science.gov (United States)

    Choudhary, Nandlal; Roy, Avijit; Guillermo, Leon M; Picton, D D; Wei, G; Nakhla, M K; Levy, L; Brlansky, R H

    2013-11-01

    Citrus leprosis virus C (CiLV-C), a causal agent for citrus leprosis disease, is present in South and Central America and is a threat for introduction into the U.S. citrus industry. A specific, inexpensive and reliable antibody based detection system is needed for the rapid identification of CiLV-C. The CiLV-C is very labile and has not been purified in sufficient amount for antibody production. The p29 gene of CiLV-C genome that codes for the putative coat protein (PCP) was codon optimized for expression in Escherichia coli and synthesized in vitro. The optimized gene was sub-cloned into the bacterial expression vector pDEST17 and transferred into E. coli BL21AI competent cells. The expression of PCP containing N-terminal His-tag was optimized by induction with l-arabinose. Induced cells were disrupted by sonication and expressed PCP was purified by affinity chromatography using Ni-NTA agarose. The purified expressed PCP was then used as an immunogen for injections into rabbits to produce polyclonal antibody (PAb). The PAb specific to the expressed PCP was identified using Western blotting. The antibody was successfully used to detect CiLV-C in the symptomatic CiLV-C infected tissues using double antibody sandwich-enzyme-linked-immunosorbent (DAS-ELISA), indirect ELISA and dot-blot immunoassay (DBIA) formats.

  5. EFFECT OF POMEGRANATE ELLAGIC ACID (PEA) ON THE EXPRESSION OF LIPOGENESIS AND LIPOLYSIS GENES IN HYPERLIPIDEMIC HAMSTERS%石榴鞣花酸对高脂血仓鼠肝脏脂质合成与分解基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    刘润; 李建科; 程玉江; 王丽芳; 霍天博; 薛佳宜

    2015-01-01

    目的:观察石榴鞣花酸(PEA)对饮食诱导性高脂仓鼠脂质合成与分解相关基因的影响,探讨PEA降脂的作用机制。方法采用HPLC法测定PEA主要成分;54只雄性仓鼠随机均分为6组:正常对照组(NG,8w普通饲料);五个高脂组(HFG):模型对照组(MG)、药物对照组(辛伐他汀1.77mg/kg bw ,SG)、PEA低(PEA-L 44 mg/kg bw)、中(PEA-M 88 mg/kg bw)、高(PEA-H 177 mg/kg bw;)剂量组,后5组为前4w高脂饲料,后4w普通饲料;4w高脂模型建立成功后,以灌胃方式连续给予仓鼠对应处理,第4w、8w检测仓鼠血清 TC、TG含量;采用 RT-PCR法测定肝脏中脂质代谢调控因子固醇调节元件结合蛋白-1c(SREBP-1c)mRNA表达和脂肪酸合成酶(FAS)以及脂蛋白脂肪酶(LPL)mRNA表达量。结论 PEA可以降低血液中TC、TG的含量,升高HDL-C的含量,主要通过抑制SREBP-1c mRNA、FAS mRNA的基因表达,促进LPL mRNA的基因表达实现降脂功能。PEA降脂效果存在剂量依赖关系,高剂量PEA 效果较好。%Objective The study was aimed to further investigate the effects of pomegranates ellagic acid (PEA) on lipid metabolism and relevant gene expressions in hyperlipidemic hamster.Method 54 male hamsters were randomly divided into six groups of equal size: normal group (NG), model group (MG), simvastatin group (SG) with 1.77mg/kg simvastatin, PEA low group (PEA-L) with PEA 44 mg/kg, PEA medium group (PEA-M) with PEA 88 mg/kg, PEA high group (PEA-H) with PEA 177 mg/kg. The last 4 groups were treated with simvastatin or PEA for 4 weeks after 4 weeks, respectively. Except NG was given normal diet, other 5 groups were fed a high fat diet at the first 4 weeks and switched to normal diet for the next 4 weeks. At the end of weeks 4 and 8, plasma total cholesterol (TC), triglyceride (TG) and high-density lipoprotein cholesterol (HDL-C) were detected by kits. Real time PCR was used to analyze the mRNA expression of

  6. Molecular cloning and characterization of the pyrB1 and pyrB2 genes encoding aspartate transcarbamoylase in pea (Pisum sativum L.).

    Science.gov (United States)

    Williamson, C L; Slocum, R D

    1994-05-01

    We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate transcarbamoylases (ATCases) by complementation of an Escherichia coli delta pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode polypeptides of 386 and 385 amino acid residues, respectively, both of which exhibit typical chloroplast transit peptide sequences. Wheat germ ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root tissues that is similar in size to other plant ATCase polypeptides and to the catalytic polypeptides of bacterial ATCases. Northern analyses indicate that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are differentially expressed in pea tissues. The small transcript size and data from biochemical studies indicate that plant ATCases are simple homotrimers of 36- to 37-kD catalytic subunits, rather than part of a multifunctional enzyme containing glutamine-dependent carbamoylphosphate synthetase and dihydroorotase activities, as is seen in other eukaryotes. In the pea ATCases, the carbamoylphosphate- and aspartate-binding domains are highly homologous to those of other prokaryotic and eukaryotic ATCases and critical active-site residues are completely conserved. The pea ATCases also exhibit a putative pyrimidine-binding site, consistent with the known allosteric regulation of plant ATCases by UMP in vitro. PMID:8029359

  7. Antigen-specific monoclonal antibodies isolated from B cells expressing constitutively active STAT5.

    Directory of Open Access Journals (Sweden)

    Ferenc A Scheeren

    Full Text Available BACKGROUND: Fully human monoclonal antibodies directed against specific pathogens have a high therapeutic potential, but are difficult to generate. METHODOLOGY/PRINCIPAL FINDINGS: Memory B cells were immortalized by expressing an inducible active mutant of the transcription factor Signal Transducer and Activator of Transcription 5 (STAT5. Active STAT5 inhibits the differentiation of B cells while increasing their replicative life span. We obtained cloned B cell lines, which produced antibodies in the presence of interleukin 21 after turning off STAT5. We used this method to obtain monoclonal antibodies against the model antigen tetanus toxin. CONCLUSIONS/SIGNIFICANCE: Here we describe a novel and relatively simple method of immortalizing antigen-specific human B cells for isolation of human monoclonal antibodies. These results show that STAT5 overexpression can be employed to isolate antigen specific antibodies from human memory B cells.

  8. 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 level

  9. Mutation breeding in peas

    International Nuclear Information System (INIS)

    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)

  10. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies.

    Science.gov (United States)

    Villani, Maria Elena; Morgun, Bogdan; Brunetti, Patrizia; Marusic, Carla; Lombardi, Raffaele; Pisoni, Ivan; Bacci, Camilla; Desiderio, Angiola; Benvenuto, Eugenio; Donini, Marcello

    2009-01-01

    The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach. PMID:18793269

  11. Plant pharming of a full-sized, tumour-targeting antibody using different expression strategies.

    Science.gov (United States)

    Villani, Maria Elena; Morgun, Bogdan; Brunetti, Patrizia; Marusic, Carla; Lombardi, Raffaele; Pisoni, Ivan; Bacci, Camilla; Desiderio, Angiola; Benvenuto, Eugenio; Donini, Marcello

    2009-01-01

    The aims of this work were to obtain a human antibody against the tumour-associated antigen tenascin-C (TNC) and to compare the yield and quality of plant-produced antibody in either stable transgenics or using a transient expression system. To this end, the characterization of a full-sized human immunoglobulin G (IgG) [monoclonal antibody H10 (mAb H10)], derived from a selected single-chain variable fragment (scFv) and produced in plants, is presented. The human mAb gene was engineered for plant expression, and Nicotiana tabacum transgenic lines expressing both heavy (HC) and light (LC) chain were obtained and evaluated for antibody expression levels, in vivo assembly and functionality. Affinity-purified H10 from transgenics (yield, 0.6-1.1 mg/kg fresh weight) revealed that more than 90% of HC was specifically degraded, leading to the formation of functional antigen-binding fragments (Fab). Consequently, H10 was transiently expressed in Nicotiana benthamiana plants through an Agrobacterium-mediated gene-transfer system. Moreover, the use of the p19 silencing suppressor gene from artichoke mottled crinkle virus raised antibody expression levels by an order of magnitude (yields of purified H10, 50-100 mg/kg fresh weight). Approximately 75% of purified protein consisted of full-sized antibody functionally binding to TNC (K(D) = 14 nm), and immunohistochemical analysis on tumour tissues revealed specific accumulation around tumour blood vessels. The data indicate that the purification yields of mAb H10, using a transient expression system boosted by the p19 silencing suppressor, are exceptionally high when compared with the results reported previously, providing a technique for the over-expression of anticancer mAbs by a rapid, cost-effective, molecular farming approach.

  12. 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.

  13. Production of Antibodies against Multipass Membrane Proteins Expressed in Human Tumor Cells Using Dendritic Cell Immunization

    OpenAIRE

    Takahiko Tamura; Joe Chiba

    2009-01-01

    Antibody mediated therapeutic strategies against human malignant tumors have been widely authorized and clinically applied to cancer patients. In order to develop methods to generate antibodies reactive to the extracellular domains of multipass plasma membrane proteins specifically expressed in malignant tumors, we examined the use of dendritic cells (DCs) for immunization. DCs were transduced with genes encoding the human six transmembrane epithelial antigen of prostate 1 (STEAP1), STEAP4, a...

  14. Human single chain antibody to vascular endothelial growth factor: gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    阎锡蕴[1; 汤健[2; 吴小平[3; 王凤采[4; 李建生[5; 杨东玲[6

    2000-01-01

    Using antibody phage display technique, a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned. The antibody expression reached 45% of the total bacterial proteins. The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph. ELISA analysis showed that the antibody not only specifically bound to human VEGF, but also competitively inhibited VEGF reacting with its receptors. In order to raise the affinity of the single chain antibody, its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed, from which a mutant with higher affinity was screened out. The three-dimensional structure and binding affinity of wild type and mutant antibody were compared. Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  15. Human single chain antibody to vascular endothelial growth factor:gene cloning, high-level expression, affinity maturation and bioactivity

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Using antibody phage display technique,a human single chain antibody to vascular endothelial growth factor (VEGF) has been cloned.The antibody expression reached 45% of the total bacterial proteins.The purification and refolding of the antibody were completed in one step by using gel filtration chromatograph.ELISA analysis showed that the antibody not only specifically bound to human VEGF,but also competitively inhibited VEGF reacting with its receptors.In order to raise the affinity of the single chain antibody,its heavy chain variable region was randomly mutated using error-prone PCR and an antibody mutant library was constructed,from which a mutant with higher affinity was screened out.The three-dimensional structure and binding affinity of wild type and mutant antibody were compared.Our study provided a potential reagent for tumor angiogenic therapy and a significant model for antibody high-level expression and affinity maturation.

  16. Transport processes in pea seed coats

    NARCIS (Netherlands)

    Dongen, Joost Thomas van

    2002-01-01

    The research described in this thesis concerns transport processes in coats of developing pea seeds. The scope of the investigation ranges from seed coat anatomy, via transport studies to the cloning of cDNA encoding proteinaceous membrane pores, and the heterologous expression of these protei

  17. Primary structure and functional scFv antibody expression of an antibody against the human protooncogen c-myc.

    Science.gov (United States)

    Fuchs, P; Breitling, F; Little, M; Dübel, S

    1997-06-01

    The immunoglobulin heavy- and light-chain variable region (Vh and Vl) genes were isolated from Myc1-9E10 hybridoma cells, which secreted monoclonal antibody against human oncogen c-myc. The expression vector pOPE52-c-myc was constructed for the recombinant production in E. coli. A 30 kDa single chain fragment (scFv) expression product was found in the periplasmic space by SDS-PAGE and immunoblotting. A significant fraction was processed correctly as demonstrated with an antiserum recognizing the processed aminoterminus only. The specific binding of the scFv fragment to the peptide epitope of the maternal monoclonal antibody was demonstrated and the primary sequence of the variable regions was determined. Sequence comparison with previously published partial Vh and Vl sequences from this hybridoma cell line revealed a genetic heterogeneity for the light chain variable region. The potential use of this scFv as a new tool for detection and purification of tagged proteins, for adding costimulatory signals to the surface of cancer cells as well as for analyzing c-myc function in the living cell by cytoplasmic expression is discussed.

  18. Cloning, Expression and Polyclonal Antibody Preparation of the Asialoglycoprotein Receptor of Marmota Himalayan

    Institute of Scientific and Technical Information of China (English)

    YANG Yan; HUANG Huang; ZHANG Zhenghua; WANG Baoju; TIAN Yongjun; LU Mengji; YANG Dongliang

    2007-01-01

    The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E. coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

  19. 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.

  20. PEA3activates CXCL12transcription in MCF-7breast cancer cells%PEA3 activates CXCL12 transcription in MCF-7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Li; CHEN Bo-bin; LI Jun-jie; JIN Wei; SHAO Zhi-min

    2011-01-01

    Objective To explore the activity of PEA3 ( polyomavirus enhancer activator 3 ) on CXCL12 (Chemokine CXC motif ligand 12) transcription and to reveal the role of PEA3 involved in CXCL12-mediated metastasis and angiogenesis in breast cancer. Methods Methods such as cell transfection, ChIP assay (chromatin immunoprecipitation ), and siRNA (small interfering RNA) were applied to demonstrate and confirm the interaction between PEA3 and CXCL12. Results Over-expression of PEA3 could increase the CXCL12 mRNA level and the CXCL12 promoter activity in human MCF-7 breast cancer cells. ChIP assay demonstrated that PEA3 could bind to the CXCL12 promoter in the cells transfected with PEA3 expression vector. PEA3 siRNA decreased CXCL12 promoter activity and the binding of PEA3 to the CXCL12 promoter in MCF-7 cells. Conclusions PEA3 could activate CXCL12 promoter transcription. It may be a potential mechanism of tumor angiogenesis and metastasis regarding of PEA3 and CXCL12.

  1. Iron as the Key Modulator of Hepcidin Expression in Erythroid Antibody-Mediated Hypoplasia

    Directory of Open Access Journals (Sweden)

    J. C. Fernandes

    2014-01-01

    Full Text Available Erythroid hypoplasia (EH is a rare complication associated with recombinant human erythropoietin (rHuEPO therapies, due to development of anti-rHuEPO antibodies; however, the underlying mechanisms remain poorly clarified. Our aim was to manage a rat model of antibody-mediated EH induced by rHuEPO and study the impact on iron metabolism and erythropoiesis. Wistar rats treated during 9 weeks with a high rHuEPO dose (200 IU developed EH, as shown by anemia, reduced erythroblasts, reticulocytopenia, and plasmatic anti-rHuEPO antibodies. Serum iron was increased and associated with mRNA overexpression of hepatic hepcidin and other iron regulatory mediators and downregulation of matriptase-2; overexpression of divalent metal transporter 1 and ferroportin was observed in duodenum and liver. Decreased EPO expression was observed in kidney and liver, while EPO receptor was overexpressed in liver. Endogenous EPO levels were normal, suggesting that anti-rHuEPO antibodies blunted EPO function. Our results suggest that anti-rHuEPO antibodies inhibit erythropoiesis causing anemia. This leads to a serum iron increase, which seems to stimulate hepcidin expression despite no evidence of inflammation, thus suggesting iron as the key modulator of hepcidin synthesis. These findings might contribute to improving new therapeutic strategies against rHuEPO resistance and/or development of antibody-mediated EH in patients under rHuEPO therapy.

  2. Preparation of Polyclonal Antibody and Expression Analysis of GR in Tomato

    Science.gov (United States)

    Xie, Yuanhong; Zhu, Benzhong; Luo, Yunbo; Chen, Xiangning; Zhang, Hongxing

    The fruit ripening of Green-ripe (Gr) mutant tomato was inhibited dramatically. To determine the expression patterns of Gr in tomato, we first produced the polyclonal antibody of Gr protein. RT-PCR was used to amplify the Gr gene from green ripe tomato fruit. And the PCR product was subcloned into prokaryotic protein expression vectors pET-30a to generate recombinant plasmid. The Gr protein was induced by IPTG in BL21 (DE3) and purified by Ni-NTA agarose column. The anti-Gr serum was produced by immunizing rabbits, and the titer of the anti-Gr serum was above 5000 by ELISA analysis. Purified by the DEAE-52 ion-column, the high purification level of anti-Gr polyclonal antibody was obtained. Furthermore, RT-CPR was used in the RNA level to demonstrate that the expression of Gr gene was specialized in some cultures of tomato. For example, the expressions of Gr were higher in seed, flower and green ripe fruit than others, and the expression level were reduced by exogenous ethylene treatment in the flower and green ripe fruit. Moreover, Polyclonal antibody of Gr was used to investigate the expression pattern of Gr in protein level by the Western blotting. Our results show that the expression level of Gr in protein level was complied with the expressions in RNA. So, we suggested that the regulation of Gr was transcriptional.

  3. Construction of expression vectors and study on single-chain antibody and reshaping single-domain antibody against CD3

    Institute of Scientific and Technical Information of China (English)

    刘喜富; 萧飒; 顾征; 王勇; 张卫国; 陈艾; 林晴; 黄华梁; 孙健; 陈润生; 沈倍奋; 陈兴

    1997-01-01

    Two vectors, pWA180 and pROH80, for expression of single-chain Fv fragments (ScFv) were con-siruciea. (?)ne anti-CD3 VH and VL genes were amplified from UCHTl cells by RT-PCR and sequenced. Both genes were cloned in pWA180 to express native ScFv and pROH80 for GST-ScFv fusion protein expression. The expression products were analysed by ELISA and Western blot. The combining site of OKT3 was modeled. Human [g LS1 and Nd were selected as acceptors of CDRs of OKT3 VL and VH to construct a reshaping antibody against CD3. By com-paring OKT3, LS1 and Nd with their own family sequences, some residues were changed and the reshaping VL and VH genes were designed. The full VH gene was assembled in three steps with eight chemically synthesized oligonu-cleotide fragments using overlapping PCR and sequenced. The VH gene was expressed as active protein in pCOMB3 and as inclusion bodies in pGEX-4T-l by ELISA and Western blot analysis.

  4. Protein expression and preparation of polydonal antibody of AD-004 and study on its expression in the adrenal and testis

    Institute of Scientific and Technical Information of China (English)

    乔洁

    2006-01-01

    Objective To prepare rabbit antibody against mouse AD-004 by AD-004 expressed in the prokaryotic expression system and to identify its distribution in the testis and adrenal. Methods The full-length cDNA of mouse AD-004 was cloned into PET28 plasmid, and the protein was induced in E. coli BL21 bacteria by adding IPTG and then purified by Ni2+ -NTA column. The purified protein was used as an immunogene to prepare polyclonal

  5. 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.

  6. 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.

  7. Response of early-weaned pigs to an enterotoxigenic Escherichia coli (K88) challenge when fed diets containing spray-dried porcine plasma or pea protein isolate plus egg yolk antibody, zinc oxide, fumaric acid, or antibiotic.

    Science.gov (United States)

    Owusu-Asiedu, A; Nyachoti, C M; Marquardt, R R

    2003-07-01

    The effect of feeding diets containing either spray-dried porcine plasma (SDPP) or pea protein-isolate (PPI) supplemented with either egg yolk antibodies (EYA) from hens immunized with enterotoxigenic Escherichia coli (ETEC) (K88 and F18) antigens, ZnO, fumaric acid (FA), or carbadox (AB) on pig performance, incidence of scours, and gut morphology was studied in a 14-d experiment. Ninety 10-d-old weaned pigs were assigned to six dietary treatments in a completely randomized design to give five pens per treatment with three pigs per pen. The diets were SDPP without EYA (SDPP - EYA), PPI without EYA (PPI - EYA), PPI with EYA (PPI + EYA), PPI with ZnO (PPI + ZnO), PPI with FA (PPI + FA), or PPI with AB (PPI + AB). Diets were formulated to similar nutrient levels, with AB, EYA, FA, and ZnO at 0.25, 0.5, 2.0, and 0.4% of the diet, respectively. Pigs were weighed and bled on d 0, 7, and 14 to determine plasma urea N (PUN). Pigs were orally challenged with a 6-mL dose of 10(10) cfu/mL ETEC (K88) on d 7. On d 14, three pigs per treatment were killed to obtain sections of the small intestine for histological measurements. Weekly feed intake, BW changes, and gain:feed were determined. Incidence of scours and scour scores were monitored and fecal swabs were taken before and after ETEC challenge for PCR test to detect ETEC (K88). Feeding SDPP or supplementing PPI-based diets with EYA, ZnO, FA, or AB did not affect (P > 0.05) ADG, ADFI (as-fed basis), or gain:feed throughout the study. However, pigs fed PPI - EYA tended to have lower (P = 0.08) ADFI during wk 2 (137.9 g/d) and lower (P pigs 8 h after the ETEC challenge, it lasted only 3 to 5 d in pigs fed SDPP or PPI supplemented with EYA, ZnO, FA, or AB. Pigs fed PPI - EYA continued to have severe diarrhea, resulting in 40% mortality vs. 13% or less in the other groups. The PCR results showed that 81% of PPI-fed pigs continued to shed ETEC K88 7 d after ETEC challenge. Pigs fed PPI-EYA had shorter villi (P pigs to efficiently

  8. Pea3 transcription factors and wnt1-induced mouse mammary neoplasia.

    Directory of Open Access Journals (Sweden)

    Rebecca Baker

    Full Text Available The role of the PEA3 subfamily of Ets transcription factors in breast neoplasia is controversial. Although overexpression of PEA3 (E1AF/ETV4, and of the related factors ERM (ETV5 and ER81 (ETV1, have been observed in human and mouse breast tumors, PEA3 factors have also been ascribed a tumor suppressor function. Here, we utilized the MMTV/Wnt1 mouse strain to further interrogate the role of PEA3 transcription factors in mammary tumorigenesis based on our previous observation that Pea3 is highly expressed in MMTV/Wnt1 mammary tumors. Pea3 expression in mouse mammary tissues was visualized using a Pea3(NLSlacZ reporter strain. In normal mammary glands, Pea3 expression is predominantly confined to myoepithelial cells. Wnt1 transgene expression induced marked amplification of this cell compartment in nontumorous mammary glands, accompanied by an apparent increase in Pea3 expression. The pattern of Pea3 expression in MMTV/Wnt1 mammary glands recapitulated the cellular profile of activated beta-catenin/TCF signaling, which was visualized using both beta-catenin immunohistochemistry and the beta-catenin/TCF-responsive reporter Axin2(NLSlacZ. To test the requirement for PEA3 factors in Wnt1-induced tumorigenesis, we employed a mammary-targeted dominant negative PEA3 transgene, DeltaNPEA3En. Expression of DeltaNPEA3En delayed early-onset tumor formation in MMTV/Wnt1 virgin females (P = 0.03, suggesting a requirement for PEA3 factor function for Wnt1-driven tumor formation. Consistent with this observation, expression of the DeltaNPEA3En transgene was profoundly reduced in mammary tumors compared to nontumorous mammary glands from bigenic MMTV/Wnt1, MMTV/DeltaNPEA3En mice (P = 0.01. Our data provide the first description of Wnt1-mediated expansion of the Pea3-expressing myoepithelial compartment in nontumorous mammary glands. Consistent with this observation, mammary myoepithelium was selectively responsive to Wnt1. Together these data suggest the MMTV

  9. Pea (Pisum sativum L.)

    Science.gov (United States)

    Pea belongs to the Leguminosae plant family, the third largest flowering plant family with 800 genera and over 18,000 species. Tribe Fabeae is considered one of the youngest groups in the legumes and Bayesian molecular clock and ancestral range analysis suggest a crown age of 23 – 16 Mya, in the mi...

  10. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK.

    Science.gov (United States)

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK.

  11. Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK

    Science.gov (United States)

    Damm-Welk, Christine; Siddiqi, Faraz; Fischer, Matthias; Hero, Barbara; Narayanan, Vignesh; Camidge, David Ross; Harris, Michael; Burke, Amos; Lehrnbecher, Thomas; Pulford, Karen; Oschlies, Ilske; Siebert, Reiner; Turner, Suzanne; Woessmann, Wilhelm

    2016-01-01

    Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK. PMID:27471553

  12. A novel lipid transfer protein from the pea Pisum sativum: isolation, recombinant expression, solution structure, antifungal activity, lipid binding, and allergenic properties

    OpenAIRE

    Bogdanov, Ivan V.; Shenkarev, Zakhar O.; Finkina, Ekaterina I.; Melnikova, Daria N.; Rumynskiy, Eugene I.; Arseniev, Alexander S.; Ovchinnikova, Tatiana V.

    2016-01-01

    Background Plant lipid transfer proteins (LTPs) assemble a family of small (7–9 kDa) ubiquitous cationic proteins with an ability to bind and transport lipids as well as participate in various physiological processes including defense against phytopathogens. They also form one of the most clinically relevant classes of plant allergens. Nothing is known to date about correlation between lipid-binding and IgE-binding properties of LTPs. The garden pea Pisum sativum is widely consumed crop and i...

  13. Clinical significance of changes of expression of anti-dsDNA antibody in serum in patients with SLE

    International Nuclear Information System (INIS)

    Objective: To investigate the significance of anti-dsDNA antibody in diagnosis and treatment of SLE through measurement of changes of serum anti-dsDNA antibody expression in patients with SLE. Methods: Serum anti-dsDNA antibody was detected with radioisotope method in 60 patients with SLE and 33 controls (consisted of patients with other collagen diseases including Sjogren syndrome, systemic sclerosis, rheumatoid arthritis, polymyositis, mixed connective tissue disease, ankylosing spondylitis). Clinical manifestation and laboratory findings in the SLE patients were studied in detail. Results: (1) Serum anti-dsDNA antibody was positive in 39 of the 60 SLE patients with only two false positive cases in the 33 controls: a sensitivity of 65% and specificity of 93. 3%. (2) In SLE patients, positivity of anti-dsDNA antibody was not correlated with positivity of anti-Sm antibody (P>0.05), but was correlated with positivity of anti-SSA antibody (P<0.05). (3) Incidences of alopecia, skin rashes, oral mucosal ulcer, proteinuria were significantly higher in SLE patients with positive anti-dsDNA antibody than those in SLE patients with negative anti-dsDNA antibody (P<0.05). (4) Incidences of leukopenia and thrombocytopenia were also significantly higher in SLE patients with positive anti-dsDNA antibody (P<0.05). Conclusion: Anti-dsDNA antibody could be taken as a specific marker of SLE and the serum expression were positively correlated with the activity and severity of the disease. (authors)

  14. Attraction of pea moth Cydia nigricana to pea flower volatiles.

    Science.gov (United States)

    Thöming, Gunda; Knudsen, Geir K

    2014-04-01

    The pea moth Cydia nigricana causes major crop losses in pea (Pisum sativum) production. We investigated attraction of C. nigricana females to synthetic pea flower volatiles in a wind tunnel and in the field. We performed electroantennogram analysis on 27 previously identified pea plant volatiles, which confirmed antennal responses to nine of the compounds identified in pea flowers. A dose-dependent response was found to eight of the compounds. Various blends of the nine pea flower volatiles eliciting antennal responses were subsequently studied in a wind tunnel. A four-compound blend comprising hexan-1-ol, (E)-2-hexen-1-ol, (Z)-β-ocimene and (E)-β-ocimene was equally attractive to mated C. nigricana females as the full pea flower mimic blend. We conducted wind-tunnel tests on different blends of these four pea flower compounds mixed with a headspace sample of non-flowering pea plants. By considering the effects of such green leaf background odour, we were able to identify (Z)- and (E)-β-ocimene as fundamental for host location by the pea moths, and hexan-1-ol and (E)-2-hexen-1-ol as being of secondary importance in that context. In the field, the two isomers of β-ocimene resulted in trap catches similar to those obtained with the full pea flower mimic and the four-compound blend, which clearly demonstrated the prime significance of the β-ocimenes as attractants of C. nigricana. The high level of the trap catches of female C. nigricana noted in this first field experiment gives a first indication of the potential use of such artificial kairomones in pea moth control.

  15. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    Directory of Open Access Journals (Sweden)

    Kiefer Hans

    2009-06-01

    Full Text Available Abstract Background RAI3 is an orphan G-protein coupled receptor (GPCR that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. Methods We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA, western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147 and normal breast tissues (n = 44 using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50 as well as lymph node metastases (n = 3 for RAI3 mRNA expression. Results The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. Conclusion We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic

  16. Production and characterisation of monoclonal antibodies against RAI3 and its expression in human breast cancer

    International Nuclear Information System (INIS)

    RAI3 is an orphan G-protein coupled receptor (GPCR) that has been associated with malignancy and may play a role in the proliferation of breast cancer cells. Although its exact function in normal and malignant cells remains unclear and evidence supporting its role in oncogenesis is controversial, its abundant expression on the surface of cancer cells would make it an interesting target for the development of antibody-based therapeutics. To investigate the link with cancer and provide more evidence for its role, we carried out a systematic analysis of RAI3 expression in a large set of human breast cancer specimens. We expressed recombinant human RAI3 in bacteria and reconstituted the purified protein in liposomes to raise monoclonal antibodies using classical hybridoma techniques. The specific binding activity of the antibodies was confirmed by enzyme-linked immunosorbent assay (ELISA), western blot and immunocytochemistry. We carried out a systematic immunohistochemical analysis of RAI3 expression in human invasive breast carcinomas (n = 147) and normal breast tissues (n = 44) using a tissue microarray. In addition, a cDNA dot blot hybridisation assay was used to investigate a set of matched normal and cancerous breast tissue specimens (n = 50) as well as lymph node metastases (n = 3) for RAI3 mRNA expression. The anti-RAI3 monoclonal antibodies bound to recombinant human RAI3 protein with high specificity and affinity, as shown by ELISA, western blot and ICC. The cDNA dot blot and immunohistochemical experiments showed that both RAI3 mRNA and RAI3 protein were abundantly expressed in human breast carcinoma. However, there was no association between RAI3 protein expression and prognosis based on overall and recurrence-free survival. We have generated a novel, highly-specific monoclonal antibody that detects RAI3 in formaldehyde-fixed paraffin-embedded tissue. This is the first study to report a systematic analysis of RAI3 expression in normal and cancerous human

  17. Prokaryotic Expression of Glycoprotein Gene of Infectious Hnematopoietic Necrosis Virus and Polyclonal Antibody Preparation

    Institute of Scientific and Technical Information of China (English)

    Liu; Xueguang; Zheng; Huaidong; Guo; Xinshuo; Luo; Jin; Lin; Cuicui; Wang; Qiuyu

    2014-01-01

    [Objective]The aim is to perform prokaryotic expression of the glycoprotein gene of infectious hnematopoietic necrosis virus and polyclonal antibody preparation. [Methods]Glycoprotein gene( G) of infectious hematopoietic tissue( IHNV) was synthesized,cloned to prokaryotic expression system pET-30a vector,yielding the recombinant plasmid pET-30a-IHNV-G. The yielded pET-30a-IHNV-G was transformed into E. coli strain BL21( DE3) plySs. [Results] SDSPAGE and Western blot results showed that protein G successfully expressed in E. coli at 37 ℃,1 mmol /L IPTG induction for 4 h. The molecular weight of fusion G protein was 57 KD. The polyclonal antibody was prepared by immunizing mice with the product of gel purification. ELISA analysis showed that the serum titer reached 1∶10 000. [Conclusion]The expressed G protein and the serum with polyclonal antibody obtained in this study provided the theoretical basis for the development of IHNV vaccine and detection of colloidal gold test strip.

  18. Polyclonal antibody production and expression of CREG protein in human vascular smooth muscle cells

    Institute of Scientific and Technical Information of China (English)

    Yaling HAN; Haiwei LIU; Jian KANG; Xiaozeng WANG; Ye HU; Lianyou ZHAO; Shaohua LI

    2005-01-01

    Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.

  19. PEA3 activates CXCR4 transcription in MDA-MB-231 and MCF7 breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Shengmei Gu; Li Chen; Qi Hong; Tingting Yan; Zhigang Zhuang; Qiaoqiao wang; Wei Jin; Hua Zhu; Jiong Wu

    2011-01-01

    CXC chemokine receptor 4 (CXCR4) is a cell surface receptor that has been shown to mediate the metastasis of many solid tumors including lung,breast,kidney,and prostate tumors.In this study,we found that overexpression of ets variant gene 4 (PEA3) could elevate CXCR4 mRNA level and CXCR4 promoter activity in human MDA-MB-231 and MCF-7 breast cancer cells.PEA3 promoted CXCR4 expression and breast cancer metastasis.Chromatin immunoprecipitation assay demonstrated that PEA3 could bind to the CXCR4 promoter in the cells transfected with PEA3 expression vector.PEA3 siRNA attenuated CXCR4 promoter activity and the binding of PEA3 to the CXCR4 promoter in MDA-MB-231 and MCF-7 cells.These results indicated that PEA3 could activate CXCR4 promoter transcription and promote breast cancer metastasis.

  20. Construction and Expression of Human Survivin and Preparation of Its Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hai-hong; ZHANG Xi-zhen; ZHAO Dong-hai; SHI He-liang; YU Yong-hui; WU Yong-ge; YU Xiang-hui; KONG Wei

    2008-01-01

    Survivin,a novel member of inhibitor ofapoptosis(IAP) protein family,is aberrantly expressed in cancer but undetectable in normal,differentiated adult tissues.The cancer-specific expression of survivin,coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment.Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B.The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21,and the relative molecule mass(Mr) of expressed fusion protein was approximately 21000.The recombinantprotein was purified through Ni2~ affinity chromatography column and characterized by SDS-PAGE and Western blot.The purified recombinant protein was then injected into rabbits,and antisurvivin polyclonal antibody with a high titer was obtained.

  1. Prokaryotic expression of soluble Arabidopsis protein AtERF1 and preparation of its polyclonal antibodies

    Directory of Open Access Journals (Sweden)

    ZHANG Yu

    2013-08-01

    Full Text Available AtERF1 encodes a member of the ERF subfamily B-3 of ERF/AP2 transcription factor family.It has been demonstrated almost every member of the B3 subgroup of AP2/ERF genes is involved in defense responses in Arabidopsis.Codon usage within a gene is a critical determinant of achievable protein expression level in E.coli. Gene optimization,therefore,is an effective method for synthetic genes with the aim of enhancing soluble expression,particular in heterologous hosts.In this paper,the AtERF1 protein of Arabidopsis thaliana was expressed in Escherichia coli using its optimized DNA sequence for E.coli. and yielded high level of soluble AtERF-1 protein in recombinant E.coli. The AtERF1 protein was used as an antigen to immune rabbits and obtains high titer antibodies.The immunological specificity of the polyclonal antibodies to AtERF1 was confirmed by Western blot assay.The prepared antibody in this work would facilitate the further functional investigation of AtERF1 in biochemical levels in Arabidopsis anther development.

  2. 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

  3. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells

    OpenAIRE

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven CL; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC ...

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

    Institute of Scientific and Technical Information of China (English)

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

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Eerd, Julliette E.M. van; Geus-Oei, Lioe-Fee de; Oyen, Wim J.G.; Corstens, Frans H.M.; Boerman, Otto C. [Radboud University Nijmegen Medical Centre, Department of Nuclear Medicine, HB Nijmegen (Netherlands)

    2006-11-15

    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 {sup 131}I-15D3 or {sup 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 {sup 111}In-DTPA-15D3 than with {sup 131}I-15D3. After injection of {sup 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 {sup 111}In-DTPA-15D3). The {sup 111}In-labelled monoclonal anti-P-gp antibody clearly visualised P-gp expression in a human uterine sarcoma tumour in nude mice. (orig.)

  6. Decreased humoral antibody episodes of acute renal allograft rejection in recipients expressing the HLA-DQβ1*0202 allele.

    Science.gov (United States)

    Mannam, Venkat K R; Santos, Mark; Lewis, Robert E; Cruse, Julius M

    2012-10-01

    The present investigation was designed to show the effect of human leukocyte antigen (HLA) class II molecular allelic specificities in the recipient on the induction of humoral antibody rejection, identified by C4d peritubular capillary staining, as well as specific antibody identified by Luminex technology. Major histocompatibility complex (MHC) class II molecules are expressed on dendritic cells, macrophages, and B lymphocytes and they present antigenic peptides to CD4 positive T lymphocytes. Human renal peritubular and glomerular capillaries express class II MHC molecules upon activation. Expression of class II molecules on renal microvascular endothelial cells exposes them to possible interaction with specific circulating antibodies. We hypothesize that HLA-DQβ1*0202 expression in recipients decreases the likelihood of antibody-mediated renal allograft rejection. We found that 80% (=25) of DQ2 positive haplotype recipients failed to induce humoral antibody renal allograft rejection and 20% (n=25) of DQ2 positive haplotype recipients induced humoral antibody renal allograft rejection (p=0.008). By contrast, 48% (n=46) of DQ2 negative haplotype recipients failed to induce a humoral antibody component of renal allograft rejection and 52% (n=46) of DQ2 negative haplotype recipients induced humoral antibody-mediated renal allograft rejection. Our results suggest that recipients who express the DQβ1*0202 allele are less likely to induce a humoral antibody component of acute renal allograft rejection than are those expressing DQ1, DQ3, or DQ4 alleles. DQβ1*0202 allele expression in recipients could possibly be protective against acute humoral allograft rejection and might serve as a future criterion in recipient selection and in appropriate therapy for acute renal rejection episodes.

  7. Expression of rice dwarf phytoreovirus Pns6 and the specificity analysis of its monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18℃) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃ after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed. Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th-509th amino acids) of Pns6, which is confirms bioinformatics analysis.

  8. Expression of rice dwarf phytoreovirus Pns6 and the specificity analysis of its monoclonal antibodies

    Institute of Scientific and Technical Information of China (English)

    JI Xu; WEI ChunHong; LI Yi

    2009-01-01

    The genome of rice dwarf phytoreovirus (RDV) is composed of 12 double-stranded RNA segments, of which segment S6 encodes a non-structural protein Pns6 identified as the movement protein. In this report, Pns6 with a 6-histidine tag at the N-terminal was expressed in E. coli after induction under low temperature (18℃) and low concentration (0.4 mmol/L and 0.2 mmol/L) of IPTG, and then purified by Ni-chelated affinity chromatography. Stability analysis indicated that the expressed HisPns6 protein was stable at 37℃ after 24 h treatment. This recombinant protein was then used to make monoclonal antibody. Total 18 hybridoma clones were obtained. The specificity of antibodies was tested by Western blot using native Pns6 extracted from RDV-infected rice leaves, and 15 positive clones were confirmed.Mapping of the antigenic sites of Pns6 using antibodies showed that the most sensitive antigen determinant is located in the C-terminal region (the 296th-509th amino acids) of Pns6, which is confirms bioinformatics analysis.

  9. PED/PEA-15在前列腺癌中的表达及对前列腺癌细胞凋亡的影响%Expression of PED/PEA-15 in Prostate Cancer and Its Effect on Apoptosis of Prostate Cancer Cell

    Institute of Scientific and Technical Information of China (English)

    胡晓勇; 陈晓春; 朱朝辉; 曾甫清; 鲁功成

    2006-01-01

    目的通过构建PED/PEA-15特异的siRNA载体,探讨PED/PEA-15对前列腺癌细胞(PC-3)凋亡的影响.方法应用免疫组化SP法检测前列腺癌和正常前列腺组织中PED/PEA-15的表达;用RT-PCR法检测PC-3细胞中PED/PEA-15的表达.体外合成PED/PEA-15特异性siRNA序列,并克隆入真核表达载体psiRNA-hH1neo.以脂质体法转染psiRNA-PED/PEA-15至PC-3细胞中,以RT-PCR和Western blot法检测psiRNA-PED/PEA-15对PED/PEA-15表达的影响;用MTT和流式细胞术检测其对PC-3细胞凋亡的影响.结果免疫组化和RT-PCR均显示PED/PEA-15在前列腺癌中高表达;正常前列腺组织没有或只有很弱的表达.酶切和DNA测序证实合成的siRNA基因序列正确,并已被准确克隆入psiRNA-hH1neo载体.psiRNA-PED/PEA-15可特异性抑制PC-3细胞中PED/PEA-15的表达.转染psiRNA-PED/PEA-15的PC-3细胞凋亡显著增加(P<0.05).结论PED/PEA-15可阻抑前列腺癌细胞凋亡,其表达对前列腺癌的发展有重要作用.

  10. 29 CFR 780.139 - Pea vining.

    Science.gov (United States)

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Pea vining. 780.139 Section 780.139 Labor Regulations... âsuch Farming Operationâ-of the Farmer § 780.139 Pea vining. Vining employees of a pea vinery located on a farm, who vine only the peas grown on that particular farm, are engaged in agriculture. If...

  11. Expression of HSV-1 ICP0 Antigen Peptide in Prokaryotic Cells and Preparation of Specific Antibody

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    As an immediate-early protein of herpes simplex virus, infected-cell polypeptide 0 (ICP0) exhibits complicated interactions with host cells, and its regulatory function on gene expression is of great importance. Since the ICP0 encoding sequence contains many rare codons which are absent in E.coli, and ICP0 is highly unstable in prokaryotic cells, expression of entire ICP0 in prokaryotic cells has never been reported. In order to further investigate the function of ICP0, a recombinant plasmid was constructed by subcloning a cDNA fragment encoding an amino-terminal of 105 residues of the ICP0 protein into pGEX-5x-1 vector. The resulting GST-105 fusion antigen peptide was expressed with high efficiency in E.coli. Antibodies prepared after the immunization of mice with purified fusion protein can recognize not only the denatured ICP0 protein, but also the native ICP0 protein with normal biological conformation.

  12. Expression of PED/PEA-15 and XIAP in prostate cancer cells and their effects on prostate cancer cell (PC-3) apoptosis%抗凋亡因子XIAP和PED/PEA-15在前列腺癌(PC-3)中的表达及对细胞凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    胡晓勇; 陈晓春; 朱朝辉; 曾甫清; 鲁功成

    2006-01-01

    目的检测抗凋亡因子XIAP与PED/PEA-15在前列腺癌细胞(PC-3)中的表达,探讨二者对前列腺癌细胞凋亡的影响.方法应用半定量RT-PCR法检测前列腺癌细胞(PC-3)中PED/PEA-15和XIAP的表达.设计并构建PED/PEA-15和XIAP特异的siRNA载体,以脂质体法转染二者的siRNA载体至前列腺癌细胞(PC-3)中,半定量RT-PCR法检测特异siRNA载体对PED/PEA-15和XIAP转录的影响;光镜观察细胞形态改变;流式细胞法检测细胞凋亡的变化.结果半定量RT-PCR显示PED/PEA-15和XIAP均在前列腺癌细胞(PC-3)中高表达.酶切和DNA测序证实XIAP和PED/PEA-15 siRNA载体构建成功.共转染XIAP和PED/PEA-15 siRNA载体入PC-3细胞,可导致XIAP和PED/PEA-15的转录抑制,并增加PC-3细胞对阿霉素的敏感性,凋亡明显增加,处理组凋亡率为79%,对照组为46%,两组差异有统计学意义(P<0.05).结论PED/PEA-15和XIAP在前列腺癌的凋亡中可起重要作用.

  13. Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma

    Institute of Scientific and Technical Information of China (English)

    Jin-Liang Xing; Xiang-Min Yang; Xi-Ying Yao; Fei Song; Zhi-Nan Chen

    2004-01-01

    AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody.METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then,the competent E. colicells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting,ELISA and HPLC.RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 μg/mL. The renatured cFab could specifically bind to related antigen with high affinity.CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

  14. Yellow pea fiber improves glycemia and reduces Clostridium leptum in diet-induced obese rats.

    Science.gov (United States)

    Eslinger, Amanda J; Eller, Lindsay K; Reimer, Raylene A

    2014-08-01

    Numerous studies have demonstrated the impact of functional fibers on gut microbiota and metabolic health, but some less well-studied fibers and/or fractions of foods known to be high in fiber still warrant examination. The aim of this study was to assess the effect of yellow pea-derived fractions varying in fiber and protein content on metabolic parameters and gut microbiota in diet-induced obese rats. We hypothesized that the yellow pea fiber (PF) fraction would improve glycemia and alter gut microbiota. Rats were randomized to 1 of 5 isoenergetic dietary treatments for 6 weeks: (1) control; (2) oligofructose (OFS); (3) yellow PF; (4) yellow pea flour (PFL); or (5) yellow pea starch (PS). Glycemia, plasma gut hormones, body composition, hepatic triglyceride content, gut microbiota, and messenger RNA expression of genes related to hepatic fat metabolism were examined. Pea flour attenuated weight gain compared with control, PF, and PS (P Pea flour, PS, and OFS had significantly lower final percent body fat compared with control. Oligofructose but not the pea fraction diets reduced food intake compared with control (P Pea fiber resulted in lower fasting glucose and glucose area under the curve compared with control. Changes in gut microbiota were fraction specific and included a decrease in Firmicutes (percent) for OFS, PF, and PFL compared with control (P yellow pea-derived fractions are able to distinctly modulate metabolic parameters and gut microbiota in obese rats. PMID:25156790

  15. Yellow pea fiber improves glycemia and reduces Clostridium leptum in diet-induced obese rats.

    Science.gov (United States)

    Eslinger, Amanda J; Eller, Lindsay K; Reimer, Raylene A

    2014-08-01

    Numerous studies have demonstrated the impact of functional fibers on gut microbiota and metabolic health, but some less well-studied fibers and/or fractions of foods known to be high in fiber still warrant examination. The aim of this study was to assess the effect of yellow pea-derived fractions varying in fiber and protein content on metabolic parameters and gut microbiota in diet-induced obese rats. We hypothesized that the yellow pea fiber (PF) fraction would improve glycemia and alter gut microbiota. Rats were randomized to 1 of 5 isoenergetic dietary treatments for 6 weeks: (1) control; (2) oligofructose (OFS); (3) yellow PF; (4) yellow pea flour (PFL); or (5) yellow pea starch (PS). Glycemia, plasma gut hormones, body composition, hepatic triglyceride content, gut microbiota, and messenger RNA expression of genes related to hepatic fat metabolism were examined. Pea flour attenuated weight gain compared with control, PF, and PS (P Pea flour, PS, and OFS had significantly lower final percent body fat compared with control. Oligofructose but not the pea fraction diets reduced food intake compared with control (P Pea fiber resulted in lower fasting glucose and glucose area under the curve compared with control. Changes in gut microbiota were fraction specific and included a decrease in Firmicutes (percent) for OFS, PF, and PFL compared with control (P pea-derived fractions are able to distinctly modulate metabolic parameters and gut microbiota in obese rats.

  16. 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.

  17. Reduced toxicity of expression, in Escherichia coli, of antipollutant antibody fragments and their use as sensitive diagnostic molecules.

    Science.gov (United States)

    Strachan, G; Williams, S; Moyle, S P; Harris, W J; Porter, A J

    1999-09-01

    Single-chain antibody fragments (scAb), specific for the chlorophenoxy acid herbicide mecoprop, have been expressed and purified from the bacterium Escherichia coli. Co-expression with the colE1-compatible, arabinose-inducible, skp expression vector pHELP1 prevented bacterial lysis and significantly increased both total and functional expression yield. The periplasmic protein, SKP, may have a role as a generic detoxification protein. Surface plasmon resonance (BIAcore 2000) analysis confirmed that the purified scAb retained similar binding kinetics to the monoclonal antibody (Mab) from which it was cloned. In competition ELISA, the bacterial scAb showed the same specificity for mecoprop and a related herbicide, MCPA, as the Mab but an increase in sensitivity for free antigen in all ELISA formats. Bacterially expressed antibody fragments provide a simple, sensitive and cost-effective alternative to the traditional production of diagnostic Mabs via tissue culture.

  18. 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....... None of the variables studied were associated with clinical disease activity. GA treatment resulted in the development of IgG and IgG4 anti-GA antibodies during the first months of treatment, persisting during long-term treatment. Conclusions: The observed relationship between the expression of m...

  19. Construction and expression of D-dimer and GPIIb/IIIa single-chain bispecific antibody

    OpenAIRE

    DAN, ZHAOKUI; Tan, Zui; Xia, Hongli; WU, GAN

    2013-01-01

    The aim of this study was to construct a plasmid expressing glycoprotein IIb-IIIa (GPIIb/IIIa) and D-dimer single-chain bispecific antibody for the targeted therapy of thrombosis. The phosphorylated gene encoding the anti-GPIIb/IIIa single-chain variable fragment (scFv) and the gene encoding the anti-D-dimer scFv were amplified by PCR and linked in tandem by blunt-end ligation. The recombinant plasmid was transfected into the competent cell line HB2151 and identified by PCR and DNA sequencing...

  20. 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

  1. 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

  2. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    Science.gov (United States)

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy. PMID:27112931

  3. A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

    Science.gov (United States)

    Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

    2016-12-01

    Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

  4. GROWTH, INSTABILITY AND FORECASTING OF PIGEON PEA, CHICKPEA AND FIELD PEA PULSE PRODUCTION IN BANGLADESH

    OpenAIRE

    Rahman, Niaz Md. Farhat; Imam, M. F.

    2008-01-01

    The study tried to find out the appropriate models using latest model selection criteria that could describe the best growth pattern of pigeon pea, chickpea and field pea pulse production. The study also tried to measure the instability, growth rates of pigeon pea, chickpea and field pea pulse production and to determine the efficient time series models, to forecast the future pigeon pea, chickpea and field pea pulse production in Bangladesh. Forecasting attempts have been made to achieve the...

  5. P53 FUSION PROTEIN EXPRESSION IN PROKARYOTE AND PREPARATION OF MONOCLONAL ANTIBODY TO P53

    Institute of Scientific and Technical Information of China (English)

    Liu Caiyun; Shou Chengchao; Sun Sulian; ZhangLei; Zeng Li

    1998-01-01

    Objective: Conventional immunohistochemistry (IHC) is available to assess P53 mutations, and expensive imported anti-P53 monoclonal antibody has been used in China, it is necessary to study a new monoclonal antibody.Methods: The P53 DNA fragment enconding N-terminal 180 amiao acide was obtained by PCR and was cloned into PGEX-2T plasmid expressing glutathione S-transferase (GST). The P53-GST fusion protein expressed by JM109was used for immunizing BALB/C mice. We have raised one hybridoma strain secreting McAb to human P53(named M126). Results: The IHC analysis of 52paraffin-embedded sections from human breast cancer with M126 and PAB1801 (Zymed Co.) has showed that the positive immunoreactions were 25 cases (48%) and 22cases (42.3%) respectively. The staining of M126 was stronger and preferable to PAB1801. Conclusion: M126can be instead of PAB1801 for studying immunohistochemical analysis on P53 Protein.

  6. Expression of Intracellular Domain of Epidermal Growth Factor Receptor and Generation of Its Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    Ying Lin; Zhiduo Liu; Jianmin Jiang; Ziqing Jiang; Yongyong Ji; Bing Sun

    2004-01-01

    To prepare monoclonal antibody specific to epidermal growth factor receptor (EGFR) intracellular domain, its gene was amplified from total RNA of A431 cell by RT-PCR. Then the gene was cloned into prokaryotic vector pET30a(+). The recombinant plasmid was transformed into E. Coli BL21 (DE3) strain for protein expression.Recombinant protein was induced with IPTG and purified using Ni2+-NTA agarose. Then the anti-EGFR monoclonal antibody (nAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked inmunoabsorbent assay (ELISA). Totally 4 hybridoma clones were obtained and these mAbs were IgG1 (3 clones) and IgG2a (1 clone), respectively. Their light chains were all kappa chains.Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line. The mAbs will be useful in the study of EGFR-mediated signal transduction.

  7. Expression of Intracellular Domain of Epidermal Growth Factor Receptor and Generation of Its Monoclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    YingLin; ZhiduoLiu; JianminJiang; ZiqingJiang; Yongyongji; BingSun

    2004-01-01

    To prepare monoclonal antibody specific to epidermal growth factor receptor (EGFR) intracellular domain, its gene was amplified from total RNA of A431 cell by RT-PCR. Then the gene was cloned into prokaryotic vector pET30a(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) strain for protein expression. Recombinant protein was induced with IPTG and purified using Nie2+-NTA agarose. Then the anti-EGFR monoclonal antibody (mAb) was prepared with classical hybridoma technique. Positive clones were selected using indirect enzyme-linked immunoabsorbent assay (ELISA). Totally 4 hybridoma clones were obtained and these mAbs were IgG1 (3 clones) and IgG2a (1 clone), respectively. Their light chains were all kappa chains. Western blotting analysis and confocal immunofluorescence assays demonstrated that mAbs could specifically recognize EGFR expressing on A431 carcinoma cell line. The mAbs will be useful in the study of EGFR-mediated signal transduction. Cellular & Molecular Immunology. 2004;1(2):137-141.

  8. Prospective study on the expression of cancer testis genes and antibody responses in 100 consecutive patients with primary breast cancer.

    NARCIS (Netherlands)

    Mischo, A.; Kubuschok, B.; Ertan, K.; Preuss, K.D.; Romeike, B.; Regitz, E.; Schormann, C.; Bruijn, D.R.H. de; Wadle, A.; Neumann, F.; Schmidt, W.; Renner, C.; Pfreundschuh, M.

    2006-01-01

    To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohis

  9. Identification of antigens by monoclonal antibody PD4 and its expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    Jin-Ying Ning; Guo-Xun Sun; Su Huang; Hong Ma; Ping An; Lin Meng; Shu-Mei Song; Jian Wu; Cheng-Chao Shou

    2003-01-01

    AIM: To clone and express the antigen of monoclonal antibody (Mab) PD4 for further investigation of its function.METHODS: MGC803 cDNA expression library was constructed and screened with PD4 as probes to clone the antigen. After failed in the library screening, immunoprecipitation and SDSpolyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Nycoplasma hyorhinis (M. Hyorhinis) was further confirmed with Western blot analysis by infecting M. Hyorhinis-free HeLa cells and eliminating the M. Hyorhinis from MGC803cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations.Tmmunofluorescence assay was used to demonstrate if p37protein could directly bind to gastric tumor cell AGS.RESULTS: The cDNA library constructed with MGC803 cells was screened by Mab PD4 as probes. Unfortunately, the positive clones identified with Mab PD4 were also reacted with unrelated antibodies. Then, immunoprecipitation was performed and the purified antigen was identified to be a membrane protein of Mycoplasrna hyorhinis (M. Hyorhinis)by sequencing of N-terminal amino acid residues. The membrane protein was intensively verified with Western blot by eliminating M. Hyorhinis from MGC803 cells and by infecting M. Hyorhinis-free HeLa cells. The full p37 gene was cloned and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence demonstrated that p37protein could directly bind to gastric tumor cell AGS.CONCLUSION: The antigen recognized by Mab PD4 is from M. Hyorhinis, which suggests the actions involved in Mab PD4 is possibly mediated by p37 protein or M. Hyorhinis. As p37 protein can bind directly to tumor cells, the pathogenic role of p37 involved in tumorigenesis justifies further investigation.

  10. 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

  11. 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.

  12. Conditional expression of full-length humanized anti-prion protein antibodies in Chinese hamster ovary cells.

    Science.gov (United States)

    Mueller, Daniel A; Heinig, Lars; Ramljak, Sanja; Krueger, Astrid; Schulte, Reiner; Wrede, Arne; Stuke, Andreas W

    2010-12-01

    Because of their high antigen specificity and metabolic stability, genetically engineered human monoclonal antibodies are on the way to becoming one of the most promising medical diagnostics and therapeutics. In order to establish an in vitro system capable of producing such biosimilar antibodies, we used human constant chain sequences to design the novel human antibody expressing vector cassette pMAB-ABX. A bidirectional tetracycline (tet)-controllable promotor was used for harmonized expression of immunoglobulin type G (IgG) heavy and light chains. As an example we used anti-prion protein (anti-PrP) IgGs. Therefore, the variable heavy (V(H)) and light chain (V(L)) sequences of anti-PrP antibodies, previously generated in our laboratory by DNA immunization of prion protein knock-out mice, were isolated from murine hybridoma cell lines and inserted into pMAB-ABX vector. After transfection of Chinese hamster ovary (CHO) cells, a number of stable antibody producing cell clones were selected. One cell line (pMAB-ABX-13F10/3B5) stably expressing the recombinant humanized antibody (rechuAb) 13F10/3B5 was selected for detailed characterization by Western blot, immunofluorescence, and flow cytometric analyses. The full-length recombinant humanized IgG antibody showed a high level of expression in the cytoplasm. In conclusion, the new cell system described here is a suitable tool to produce functional intact full-length humanized IgG antibodies. PMID:21087094

  13. SPECT imaging of neuropilin receptor type-1 expression with 131I-labeled monoclonal antibody.

    Science.gov (United States)

    Dou, Xiaofeng; Yan, Jianghua; Zhang, Yafei; Liu, Peng; Jiang, Yizhen; Lv, Sha; Zeng, Fanwei; Chen, Xiaoli; Wang, Shengyu; Zhang, Haipeng; Wu, Hua; Zhang, Hong; Ouyang, Lin; Su, Xinhui

    2016-09-01

    As a novel co-receptor for vascular endothelial growth factor (VEGF), neuropilin receptor type-1 (NRP-1) is overexpressed in several cancers and metastases, and serves as an attractive target for cancer molecular imaging and therapy. Previous single photon emission computerized tomography (SPECT) studies demonstrated that the small NRP-1-targeting peptides 99mTc-MA-ATWLPPR and 99mTc-CK3 showed poor tumor imaging quality, because of their rapid blood clearance and very low tumor uptake. Compared with small peptides, monoclonal antibodies (mAbs) can improve imaging of NRP-1-expression, due to their high affinity, specificity and slow extraction. A6-11-26 is a novel monoclonal antibody against NRP-1 b1b2 domain that exhibits inhibition of tumor growth in NPR-1-expressing preclinical models. The aim of the present study was to develop the 131I-labeled anti-NRP-1 monoclonal antibody A6-11-26 as a SPECT probe for imaging of NRP-1-positive tumor. An anti-NRP-1 monoclonal antibody (A6-11-26) was produced by hybridomas and was labeled with iodine-131 by the iodogen method. In vitro, the radiolabeling efficiency, radiochemical purity, immunoreactive fraction and stability were assessed. Binding affinity and specificity of 131I‑A6-11-26 to NRP-1 were evaluated using human glioblastoma U87MG cells. In vivo, biodistribution and SPECT/CT studies were conducted on mice bearing U87MG xenografts after the injection of 131I-A6-11-26 with or without co-injection of unlabeled A6-11-26 antibody. A6-11-26 was generated successfully by hybridoma with high purity (>95%) and was labeled with iodine-131 within 60 min with high labelling efficiency (95.46±3.34%), radiochemical purity (98.23±1.41%). 131I-A6-11-26 retained its immunoreactivity and also displayed excellent stability in mouse serum and PBS solution during 1 to 96 h. Cell uptake assays showed high NRP-1-specific uptake (15.80±1.30% applied activity at 6 h) in U87MG cells. 131I-A6-11-26 bound to NRP-1 with low nanomolar

  14. Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    Jiong Cai; Fang Li; Shizhen Wang

    2008-01-01

    BACKGROUND: Studies have shown that monoclonal or polyclonal antibody injections ofamyloid β peptide arc effective in removing amyloid β peptide overload in the brain.OBJECTIVE: Based on successful screening of a human single-chain fragment variable antibody specific to amyloid β peptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide.DESIGN, TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006.MATERIALS: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library.METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a Teasy plasmid for pT-seFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvA β was cut by EcoRl and Notl endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvA β expression vector, which was confirmed by gene sequencing. Linearized pPICgK-scFvA β was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5 % methanol to express human single-chain fragment variable antibody specific to amyloid β peptide.MAIN OUTCOME MEASURES: Protein electrophoresis was used to identify PCR products, gene sequencing was uscd to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris.RESULTS: Gene sequencing confirmed pPICgK-scFvA β orientation. Rccomhinants were obtained by lineadzed pPIC9K-scFvA β transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size

  15. Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeast.

    Science.gov (United States)

    Doyle, Patrick J; Saeed, Hanaa; Hermans, Anne; Gleddie, Steve C; Hussack, Greg; Arbabi-Ghahroudi, Mehdi; Seguin, Charles; Savard, Marc E; Mackenzie, C Roger; Hall, J Christopher

    2009-12-11

    15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops. PMID:19783651

  16. Treponema pallidum-specific antibody expression for the diagnosis of different stages of syphilis

    Institute of Scientific and Technical Information of China (English)

    SUN Ran; LAI Di-hui; REN Rong-xin; LIAN Shi; ZHANG Hai-ping

    2013-01-01

    Background Tp15,Tp17,Tp45,and Tp47 are outer-membrane proteins found in Treponema pallidum,the etiologic agent of syphilis.These proteins are potent antigens and are potential markers for the serological detection of syphilis.The present study analyzed antibodies to these protein antigens (TP-IgM and TP-IgG) in human serum and investigated the expression of these antibodies during different stages of syphilis.Methods Serum samples were collected from 69 subjects (male 45,female 24) diagnosed with syphilis and analyzed by Western blotting for the expression of IgM and IgG against the four protein antigens.Expression levels of the target antibodies were compared during the same stage of syphilis as well as between different stages of this disease.Results In subjects with primary syphilis,the positive rate of Tp45 IgM was higher than that of other TP-IgM.Tp15 IgM was detected only in subjects with tertiary syphilis.Similarly,the seroprevalence of Tp45 IgG in primary syphilis was higher than for other TP-IgG No target TP-IgM was detected in subjects with latent syphilis.In subjects with secondary syphilis,the expression level of Tp15 IgG (138.73±20.16) was higher than for other target TP-IgG In subjects with tertiary syphilis,all target TP-IgG were detected.In subjects with tertiary or latent syphilis,the expression levels of Tp45 IgG (121.33±11.04 and 110.10±40.19,respectively) were higher than those of other target TP-IgG.The expression levels of all Tp-IgM were similar before or after anti-syphilis treatment.In comparison,the expression levels of all TP-IgG decreased compared with the pre-treatment levels,and this decrease was statistically significant (both P <0.05) for Tp17 IgG and Tp47 IgG.Conclusions After Treponema pallidum infection,Tp45 IgM appeared first and Tp15 IgM occurred during later stages.The positive rates of all TP-IgG increased with the duration of this disease.Anti-syphilis treatment reduced the expression levels of Tp17 IgG and Tp47 Ig

  17. 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.

  18. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells.

    Science.gov (United States)

    Chng, Jake; Wang, Tianhua; Nian, Rui; Lau, Ally; Hoi, Kong Meng; Ho, Steven C L; Gagnon, Peter; Bi, Xuezhi; Yang, Yuansheng

    2015-01-01

    Linking the heavy chain (HC) and light chain (LC) genes required for monoclonal antibodies (mAb) production on a single cassette using 2A peptides allows control of LC and HC ratio and reduces non-expressing cells. Four 2A peptides derived from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A), respectively, were compared for expression of 3 biosimilar IgG1 mAbs in Chinese hamster ovary (CHO) cell lines. HC and LC were linked by different 2A peptides both in the absence and presence of GSG linkers. Insertion of a furin recognition site upstream of 2A allowed removal of 2A residues that would otherwise be attached to the HC. Different 2A peptides exhibited different cleavage efficiencies that correlated to the mAb expression level. The relative cleavage efficiency of each 2A peptide remains similar for expression of different IgG1 mAbs in different CHO cells. While complete cleavage was not observed for any of the 2A peptides, GSG linkers did enhance the cleavage efficiency and thus the mAb expression level. T2A with the GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Stably amplified CHO DG44 pools generated using GT2A had titers 357, 416 and 600 mg/L for the 3 mAbs in shake flask batch cultures. Incomplete cleavage likely resulted in incorrectly processed mAb species and aggregates, which were removed with a chromatin-directed clarification method and protein A purification. The vector and methods presented provide an easy process beneficial for both mAb development and manufacturing. PMID:25621616

  19. Antibody Reactivity of B Cells in Lupus Patients with Increased Disease Activity and ARID3a Expression

    Directory of Open Access Journals (Sweden)

    Julie M. Ward

    2015-11-01

    Full Text Available Earlier studies showed that the DNA-binding protein, Bright/ARID3a bound to a subset of human and mouse immunoglobulin heavy chain promoters where it enhanced expression. Indeed, mice with transgenic expression of ARID3a in all B lymphocytes have expanded MZ B cells and produce anti-nuclear antibodies (ANAs. Consistent with our findings in mice, we observed that human systemic lupus erythematosus (SLE patients had expanded numbers of peripheral blood ARID3a+ B cells that were associated with increased disease activity (p = 0.0038. We hypothesized that ARID3a+ naïve B cells would eventually produce autoantibodies, explaining associations between ARID3a expression and disease activity in lupus. Unlike healthy controls, ARID3a was expressed in the naïve B cell population in SLE patients, and we hypothesized that these might represent expansions of autoreactive cells. Therefore, monoclonal antibodies were generated from single-sorted naïve B cells derived from patients with normal (ARID3aN and high (ARID3aH numbers of ARID3a+ B cells. We found that ARID3a expression did not correlate with autoantibody expression. Furthermore, measures of antigen specificities of autoreactive antibodies did not reveal skewing toward particular proteins. These data suggest that the association of increased disease activity in SLE with numbers of ARID3a+ B lymphocytes may be mediated by an antibody-independent mechanism.

  20. Antibody microarray analyses of signal transduction protein expression and phosphorylation during porcine oocyte maturation.

    Science.gov (United States)

    Pelech, Steven; Jelinkova, Lucie; Susor, Andrej; Zhang, Hong; Shi, Xiaoqing; Pavlok, Antonin; Kubelka, Michal; Kovarova, Hana

    2008-07-01

    Kinex antibody microarray analyses was used to investigate the regulation of 188 protein kinases, 24 protein phosphatases, and 170 other regulatory proteins during meiotic maturation of immature germinal vesicle (GV+) pig oocytes to maturing oocytes that had completed meiosis I (MI), and fully mature oocytes arrested at metaphase of meiosis II (MII). Increases in apparent protein levels of protein kinases accounted for most of the detected changes during the GV to MI transition, whereas reduced protein kinase levels and increased protein phosphorylation characterized the MI to MII transition. During the MI to MII period, many of the MI-associated increased levels of the proteins and phosphosites were completely or partially reversed. The regulation of these proteins were also examined in parallel during the meiotic maturation of bovine, frog, and sea star oocytes with the Kinex antibody microarray. Western blotting analyses confirmed altered expression levels of Bub1A, IRAK4, MST2, PP4C, and Rsk2, and the phosphorylation site changes in the kinases Erk5 (T218 + Y220), FAK (S722), GSK3-beta (Y216), MEK1 (S217 + S221) and PKR1 (T451), and nucleophosmin/B23 (S4) during pig oocyte maturation.

  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.

  2. Prokaryotic Expression and Purification of HIV-1 Vif and hAPOBEC3G, Preparation of Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    Lan LI; Yi-shu YANG; Ze-lin LI; Yi ZENG

    2008-01-01

    To prepare HIV-1 Vif and hAPOBEC3G and to produce their antibodies, the full length gene fragment of HIV-1 Vif was amplified by PCR from a plasmid of HIV-1 NL4.3 cDNA, and the APOBEC3G gene was obtained by RT-PCR from the total RNA of H9 cells. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-32a). Recombinant pET-vif and pET-APOBEC3G were expressed respectively in Eserichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine tag at the C-terminus for convenient purification and detection. To express and purify the HIV-1 Vif and hAPOBEC3G in E. coli cells, the accuracy of inserted gene and specificity of proteins were detected by the two enzyme digestion method, SDS-PAGE, and Western blotting. Rabbits were then immunized by Vif or APOBEC3G protein and serum samples were tested by indirect ELISA to determine the level of antibodies. Immunoenzyme and immunofluorescence assays were performed to identify the specificity of polyclonal antibodies. The titer of the anti-Vif antibodies was 1:204800, and that of the anti-APOBEC3G antibodies was 1:102400. Thus the antibodies could detect the antigen expression in the cells, demonstrating that fusion proteins with high purity and their corresponding polyclonal antibodies with high titer and specificity were achieved.

  3. Polyclonal antibody preparation and expression in liver tissues of transactivated protein 5 of hepatitis C virus nonstructural 5A

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To prepare polyclonal antibody of transactivated protein 5 of hepatitis C virus nonstructural 5A (NA5ATP5) and to explore its expression in the liver tissues. Methods In Escherichia coli BL21,the prokaryotic expression vector pET32a(+)-NS5ATP5 was induced by isopropyl-β-D-thiogalactoside (IPTG),and it was analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. And the purified protein was used to immunize the rabbit to prepare polyclonal antibody,wi...

  4. 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

    OpenAIRE

    Maritza Berti; Manuel Pinto

    2012-01-01

    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 pere...

  5. 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

  6. 78 FR 63160 - United States Standards for Feed Peas, Split Peas, and Lentils

    Science.gov (United States)

    2013-10-23

    ... Peas, and Lentils AGENCY: Grain Inspection, Packers and Stockyards Administration, USDA ACTION: Notice... Peas, and Lentils under the Agriculture Marketing Act (AMA) of 1946. To ensure that the standards and... current U.S. Standards for Feed Peas, Split Peas, and Lentils are meeting the needs in today's...

  7. THE PRESENCE OF ANTI-PHOSPHATIDYLETHANOLAMINE ANTIBODIES IN ACUTE MYOCARDIAL INFARCTION

    OpenAIRE

    Abdolreza Sotoodeh Jahromi; Mohammad Shojaei; Mohammad Reza Farjam; Abdolhossien Madani

    2013-01-01

    Acute Myocardial Infarction (AMI) is a clinical manifestation of coronary atherothrombosis and is the important causes of death. Many factors play a role in AMI. Anti-Phospholipid (aPL) antibodies may act in the induction of immunological response leading to the development of AMI. Anti-Phosphatidylethanolamine (aPEA) antibody has been detected in various autoimmune diseases and anti-phospholipid antibody syndrome. The study of aPEA antibody in AMI might shed light on etiologic mechanisms in ...

  8. Adenovirus-expressed preS2 antibody inhibits hepatitis B virus infection and hepatic carcinogenesis

    Institute of Scientific and Technical Information of China (English)

    Qian Zhang; Zhi-Qing Li; Hu Liu; Jia-He Yang

    2012-01-01

    AIM: To investigate the inhibitory effect of hepatitis B virus (HBV) preS2 antibody (preS2Ab) against HBV infection and HBV-associated hepatic carcinogenesis. METHODS: An adenoviral vector carrying the fulllength light and heavy chains of the HBV preS2Ab gene, Ad315-preS2Ab, was constructed. Enzyme linked immunosorbent assay (ELISA) and Western blotting analyses were used to determine the preS2Ab expression levels in vitro . Immunofluorescent techniques were used to examine the binding affinity between the expressed HBV preS2Ab and HBV-positive liver cells. ELISAs were also used to determine hepatitis B surface antigen (HBsAg) levels to assess the inhibitory effect of the preS2Ab against HBV infection in L02 cells. The inhibitory effect of preS2Ab against hepatic carcinogenesiswas studied with diethylnitrosamine (DEN)-induced hepatocellular carcinomas (HCCs) in HBV transgenic mice. RESULTS: The expression of HBV preS2Ab increased with increases in the multiplicity of infection (MOI) of Ad315-preS2Ab in L02 cells, with 350.87 ± 17.37 μg/L of preS2Ab when the MOI was 100 plaque forming units (pfu)/cell. The expressed preS2Abs could recognize liver cells from HBV transgenic mice. ELISA results showed that L02 cells expressing preS2Ab produced less HBsAg after treatment with the serum of HBV patients than parental L02 cells expressing no preS2Ab. HBV transgenic mice treated with Ad315-preS2Ab had fewer and smaller cancerous nodes after induction with DEN than mice treated with a blank Ad315 vector or untreated mice. Additionally, the administration of Ad315-preS2Ab could alleviate hepatic cirrhosis and decrease the serum levels of alanine transaminase and aspartate transaminase. CONCLUSION: Adenovirus-mediated HBV preS2Ab expression could inhibit HBV infection in L02 cells, and then inhibit DEN-induced hepatocellular carcinogenesis and protect hepatic function in HBV transgenic mice.

  9. 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.

  10. Expression, purification of herpes simplex virus type 1 US11 Protein, and production of US11 polyclonal antibody

    OpenAIRE

    Yao Shanglong; Wuyunerdeni,; Huang Yizhong

    2011-01-01

    Abstract Background The US11 protein of herpes simplex virus type 1 (HSV-1) is a small, highly basic phosphoprotein expressed at late times during infection. To date, the function of US11 protein in cell culture and animal models is poorly understood. To further investigate the function of the US11 protein, this study was undertaken to express the US11 protein and raise a polyclonal antibody. Results The US11 gene was cloned into the prokaryotic expression vector pET-32a (+) to express His-ta...

  11. Construction and expression of a recombinant antibody-targeted plasminogen activator

    International Nuclear Information System (INIS)

    Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin β chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the γ2b constant region and the catalytic β chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

  12. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    International Nuclear Information System (INIS)

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  13. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  14. 绿脓杆菌外毒素A单链抗体基因的制备及其克隆的研究%Preparation of the Anti-PEA Monoclone Antibody and the Cloning of Its ScFv

    Institute of Scientific and Technical Information of China (English)

    崔淑芳; 汤球; 郝光荣; 曹平

    2002-01-01

    为制备中和活性较高的单抗细胞2F6的单链抗体(ScFv)基因,以绿脓杆菌外毒素A(PEA)类毒素为免疫原,用单克隆抗体技术制备可分泌具有中和活力单克隆抗体的杂交瘤细胞,再应用RT-PCR从其中一株杂交瘤细胞中扩增出抗体VH和VL基因,用Linker(G4S)3将VH和VL基因连接成ScFv基因,并将其克隆至pGEM-T载体中.经核酸序列分析证实,VH、VL和Linker基因连接正确,基因全长729bp.经计算机分析VH、VL基因符合功能性重排的鼠抗体可变区基因的特征.VH和VL基因分别属于鼠免疫球蛋白重链Ⅱ(A)和轻链κⅥ家簇.

  15. Omi/HtrA2对前列腺癌细胞株PED/PEA-15表达及PC-3细胞凋亡的影响%Effects of Omi/HtrA2 on Expression of Anti-apoptotic Protein PED/PEA-15 and Apoptosis of Prostate Cancer Cell Line PC-3

    Institute of Scientific and Technical Information of China (English)

    胡晓勇; 陈晓春; 朱朝辉; 陈朝晖; 曾甫清; 鲁功成

    2006-01-01

    背景与目的:促、抑凋亡因子间的相互作用与肿瘤的发生、发展密切相关.Omi/HtrA2是新近发现的一种凋亡调节因子,PED/PEA-15是一种广泛表达的抗凋亡蛋白.本研究旨在探讨Omi/HtrA2对PED/PEA-15表达和前列腺癌细胞PC-3凋亡的影响.方法:构建Omi/HtrA2的表达载体和siRNA载体,并用脂质体法分别将两载体转染至PC-3细胞中,Western blot和ELISA法检测Omi/HtrA2对PED/PEA-15表达和细胞凋亡的影响;Caspase-8检测试剂盒检测PED/PEA-15对Caspase-8活性的影响;Western blot、RT-PCR法检测Omi/HtrA2特异siRNA序列对其转录、翻译的影响,流式细胞仪检测siRNA导致Omi/HtrA2基因沉默后PC-3细胞凋亡的变化.结果:酶切和DNA测序证实Omi/HtrA2的表达载体和siRNA载体构建成功.通过转染Omi/HtrA2表达载体高表达Omi/HtrA2可抑制PED/PEA-1 5表达,并增加肿瘤细胞的凋亡率;抑制PED/PEA-15的表达可提高Caspase-8活性.siRNA沉默Omi/HtrA2基因后PC-3细胞对顺铂的敏感性降低.结论:Omi/HtrA2可通过抑制抗凋亡蛋白PED/PEA-15表达而在PC-3细胞凋亡中发挥重要作用.

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

    Directory of Open Access Journals (Sweden)

    H Keyvani

    2012-04-01

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

  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. Gene discovery in Eimeria tenella by immunoscreening cDNA expression libraries of sporozoites and schizonts with chicken intestinal antibodies.

    Science.gov (United States)

    Réfega, Susana; Girard-Misguich, Fabienne; Bourdieu, Christiane; Péry, Pierre; Labbé, Marie

    2003-04-01

    Specific antibodies were produced ex vivo from intestinal culture of Eimeria tenella infected chickens. The specificity of these intestinal antibodies was tested against different parasite stages. These antibodies were used to immunoscreen first generation schizont and sporozoite cDNA libraries permitting the identification of new E. tenella antigens. We obtained a total of 119 cDNA clones which were subjected to sequence analysis. The sequences coding for the proteins inducing local immune responses were compared with nucleotide or protein databases and with expressed sequence tags (ESTs) databases. We identified new Eimeria genes coding for heat shock proteins, a ribosomal protein, a pyruvate kinase and a pyridoxine kinase. Specific features of other sequences are discussed.

  19. Polymorphic Expression of a Human Superficial Bladder Tumor Antigen Defined by Mouse Monoclonal Antibodies

    Science.gov (United States)

    Fradet, Yves; Islam, Nazrul; Boucher, Lucie; Parent-Vaugeois, Carmen; Tardif, Marc

    1987-10-01

    Three mouse monoclonal antibodies (mAbs), which define a highly restricted antigen, were obtained by simultaneous immunizations with superficial papillary bladder tumor cells and mouse polyclonal serum against normal urothelium. The antigen was detected by the avidin/biotin/peroxidase method in 30/44 superficial bladder tumors (68%) but in only 4/27 infiltrating urothelial cancers (with much less intensity). No normal adult or fetal tissues tested expressed the antigen, including normal urothelium from 40 individuals, 13 of whom had a bladder tumor positive for the antigen. Only 1 of 45 nonbladder tumors showed some reactivity with one of the three mAbs. Serological tests on a large panel of human cancer cell lines and normal cultured cells were negative. The antigen is highly stable and well preserved on paraffin-embedded tissues. Electrophoretic transfer blot experiments with fresh tumor extracts showed that all three mAbs react with a determinant on a component of 300,000 Mr (pI 9.5) and 62,000 Mr (pI 6.5). The antigen shows polymorphic expression at the cellular level on tissue sections and also at a molecular level on immunoblots where the two bands are differentially detected on extracts of a series of tumors but are not visualized on normal urothelium extracts. The characteristics of this antigenic system suggest that it may provide some insights about the biology of bladder cancer. Specific detection of the antigen on 70% of superficial bladder tumors with normal cytology may be useful for their diagnosis and follow-up.

  20. Sigma-1 receptor expression in the dorsal root ganglion: Reexamination using a highly specific antibody.

    Science.gov (United States)

    Mavlyutov, Timur A; Duellman, Tyler; Kim, Hung Tae; Epstein, Miles L; Leese, Charlotte; Davletov, Bazbek A; Yang, Jay

    2016-09-01

    Sigma-1 receptor (S1R) is a unique pluripotent modulator of living systems and has been reported to be associated with a number of neurological diseases including pathological pain. Intrathecal administration of S1R antagonists attenuates the pain behavior of rodents in both inflammatory and neuropathic pain models. However, the S1R localization in the spinal cord shows a selective ventral horn motor neuron distribution, suggesting the high likelihood of S1R in the dorsal root ganglion (DRG) mediating the pain relief by intrathecally administered drugs. Since primary afferents are the major component in the pain pathway, we examined the mouse and rat DRGs for the presence of the S1R. At both mRNA and protein levels, quantitative RT-PCR (qRT-PCR) and Western confirmed that the DRG contains greater S1R expression in comparison to spinal cord, cortex, or lung but less than liver. Using a custom-made highly specific antibody, we demonstrated the presence of a strong S1R immuno-fluorescence in all rat and mouse DRG neurons co-localizing with the Neuron-Specific Enolase (NSE) marker, but not in neural processes or GFAP-positive glial satellite cells. In addition, S1R was absent in afferent terminals in the skin and in the dorsal horn of the spinal cord. Using immuno-electron microscopy, we showed that S1R is detected in the nuclear envelope and endoplasmic reticulum (ER) of DRG cells. In contrast to other cells, S1R is also located directly at the plasma membrane of the DRG neurons. The presence of S1R in the nuclear envelope of all DRG neurons suggests an exciting potential role of S1R as a regulator of neuronal nuclear activities and/or gene expression, which may provide insight toward new molecular targets for modulating nociception at the level of primary afferent neurons. PMID:27339730

  1. Evidence against the involvement of ionically bound cell wall proteins in pea epicotyl growth

    Science.gov (United States)

    Melan, M. A.; Cosgrove, D. J.

    1988-01-01

    Ionically bound cell wall proteins were extracted from 7 day old etiolated pea (Pisum sativum L. cv Alaska) epicotyls with 3 molar LiCl. Polyclonal antiserum was raised in rabbits against the cell wall proteins. Growth assays showed that treatment of growing region segments (5-7 millimeters) of peas with either dialyzed serum, serum globulin fraction, affinity purified immunoglobulin, or papain-cleaved antibody fragments had no effect on growth. Immunofluorescence microscopy confirmed antibody binding to cell walls and penetration of the antibodies into the tissues. Western blot analysis, immunoassay results, and affinity chromatography utilizing Sepharose-bound antibodies confirmed recognition of the protein preparation by the antibodies. Experiments employing in vitro extension as a screening measure indicated no effect upon extension by antibodies, by 50 millimolar LiCl perfusion of the apoplast or by 3 molar LiCl extraction. Addition of cell wall protein to protease pretreated segments did not restore extension nor did addition of cell wall protein to untreated segments increase extension. It is concluded that, although evidence suggests that protein is responsible for the process of extension, the class(es) of proteins which are extracted from pea cell walls with 3 molar LiCl are probably not involved in this process.

  2. Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli.

    Science.gov (United States)

    Shapira, R; Paster, N; Menasherov, M; Eyal, O; Mett, A; Meiron, T; Kuttin, E; Salomon, R

    1997-03-01

    Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods. PMID:9055416

  3. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability

    DEFF Research Database (Denmark)

    Assarsson, Erika; Lundberg, Martin; Holmquist, Göran;

    2014-01-01

    reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput...... detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes...

  4. Cloning and Expression of Intracellular Part of Receptor Protein Tyrosine Phosphatase RPTPα and Preparation of Its Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    CHEN Yang; YANG Su-juan; FU Yao; WANG Jia-peng; ZHAO Zhi-zhuang; FU Xue-qi

    2008-01-01

    A DNA fragment encoding the intracellular part of tyrosine phosphatase RPTPα designated as RPTPα-2D gene was amplified by PCR from a human prostate cDNA library and cloned into the pT7 E. coli expression vector. The resulting plasmid pT7-RPTPα-2D was used to transform Rosetta DE3 E. coli cells. RPTPα-2D was predominately expressed in the insoluble inclusion body and was effectively purified using preparative electrophoresis gels. Polyclonal antibodies were obtained after immunization of a rabbit with purified RPTPα-2D. The antibodies displayed a high titer and sensitivity. This study thus provided a valuable tool for further researches on RPTPa.

  5. Characterization of NADPH oxidase 5 expression in human tumors and tumor cell lines with a novel mouse monoclonal antibody

    OpenAIRE

    Antony, Smitha; Wu, Yongzhong; Hewitt, Stephen M.; Anver, Miriam R.; Butcher, Donna; Jiang, Guojian; MEITZLER, JENNIFER L.; Liu, Han; JUHASZ, AGNES; Lu, Jiamo; Roy, Krishnendu K.; James H Doroshow

    2013-01-01

    Reactive oxygen species generated by NADPH oxidase 5 (Nox5) have been implicated in physiological and pathophysiological signaling pathways, including cancer development and progression. However, because immunological tools are lacking, knowledge of the role of Nox5 in tumor biology has been limited; the expression of Nox5 protein across tumors and normal tissues is essentially unknown. Here, we report the characterization and use of a mouse monoclonal antibody against a recombinant Nox5 prot...

  6. Differential transgene expression patterns in Alzheimer mouse models revealed by novel human amyloid precursor protein-specific antibodies.

    Science.gov (United States)

    Höfling, Corinna; Morawski, Markus; Zeitschel, Ulrike; Zanier, Elisa R; Moschke, Katrin; Serdaroglu, Alperen; Canneva, Fabio; von Hörsten, Stephan; De Simoni, Maria-Grazia; Forloni, Gianluigi; Jäger, Carsten; Kremmer, Elisabeth; Roßner, Steffen; Lichtenthaler, Stefan F; Kuhn, Peer-Hendrik

    2016-10-01

    Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aβ deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aβ pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aβ deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aβ in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals. PMID:27470171

  7. 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; Kim D. Janda; 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...

  8. Cytokines Expression Profile and Kinetics of Peste des petits ruminants Virus Antigen and Antibody in Infected and Vaccinated Goats

    Institute of Scientific and Technical Information of China (English)

    Arun Patel; Kaushal Kishor Rajak; Vinayagamurthy Balamurugan; Arnab Sen; Shashi Bhusan Sudhakar; Veerakyathappa Bhanuprakash; Raj Kumar Singh; Awadh Bihari Pandey

    2012-01-01

    The present study deals with the co-ordination of cytokine(IL-4 and IFN-γ) expression and kinetics of peste des petits ruminants(PPR) virus antigen and antibody in PPRV infected and vaccinated goats.The infected animals exhibited mixed cytokine(both TH1 and TH2) responses in the initial phase of the disease.The infected and dead goats had increased IFN-γ response before their death; while IL-4 remained at the base level.The cytokine expression in recovered animals was almost similar to that of vaccinated ones,where a unique biphasic response of IL-4 expression was observed with an up-regulation of IFN-γ on 7th days post vaccination(dpv).Analysis of PPR virus antigen and antibody kinetics in different components of blood from infected and vaccinated animals revealed that the PPR virus antigen load was highest in plasma followed by serum and blood of the infected animals,whereas vaccinated animals showed only marginal positivity on 9th dpv.The antibody titer was high in serum followed by plasma and blood in both vaccinated and infected animals.Therefore,it is inferred that the presence of antigen and antibody were significant with the expression of cytokine,and that a decreased response of IL-4 was noticed during intermediate phase of the disease i.e.,7 to 12th days post infection(dpi).This indicates the ability to mount a functional TH2 response after 14th dpi could be a critical determinant in deciding the survival of the PPR infected animal.

  9. Polymerase chain reaction facilitates the cloning, CDR-grafting, and rapid expression of a murine monoclonal antibody directed against the CD18 component of leukocyte integrins.

    OpenAIRE

    Daugherty, B L; DeMartino, J A; Law, M F; Kawka, D W; Singer, I I; Mark, G E

    1991-01-01

    Two novel approaches of recombinant PCR technology were employed to graft the complementarity determining regions from a murine monoclonal antibody (mAb) onto human antibody frameworks. One approach relied on the availability of cloned human variable region templates, whereas the other strategy was dependent only on human variable region protein sequence data. The transient expression of recombinant humanized antibody was driven by the adenovirus major late promoter and was detected 48 hrs po...

  10. Magnetosome Expression of Functional Camelid Antibody Fragments (Nanobodies) in Magnetospirillum gryphiswaldense▿†

    OpenAIRE

    Pollithy, Anna; Romer, Tina; Lang, Claus; Müller, Frank D.; Helma, Jonas; Leonhardt, Heinrich; Rothbauer, Ulrich; Schüler, Dirk

    2011-01-01

    Numerous applications of conventional and biogenic magnetic nanoparticles (MNPs), such as in diagnostics, immunomagnetic separations, and magnetic cell labeling, require the immobilization of antibodies. This is usually accomplished by chemical conjugation, which, however, has several disadvantages, such as poor efficiency and the need for coupling chemistry. Here, we describe a novel strategy to display a functional camelid antibody fragment (nanobody) from an alpaca (Lama pacos) on the surf...

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

    OpenAIRE

    Markiv Anatoliy; Beatson Richard; Burchell Joy; Durvasula Ravi V; Kang Angray S

    2011-01-01

    Abstract Background Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains link...

  12. Pea (Pisum sativum) genes involved in symbiosis with nitrogen-fixing bacteria.1.Analysis of the expression of the early nodulin gene ENOD12 using the polymerase chain-reaction

    NARCIS (Netherlands)

    Zalenskii, A.O.; Kozik, A.V.; Scheres, B.J.G.; Bisseling, A.; Tikhonovich, I.A.

    1991-01-01

    The polymerase chain reaction (PCR) was used to detect the transcription products of the early nodulin gene in the pea. Single-stranded DNA copies were prepared using a primer corresponding to the terminal part of a previously sequenced cDNA clone and a total RNA isolate. The presence of amplificati

  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. Interferon-induced changes in expression of antigens defined by monoclonal antibodies on malignant and nonmalignant mononuclear hematopoietic cells

    DEFF Research Database (Denmark)

    Hokland, M; Ritz, J; Hokland, P

    1983-01-01

    The effect of alpha interferon (alpha IFN) on the expression of histocompatibility--as well as differentiation antigens on normal and malignant hematopoietic mononuclear cells--were investigated by cell cytofluorometry using a panel of monoclonal antibodies (MoAbs). An increase in the expression...... of HLA-antigens detected by beta 2-Microglobulin (beta 2-M) could be demonstrated for peripheral blood mononuclear cells, non-T cells, Null cells, activated T cells, fetal thymocytes, adherent cells, and on four malignant non-T lymphoblastoid cell lines. In contrast, no significant differences were...... observed in the expression of antigens specific for B-lymphocytes (B1), T-lymphocytes (T3, T4, T6, T8, T11), NK-cells (901) and adherent cells (Mo1-4). Likewise, the expression of Ia-antigens remained unaltered on non-T cells, Null cells, and monocytes. The only other effect of IFN was an increase...

  15. The present state of the art in expression, production and characterization of monoclonal antibodies.

    Science.gov (United States)

    Gaughan, Christopher L

    2016-02-01

    Monoclonal antibodies (MAb's) have become one the most powerful therapeutic and diagnostic tools in modern medicine. Some estimates target the worldwide market of MAb's on the order of $125 billion in the next four years. Recent advances in molecular biology, immunology, and development of robust production platforms will drive the development of more MAb's suitable to treat an ever increasing number of disease states. This circumstance combined with the fact that many of the original antibody therapies from the 1980 s and 1990 s will soon be coming off patent will attract a great deal of investment in the development of larger industrial facilities to increase monoclonal antibody to meet increasing demand. In this review, the present state of the science that underlies the development of new antibodies therapies in Chinese hamster ovary cells combined with a description of the present challenges facing the industry in terms of the limitations of output and compliance with current good manufacturing practices and FDA regulations. Also addressed are future challenges to overcome production bottlenecks, description of critical quality control attributes particular to antibodies, and detailed treatment of scale-up considerations. PMID:26299798

  16. 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.

  17. Detection of Alfalfa mosaic virus (AMV) in pea field in Iran.

    Science.gov (United States)

    Esfandiari, N; Kohi Habibi, M; Mosahebi, G H; Mozafari, J

    2005-01-01

    During the spring and summer, in 2003-2004, pea viruses were identified in twenty pea fields of Tehran. Some leaf samples were collected randomly from pea fields of Tehran. Samples were tested by Double Antibody Sandwich Enzyme Linked Immunosorbent Assay (DAS-ELISA) technique using polyclonal antiserum of Alfalfa mosaic virus (AMV), AS-0001, DSMZ, Braunschweig, Germany). The samples were extracted in 0.1 M Phosphate buffer pH 7 to 7.5 and inoculated on Chenopodium amaranticolor, Chenopodium quina, Phaseolus valgaris, Vicia faba, Vignia unguiculata. Pea cultivars were infected by AMV, causing mild mosaic, translucent veins and a diffuse green-yellow of tender parts and spots may also was involved necrosis of tissue. Infected plants grow slowly and malformed pods produce fewer ovules. In Chenopodium amranticolor, C. quina chlorotic and necrotic flecks, and Vicia faba systemic mosaic had produced. Phaselous vulgaris and Viginia unguiculata are good assay hosts for strains that produce local lesions after 3-5 days in these plants. Back inoculated on Pisum sativum and Vicia faba and tested with DAS-ELISA that had been confirmed the results. This is the first report of AMV on pea from Iran. PMID:16637206

  18. Transcriptional activation of the parsley chalcone synthase promoter in heterologous pea and yeast systems.

    Science.gov (United States)

    Kalbin; Strid; Frohnmeyer

    1999-11-01

    Introduction by electroporation of different parsley (Petroselinum crispum) CHS-promoter/beta-glucuronidase(GUS)-reporter constructs into pea (Pisum sativum L.) protoplasts leads to a high constitutive GUS-expression and to the loss of the light-inducibility seen in the homologous parsley protoplast system. These results indicate that Unit 1 of the parsley CHS-promoter is only partly responsible for the GUS-expression detected. Instead, additional cis-elements, which are located downstream within 100 bp from the transcriptional start site, mediate the de-repression in pea protoplasts. In contrast, in yeast (Saccharomyces cerevisiae) cells, the GUS expression from the heterologous CHS/GUS construct is controlled by elements between Unit 1 and -100 bp. In both pea and yeast cells, transcription factors different from those regulating UV-responsiveness in parsley, are probably mediating the constitutive expression from the heterologous construct. The results with pea protoplasts imply that protoplastation of pea leaf cells itself induces de-repression as a result of stress to the protoplasts. This notion was strengthened by the finding that mRNA levels of the endogenous chalcone synthase were drastically increased as the result of the protoplastation procedure. PMID:10580282

  19. Expression of CR2/EBV receptors on human thymocytes detected by monoclonal antibodies.

    Science.gov (United States)

    Tsoukas, C D; Lambris, J D

    1988-08-01

    The biologic effects of the third component of complement, C3, are mediated via receptors which specifically bind the enzymatic degradation products resulting from the cleavage of C3. One of the products, C3d, has been associated with binding to the second complement receptor CR2 (CD21). This receptor, which is identical to the receptor for Epstein-Barr virus (EBV), has been primarily found on cells of the B lineage, but not on mature T cells or other cells of erythroid or myeloid lineages. In the present investigation, we report the presence of CR2 on human thymocytes. Indirect immunofluorescence analysis employing monoclonal anti-CR2 antibodies revealed a range of thymocyte reactivity from 15% to 63% in thirteen experiments using cells of different donors. Reactivity was always greater with the monoclonal anti-CR2 (CD21) antibody HB-5 than with two other antibodies which recognize distinct epitopes on the CR2 molecule. Two-color immunofluorescence analysis indicated that the brightest of the HB-5-stained thymocytes also reacted with the monoclonal anti-CD1 antibody T6 (immature thymocyte marker) while some of the duller HB-5-staining cells reacted with the monoclonal anti-CD3 antibody Leu-4 (mature thymocyte marker). Immunoprecipitation of CR2 on thymocytes with antibody HB-5 and polyacrylamide gel electrophoretic analysis revealed a protein of 145 kDa molecular mass which is consistent with the size of CR2 found on B lymphocytes. These findings raise several questions regarding the biologic role of CR2-EBV receptor on cells of the T lineage. PMID:2970972

  20. 21 CFR 158.170 - Frozen peas.

    Science.gov (United States)

    2010-04-01

    ... leaf or pod material from the pea plant or other such material per sample unit as defined in paragraph... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Frozen peas. 158.170 Section 158.170 Food and... CONSUMPTION FROZEN VEGETABLES Requirements for Specific Standardized Frozen Vegetables § 158.170 Frozen...

  1. Yield potential of pigeon pea cultivars

    Science.gov (United States)

    Yield potential of twelve vegetable pigeon pea (Cajanus cajun) cultivars was evaluated at two locations in eastern Kenya during 2012 and 2013 cropping years. Pigeon pea pod numbers, seeds per pod, seed mass, grain yield and shelling percentage were quantified in three replicated plots, arranged in a...

  2. 21 CFR 155.170 - Canned peas.

    Science.gov (United States)

    2010-04-01

    ...., vine or leaf or pod material from the pea plant or other such material. (vi) Alcohol-insoluble solids... 21 Food and Drugs 2 2010-04-01 2010-04-01 false Canned peas. 155.170 Section 155.170 Food and... CONSUMPTION CANNED VEGETABLES Requirements for Specific Standardized Canned Vegetables § 155.170 Canned...

  3. Polyclonal antibody against an insect excitatory toxin BmKIT from Buthus Martensii karsch and detection of BmKIT expressed in transgenic cotton

    Institute of Scientific and Technical Information of China (English)

    Hao Chanjuan; Xu Chenggang; Zhang Zhiyun; Liang Aihua

    2008-01-01

    An insect excitatory toxin gene from Buthus martensii Karsch (BmKIT) was cloned into the expression vector, pET-28a. BmKIT was expressed as inclusion bodies in Escherichia coli BL21 (DE3) host cells. The authenticity of in vitro expressed protein was confirmed by Western blot. The inclusion body protein band in SDS-PAGE was excised and the protein, BmKIT, was extracted. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed BmKIT and was used to quantify its presence in transgenic cotton.

  4. Stereological analysis of pea protein in model samples

    Directory of Open Access Journals (Sweden)

    Zdeňka Javůrková

    2016-07-01

    Full Text Available With the growing popularity of various plant proteins used as raw materials for meat production, interest of manufacturers to extend the range of such raw materials is increasing as well. Manufacturers are trying to minimize the cost of manufacturing their products with simultaneous preserving the nutritional value of their products to the maximum extent possible. Such cheaper raw materials, which are also nutritionally rich, include pea protein. Another advantage for manufacturers is the fact that legislation does not order them to indicate pea protein presence in case of its addition, as it does for other allergenic ingredients, although this legume contains storage proteins which can cause a variety of allergic reactions, just like other legumes. Currently no method used for its qualitative determination has been described in literature, let alone its quantitative determination. Our work describes a possible method that can be applied for its quantification. It is a stereological method applied to microscopic sections stained by immunohistochemical staining based on the avidin-biotin complex using monoclonal legumin (1H9 as the primary antibody. The stereological method is based on geometry, it applies knowledge of geometry to analyze a sample of diverse origin, size and internal structure. Despite potential shortcomings in staining microscopic preparations, stereology allows us to perform quantification based on knowledge of morphology of the observed structures. This work describes a procedure of a known pea protein addition quantification in model meat products by means of Ellipse software. Pea protein quantification was performed in two ways. In the first case ten microimages of all sections prepared were examined, while in the second case one scan of the entire section was analyzed. Based on the results, Spearman's correlation coefficient was calculated, which confirmed our assumption of correlation between the protein added into the

  5. 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.

  6. THE PRESENCE OF ANTI-PHOSPHATIDYLETHANOLAMINE ANTIBODIES IN ACUTE MYOCARDIAL INFARCTION

    Directory of Open Access Journals (Sweden)

    Abdolreza Sotoodeh Jahromi

    2013-01-01

    Full Text Available Acute Myocardial Infarction (AMI is a clinical manifestation of coronary atherothrombosis and is the important causes of death. Many factors play a role in AMI. Anti-Phospholipid (aPL antibodies may act in the induction of immunological response leading to the development of AMI. Anti-Phosphatidylethanolamine (aPEA antibody has been detected in various autoimmune diseases and anti-phospholipid antibody syndrome. The study of aPEA antibody in AMI might shed light on etiologic mechanisms in the pathogenesis of coronary atherothrombosis and AMI. This study was aimed to evaluate whether prevalence of aPEA antibodies, in patients with AMI and to analyze their relationship with traditional cardiovascular risk factors. The prevalence of aPEA IgG and IgM in a well characterized group of patients with AMI as a case group and in age and sex matched healthy subjects as a control group. Sera from two groups were tested to evaluate the presence of aPEA IgG and IgM isotypes by ELISA method. The frequencies of positive test for aPEA IgG were 12.22 and 2.22% among patients and controls respectively with significant difference (p = 0.007. The aPEA IgM frequencies were 3.33 and 0.00% in patients and the controls, with significant difference (p = 0.005. According to the results of this study, aPEA antibodies have a role in AMI, independent risk factors for AMI, which may represent a link between autoimmunity and coronary atherothrombosis. Further studies with larger sample size of patients and healthy people are needed to explore the role of aPEA antibodies in coronary atherothrombosis.

  7. Molecular Prediction of Pea Footrot Disease

    Directory of Open Access Journals (Sweden)

    Ebimieowei Etebu

    2011-11-01

    Full Text Available PCR based assays were developed in this study to quantitatively predict pea footrot infections in agricultural soils prior to cultivation. Pea footrot disease due to Nectria haematococca (anamorph Fusarium solani f.sp. pisi is linked to the presence of six pea pathogenicity (PEP genes (PDA1, PEP1, PEP2, PEP3, PEP4 and PEP5. Whilst molecular assays have been used recently to selectively detect these genes in soil- DNA, quantitative molecular assay has been extended to only the PEP3 gene whose role in pea pathogenicity is yet unknown. In this research, PCR-based quantification assays were developed to quantify the two pea pathogenicity genes (PDA and PEP5 with identified roles in pea pathogenicity from soil-DNA obtained from fields with pea footrot histories. Results showed that the quantitative molecular assays developed herein were both efficient. Amplification efficiency of the Q-PCR assay for the PDA and PEP5 gene were 97 and 89%, respectively. PDA and PEP5 gene copy numbers were shown to vary significantly (p = 0.01 between fields. However, the PDA gene copy numbers were relatively higher than those of the PEP5 gene in agricultural fields. The genes, especially PEP5 gene, were comparable to and positively correlated to the number of spores of pathogenic N. haematococca, and footrot disease. The PDA gene alone in soil could not cause footrot disease in peas after 8 weeks of planting; assays directed at it alone may therefore be insufficient to predict pea footrot disease. However, the molecular assay targeting the PDA alongside the PEP5 gene offers the opportunity for quantitative prediction of pea footrot infections in agricultural soils prior to cultivation.

  8. CHO-S antibody titers >1 gram/liter using flow electroporation-mediated transient gene expression followed by rapid migration to high-yield stable cell lines.

    Science.gov (United States)

    Steger, Krista; Brady, James; Wang, Weili; Duskin, Meg; Donato, Karen; Peshwa, Madhusudan

    2015-04-01

    In recent years, researchers have turned to transient gene expression (TGE) as an alternative to CHO stable cell line generation for early-stage antibody development. Despite advances in transfection methods and culture optimization, the majority of CHO-based TGE systems produce insufficient antibody titers for extensive use within biotherapeutic development pipelines. Flow electroporation using the MaxCyte STX Scalable Transfection System is a highly efficient, scalable means of CHO-based TGE for gram-level production of antibodies without the need for specialized expression vectors or genetically engineered CHO cell lines. CHO cell flow electroporation is easily scaled from milligram to multigram quantities without protocol reoptimization while maintaining transfection performance and antibody productivity. In this article, data are presented that demonstrate the reproducibility, scalability, and antibody production capabilities of CHO-based TGE using the MaxCyte STX. Data show optimization of posttransfection parameters such as cell density, media composition, and feed strategy that result in secreted antibody titers >1 g/L and production of multiple grams of antibody within 2 weeks of a single CHO-S cell transfection. In addition, data are presented to demonstrate the application of scalable electroporation for the rapid generation of high-yield stable CHO cell lines to bridge the gap between early- and late-stage antibody development activities.

  9. Seroprevalence against Borrelia burgdorferi sensu lato and occurence of antibody co-expression with Anaplasma phagocytophilum in dogs in Latvia

    OpenAIRE

    Berzina, Inese; Matise, Ilze

    2013-01-01

    Background Lyme disease is commonly diagnosed in humans in Latvia, but up to date no studies have been performed to investigate its prevalence in dogs. The aim of this study was to evaluate if seroprevalence against B. burgdorferi sensu lato (B. burgdorferi s.l.) and co-expression of antibodies against B.burgdorferi s.l. and A. phagocytophilum is higher in dogs with clinical suspicion of tick-borne diseases compared to healthy dogs. Findings Venous blood was taken from healthy dogs (n=441) an...

  10. Comparison of thyroid transcription factor-1 expression by two monoclonal antibodies in pulmonary and non-pulmonary primary tumors

    OpenAIRE

    Matoso, Andres; Singh, Kamaljeet; JACOB, RAFIK; Greaves, Wesley O.; Tavares, Rosemarie; Noble, Lelia; Resnick, Murray B.; DeLellis, Ronald A.; Wang, Li J.

    2010-01-01

    Thyroid transcription factor-1 (TTF-1) is a transcription factor that plays a role in the development and physiology of the thyroid and lungs. Expression of TTF-1 is used as a marker of lung and thyroid clinically. Commercially available clones of TTF-1 monoclonal antibodies, 8G7G3/1 and SPT24, have been reported to have different sensitivities for the detection of neoplasms of different origins. Although they are used extensively in daily practice, a comprehensive comparative study of these ...

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

    OpenAIRE

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

    2015-01-01

    Background Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequen...

  12. A Novel PET Imaging Using 64Cu-Labeled Monoclonal Antibody against Mesothelin Commonly Expressed on Cancer Cells

    Directory of Open Access Journals (Sweden)

    Kazuko Kobayashi

    2015-01-01

    Full Text Available Mesothelin (MSLN is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

  13. Co-expression of anti-rotavirus proteins (llama VHH antibody fragments in Lactobacillus: development and functionality of vectors containing two expression cassettes in tandem.

    Directory of Open Access Journals (Sweden)

    Gökçe Günaydın

    Full Text Available Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3 as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11, by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules.

  14. The monoclonal antibody SM5-1 recognizes a fibronectin variant which is widely expressed in melanoma

    Directory of Open Access Journals (Sweden)

    Guo Yajun

    2006-01-01

    Full Text Available Abstract Background Previously we have generated the monoclonal antibody SM5-1 by using a subtractive immunization protocol of human melanoma. This antibody exhibits a high sensitivity for primary melanomas of 99% (248/250 tested and for metastatic melanoma of 96% (146/151 tested in paraffin embedded sections. This reactivity is superior to the one obtained by HMB-45, anti-MelanA or anti-Tyrosinase and is comparable to anti-S100. However, as compared to anti-S100, the antibody SM5-1 is highly specific for melanocytic lesions since 40 different neoplasms were found to be negative for SM5-1 by immunohistochemistry. The antigen recognized by SM5-1 is unknown. Methods In order to characterize the antigen recognized by mAb SM5-1, a cDNA library was constructed from the metastatic human melanoma cell line SMMUpos in the Uni-ZAP lambda phage and screened by mAb SM5-1. The cDNA clones identified by this approach were then sequenced and subsequently analyzed. Results Sequence analysis of nine independent overlapping clones (length 3100–5600 bp represent fibronectin cDNA including the ED-A, but not the ED-B region which are produced by alternative splicing. The 89aa splicing variant of the IIICS region was found in 8/9 clones and the 120aa splicing variant in 1/9 clones, both of which are included in the CS1 region of fibronectin being involved in melanoma cell adhesion and spreading. Conclusion The molecule recognized by SM5-1 is a melanoma associated FN variant expressed by virtually all primary and metastatic melanomas and may play an important role in melanoma formation and progression. This antibody is therefore not only of value in immunohistochemistry, but potentially also for diagnostic imaging and immunotherapy.

  15. Genetic analysis of pod dehiscence in pea (Pisum sativum L.).

    Science.gov (United States)

    Weeden, Norman F; Brauner, Soren; Przyborowski, Jerzy A

    2002-01-01

    The inheritance of the dehiscent pod character was investigated in two recombinant inbred populations using a simplified correlation analysis. The approach identified three regions on the pea genome that affect the expression of pod dehiscence. The region on linkage group III corresponded to the expected position of Dpo, a gene known to influence pod dehiscence. A locus on linkage group V appeared to have a slightly smaller effect on expression of the phenotype. The third region was observed only in one cross, had a greater effect than Dpo, and was postulated to be yellow pod allele at the Gp locus

  16. Protective Effects of Ultramicronized Palmitoylethanolamide (PEA-um) in Myocardial Ischaemia and Reperfusion Injury in VIVO.

    Science.gov (United States)

    Di Paola, Rosanna; Cordaro, Marika; Crupi, Rosalia; Siracusa, Rosalba; Campolo, Michela; Bruschetta, Giuseppe; Fusco, Roberta; Pugliatti, Pietro; Esposito, Emanuela; Cuzzocrea, Salvatore

    2016-08-01

    Myocardial infarction is the leading cause of death, occurs after prolonged ischemia of the coronary arteries. Restore blood flow is the first intervention help against heart attack. However, reperfusion of the arteries leads to ischemia/reperfusion injury (I/R). The fatty acid amide palmitoylethanolamide (PEA) is an endogenous compound widely present in living organisms, with analgesic and anti-inflammatory properties. The present study evaluated the effect of ultramicronized palmitoylethanolamide (PEA-um) treatment on the inflammatory process associated with myocardial I/R. Myocardial ischemia reperfusion injury was induced by occlusion of the left anterior descending coronary artery for 30 min followed by 2 h of reperfusion. PEA-um, was administered (10 mg/kg) 15 min after ischemia and 1 h after reperfusion. In this study, we demonstrated that PEA-um treatment reduces myocardial tissue injury, neutrophil infiltration, adhesion molecules (ICAM-1, P-selectin) expression, proinflammatory cytokines (TNF-α, IL-1β) production, nitrotyrosine and PAR formation, nuclear factor kB expression, and apoptosis (Fas-L, Bcl-2) activation. In addition to study whether the protective effect of PEA-um on myocardial ischemia reperfusion injury is also related to the activation of PPAR-α, in a separate set of experiments it has been performed myocardial I/R in PPARα mice. Genetic ablation of peroxisome proliferator activated receptor (PPAR)-α in PPAR-αKO mice exacerbated Myocardial ischemia reperfusion injury when compared with PPAR-αWT mice. PEA-um induced cardioprotection in PPAR-α wild-type mice, but the same effect cannot be observed in PPAR-αKO mice. Our results have clearly shown a modulation of the inflammatory process, associated with myocardial ischemia reperfusion injury, following administration of PEA-um. PMID:26844976

  17. Expression Enhancement in Trastuzumab Therapeutic Monoclonal Antibody Production using Genomic Amplification with Methotrexate

    OpenAIRE

    Akbarzadeh-Sharbaf, Soudabeh; Yakhchali, Bagher; Minuchehr, Zarrin; Shokrgozar, Mohammad Ali; Zeinali, Sirous

    2013-01-01

    Background Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. Methods According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were sele...

  18. Monoclonal antibody-escape variant of dengue virus serotype 1: Genetic composition and envelope protein expression.

    Science.gov (United States)

    Chem, Y K; Chua, K B; Malik, Y; Voon, K

    2015-06-01

    Monoclonal antibody-escape variant of dengue virus type 1 (MabEV DEN-1) was discovered and isolated in an outbreak of dengue in Klang Valley, Malaysia from December 2004 to March 2005. This study was done to investigate whether DEN152 (an isolate of MabEV DEN-1) is a product of recombination event or not. In addition, the non-synonymous mutations that correlate with the monoclonal antibody-escape variant were determined in this study. The genomes of DEN152 and two new DEN-1 isolates, DENB04 and DENK154 were completely sequenced, aligned, and compared. Phylogenetic tree was plotted and the recombination event on DEN152 was investigated. DEN152 is sub-grouped under genotype I and is closely related genetically to a DEN-1 isolated in Japan in 2004. DEN152 is not a recombinant product of any parental strains. Four amino acid substitutions were unique only to DEN 152. These amino acid substitutions were (Ser)[326](Leu), (Ser)[340](Leu) at the deduced E protein, (Ile)[250](Thr) at NS1 protein, and (Thr)[41](Ser) at NS5 protein. Thus, DEN152 is an isolate of the emerging monoclonal antibody-escape variant DEN-1 that escaped diagnostic laboratory detection. PMID:26691263

  19. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    Directory of Open Access Journals (Sweden)

    Benevolo Maria

    2006-09-01

    Full Text Available Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu, a member of the human Epidermal Growth Factor (EGF receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs to ErbB-2 that involves their functional expression in (a bacteria, (b transient as well as stable transgenic tobacco plants, and (c a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6 was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T, displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1 only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology

  20. Construction, expression and characterization of the engineered antibody against tumor surface antigen, p185c-erbB-2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The c-erbB-2 proto-oncogene encodes a 185kDa protein p185, which belongs to epidermal growth factorreceptor family. Amplification of this gene has been shown to correlate with poor clinical prognosis forcertain cancer patients. The monoclonal antibody A21 which directed against p185 specifically inhibitsproliferation of tumor cells overexpressing p185, hence allows it to be a candidate for targeted therapy. Inorder to overcome several drawbacks of murine MAb, we cloned its VH and VL genes and constructed thesingle-chain Fv (scFv) through a peptide linker. The recombinant scFvA21 was expressed in Escherichiacoli and purified by the affinity column. Subsequently it was characterized by ELISA, Western blot, cellimmunohistochemistry and FACS. All these assays showed the binding activity to extracellular domain(ECD) of p185. Based on those properties of scFvA21, we further constructed the scFv-Fc fusion moleculewith a homodimer form and the recombinant product was expressed in mammalian cells. In a series ofsubsequent analysis this fusion protein showed identical antigen binding site and activity with the parentantibody. These anti-p185 engineered antibodies have promised to be further modified as a tumor targetingdrugs, with a view of application in the diagnosis and treatment of human breast cancer.

  1. A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; McEachern, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2009-09-01

    Nipah virus (NiV) is a new zoonotic paramyxovirus that emerged in 1998 and is now classified in the genus Henipavirus along with the closely related Hendra virus (HeV). NiV is highly pathogenic in several vertebrate species including humans, and the lack of available vaccines or specific treatment restricts it to biosafety level 4 (BSL4) containment. A serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and bearing the F and G proteins of NiV (VSV-NiV-GFP). The neutralization titers were obtained by counting GFP-expressing cells or by measuring fluorescence. The performance of this new assay was compared against the conventional test using live NiV with panels of sera from several mammalian species, including sera from NiV outbreaks, experimental infections, as well as HeV-specific sera. The results obtained with the VSV-NiV-GFP based test correlated with those obtained using live NiV. Using a 50% reduction in VSV-NiV-GFP infected cells as the cut-off for neutralization, this new assay demonstrated its potential as an effective tool for detecting NiV neutralizing antibodies under BSL2 containment with greater speed, sensitivity and safety as compared to the conventional NiV serum neutralization test. PMID:19433112

  2. Characterization of a Monoclonal Antibody Against CREPT, a Novel Protein Highly Expressed in Tumors

    OpenAIRE

    Ren, Fangli; Wang, Ruoke; Zhang, Yanquan; Liu, Chunxiao; Wang, Yinyin; Hu, Jim; Zhang, Linqi; Chang, Zhijie

    2014-01-01

    CREPT (cell-cycle related and expression-elevated protein in tumor), a novel gene also called RPRD1B and C20ORF77, was recently identified to promote tumorigenesis through up-regulation of the expression of genes related to cell cycle. The previous study demonstrated that CREPT is highly expressed in a variety of tumors and enhances the expression of Cyclin D1 by promoting the formation of a chromatin loop. To study the correlation of CREPT expression with clinical factors in different tumors...

  3. Studies susceptibility of peas and field peas cultivars to Ascochyta pinodes (Jones

    Directory of Open Access Journals (Sweden)

    Helena Furgał-Węgrzycka

    2013-12-01

    Full Text Available The aim of the work was to find plants resistant to Ascochyta pinodes causing leaf and pod spot-pot of peas and field peas. Fourty five cultivars of peas and field peas and 6 breeding materials were tested in the field in the period 1975-1979 on artificially inoculated field plots. Cultivars: Bartel, Birte, Bodil, Borek, Jubilat, Karo, Meteor, Rondo and Żółty Pomorski were to found be less susceptible. In laboratory and greenhouse conditions pea and field pea cultivars were examined for susceptibility. to pathotypes 3 and 5 of Ascochyta pinodes. The results obtained proved that cultivars: Bartel, Birte, Bodil, Borek, Jubilat, Karo, Meteor, Rondo and Żółty Pomorski to be less susceptible to two pathotypes of Ascochyta pinodes.

  4. Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

    Institute of Scientific and Technical Information of China (English)

    Jian-Li Wang; Yu-Ling Zheng; Ru Ma; Bao-Li Wang; Ai-Guang Guo; Yong-Qiang Jiang

    2005-01-01

    AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

  5. 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.

  6. 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

  7. Differential regulation of interleukin 4 and interleukin 5 gene expression: a comparison of T-cell gene induction by anti-CD3 antibody or by exogenous lymphokines.

    OpenAIRE

    Bohjanen, P R; Okajima, M; Hodes, R J

    1990-01-01

    Murine T helper type 2 clones were stimulated with immobilized anti-CD3 antibody or with recombinant lymphokines to compare the expression of T-cell activation genes induced by these stimuli. Immobilized anti-CD3 antibody, recombinant interleukin 2 (IL-2), and recombinant interleukin 4 (IL-4) all induced proliferation of the T helper type 2 clones 10-5-17 and D10. Proliferation of these clones induced by anti-CD3 antibody was completely inhibited by cyclosporine A, whereas cyclosporine A had ...

  8. Cationic liposomes enhance targeted delivery and expression of exogenous DNA mediated by N-terminal modified poly(L-lysine)-antibody conjugate in mouse lung endothelial cells.

    Science.gov (United States)

    Trubetskoy, V S; Torchilin, V P; Kennel, S; Huang, L

    1992-07-15

    A new and improved system for targeted gene delivery and expression is described. Transfection efficiency of N-terminal modified poly(L-lysine) (NPLL) conjugated with anti-thrombomodulin antibody 34A can be improved by adding to the system a lipophilic component, cationic liposomes. DNA, antibody conjugate and cationic liposomes form a ternary electrostatic complex which preserves the ability to bind specifically to the target cells. At the same time the addition of liposomes enhance the specific transfection efficiency of antibody-polylysine/DNA binary complex by 10 to 20-fold in mouse lung endothelial cells in culture.

  9. Expression of Two Members of the pMGA Gene Family of Mycoplasma gallisepticum Oscillates and Is Influenced by pMGA-Specific Antibodies

    OpenAIRE

    Markham, Philip F.; Glew, Michelle D.; Browning, Glenn F.; Whithear, Kevin G.; Ian D. Walker

    1998-01-01

    Certain monoclonal antibodies and polyclonal antisera directed to pMGA, the major protein of Mycoplasma gallisepticum, were tested for the ability to influence the surface phenotype of the cell population which resulted from their inclusion in growth medium. The polyclonal antiserum and one monoclonal antibody (MAb 66) resulted in an alteration of surface phenotype; specifically, populations of cells grown either on plates or in broth cultures which contained these reagents ceased the express...

  10. 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

    2001-06-01

    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.

  11. 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.

  12. 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

    continue to exhibit great discrepancy in the measured percentage of CD163-expressing blood monocytes in healthy individuals. In this study we sought to clarify this inconsistency in reported levels of CD163 surface expression by a detailed analysis of a panel of CD163 antibodies used in previous studies......, and the specificity of the CD163 monoclonal antibodies was analyzed by western blotting. Results and Discussion Flow cytometric analysis revealed that the estimated proportion of CD163-expressing human peripheral blood monocytes increased when using CD163 monoclonal antibodies recognizing epitopes in the N...... of binding in heparin-stabilized whole blood observed by flow cytometry. In contrast, RM3/1 exhibited weak binding to CD163 in the absence of calcium but high affinity binding to CD163 in the presence of calcium. R-20 and MAC2-158 were unaffected by extracellular calcium levels. The latter SRCR domain 1 m...

  13. Genotoxicological Evaluation of NUTRALYS Pea Protein Isolate

    OpenAIRE

    Chentouf Aouatif; Ph. Looten; Parvathi, M. V. S.; Raja Ganesh, S.; Paranthaman, V

    2013-01-01

    NUTRALYS Pea Protein Isolate, a protein supplement, is a high-quality source of protein which is primarily emulsifying functional protein. We evaluated the genotoxic potential of NUTRALYS isolated from dry yellow pea, using three established genotoxicity tests (AMES test in vitro chromosomal aberration test, and in vivo micronucleus test) employing OECD guidelines under GLP conditions. In the bacterial reverse mutation test, NUTRALYS did not show positive responses in strains detecting point ...

  14. Model-directed engineering of "difficult-to-express" monoclonal antibody production by Chinese hamster ovary cells.

    Science.gov (United States)

    Pybus, Leon P; Dean, Greg; West, Nathan R; Smith, Andrew; Daramola, Olalekan; Field, Ray; Wilkinson, Stephen J; James, David C

    2014-02-01

    Despite improvements in volumetric titer for monoclonal antibody (MAb) production processes using Chinese hamster ovary (CHO) cells, some "difficult-to-express" (DTE) MAbs inexplicably reach much lower process titers. These DTE MAbs require intensive cell line and process development activity, rendering them more costly or even unsuitable to manufacture. To rapidly and rationally identify an optimal strategy to improve production of DTE MAbs, we have developed an engineering design platform combining high-yielding transient production, empirical modeling of MAb synthesis incorporating an unfolded protein response (UPR) regulatory loop with directed expression and cell engineering approaches. Utilizing a panel of eight IgG1 λ MAbs varying >4-fold in volumetric titer, we showed that MAb-specific limitations on folding and assembly rate functioned to induce a proportionate UPR in host CHO cells with a corresponding reduction in cell growth rate. Derived from comparative empirical modeling of cellular constraints on the production of each MAb we employed two strategies to increase production of DTE MAbs designed to avoid UPR induction through an improvement in the rate/cellular capacity for MAb folding and assembly reactions. Firstly, we altered the transfected LC:HC gene ratio and secondly, we co-expressed a variety of molecular chaperones, foldases or UPR transactivators (BiP, CypB, PDI, and active forms of ATF6 and XBP1) with recombinant MAbs. DTE MAb production was significantly improved by both strategies, although the mode of action was dependent upon the approach employed. Increased LC:HC ratio or CypB co-expression improved cell growth with no effect on qP. In contrast, BiP, ATF6c and XBP1s co-expression increased qP and reduced cell growth. This study demonstrates that expression-engineering strategies to improve production of DTE proteins in mammalian cells should be product specific, and based on rapid predictive tools to assess the relative impact of

  15. Expression, purification and polyclonal antibody generation of p23,an Hsp90 cochaperone, in the amphioxus Branchiostoma belcheri

    Institute of Scientific and Technical Information of China (English)

    ZHAO Bosheng; ZHANG Shicui; PANG Qiuxiang; LIU Zhenhui; LIANG Yujun

    2006-01-01

    The cDNA of amphioxus p23, a highly conserved co-chaperone for Hsp90, was cloned into a bacterial expression vector pGEX-6P-1 and the GST-tagged fusion protein was produced in Eschherichia coli cells. The recombinant p23 was purified by affinity purification, and its molecular mass was estimated to be approximately 22 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminus of purified p23 was sequenced, and the resulting amino acid sequence matches exactly the predicted residues deduced from the amphioxus p23 gene. Besides, polyclonal antibodies against the recombinant p23 were generated, and these antibodies not only recognized specifically the fusion protein GST-p23 from induced E. coli cells, purified GST-p23 and p23 protein, but also reacted with the total protein extracted from the adult amphioxus and formed a single positive band. These results pave the way for identifying its tissue and subcellular localization, and may open the door to clarifying its structure and mechanisms of biological role.

  16. Auxin influences strigolactones in pea mycorrhizal symbiosis.

    Science.gov (United States)

    Foo, E

    2013-03-15

    Hormone interactions are essential for the control of many developmental processes, including intracellular symbioses. The interaction between auxin and the new plant hormone strigolactone in the regulation of arbuscular mycorrhizal symbiosis was examined in one of the few auxin deficient mutants available in a mycorrhizal species, the auxin-deficient bsh mutant of pea (Pisum sativum). Mycorrhizal colonisation with the fungus Glomus intraradices was significantly reduced in the low auxin bsh mutant. The bsh mutant also exhibited a reduction in strigolactone exudation and the expression of a key strigolactone biosynthesis gene (PsCCD8). Strigolactone exudation was also reduced in wild type plants when the auxin content was reduced by stem girdling. Low strigolactone levels appear to be at least partially responsible for the reduced colonisation of the bsh mutant, as application of the synthetic strigolactone GR24 could partially rescue the mycorrhizal phenotype of bsh mutants. Data presented here indicates root auxin content was correlated with strigolactone exudation in both mutant and wild type plants. Mutant studies suggest that auxin may regulate early events in the formation of arbuscular mycorrhizal symbiosis by controlling strigolactone levels, both in the rhizosphere and possibly during early root colonisation. PMID:23219475

  17. Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages

    OpenAIRE

    Ooms, Karen; Van Gorp, Hanne; Van Gaever, Tim; Nauwynck, Hans; Delputte, Peter

    2013-01-01

    Background: Sialoadhesin (Sn)-expressing monocytes/macrophages have been associated with several diseases like inflammatory and autoimmune disorders as well as viral infections, and they also appear to play a role in the initiation of an adaptive immune response. This makes Sn-expressing cells not only attractive targets for cell-directed therapies, but also an appealing target for vaccination. Furthermore, since Sn was shown to be an endocytic receptor, the conjugation of effector molecules ...

  18. Recombinant human antibodies: linkage of an Fab fragment from a combinatorial library to an Fc fragment for expression in mammalian cell culture.

    Science.gov (United States)

    Bender, E; Woof, J M; Atkin, J D; Barker, M D; Bebbington, C R; Burton, D R

    1993-04-01

    The combinatorial phage library approach to immunoglobulin repertoire cloning recently made it possible to isolate gene fragments encoding human immunoglobulin G1 Fabs binding with high affinity to specific antigens. Here we describe the construction of genes encoding whole human anti-tetanus toxoid antibodies based on one of these gene fragments and the efficient expression of these constructs by co-transfection of separate heavy and light chain vectors into a Chinese hamster ovary cell line constitutively expressing a viral transactivator protein. This system will be generally useful for the rapid analysis of recombinant antibodies derived from combinatorial libraries. PMID:8518367

  19. Preparation of polyclonal antibody specific for NOR1 and detection of its expression pattern in human tissues and nasopharyngeal carcinoma

    Institute of Scientific and Technical Information of China (English)

    Bo Xiang; Mei Yi; Li Wang; Wei Liu; Wenling Zhang; Jue Ouyang; Ya Peng; Wenjuan Li; Ming Zhou; Huaying Liu; Minghua Wu; Rong Wang; Xiaoling Li; Guiyuan Li

    2009-01-01

    Oxidored-nitro domain containing protein 1 (NORI)gene is a novel nitroreductase gene first isolated from nasopharyngeal carcinoma (NPC). It plays an important role in the formation of chemical carcinogen and the carcinogenesis of NPC for its nitrosation function. Overexpression of the wild-type NOR1 gene in naso-pharyngeal carcinoma cells is effective to inhibit cell growth and proliferation. In this study, for the first time, we generated a highly specific NOR1 antibody and ana-lyzed NOR1 distribution in the human tissues and NPC biopsies. The results showed that NOR1 protein is predominantly expressed in human nasopharynx and tracheal tissues. Human heart, liver, spleen, stomach, colon, kidney, skeletal muscle, thymus, and pancreas are aH deficient of NORI protein. More importantly, we per-formed immunohistochemistry assay of NOR1 protein expression in the NPC tissues, and the result showed that NOR1 protein is frequently down-expressed in NPC. These data shed light on the selectivity of potential physiological functions of NOR1 and provides an indis-pensable reference to the carcinogenesis process of NPC and to identify or validate tissue-specific drug targets.

  20. Induction of neutralizing antibodies by varicella-zoster virus gpII glycoprotein expressed from recombinant vaccinia virus.

    Science.gov (United States)

    Massaer, M; Haumont, M; Place, M; Bollen, A; Jacobs, P

    1993-03-01

    The gpII glycoprotein of varicella-zoster virus (VZV) was produced in CV1 cells via vaccinia virus recombinants. Two different DNA constructs were expressed: the first one encodes the complete gpII protein (gpII s+a+) and the second a truncated species lacking the membrane anchorage domain (gpII s+a-). To achieve expression both coding sequences had to be engineered at the 5' end by substituting the unusually short (24 bp) natural signal sequence by a more conventional one encoding 29 amino acids. Recombinant gpII proteins were detected in vaccinia virus-infected cells by ELISA and immunoprecipitation. Both forms of recombinant gpII proteins were produced as glycosylated single-chain molecules of respectively 110K and 90K. Upon reduction these were only partially converted into subunits. A rabbit infected with the vaccinia virus recombinant expressing the complete gpII produced antibodies which recognized VZV antigens and neutralized VZV infectivity in vitro, independent of complement.

  1. Characterisation of a pea hsp70 gene which is both developmentally and stress-regulated.

    Science.gov (United States)

    Dhankher, O P; Drew, J E; Gatehouse, J A

    1997-05-01

    A pea pod cDNA library was screened for sequences specific to lignifying tissue. A cDNA clone (pLP19) encoding the C-terminal region of a hsp70 heat shock protein hybridised only to pod mRNA from pea lines where pod lignification occurred. Expression of pLP19 was induced by heat shock in leaves, stems and roots of pea and chickpea plants. Four different poly(A) addition sites were observed in cDNAs derived from the same gene as pLP19. This gene was fully sequenced; unlike most hsp70 genes, it contains no introns. The 5'-flanking sequence contains heat shock elements and other potential regulatory sequences.

  2. Molecular cloning of GA-suppressed G2 pea genes by cDNA RDA

    Institute of Scientific and Technical Information of China (English)

    朱玉贤; 张翼凤; 李慧英

    1997-01-01

    GA-treated and non-treated G2 pea cDNAs were compared using a newly developed method called cDNA representational difference analysis (cDNA-RDA), and several GA-suppressed mRNAs were found. After cloning of the larger fragments PGAS1-3 ( pea GA-suppressed cDNA 1-3), they were demonstrated to be expressed only in pea tissue not treated with GA3 through Northern analysis. Compared with subtractive hybridization and differ-ential display techniques, this method not only can be easily manipulated but also has a relatively low rate of false posi-tive and is highly repetitive. It is the major progress in molecular cloning techniques.

  3. 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.

  4. The pea gene NA encodes ent-kaurenoic acid oxidase.

    Science.gov (United States)

    Davidson, Sandra E; Elliott, Robert C; Helliwell, Chris A; Poole, Andrew T; Reid, James B

    2003-01-01

    The gibberellin (GA)-deficient dwarf na mutant in pea (Pisum sativum) has severely reduced internode elongation, reduced root growth, and decreased leaflet size. However, the seeds develop normally. Two genes, PsKAO1 and PsKAO2, encoding cytochrome P450 monooxygenases of the subfamily CYP88A were isolated. Both PsKAO1 and PsKAO2 had ent-kaurenoic acid oxidase (KAO) activity, catalyzing the three steps of the GA biosynthetic pathway from ent-kaurenoic acid to GA(12) when expressed in yeast (Saccharomyces cerevisiae). In addition to the intermediates ent-7alpha-hydroxykaurenoic acid and GA(12)-aldehyde, some additional products of the pea KAO activity were detected, including ent-6alpha,7alpha-dihydroxykaurenoic acid and 7beta-hydroxykaurenolide. The NA gene encodes PsKAO1, because in two independent mutant alleles, na-1 and na-2, PsKAO1 had altered sequences and the five-base deletion in PsKAO1 associated with the na-1 allele cosegregated with the dwarf na phenotype. PsKAO1 was expressed in the stem, apical bud, leaf, pod, and root, organs in which GA levels have previously been shown to be reduced in na plants. PsKAO2 was expressed only in seeds and this may explain the normal seed development and normal GA biosynthesis in seeds of na plants.

  5. 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

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

    International Nuclear Information System (INIS)

    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

  7. Lentil root protoplasts: a transient expression system suitable for coelectroporation of monoclonal antibodies and plasmid molecules

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Maccarrone, M.; Veldink, G.A.; Finazzi Agrò, A.

    1995-01-01

    Protoplasts were isolated from lentil (Lens culinaris) roots and their suitability as a transient expression system was investigated. After transfecting the protoplasts with the -glucuronidase (GUS) gene by either electroporation or polyethylene glycol (PEG), the specific activity of the reporter en

  8. 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.

  9. Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen.

    OpenAIRE

    Lagranderie, M; Lo-Man, R; Dériaud, E; Gicquel, B; Gheorghiu, M; Leclerc, C

    1997-01-01

    Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various...

  10. Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

    Directory of Open Access Journals (Sweden)

    Hafez Heydari Zarnagh

    2015-04-01

    Full Text Available Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3 cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

  11. PEA: Polymorphic Encryption Algorithm based on quantum computation

    OpenAIRE

    Komninos, N.; Mantas, G.

    2011-01-01

    In this paper, a polymorphic encryption algorithm (PEA), based on basic quantum computations, is proposed for the encryption of binary bits. PEA is a symmetric key encryption algorithm that applies different combinations of quantum gates to encrypt binary bits. PEA is also polymorphic since the states of the shared secret key control the different combinations of the ciphertext. It is shown that PEA achieves perfect secrecy and is resilient to eavesdropping and Trojan horse attacks. A securit...

  12. Pneumococcal Surface Protein A Is Expressed In Vivo, and Antibodies to PspA Are Effective for Therapy in a Murine Model of Pneumococcal Sepsis

    OpenAIRE

    Swiatlo, E.; J. King; Nabors, G S; Mathews, B; Briles, D. E.

    2003-01-01

    Pneumococcal surface protein A (PspA) is an immunogenic protein expressed on the surface of all strains of Streptococcus pneumoniae (pneumococcus) and induces antibodies which protect against invasive infection in mice. Pneumococci used for infectious challenge in protection studies are typically collected from cultures grown in semisynthetic medium in vitro. The purpose of these studies is to confirm that PspA is expressed by pneumococci during growth in vivo at ...

  13. Pea Plants Show Risk Sensitivity.

    Science.gov (United States)

    Dener, Efrat; Kacelnik, Alex; Shemesh, Hagai

    2016-07-11

    Sensitivity to variability in resources has been documented in humans, primates, birds, and social insects, but the fit between empirical results and the predictions of risk sensitivity theory (RST), which aims to explain this sensitivity in adaptive terms, is weak [1]. RST predicts that agents should switch between risk proneness and risk aversion depending on state and circumstances, especially according to the richness of the least variable option [2]. Unrealistic assumptions about agents' information processing mechanisms and poor knowledge of the extent to which variability imposes specific selection in nature are strong candidates to explain the gap between theory and data. RST's rationale also applies to plants, where it has not hitherto been tested. Given the differences between animals' and plants' information processing mechanisms, such tests should help unravel the conflicts between theory and data. Measuring root growth allocation by split-root pea plants, we show that they favor variability when mean nutrient levels are low and the opposite when they are high, supporting the most widespread RST prediction. However, the combination of non-linear effects of nitrogen availability at local and systemic levels may explain some of these effects as a consequence of mechanisms not necessarily evolved to cope with variance [3, 4]. This resembles animal examples in which properties of perception and learning cause risk sensitivity even though they are not risk adaptations [5]. PMID:27374342

  14. Effect of pigeon pea (Cajanus cajan L.) on high-fat diet-induced hypercholesterolemia in hamsters.

    Science.gov (United States)

    Dai, Fan-Jhen; Hsu, Wei-Hsuan; Huang, Jan-Jeng; Wu, She-Ching

    2013-03-01

    Obesity is associated with increased systemic and airway oxidative stress, which may result from a combination of adipokine imbalance and antioxidant defenses reduction. Obesity-mediated oxidative stress plays an important role in the pathogenesis of dyslipidemia, vascular disease, and nonalcoholic hepatic steatosis. The antidyslipidemic activity of pigeon pea were evaluated by high-fat diet (HFD) hamsters model, in which the level of high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), total cholesterol (TC), and total triglyceride (TG) were examined. We found that pigeon pea administration promoted cholesterol converting to bile acid in HFD-induced hamsters, thereby exerting hypolipidemic activity. In the statistical results, pigeon pea significantly increased hepatic carnitine palmitoyltransferase-1 (CPT-1), LDL receptor, and cholesterol 7α-hydroxylase (also known as cytochrome P450 7A1, CYP7A1) expression to attenuate dyslipidemia in HFD-fed hamsters; and markedly elevated antioxidant enzymes in the liver of HFD-induced hamsters, further alleviating lipid peroxidation. These effects may attribute to pigeon pea contained large of unsaturated fatty acids (UFA; C18:2) and phytosterol (β-sitosterol, campesterol, and stigmasterol). Moreover, the effects of pigeon pea on dyslipidemia were greater than β-sitosterol administration (4%), suggesting that phytosterone in pigeon pea could prevent metabolic syndrome. PMID:23287313

  15. 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. PMID:11158527

  16. 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.

  17. Effect of pigeon pea (Cajanus cajan L.) on high-fat diet-induced hypercholesterolemia in hamsters.

    Science.gov (United States)

    Dai, Fan-Jhen; Hsu, Wei-Hsuan; Huang, Jan-Jeng; Wu, She-Ching

    2013-03-01

    Obesity is associated with increased systemic and airway oxidative stress, which may result from a combination of adipokine imbalance and antioxidant defenses reduction. Obesity-mediated oxidative stress plays an important role in the pathogenesis of dyslipidemia, vascular disease, and nonalcoholic hepatic steatosis. The antidyslipidemic activity of pigeon pea were evaluated by high-fat diet (HFD) hamsters model, in which the level of high-density lipoprotein-cholesterol (HDL-C), low-density lipoprotein-cholesterol (LDL-C), total cholesterol (TC), and total triglyceride (TG) were examined. We found that pigeon pea administration promoted cholesterol converting to bile acid in HFD-induced hamsters, thereby exerting hypolipidemic activity. In the statistical results, pigeon pea significantly increased hepatic carnitine palmitoyltransferase-1 (CPT-1), LDL receptor, and cholesterol 7α-hydroxylase (also known as cytochrome P450 7A1, CYP7A1) expression to attenuate dyslipidemia in HFD-fed hamsters; and markedly elevated antioxidant enzymes in the liver of HFD-induced hamsters, further alleviating lipid peroxidation. These effects may attribute to pigeon pea contained large of unsaturated fatty acids (UFA; C18:2) and phytosterol (β-sitosterol, campesterol, and stigmasterol). Moreover, the effects of pigeon pea on dyslipidemia were greater than β-sitosterol administration (4%), suggesting that phytosterone in pigeon pea could prevent metabolic syndrome.

  18. Auxin-cytokinin and auxin-gibberellin interactions during morphogenesis of the compound leaves of pea (Pisum sativum).

    Science.gov (United States)

    DeMason, Darleen A

    2005-09-01

    A number of mutations that alter the form of the compound leaf in pea (Pisum sativum) has proven useful in elucidating the role that auxin might play in pea leaf development. The goals of this study were to determine if auxin application can rescue any of the pea leaf mutants and if gibberellic acid (GA) plays a role in leaf morphogenesis in pea. A tissue culture system was used to determine the effects of various auxins, GA or a GA biosynethesis inhibitor (paclobutrazol) on leaf development. The GA mutant, nana1 (na1) was analyzed. The uni-tac mutant was rescued by auxin and GA and rescue involved both a conversion of the terminal leaflet into a tendril and an addition of a pair of lateral tendrils. This rescue required the presence of cytokinin. The auxins tested varied in their effectiveness, although methyl-IAA worked best. The terminal tendrils of wildtype plantlets grown on paclobutrazol were converted into leaflets, stubs or were aborted. The number of lateral pinna pairs produced was reduced and leaf initiation was impaired. These abnormalities resembled those caused by auxin transport inhibitors and phenocopy the uni mutants. The na1 mutant shared some morphological features with the uni mutants; including, flowering late and producing leaves with fewer lateral pinna pairs. These results show that both auxin and GA play similar and significant roles in pea leaf development. Pea leaf morphogenesis might involve auxin regulation of GA biosynthesis and GA regulation of Uni expression. PMID:15809864

  19. Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids.

    Science.gov (United States)

    Zhang, Lizhi; Garneau, Matthew G; Majumdar, Rajtilak; Grant, Jan; Tegeder, Mechthild

    2015-01-01

    The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic-active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane-localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element-companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.

  20. Prokaryotic expression, purification, polyclonal antibody preparation, and tissue distribution of porcine Six1

    OpenAIRE

    XU, MENG; CHEN, Xiaoling; Huang, Zhiqing; WEN, WANXUE; Chang, Shuai; WANG, XIAOYAN; Chen, Daiwen; Yu, Bing; Luo, Junqiu; Liu, Guangmang

    2015-01-01

    Sine oculis homeobox 1 (Six1), a member of the Six homeoproteins, plays an important role in skeletal myogenesis and the specification of myofiber diversity. In this study, in order to scale up the production of recombinant porcine Six1 (pSix1), a pET-30a(+)-pSix1 plasmid was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant pSix1 could be induced for efficient expression with 2 mM IPTG for 2 h at 30 °C, yielding approximately 4.6 mg/L. The protein was then purifie...

  1. 7 CFR 457.140 - Dry pea crop insurance provisions.

    Science.gov (United States)

    2010-01-01

    ... insured crop: (1) If you do not have any insured fall-planted dry pea acreage covered under the Winter... closing date; or (2) If you have any insured fall-planted dry pea acreage covered under the Winter... dry peas not covered by the Winter Coverage Option will begin on the earlier of April 15 or the...

  2. Pea proteins for piglets: effects on digestive processes.

    NARCIS (Netherlands)

    le Guen, M.P.

    1993-01-01

    White-flowered pea ( Pisum sativum ) contains 20 to 25% crude protein (Nx6.25). The pea proteins consist in globulins (60%) and albumins (25%). Because the pea albumin proteins have biological activities due to their metabolic roles in the seed, some of them are called antinutritional factors (ANFs)

  3. 7 CFR 457.137 - Green pea crop insurance provisions.

    Science.gov (United States)

    2010-01-01

    .... Dry peas. Green peas that have matured to the dry form for use as food, feed, or seed. Good farming... the Special Provisions. Processor. Any business enterprise regularly engaged in canning or freezing... peas genetically developed to be shelled prior to eating, canning or freezing. 2. Unit Division (a)...

  4. Control of winter forage pea diseases by pea-oat intercropping under field conditions

    Directory of Open Access Journals (Sweden)

    Dalibor Živanov

    2014-06-01

    Full Text Available A field experiment was conducted at the experimental field of the Institute of Field and Vegetable Crops in Novi Sad to investigate the effect of forage winter pea and winter oat intercropping on ascochyta blight and powdery mildew infections. Seeding rations of pea and oat in Treatment 1 (50:50% and Treatment 2 (75:25%, respectively reduced ascochyta leaf infection by 32.5% and 12.8%, and powdery mildew infection by 12.3% and 17.5%, respectively, compared to pea monoculture used as a control (Treatment 3. The same seeding rations in Treatment 1 and 2 reduced ascochyta blight on pea plants by 37.2% and 18.3%, respectively. However, there were no significant differences between the treatments in reducing powdery mildew on plants. The effects of different treatments on the average number of pods per plant, seed per pod, shriveled pods and seed weight were analyzed using Spearman’s correlation coefficient. Negative but not statistically significant effects on those measured parameters were registered in Treatments 2 and 3, while Treatment 1 showed positive effects on all parameters except shriveled pods. According to all data obtained in this research, the intercropping mixture of pea and oat at 50:50% seeding ratio had the best effect on the measured parameters while the intercropping mixture of pea and oat at 75:25% seeding ratio had low to moderate effect in comparison with pea monocrop.

  5. PED/PEA-15 inhibits hydrogen peroxide-induced apoptosis in Ins-1E pancreatic beta-cells via PLD-1.

    Directory of Open Access Journals (Sweden)

    Francesca Fiory

    Full Text Available The small scaffold protein PED/PEA-15 is involved in several different physiologic and pathologic processes, such as cell proliferation and survival, diabetes and cancer. PED/PEA-15 exerts an anti-apoptotic function due to its ability to interfere with both extrinsic and intrinsic apoptotic pathways in different cell types. Recent evidence shows that mice overexpressing PED/PEA-15 present larger pancreatic islets and increased beta-cells mass. In the present work we investigated PED/PEA-15 role in hydrogen peroxide-induced apoptosis in Ins-1E beta-cells. In pancreatic islets isolated from Tg(PED/PEA-15 mice hydrogen peroxide-induced DNA fragmentation was lower compared to WT islets. TUNEL analysis showed that PED/PEA-15 overexpression increases the viability of Ins-1E beta-cells and enhances their resistance to apoptosis induced by hydrogen peroxide exposure. The activity of caspase-3 and the cleavage of PARP-1 were markedly reduced in Ins-1E cells overexpressing PED/PEA-15 (Ins-1E(PED/PEA-15. In parallel, we observed a decrease of the mRNA levels of pro-apoptotic genes Bcl-xS and Bad. In contrast, the expression of the anti-apoptotic gene Bcl-xL was enhanced. Accordingly, DNA fragmentation was higher in control cells compared to Ins-1E(PED/PEA-15 cells. Interestingly, the preincubation with propranolol, an inhibitor of the pathway of PLD-1, a known interactor of PED/PEA-15, responsible for its deleterious effects on glucose tolerance, abolishes the antiapoptotic effects of PED/PEA-15 overexpression in Ins-1E beta-cells. The same results have been obtained by inhibiting PED/PEA-15 interaction with PLD-1 in Ins-1E(PED/PEA-15. These results show that PED/PEA-15 overexpression is sufficient to block hydrogen peroxide-induced apoptosis in Ins-1E cells through a PLD-1 mediated mechanism.

  6. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies

    Science.gov (United States)

    Ricklin, Meret E.; Vielle, Nathalie J.; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4+ T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.

  7. Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein.

    Science.gov (United States)

    Sharma, Susheel Kumar; Kumar, P Vignesh; Baranwal, Virendra Kumar

    2014-10-01

    A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate-enzyme linked immunosorbent assay (ACP-ELISA). The antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.

  8. Automorphosis and auxin polar transport of etiolated pea seedlings under microgravity conditions.

    Science.gov (United States)

    Hoshino, Tomoki; Miyamoto, Kensuke; Ueda, Junichi

    2004-11-01

    On STS-95 space experiment, etiolated pea (Pisum sativum L. cv. Alaska) seedlings showed automorphosis and activities of auxin polar transport in epicotyls were substantially suppressed. These results together with the fact that inhibitors of auxin polar transport induced automorphosis-like growth and development strongly suggested that there are close relationships between automorphosis and auxin polar transport in etiolated pea seedlings. In order to know how gravistimuli control auxin polar transport at molecular levels, we isolated novel cDNAs of PsPIN2 and PsAUX1 encoding putative auxin efflux and influx carriers from etiolated pea seedlings. Significantly high levels in homology were found on nucleotide and deduced amino acid sequences among PsPIN2, PsPIN1 (accession no. AY222857) and AtPINs, and between PsAUX1 and AtAUX1. Exogenously applied auxin substantially enhanced the expression of PsAUX1 and PsPIN2 as well as PsPIN1. Simulated microgravity conditions on a 3-dimensional clinostat remarkably increased gene expression of PsPIN1 and PsAUX1 in the hook and the 1st internode of pea epicotyls, while the increase of expression of PsPIN2 in both organs was not so much. These results suggest that PsPINs and PsAUX1 are auxin-inducible genes, and the expression of PsPINs and PsAUX1 is under the control of gravistimulation. A possible role of these genes in regulating auxin transport relevant to automorphosis of etiolated pea seedlings is also discussed. PMID:15858337

  9. Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining.

    Science.gov (United States)

    Lin, Gee-Way; Chang, Chun-che

    2016-01-01

    The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H2O2) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids. PMID:26862939

  10. Pea VEGETATIVE2 Is an FD Homolog That Is Essential for Flowering and Compound Inflorescence Development.

    Science.gov (United States)

    Sussmilch, Frances C; Berbel, Ana; Hecht, Valérie; Vander Schoor, Jacqueline K; Ferrándiz, Cristina; Madueño, Francisco; Weller, James L

    2015-04-01

    As knowledge of the gene networks regulating inflorescence development in Arabidopsis thaliana improves, the current challenge is to characterize this system in different groups of crop species with different inflorescence architecture. Pea (Pisum sativum) has served as a model for development of the compound raceme, characteristic of many legume species, and in this study, we characterize the pea VEGETATIVE2 (VEG2) locus, showing that it is critical for regulation of flowering and inflorescence development and identifying it as a homolog of the bZIP transcription factor FD. Through detailed phenotypic characterizations of veg2 mutants, expression analyses, and the use of protein-protein interaction assays, we find that VEG2 has important roles during each stage of development of the pea compound inflorescence. Our results suggest that VEG2 acts in conjunction with multiple FLOWERING LOCUS T (FT) proteins to regulate expression of downstream target genes, including TERMINAL FLOWER1, LEAFY, and MADS box homologs, and to facilitate cross-regulation within the FT gene family. These findings further extend our understanding of the mechanisms underlying compound inflorescence development in pea and may have wider implications for future manipulation of inflorescence architecture in related legume crop species.

  11. Detection of MUC1-Expressing Ovarian Cancer by C595 Monoclonal Antibody-Conjugated SPIONs Using MR Imaging

    Directory of Open Access Journals (Sweden)

    Daryoush Shahbazi-Gahrouei

    2013-01-01

    Full Text Available The aim of this study is to find out the development and application of MUC1-expressing ovarian cancer (OVCAR3 by C595 monoclonal antibody-conjugated superparamagnetic iron oxide nanoparticles (SPIONs using MR imaging. At the end, its use as a nanosized contrast agent MR imaging probe for ovarian cancer detection was investigated. The strategy is to use SPIONs attached to C595 mAb that binds to the MUC1, to specifically detect ovarian cancer cells. Anticancer effects and MR imaging parameters of the prepared nanoconjugate was investigated both under in vitro and in vivo experiments. The characterization of nanoconjugate includes its size, cell toxicity, flow cytometry, Prussian blue staining test and its cellular uptake as well as its biodistribution, and MR imaging was also investigated. The findings of the study showed good tumor accumulation and detection, no in vivo toxicity, and potential selective antiovarian cancer activity. Overall, based on the findings SPIONs-C595 nanosized probe is a selective ovarian molecular imaging modality. Further subsequent clinical trials appear warranted.

  12. Characterization of antigen-expressing Plasmodium falciparum cDNA clones that are reactive with parasite inhibitory antibodies.

    Science.gov (United States)

    Horii, T; Bzik, D J; Inselburg, J

    1988-07-01

    A Plasmodium falciparum (FCR3 strain) lambda gt11 cDNA expression library was constructed from trophozoite and schizont poly(A) RNA and was screened immunologically with a pooled human immune serum from Nigeria to form a gene bank of 288 positive clones. The gene bank was subsequently screened with parasite inhibitory mouse monoclonal antibodies (mMAb) and with individual human Liberian sera. Two mMAb, 43E5 and 5H10, strongly reacted with 8 and 3 cDNA clones, respectively. Several of those clones also weakly cross-reacted with the other mMAb. Two of those weakly cross-reactive clones, cDNA#366 and cDNA#22, were shown to be located in different chromosomal regions of the parasite by Southern hybridization and so appeared to represent two different parasite genes. The genomic organization of both cDNA#366 and cDNA#22 sequences were identical in the FCR3 and the Honduras-1 strain. The nucleotide sequence of cDNA#366 and the amino acid sequence it coded for were homologous to a partial DNA and amino acid sequence previously reported for a P. falciparum (Camp strain) exoantigen designated p126. The mRNA for cDNA#366 appeared to represent an abundant message in blood stage trophozoites and schizonts. PMID:2456465

  13. Simple immunoblot and immunohistochemical detection of Penaeus stylirostris densovirus using monoclonal antibodies to viral capsid protein expressed heterologously.

    Science.gov (United States)

    Sithigorngul, Paisarn; Hajimasalaeh, Warunee; Longyant, Siwaporn; Sridulyakul, Pattarin; Rukpratanporn, Sombat; Chaivisuthangkura, Parin

    2009-12-01

    Penaeus stylirostris densovirus (PstDNV), called formerly infectious hypodermal and hematopoietic necrosis virus (IHHNV), is an important shrimp pathogen which can cause mortality in the blue shrimp Penaeus (Litopenaeus) stylirostris and stunting in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. Five monoclonal antibodies (MAbs) were produced against the 37kDa capsid protein 3 (CP3) of PstDNV expressed heterologously in the form of a fusion protein with glutathione-S-transferase called GST-CP3. All MAbs belonged to the IgG2b subclass and could bind to GST-CP3 at 300 pg/spot in immunodot-blot tests. They could detect CP3 in naturally infected shrimp extracts by Western blotting and dot blotting and in shrimp tissues by immunohistochemistry without cross-reactivity to extracts from uninfected shrimps or shrimps infected with several other viruses. Although dot blot assay sensitivity was approximately 1000 times lower than that of one step PCR for PstDNV, it easily detected PstDNV infections in field samples of Penaeus monodon and Penaeus vannamei. PMID:19654023

  14. Review of the health benefits of peas (Pisum sativum L.).

    Science.gov (United States)

    Dahl, Wendy J; Foster, Lauren M; Tyler, Robert T

    2012-08-01

    Pulses, including peas, have long been important components of the human diet due to their content of starch, protein and other nutrients. More recently, the health benefits other than nutrition associated with pulse consumption have attracted much interest. The focus of the present review paper is the demonstrated and potential health benefits associated with the consumption of peas, Pisum sativum L., specifically green and yellow cotyledon dry peas, also known as smooth peas or field peas. These health benefits derive mainly from the concentration and properties of starch, protein, fibre, vitamins, minerals and phytochemicals in peas. Fibre from the seed coat and the cell walls of the cotyledon contributes to gastrointestinal function and health, and reduces the digestibility of starch in peas. The intermediate amylose content of pea starch also contributes to its lower glycaemic index and reduced starch digestibility. Pea protein, when hydrolysed, may yield peptides with bioactivities, including angiotensin I-converting enzyme inhibitor activity and antioxidant activity. The vitamin and mineral contents of peas may play important roles in the prevention of deficiency-related diseases, specifically those related to deficiencies of Se or folate. Peas contain a variety of phytochemicals once thought of only as antinutritive factors. These include polyphenolics, in coloured seed coat types in particular, which may have antioxidant and anticarcinogenic activity, saponins which may exhibit hypocholesterolaemic and anticarcinogenic activity, and galactose oligosaccharides which may exert beneficial prebiotic effects in the large intestine. PMID:22916813

  15. Transcriptional profiling of the pea shoot apical meristem reveals processes underlying its function and maintenance

    Directory of Open Access Journals (Sweden)

    Singh Mohan B

    2008-06-01

    Full Text Available Abstract Background Despite the importance of the shoot apical meristem (SAM in plant development and organ formation, our understanding of the molecular mechanisms controlling its function is limited. Genomic tools have the potential to unravel the molecular mysteries of the SAM, and legume systems are increasingly being used in plant-development studies owing to their unique characteristics such as nitrogen fixation, secondary metabolism, and pod development. Garden pea (Pisum sativum is a well-established classic model species for genetics studies that has been used since the Mendel era. In addition, the availability of a plethora of developmental mutants makes pea an ideal crop legume for genomics studies. This study aims to utilise genomics tools in isolating genes that play potential roles in the regulation of SAM activity. Results In order to identify genes that are differentially expressed in the SAM, we generated 2735 ESTs from three cDNA libraries derived from freshly micro-dissected SAMs from 10-day-old garden peas (Pisum sativum cv Torsdag. Custom-designed oligonucleotide arrays were used to compare the transcriptional profiles of pea SAMs and non-meristematic tissues. A total of 184 and 175 transcripts were significantly up- or down-regulated in the pea SAM, respectively. As expected, close to 61% of the transcripts down-regulated in the SAM were found in the public database, whereas sequences from the same source only comprised 12% of the genes that were expressed at higher levels in the SAM. This highlights the under-representation of transcripts from the meristematic tissues in the current public pea protein database, and demonstrates the utility of our SAM EST collection as an essential genetic resource for revealing further information on the regulation of this developmental process. In addition to unknowns, many of the up-regulated transcripts are known to encode products associated with cell division and proliferation

  16. Pichia pastoris-expressed Dengue 3 Envelope-based Virus-like Particles Elicit Predominantly Domain III-Focused High Titer Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Lav eTripathi

    2015-09-01

    Full Text Available Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3 and -4 cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE, by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs, in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed towards domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential towards heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way towards developing a DENV E-based, inexpensive, safe and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

  17. 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.

  18. Thyroid Antibodies

    Science.gov (United States)

    ... be limited. Home Visit Global Sites Search Help? Thyroid Antibodies Share this page: Was this page helpful? Also known as: Thyroid Autoantibodies; Antithyroid Antibodies; Antimicrosomal Antibody; Thyroid Microsomal Antibody; ...

  19. Production and characterization of chimeric monoclonal antibodies against Burkholderia pseudomallei and B. mallei using the DHFR expression system.

    Directory of Open Access Journals (Sweden)

    Hyung-Yong Kim

    Full Text Available Burkholderia pseudomallei (BP and B. mallei (BM are closely related gram-negative, facultative anaerobic bacteria which cause life-threatening melioidosis in human and glanders in horse, respectively. Our laboratory has previously generated and characterized more than 100 mouse monoclonal antibodies (MAbs against BP and BM, according to in vitro and in vivo assay. In this study, 3 MAbs (BP7 10B11, BP7 2C6, and BP1 7F7 were selected to develop into chimeric mouse-human monoclonal antibodies (cMAbs against BP and/or BM. For the stable production of cMAbs, we constructed 4 major different vector systems with a dihydrofolate reductase (DHFR amplification marker, and optimized transfection/selection conditions in mammalian host cells with the single-gene and/or double-gene expression system. These 3 cMAbs were stably produced by the DHFR double mutant Chinese hamster ovarian (CHO-DG44 cells. By ELISA and Western blot analysis using whole bacterial antigens treated by heat (65°C/90 min, sodium periodate, and proteinase K, the cMAb BP7 10B11 (cMAb CK1 reacted with glycoproteins (34, 38, 48 kDa in BP; 28, 38, 48 kDa in BM. The cMAb BP7 2C6 (cMAb CK2 recognized surface-capsule antigens with molecular sizes of 38 to 52 kDa, and 200 kDa in BM. The cMAb CK2 was weakly reactive to 14∼28, 200 kDa antigens in BP. The cMAb BP1 7F7 (cMAb CK3 reacted with lipopolysaccharides (38∼52 kDa in BP; 38∼60 kDa in B. thailandensis. Western blot results with the outer surface antigens of the 3 Burkholderia species were consistent with results with the whole Burkholderia cell antigens, suggesting that these immunodominant antigens reacting with the 3 cMAbs were primarily present on the outer surface of the Burkholderia species. These 3 cMAbs would be useful for analyzing the role of the major outer surface antigens in Burkholderia infection.

  20. Delineation of stage specific expression of Plasmodium falciparum EBA-175 by biologically functional region II monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    B Kim Lee Sim

    Full Text Available BACKGROUND: The malaria parasite Plasmodium falciparum EBA-175 binds its receptor sialic acids on glycophorin A when invading erythrocytes. The receptor-binding region (RII contains two cysteine-rich domains with similar cysteine motifs (F1 and F2. Functional relationships between F1 and F2 domains and characterization of EBA-175 were studied using specific monoclonal antibodies (mAbs against these domains. METHODS AND FINDINGS: Five mAbs specific for F1 or F2 were generated. Three mAbs specific for F2 potently blocked binding of EBA-175 to erythrocytes, and merozoite invasion of erythrocytes (IC(50 10 to 100 µg/ml IgG in growth inhibition assays. A mAb specific for F1 blocked EBA-175 binding and merozoite invasion less effectively. The difference observed between the IC(50 of F1 and F2 mAbs was not due to differing association and disassociation rates as determined by surface plasmon resonance. Four of the mAbs recognized conformation-dependent epitopes within F1 or F2. Used in combination, F1 and F2 mAbs blocked the binding of native EBA-175 to erythrocytes and inhibited parasite invasion synergistically in vitro. MAb R217, the most potent, did not recognize sporozoites, 3-day hepatocyte stage parasites, nor rings, trophozoites, gametocytes, retorts, ookinetes, and oocysts but recognized 6-day hepatocyte stage parasites, and schizonts. Even though efficient at blocking binding to erythrocytes and inhibiting invasion into erythrocytes, MAb R217 did not inhibit sporozoite invasion and development in hepatocytes in vitro. CONCLUSIONS: The role of the F1 and F2 domains in erythrocyte invasion and binding was elucidated with mAbs. These mAbs interfere with native EBA-175 binding to erythrocyte in a synergistic fashion. The stage specific expression of EBA-175 showed that the primary focus of activity was the merozoite stage. A recombinant RII protein vaccine consisting of both F1 and F2 domains that could induce synergistic activity should be

  1. 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.

  2. Cloning, expression and purification of binding domains of lethal factor and protective antigen of Bacillus anthracis in Escherichia coli and evaluation of their related murine antibody.

    Science.gov (United States)

    Rezaee, Mehdi; Honari, Hossein; Kooshk, Mohammad Reza Ashrafi

    2014-01-01

    Anthrax is common disease between human and animals caused by Bacillus anthracis. The cell binding domain of protective antigen (PAD4) and the binding domain of lethal factor (LFD1) have high immunogenicity potential and always were considered as a vaccine candidate against anthrax. The aims of this study are cloning and expressing of PAD4 and LFD1 in Escherichia coli, purification of the recombinant proteins and determination of their immunogenicity through evaluating of the relative produced polyclonal antibodies in mice. PAD4 and LFD1 genes were cloned in pET28a(+) vector and expressed in E. coli Bl21(DE3)PlysS. Expression and purification of the two recombinant proteins were confirmed by SDS-PAGE and Western blotting techniques. The PAD4 and LFD1 were purified using Ni(+)-NTA affinity chromatography (95-98 %), yielding 37.5 and 45 mg/l of culture, respectively. The antigens were injected three times into mice and production of relative antibodies was evaluated by ELISA test. The results showed that both PAD4 and LFD1 are immunogenic, but LFD1 has higher potential to stimulate Murine immune system. With regard to the high level of LFD1 and PAD4 expression and also significant increment in produced polyclonal antibodies, these recombinant proteins can be considered as a recombinant vaccine candidate against anthrax. PMID:24430302

  3. Structural characterization of expressed monoclonal antibodies by single sample mass spectral analysis after IdeS proteolysis.

    Science.gov (United States)

    Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

    2016-08-26

    Simple and rapid methods for analysis of monoclonal antibody structure and post-translational modifications are increasingly needed due to the explosion of therapeutic monoclonal antibodies and monoclonal antibody applications. Mass spectral analysis is a powerful method for characterizing monoclonal antibodies. Recent discovery and commercialization of the Immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS protease) has facilitated and improved the generation of antibody fragments of suitable size to allow characterization of the structure of the entire antibody molecule via analysis of just a few fragments. In this study, we coupled IdeS fragmentation and simultaneous reduction and alkylation of the resultant fragments using tributylphosphine and iodoacetamide to prepare samples in about 2 h. Following simple introduction of a single, unseparated mixture of alkylated fragments into a mass spectrometer, detailed structural information is obtained, covering the entire antibody molecule. The large majority of the glycoforms present on the single, conserved N-linked glycosylation site of the heavy chain is elucidated, although some of the very low abundance glycoforms are not determined by this protocol. The ease, simplicity, speed, and power of this method make it attractive for analysis of the developmental stages and production batches of therapeutic monoclonal antibodies. PMID:27342663

  4. CEI-PEA Alert, Summer 2006

    Science.gov (United States)

    Center for Educational Innovation - Public Education Association, 2006

    2006-01-01

    The "CEI-PEA Alert" is an advocacy newsletter that deals with topics of interest to all concerned with the New York City public schools. This issue includes: (1) Practical Skills & High Academic Standards: Career Technical Education; (2) Parents: Help Your Children Gain "Soft Skills" for the Workforce; (3) Culinary Arts Motivate High School…

  5. CEI-PEA Alert, Fall 2005

    Science.gov (United States)

    Center for Educational Innovation - Public Education Association, 2005

    2005-01-01

    The "CEI-PEA Alert" is an advocacy newsletter that deals with topics of interest to all concerned with the New York City public schools. This issue includes: (1) Chancellor Joel I. Klein Announces New Accountability System for NYC Schools; (2) Students Achieve Record-High Scores!; (3) Use Data to Help Your Child Improve Performance; (4) Are…

  6. 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

    OpenAIRE

    Nozomi Satoh; Tatsuya Kon; Noriko Yamagishi; Tsubasa Takahashi; Tomohide Natsuaki; Nobuyuki Yoshikawa

    2014-01-01

    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 sym...

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

    Science.gov (United States)

    2010-01-01

    ... provisions of this subpart: (a) The peas must be shelled from the pod. (b) The peas must be washed in... 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...

  8. 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.

  9. 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. PMID:27117847

  10. Expression of the HNK-1 carbohydrate epitope in human retina and retinoblastoma. An immunohistochemical study with the anti-Leu-7 monoclonal antibody.

    OpenAIRE

    KivelÀ, Tero Tapani

    1986-01-01

    Fifty formalin-fixed and paraffin-embedded retinoblastoma specimens and five normal human eyes were studied with the monoclonal anti-Leu-7 antibody, directed against the HNK-1 carbohydrate epitope that is shared by human natural killer cells and many neuronal, glial and neuroectodermal cells. The laboratory method was a sensitive immunohistochemical staining procedure, and neuroectodermal tumours that usually express this epitope were used as positive controls. In the human retina, MÃŒller ce...

  11. Experimental and In Silico Modelling Analyses of the Gene Expression Pathway for Recombinant Antibody and By-Product Production in NS0 Cell Lines

    OpenAIRE

    Emma J Mead; Lesley M Chiverton; Sarah K Spurgeon; Martin, Elaine B.; Montague, Gary A.; C Mark Smales; Tobias von der Haar

    2012-01-01

    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 va...

  12. Human teratomas express differentiated neural antigens. An immunohistochemical study with anti-neurofilament, anti-glial filament, and anti-myelin basic protein monoclonal antibodies.

    OpenAIRE

    Trojanowski, J Q.; Hickey, W. F.

    1984-01-01

    Monoclonal antibodies specific for neurofilament proteins, glial filament protein, or myelin basic protein were used with immunohistochemistry for evaluation of a series of 14 human benign and malignant teratomas for the presence of these neural specific antigens. The results indicate that human teratomas can express all of these neural antigens, reflecting the presence of differentiated neurons, astrocytes, and oligodendroglia, respectively. Although the tumors were selected because neural t...

  13. Purification and properties of asparaginase from the testa of immature seeds of pea (Pisum sativum L.

    Directory of Open Access Journals (Sweden)

    Chagas Eliana P.

    2001-01-01

    Full Text Available A K+-dependent asparaginase (E.C. 3.5.1.1. was purified 1328-fold from the testas of immature pea seeds (Pisum sativum L., var. Bolero and characterized. Antibodies raised against purified asparaginase cross-reacted with the putative asparaginase band in Western blot analyses of semi-purified extracts. However, for crude extracts of pea testas, a cross-reaction was obtained with at least four protein bands, one of which was asparaginase protein. Affinity-purified antibodies to the four strongest bands of crude extracts were fairly specific for the bands from which they were purified, suggesting a mixture of specific antibodies. The Mr of asparaginase was 69,000 by Sephacryl S200 chromatography and also by mobility on native PAGE relative to BSA. There was no evidence for dissociation into subunits on SDS-PAGE, suggesting a monomeric protein of Mr 69,000. Other properties include an apparent Km of 2.4 mM, pI between 4.5 and 5, and competitive inhibition by aspartate and glycine.

  14. EFFECT OF VL AND VH CONSENSUS SEQUENCE-SPECIFIC PRIMERS ON THE BINDING AND EXPRESSION OF A MINI- MOLECULE ANTIBODY DIRECTED TOWARDS HUMAN GASTRIC CANCER

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective. To construct ScFv and Fab from murine anti-gastric cancer monoclonal antibody( mAb)3H11. Methods. At first,3H11 ScFv and Fab were constructed with V genes PCR amplified by degenerate primers for FRI. The bacterial expressed 3H11 Ab fragments showed no antigen binding activity. Then, phage antibody library andrandom mutated library were constructed from 3H1 1 hybridoma cells and panning selection was performed. Again the i-dentification of positive clone was failed. Finally the N-terminal sequences of V regions were resumed to 3H1 1 original sequences by site-directed mutagenesis via PCR. Results. Binding activity to gastric cancer cells was detected only from N-terminal sequence corrected 3H11 ScFv and Fab,though the expression of the Ab fiagments was not affected. Correction of either VL or VH N-terminal se-quences could partially resume the antigen binding activity. Conclusion. Sequence changes of V region N terminal introduced by PCR may seriously affect antigen binding without affecting the expression of antibody. Received for publication Oct. 26,1998.

  15. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Directory of Open Access Journals (Sweden)

    Ryan Haryadi

    Full Text Available Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig heavy chain (HC and kappa light chain (LC was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.

  16. Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells.

    Science.gov (United States)

    Haryadi, Ryan; Ho, Steven; Kok, Yee Jiun; Pu, Helen X; Zheng, Lu; Pereira, Natasha A; Li, Bin; Bi, Xuezhi; Goh, Lin-Tang; Yang, Yuansheng; Song, Zhiwei

    2015-01-01

    Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells. PMID:25706993

  17. 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......-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...

  18. Increased expression of Matrix Metalloproteinase 9 in liver from NZB/W F1 mice received antibody against human parvovirus B19 VP1 unique region protein

    Directory of Open Access Journals (Sweden)

    Hsu Gwo-Jong

    2009-01-01

    Full Text Available Abstract Background Human parvovirus B19 infection has been postulated to the anti-phospholipid syndrome (APS in autoimmunity. However, the influence of anti-B19-VP1u antibody in autoimmune diseases is still obscure. Methods To elucidate the effect of anti-B19-VP1u antibodies in systemic lupus erythematosus (SLE, passive transfer of rabbit anti-B19-VP1u IgG was injected intravenously into NZB/W F1 mice. Results Significant reduction of platelet count and prolonged thrombocytopenia time were detected in anti-B19-VP1u IgG group as compared to other groups, whereas significant increases of anti-B19-VP1u, anti-phospholipid (APhL, and anti-double strand DNA (dsDNA antibody binding activity were detected in anti-B19-VP1u group. Additionally, significant increases of matrix metalloproteinase-9 (MMP9 activity and protein expression were detected in B19-VP1u IgG group. Notably, phosphatidylinositol 3-phosphate kinase (PI3K and phosphorylated extracellular signal-regulated kinase (ERK proteins were involved in the induction of MMP9. Conclusion These experimental results firstly demonstrated the aggravated effects of anti-B19-VP1u antibody in disease activity of SLE.

  19. Genetically Modified α-Amylase Inhibitor Peas Are Not Specifically Allergenic in Mice

    OpenAIRE

    Rui-Yun Lee; Daniela Reiner; Gerhard Dekan; 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 ...

  20. Field Pea and Lentil Marketing Strategies for Northern Plains Producers

    OpenAIRE

    Flaskerud, George

    2006-01-01

    Marketing strategies are analyzed for field pea and lentil producers in the Northern Plains. Seasonal price patterns were derived from the 1999-2003 marketing years. Correlations indicate that corn futures may provide risk reduction for cross-hedging pea prices. Relationships were too weak to consider a cross-hedge for lentils. Combining a pre-harvest strategy with a marketing loan strategy offered the best total net price for the pea crop in 2004. No one marketing loan strategy performed bes...

  1. Genotoxicological Evaluation of NUTRALYS Pea Protein Isolate.

    Science.gov (United States)

    Aouatif, Chentouf; Looten, Ph; Parvathi, M V S; Raja Ganesh, S; Paranthaman, V

    2013-01-01

    NUTRALYS Pea Protein Isolate, a protein supplement, is a high-quality source of protein which is primarily emulsifying functional protein. We evaluated the genotoxic potential of NUTRALYS isolated from dry yellow pea, using three established genotoxicity tests (AMES test in vitro chromosomal aberration test, and in vivo micronucleus test) employing OECD guidelines under GLP conditions. In the bacterial reverse mutation test, NUTRALYS did not show positive responses in strains detecting point and frame shift mutations. In the chromosomal aberration test, NUTRALYS did not induce chromosome aberrations in the presence and absence of metabolic activation. In the bone marrow micronucleus test, NUTRALYS did not induce significant increases of micronucleated immature (polychromatic) erythrocytes in bone marrow of test animals. PMID:23762639

  2. Water deficit down-regulates miR398 and miR408 in pea (Pisum sativum L.).

    Science.gov (United States)

    Jovanović, Živko; Stanisavljević, Nemanja; Mikić, Aleksandar; Radović, Svetlana; Maksimović, Vesna

    2014-10-01

    MicroRNAs (miRNAs), recently recognized as important regulator of gene expression at posttranscriptional level, have been found to be involved in plant stress responses. The observation that some miRNAs are up- or down regulated by stress implies that they could play vital roles in plant resistance to abiotic and biotic stress. We investigated the effect of water stress treatment during 10 days on expression of conserved miRNAs-miR398a/b and miR408 in pea plants. This time frame reflects the changes as close as possible to the changes where water stress causes visible effects under field condition. It was observed that dehydration strongly down regulates the expression of both miR398a/b and miR408 in pea roots and shoots. The down-regulation of miR398a/b and the up-regulation of potential target genes - copper superoxide dismutase, CSD1, highlight the involvement of this miRNA in pea stress response. To the contrary, the mRNA level of cytochrome c oxidase subunit 5 (COX5b) did not change in roots and shoots of water-stressed plants, compared to control (well) hydrated plants. This suggests that COX5b is not the target of miR398, or that its expression is regulated by some other mechanism. P1B-ATPase expression increased during water deficit only in the shoots of pea; in the roots there were no changes in expression. Our results help to understand the possible role of investigated miRNAs and their contribution to pea capacity to cope with water deficit.

  3. 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.

  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...... 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...

  5. Anti-citrullinated protein antibodies suppress let-7a expression in monocytes from patients with rheumatoid arthritis and facilitate the inflammatory responses in rheumatoid arthritis.

    Science.gov (United States)

    Lai, Ning-Sheng; Yu, Hui-Chun; Yu, Chia-Li; Koo, Malcolm; Huang, Hsien-Bin; Lu, Ming-Chi

    2015-12-01

    We hypothesized that anti-citrullinated protein antibodies (ACPAs) could affect the expression of miRNAs in monocytes and contribute to the inflammatory responses in rheumatoid arthritis (RA). The expression profiles of 270 human miRNAs, co-cultured with ACPAs or human immunoglobulin G (IgG), were analyzed using real-time polymerase chain reaction. Ten miRNAs exhibited differential expression in U937 cells after co-cultured with ACPAs compared with human IgG. The expression levels of these miRNAs were investigated in monocytes from 21 ACPA-positive RA patients and 13 controls. Among these miRNAs, the expression levels of let-7a was decreased in monocytes from ACPA-positive RA patients. The expression levels of let-7a showed a negative correlation with positivity of rheumatoid factor in patients sampled. We found that transfection of U937 cells with let-7a mimic suppressed K-Ras protein expression. In the ACPA-mediated signaling pathway, transfection of U937 cells with let-7a mimic suppressed the ACPA-enhanced phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and the expression and secretion of interleukin (IL)-1β. In conclusion, ACPA-mediated decreased let-7a expression in monocytes from ACPA-positive RA patients. Decreased let-7a expression was associated with the positivity of RF in ACPA-positive RA patients. The decreased expression of let-7a could facilitate the inflammatory pathway via enhanced ACPA-mediated phosphorylation of ERK1/2 and JNK and increased expression of IL-1β through an increase in the expression of Ras proteins.

  6. Taxonomy Icon Data: pea aphid [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available pea aphid Acyrthosiphon pisum Arthropoda Acyrthosiphon_pisum_L.png Acyrthosiphon_pisum_NL.png Acyrthosiph...on_pisum_S.png Acyrthosiphon_pisum_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Acyrthosiph...on+pisum&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Acyrthosiphon+pisum&t=NL http:...//biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Acyrthosiphon+pisum&t=S http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Acyrthosiphon+pisum&t=NS ...

  7. CHICK PEAS EFFICIENCY IN HENS FEEDING

    OpenAIRE

    Nikolaev S. I.; Karapetyan A. K.; Kornilova E. V.; Struk M. V.

    2015-01-01

    This article presents the results of the chick peas use instead of sunflower cake, in feeding young and adult livestock hens-layers of the cross "Hajseks brown". The researches were carried out in the JSC "Agrofirm Vostok" of the Nikolayevskiy district in the Volgograd region. The sunflower cake replacement with legumes - chickpeas as the part of the experimental animal fodder for young and adult livestock hens-layers had a positive influence on productivity, physiological state of the birds,...

  8. 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.

    Science.gov (United States)

    Nielsen, Morten A; Resende, Mafalda; de Jongh, Willem A; Ditlev, Sisse B; Mordmüller, Benjamin; Houard, Sophie; Ndam, Nicaise Tuikue; Agerbæk, Mette Ø; Hamborg, Mette; Massougbodji, Achille; Issifou, Saddou; Strøbæk, Anette; Poulsen, Lars; Leroy, Odile; Kremsner, Peter G; Chippaux, Jean-Philippe; Luty, Adrian J F; Deloron, Philippe; Theander, Thor G; Dyring, Charlotte; Salanti, Ali

    2015-01-01

    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 that induce a

  9. 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

  10. Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions.

    Science.gov (United States)

    Ma, Biao; Osborn, Michael J; Avis, Suzanne; Ouisse, Laure-Hélène; Ménoret, Séverine; Anegon, Ignacio; Buelow, Roland; Brüggemann, Marianne

    2013-12-31

    Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3' end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes.

  11. 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.

  12. Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-06-01

    Full Text Available Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

  13. Simple and Efficient Method for Measuring Anti-Toxoplasma Immunoglobulin Antibodies in Human Sera Using Complement-Mediated Lysis of Transgenic Tachyzoites Expressing β-Galactosidase

    Science.gov (United States)

    Dando, Caroline; Gabriel, Katie E.; Remington, Jack S.; Parmley, Stephen F.

    2001-01-01

    A simple and efficient method using transgenic Toxoplasma gondii tachyzoites expressing β-galactosidase was developed for detection of specific antibodies against the parasite in sera of patients. The titers obtained with the new test were similar to those obtained with the Sabin-Feldman dye test run in parallel. Although significant changes in endpoint titers were not observed when sera drawn sequentially at 2- to 3-week intervals were tested with both procedures, apparent differences in antibody affinity were observed with the new test which were not perceptible with the Sabin-Feldman dye test. Like the Sabin-Feldman dye test, the new test is based on complement lysis of tachyzoites, but it is much easier to perform and the reaction is read colorimetrically instead of visually. PMID:11376045

  14. 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.

  15. Roles for auxin during morphogenesis of the compound leaves of pea ( Pisum sativum).

    Science.gov (United States)

    DeMason, Darleen A; Chawla, Rekha

    2004-01-01

    The goal of this study was to explore the impact of the plant growth regulator auxin on the development of compound leaves in pea. Wildtype ( WT) plantlets, as well as those of two leaf mutants, acacia ( tl) and tendrilled acacia ( uni-tac) of pea ( Pisum sativum L.), were grown on media containing the auxin-transport inhibitors 2,3,5-triiodobenzoic acid (TIBA), N-(1-naphthyl)phthalamic acid (NPA), or the auxin antagonist, p-chlorophenoxyisobutyric acid (PCIB). The resulting plantlets were carefully analyzed morphologically, by scanning electron microscopy and for Uni gene expression using quantitative reverse transcription-polymerase chain reaction. Auxin transport was measured in WT leaf parts using [(14)C]indole-3-acetic acid. Relative Uni gene expression was determined in shoot tips of a range of leaf-form mutants. Morphological abnormalities were observed for all genotypes examined. The terminal tendrils on WT plants were converted to leaflets, stubs or were aborted. The number of pinna pairs produced on leaves was reduced, with the distal forms being eliminated before the proximal ones. Some leaves were converted to simple, including tri-and bilobed, forms. These treatments phenocopy the uni-tac and unifoliata ( uni) mutants of pea. In the most extreme situations, leaf blades were completely lost leaving only a pair of stipules or scale leaves. Polar auxin transport was basipetal for all leaf parts. Uni gene expression in shoot tips was significantly reduced in 60 microM NPA and TIBA. Uni mRNA was more abundant in tl, af and af tl and reduced in the uni mutants compared to WT. These results indicate that an auxin gradient plays fundamental roles in controlling morphogenesis in the compound leaves of pea and specifically it: (i). is the driving force for leaf growth and pinna determination; (ii). is necessary for pinna initiation; and (iii). controls subsequent pinna development. PMID:12942326

  16. T cell-independent type I antibody response against B cell epitopes expressed repetitively on recombinant virus particles

    OpenAIRE

    Fehr, Thomas; Skrastina, Dace; Pumpens, Paul; Zinkernagel, Rolf M.

    1998-01-01

    Recombinant viral or virus-like particles offer new tools for vaccine development. This study investigated hepatitis B core antigen (HBcAg) capsids and RNA phage Qβ coats as carriers of a foreign epitope to induce antibody responses in mice. HBcAg capsids were shown to induce T cell-independent (TI) antibodies. We found that these particles behave as antigen-specific TI type 1 (TI-1) Ag comparable to other rigidly structured viruses. When a 5-aa long epitope of the pre-S1 domain of hepatitis ...

  17. Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    XU Yunjian (徐云剑); GU Xuesong (顾雪松); LI Jun (李珺); LI Qing (李 晴); Peter J. Davies; ZHU Yuxian (朱玉贤)

    2003-01-01

    The AAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficient E. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with the AAIR expression vector. Further study revealed that overexpression of the pea AAIR cDNA in acetohydroxy acid isomeroreductase deficient E. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of the AAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulate AAIR gene expression.

  18. Heavy chain only antibodies

    DEFF Research Database (Denmark)

    Moghimi, Seyed Moein; Rahbarizadeh, Fatemeh; Ahmadvand, Davoud;

    2013-01-01

    Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen-...... for combating HER2+ breast cancer. © 2013 by Tabriz University of Medical Sciences.......Unlike conventional antibodies, heavy chain only antibodies derived from camel contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Cloned and isolated VHHs possess unique properties that enable them to excel conventional therapeutic antibodies and their smaller antigen......-binding fragments in cancer targeting and therapy. VHHs express low immunogenicity, are highly robust and easy to manufacture and have the ability to recognize hidden or uncommon epitopes. We highlight the utility of VHH in design of new molecular, multifunctional particulate and immune cell-based systems...

  19. Expression and Characterization of the Extracellular Domain of Human HER2 from Escherichia Coli, and Production of Polyclonal Antibodies Against the Recombinant Proteins.

    Science.gov (United States)

    Sun, Yong; Feng, Xue; Qu, Jiao; Han, Wenqi; Liu, Zi; Li, Xu; Zou, Ming; Zhen, Yuhong; Zhu, Jie

    2015-06-01

    Human epidermal growth factor receptor 2 (HER2) is a member of the epidermal growth factor receptor (EGFR) family. In this study, the whole extracellular domain gene of HER2 was amplified by RT-PCR from human breast cancer cell line SK-BR-3. The genes of membrane-distal region (A) and membrane proximal region (B) of HER2 extracellular domain were amplified from the cloned template, and then inserted into the expression vector pET-28a and pET-30a, respectively. The recombinant expression vectors were transformed into Escherichia coli BL21 (DE3) cells and induced by isopropyl-b-D-thiogalactopyranoside (IPTG) for expression of proteins His-A and His-B. The expressed proteins were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. The optimization of culture conditions led us to accomplish the recombinant protein induction with 1.0 mM IPTG at 37 °C for 8 h, and both proteins were expressed in the insoluble form. Both proteins were purified under the denaturing condition using Ni-NTA sepharose column. Balb/c mice were immunized with the purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by ELISA testing and had good specificity by western blot detection. The HER2 ECD proteins His-A and His-B could be expressed in E. coli and were suitable for production of high titer antibodies against HER2 ECD. PMID:25906688

  20. 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 demonstr...

  1. 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

    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...

  2. 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.

  3. Cloning and expression of a truncated pigeon circovirus capsid protein suitable for antibody detection in infected pigeons.

    Science.gov (United States)

    Daum, Iris; Finsterbusch, Tim; Härtle, Stefan; Göbel, Thomas W; Mankertz, Annette; Korbel, Rüdiger; Grund, Christian

    2009-04-01

    Infections with pigeon circovirus (PiCV) (also termed columbid circovirus) occur in meat and racing pigeons (Columba livia) of all ages and have been reported worldwide. A PiCV infection is associated with immunosuppression and the development of young pigeon disease syndrome. An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of virus-specific serum antibody was developed for research purposes. In the absence of a method to propagate PiCV in cell culture, the assay was based on a recombinant truncated capsid protein (rCapPiCV) produced by overexpression in Escherichia coli. A 6xHis-Tag was fused to the N-terminus of the protein to facilitate purification by metal affinity chromatography and detection by anti-His antibody. PiCV-negative and PiCV-positive control sera were generated by inoculation of pigeons with tissue homogenate containing PiCV, followed by five weekly blood sample collections. Western blotting of the immune serum revealed a specific protein band of approximately 32 kDa, which was absent in the pre-immune sera. Using rCapPiCV as antigen in an indirect ELISA, PiCV-specific antibody was detected in sera of the experimentally PiCV-infected pigeons collected at 1 to 5 weeks post infection. By testing 118 field sera collected in the years 1989, 1991, 1994 and 2008 in the rCapPiCV ELISA, virus-specific antibody was detected in 89 (75%) of the sera. The results obtained demonstrate that the rCapPiCV-based indirect ELISA is able to detect PiCV-specific antibodies in pigeon sera and may be a useful tool for PiCV serodiagnosis.

  4. 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.

  5. 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...

  6. 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

  7. Fibril Formation from Pea Protein and Subsequent Gel Formation

    NARCIS (Netherlands)

    Munialo, XC.D.; Martin, A.H.; Linden, E. van der; Jongh, H.H.J de

    2014-01-01

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20

  8. Fibril formation from pea protein and subsequent gel formation.

    Science.gov (United States)

    Munialo, Claire Darizu; Martin, Anneke H; van der Linden, Erik; de Jongh, Harmen H J

    2014-03-19

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

  9. Construction and bacterial expression of a recombinant single-chain antibody fragment against Wuchereria bancrofti SXP-1 antigen for the diagnosis of lymphatic filariasis.

    Science.gov (United States)

    Kamatchi, R; Charumathi, J; Ravishankaran, R; Kaliraj, P; Meenakshisundaram, S

    2016-01-01

    Global programmes to eliminate lymphatic filariasis (GPELF) require mapping, monitoring and evaluation using filarial antigen diagnostic kits. To meet this objective, a functional single-chain fragment variable (ScFv) specific for filarial Wuchereria bancrofti SXP-1 (Wb-SXP-1) antigen was constructed for the diagnosis of active filarial infection, an alternative to the production of complete antibodies using hybridomas. The variable heavy chain (VH) and the variable light chain (kappa) (Vκ) genes were amplified from the mouse hybridoma cell line and were linked together with a flexible linker by overlap extension polymerase chain reaction (PCR). The ScFv construct (Vκ-Linker-VH) was expressed as a fusion protein with N-terminal His tag in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) without the addition of reducing agents. Immunoblotting and sandwich enzyme-linked immunosorbent assay (ELISA) were used to analyse the antigen binding affinity of purified ScFv. The purified ScFv was found to recognize recombinant and native Wb-SXP-1 antigen in microfilariae (Mf)-positive patient sera. The affinity of ScFv was comparable with that of the monoclonal antibody. The development of recombinant ScFv to replace monoclonal antibody for detection of filarial antigen was achieved. The recombinant ScFv was purified, on-column refolded and its detection ability validated using field samples. PMID:26693887

  10. OX48, a monoclonal antibody against a 70,000 MW rat activation antigen expressed by T cells bearing the high-affinity interleukin-2 receptor.

    Science.gov (United States)

    Somoza, C; Fernández-Ruiz, E; Rebollo, A; Sanz, E; Ramírez, F; Silva, A

    1990-01-01

    The monoclonal antibody (mAb) OX48 recognizes a 70,000 MW cell-surface protein present in a small percentage of activated rat T cells and in CD8+ rat x BW5147 interleukin-2 (IL-2)-dependent T-cell hybridomas, but not in resting spleen cells or in IL-2-independent T-cell hybrids. OX48 antibody added simultaneously with concanavalin A (Con A) to resting spleen cells inhibits the cell proliferation and reduces the IL-2 production. However, addition of IL-2 does not restore the mitogenic response. Growth of rat blast T cells or IL-2-dependent hybrids is not affected by the OX48 antibody. There is a close correlation between the expression of high-affinity IL-2 receptors (IL-2R) and the OX48 antigen in T-cell hybridomas. In spite of this striking correlation, OX48 mAb does not inhibit the binding of 125I-IL-2 to the IL-2-dependent hybrids, and is unable to immunoprecipitate any of the proteins chemically cross-linked to 125I-IL-2. Therefore, the OX48 molecule represents a new rat activation antigen, undefined in other species, and probably involved in the early steps of T-cell activation. Images Figure 5 Figure 7 PMID:2373518

  11. Increased antibody responses to human papillomavirus type 16 L1 protein expressed by recombinant vaccinia virus lacking serine protease inhibitor genes.

    Science.gov (United States)

    Zhou, J; Crawford, L; McLean, L; Sun, X Y; Stanley, M; Almond, N; Smith, G L

    1990-09-01

    The L1 gene of human papillomavirus type 16 (HPV-16) driven by the vaccinia virus major late 4b gene promoter has been inserted into three different sites of the vaccinia virus genome. Insertion into the thymidine kinase (TK) gene was achieved by selection of TK- mutants in BUdR on TK- cells. Insertion into two vaccinia virus serine protease inhibitor (serpin) genes was achieved by co-insertion of the Escherichia coli xanthine guanine phosphoribosyltransferase gene linked to the vaccinia virus 7.5K promoter and selection of mycophenolic acid-resistant recombinant viruses. Each recombinant virus expressed a 57K L1 protein at similar levels and with similar kinetics. However, immunization of mice with these recombinant viruses induced different levels of antibody to the L1 protein. Viruses lacking serpin genes B13R and B24R induced significantly higher antibody levels than did viruses lacking the TK gene. The presence of functional B13R and B24R gene products is therefore somehow immunosuppressive at least for antibody responses to the L1 protein of HPV-16.

  12. 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

  13. Construction of a prokaryotic expression system of vacA gene and detection of vatA gene,VacA protein in Helicobacter pylori isolates and ant-VacA antibody in patients'sera

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Ya-Fei Mao

    2004-01-01

    AIM: To construct a recombinant prokaryotic expression vector inserted with Helicobacter pylori vacA gene and identify the immunity of the expressed recombinant protein,and to determine prevalence of vacA-carryinglVacA expressing Hpyloriisolates and seroprevalence of specific ant-VacA antibody in H pyloriinfected patients.METHODS: Polymerase chain reaction technique was used to amplify complete vacA gene of H pyloristrain NCTC11637 and to detect vacA gene in 109 H pylori isolates. The amplification product of the complete vacA gene was sequenced after T-A cloning. A recombinant expression vector inserted with a complete vacA gene fragment, named as pET32a-vacA, was constructed. Expression of the target recombinant protein VacA (rVacA) was examined by SDSPAGE. Western blot using commercial antibodies against whole cell of H pyloriand an immunodiffusion assay using self-prepared rabbit anti-rVacA antibody were applied to determine immunoreaction and antigenicity of rVacA. Two ELISA methods were established to detect VacA expression in H pyloriisolates and the specific anti-VacA antibody in sera from 125 patients infected with H pylori.RESULTS: In comparison with the reported corresponding sequences, homologies of nucleotide and putative amino acid sequences of the cloned vacA gene were 99.82% and 100%, respectively. The constructed recombinant prokaryotic expression system efficiently produced rVacA. rVacA was able to combine with the commercial antibodies against whole cell of H pyloriand to induce the immunized rabbit to produce specific antibody with an immunodiffusion titer of 1:4. All tested H pyloriisolates carried vacA gene, but only 66.1% expressed Vac A protein. Of the serum samples tested,42.4% were positive for specific anti-VacA antibody.CONCLUSION: A prokaryotic expression system of H pylori vacA gene was successfully constructed. The expressed rVacA can be used to detect specific anti-VacA antibody in human and to prepare antiserum in animals. The high

  14. Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance.

    Science.gov (United States)

    Avelange-Macherel, Marie-Hélène; Payet, Nicole; Lalanne, David; Neveu, Martine; Tolleter, Dimitri; Burstin, Judith; Macherel, David

    2015-07-01

    LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms. PMID:25367071

  15. Variability within a pea core collection of LEAM and HSP22, two mitochondrial seed proteins involved in stress tolerance.

    Science.gov (United States)

    Avelange-Macherel, Marie-Hélène; Payet, Nicole; Lalanne, David; Neveu, Martine; Tolleter, Dimitri; Burstin, Judith; Macherel, David

    2015-07-01

    LEAM, a late embryogenesis abundant protein, and HSP22, a small heat shock protein, were shown to accumulate in the mitochondria during pea (Pisum sativum L.) seed development, where they are expected to contribute to desiccation tolerance. Here, their expression was examined in seeds of 89 pea genotypes by Western blot analysis. All genotypes expressed LEAM and HSP22 in similar amounts. In contrast with HSP22, LEAM displayed different isoforms according to apparent molecular mass. Each of the 89 genotypes harboured a single LEAM isoform. Genomic and RT-PCR analysis revealed four LEAM genes differing by a small variable indel in the coding region. These variations were consistent with the apparent molecular mass of each isoform. Indels, which occurred in repeated domains, did not alter the main properties of LEAM. Structural modelling indicated that the class A α-helix structure, which allows interactions with the mitochondrial inner membrane in the dry state, was preserved in all isoforms, suggesting functionality is maintained. The overall results point out the essential character of LEAM and HSP22 in pea seeds. LEAM variability is discussed in terms of pea breeding history as well as LEA gene evolution mechanisms.

  16. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems

    OpenAIRE

    Benevolo Maria; Natali Pier; Martayan Aline; Fraioli Rocco; Tornambé Andrea; Sperandei Maria; Di Donato Monica; Novelli Flavia; Pietraforte Immacolata; Lombardi Alessio; Galeffi Patrizia; Mottolese Marcella; Ylera Francisco; Cantale Cristina; Giacomini Patrizio

    2006-01-01

    Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach ...

  17. 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...

  18. 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

  19. 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. PMID:26850143

  20. 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.

  1. Expression of Bovine Viral Diarrhea Virus Envelope Glycoprotein E2 in Yeast Pichia pastoris and its Application to an ELISA for Detection of BVDV Neutralizing Antibodies in Cattle.

    Science.gov (United States)

    Behera, Sthita Pragnya; Mishra, Niranjan; Nema, Ram Kumar; Pandey, Pooja Dubey; Kalaiyarasu, Semmannan; Rajukumar, Katherukamem; Prakash, Anil

    2015-01-01

    The aim of this article is to express envelope glycoprotein E2 of bovine viral diarrhea virus (BVDV) in yeast Pichia pastoris and its utility as a diagnostic antigen in ELISA. The BVDV E2 gene was cloned into the pPICZαA vector followed by integration into the Pichia pastoris strain X-33 genome for methanol-induced expression. SDS-PAGE and Western blot results showed that the recombinant BVDV E2 protein (72 kDa) was expressed and secreted into the medium at a concentration of 40 mg/L of culture under optimized conditions. An indirect ELISA was then developed by using the yeast-expressed E2 protein. Preliminary testing of 300 field cattle serum samples showed that the E2 ELISA showed a sensitivity of 91.07% and a specificity of 92.02% compared to the reference virus neutralization test. The concordance between the E2 ELISA and VNT was 91.67%. This study demonstrates feasibility of BVDV E2 protein expression in yeast Pichia pastoris for the first time and its efficacy as an antigen in ELISA for detecting BVDV neutralizing antibodies in cattle.

  2. 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 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.

  3. Expression of E1AF/PEA3, an Ets-related transcription factor in human non-small-cell lung cancers: its relevance in cell motility and invasion.

    Science.gov (United States)

    Hiroumi, H; Dosaka-Akita, H; Yoshida, K; Shindoh, M; Ohbuchi, T; Fujinaga, K; Nishimura, M

    2001-09-01

    Cell invasion and metastasis characterize the malignant potential of non-small-cell lung cancers (NSCLCs). We have previously reported that E1AF, a member of the Ets-related transcription factor family, confers invasive phenotype on breast cancer and oral squamous-cell carcinoma cell lines. In our study, we analyzed the E1AF expression in cell lines and resected tumors of NSCLCs by Northern blot and in situ hybridization analyses and found that 15 of 17 cell lines and 12 of 19 tumors expressed E1AF mRNA while normal lung tissue and concomitant normal cells within tumors did not. To examine the biologic importance of E1AF in NSCLCs, we introduced the E1AF gene into VMRC-LCD and NCI-H226, NSCLC cell lines lacking E1AF expression, and examined cell motility and invasion activities. E1AF-transfected VMRC-LCD cells showed increased cell motility that was 2-fold that of parental and vector-transfected control cells (p H226 (p H226 cells but not in their parental or vector-transfected control cells. Ets-1 mRNA expression was found in E1AF-transfected VMRC-LCD cells but not in parental or vector-transfected cells. HGF further induced expression of the Ets-1 and urokinase-type plasminogen activator (uPA) genes specifically in E1AF-transfected cells. These findings suggest that E1AF plays a substantial role in the cell motility and invasion of NSCLCs. PMID:11519038

  4. OLIGOSACCHARIDE LEVELS IN IMMATURE AND NATURE SEEDS FROM SEVERAL VARIETIES OF PIGEON PEAS (Cajanus cajan)

    OpenAIRE

    Jairo Osvaldo CAZETTA; Hugo Candido SILVA; Gilberto Leite BRAGA; Roberto de CARVALHO

    2009-01-01

    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....

  5. Breeding strategy for improvement of colour quality and carotenoid levels in dry pea seeds

    Directory of Open Access Journals (Sweden)

    Eszter Nemeskéri

    2006-08-01

    Full Text Available The purpose of this study was to show the relationship between the yellow pigments of pea (Pisum sativum L. seeds and climatic conditions, and to identify effective selection methods for improving seed colour quality. Three dry pea cultivars with different yellow hues of seeds and leaves and their progenies were grown in non-irrigated field experiments. A colour scale from 1 to 9 was created to measure seed colour. Drought during seed development caused a significant decrease in the xanthophyll content of pea seeds. Based on heritability estimates, the potential for selection for increased xanthophyll content in seeds (h2=0.78 was greater than for carotene (h2=0.32. The “yellow index,” defined as the ratio of carotene and xanthophylls, expressed the intensity of yellow colour of the seeds. The weak relation between seed colour values and yellow index (R2=0.445 should allow simultaneous selection for seeds with deep-yellow colour and higher carotene content.

  6. LGR5 expressing cells of hair follicle as potential targets for antibody mediated anti-cancer laser therapy

    Science.gov (United States)

    Popov, Boris V.

    2013-02-01

    Near infrared laser immunotherapy becomes now a new promising research field to cure the patients with cancers. One of the critical limitation in medical application of this treatment is availability of the specific markers for delivery of laser-sensitive nanoparticles. When coupled to antibodies to the cancer stem cells markers these nanoparticles may be delivered to the cancer tissue and mediate the laser induced thermolysis of the cancer stem cells that initiate and drive growth of cancer. This paper addresses the Lgr5 cell surface marker mediating the Wnt/β-catenin signal transduction as a potential target for anti-cancer laser immunotherapy of skin cancers.

  7. IL-6 blockade by monoclonal antibodies inhibits apolipoprotein (a) expression and lipoprotein (a) synthesis in humans[S

    OpenAIRE

    Müller, Nike; Schulte, Dominik M.; Türk, Kathrin; Freitag-Wolf, Sandra; Hampe, Jochen; Zeuner, Rainald; Johann O Schröder; Gouni-Berthold, Ioanna; Heiner K Berthold; Krone, Wilhelm; Rose-John, Stefan; Schreiber, Stefan; Laudes, Matthias

    2015-01-01

    Lipoprotein (a) [Lp(a)] is a highly atherogenic lipid particle. Although earlier reports suggested that Lp(a) levels are mostly determined by genetic factors, several recent studies have revealed that Lp(a) induction is also caused by chronic inflammation. Therefore, we aimed to examine whether cytokine blockade by monoclonal antibodies may inhibit Lp(a) metabolism. We found that interleukin 6 (IL-6) blockade by tocilizumab (TCZ) reduced Lp(a) while TNF-α-inhibition by adalimumab in humans ha...

  8. Var2CSA DBL6-epsilon domain expressed in HEK293 induces limited cross-reactive and blocking antibodies to CSA binding parasites

    Directory of Open Access Journals (Sweden)

    Scherf Artur

    2008-09-01

    Full Text Available Abstract Background Pregnancy-associated malaria (PAM is a serious consequence of Plasmodium falciparum-infected erythrocytes sequestration in the placenta through the adhesion to the placental receptor chondroitin sulfate A (CSA. Although women become resistant to PAM as they acquire transcending inhibitory immunity against CSA-binding parasites, hundreds of thousands of lives could be saved if a prophylactic vaccine targeting the surface proteins of placental parasites could be designed. Recent works point to the variant protein var2CSA as the key target for the development of a pregnancy-associated malaria vaccine. However, designing such a prophylactic vaccine has been hindered by the difficulty in identifying regions of var2CSA that could elicit broadly neutralizing and adhesion-blocking antibodies. Methods Var2CSA is a very large protein with an estimated molecular weight of 350 kDa, and can be divided into six cysteine rich Duffy binding-like domains (DBL. The human embryonic kidney 293 cell line (HEK293 was used to produce secreted soluble recombinant forms of var2CSA DBL domains. The Escherichia coli expression system was also assessed for the domains not expressed or expressed in low amount in the HEK293 system. To investigate whether var2CSA binding DBL domains can induce biologically active antibodies recognizing the native var2CSA and blocking the interaction, mice were immunized with the refolded DBL3-X or the HEK293 secreted DBL6-ε domains. Results Using the HEK293 expression system, DBL1-X, DBL4-ε and DBL6-ε were produced at relatively high levels in the culture supernatant, while DBL3-X and DBL5-ε were produced at much lower levels. DBL2-X and DBL3-X domains were obtained after refolding of the inclusion bodies produced in E. coli. Importantly, mice antisera raised against the recombinant DBL6-ε domain, specifically reacted against the surface of CSA-binding parasites and revealed adhesion blocking activity. Conclusion This

  9. The physiology of sterol nutrition in the pea aphid Acyrthosiphon pisum.

    Science.gov (United States)

    Bouvaine, Sophie; T Behmer, Spencer; Lin, George G; Faure, Marie-Line; Grebenok, Robert J; Douglas, Angela E

    2012-11-01

    The phloem sap of fava bean (Vicia faba) plants utilized by the pea aphid Acyrthosiphon pisum contains three sterols, cholesterol, stigmasterol and sitosterol, in a 2:2:1 ratio. To investigate the nutritional value of these sterols, pea aphids were reared on chemically-defined diets containing each sterol at 0.1, 1 and 10μgml(-1) with a sterol-free diet as control. Larval growth rate and aphid lifespan did not vary significantly across the diets, indicating that sterol reserves can buffer some performance indices against a shortfall in dietary sterol over at least one generation. However, lifetime reproductive output was depressed in aphids on diets containing stigmasterol or no sterol, relative to diets supplemented with cholesterol or sitosterol. The cholesterol density of embryos in teneral adults was significantly higher than in the total body; and the number and biomass of embryos in aphids on diets with stigmasterol and no sterols were reduced relative to diets with cholesterol or sitosterol, indicating that the reproductive output of the pea aphid can be limited by the amount and composition of dietary sterol. In a complementary RNA-seq analysis of pea aphids reared on plants and diets with different sterol contents, 7.6% of the 17,417 detected gene transcripts were differentially expressed. Transcript abundance of genes with annotated function in sterol utilization did not vary significantly among treatments, suggesting that the metabolic response to dietary sterol may be mediated primarily at the level of enzyme function or metabolite concentration. PMID:22878342

  10. Algae as Protein Factories: Expression of a Human Antibody and the Respective Antigen in the Diatom Phaeodactylum tricornutum

    OpenAIRE

    Franziska Hempel; Julia Lau; Andreas Klingl; Maier, Uwe G.

    2011-01-01

    Microalgae are thought to offer great potential as expression system for various industrial, therapeutic and diagnostic recombinant proteins as they combine high growth rates with all benefits of eukaryotic expression systems. Moreover, microalgae exhibit a phototrophic lifestyle like land plants, hence protein expression is fuelled by photosynthesis, which is CO(2)-neutral and involves only low production costs. So far, however, research on algal bioreactors for recombinant protein expressio...

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

    International Nuclear Information System (INIS)

    Symbiotic nitrogen fixation and nitrate uptake by pea plants (Pisum sativum L.) were studied in field and pot experiments using the 15N 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 15N-technique. Maximum N2 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 N2 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 NO3-N ha-1 inhibited the N2 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)

  12. High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector.

    Science.gov (United States)

    Gurkan, Cemal; Symeonides, Stefan N; Ellar, David J

    2004-02-01

    The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the recombinant production of a wide variety of proteins. Initial success with this system was greatly facilitated by the development of versatile expression vectors that were almost exclusively based on the strong, tightly regulated promoter of the P. pastoris major alcohol oxidase gene ( AOX1 ). For example, pIB4 is an Escherichia coli - P. pastoris shuttle vector that also uses the AOX1 promoter to allow intracellular expression of endogenous and foreign genes in the latter organism. Since the eukaryotic advantages of P. pastoris would be best harnessed through the secretory targeting of the recombinant proteins, we modified the pIB4 vector by adding the Saccharomyces cerevisiae alpha-factor secretion signal immediately upstream of its multiple cloning site. Here we describe the construction of this modified vector, pIB4alpha, and its successful use for the high-level expression and secretion of a functional single-chain antibody fragment (scFv), C6.5, which targets p185(HER-2), a cell-surface glycoprotein overexpressed in about 30% of human breast and ovarian cancers. The PCR strategy used for the subcloning of the C6.5 construct into pIB4alpha also introduced a short DNA sequence coding for a C-terminal hexahistidine tag, which allowed subsequent purification of the secreted scFv, by immobilized-metal-affinity chromatography, to a yield of 70 mg x l(-1) of shake-flask culture. In conclusion, our results suggest that the secretion expression vector pIB4alpha not only complements the original pIB4 vector for intracellular expression in P. pastoris, but might also constitute an attractive alternative to the commercially available secretion expression vectors. PMID:12962542

  13. Prokaryotic Expression, Purification, Antibody Production of a NH2-Terminal Fragment of mCLCA3 Protein and Analysis of mClCA3 Protein Expression in Asthmatic Mouse Lung

    Institute of Scientific and Technical Information of China (English)

    HOU Xia; WU Qing-tian; ZHANG Yu-ping; ZHU Na; LIU Yan-li; FENG Xue-chao; MA Tong-hui

    2007-01-01

    mCLCA3 is a member of calcium activated chloride channel(CACC) family that may play an important role in mucin packaging and secretion in asthmatic and cystic fibrosis lung. To study the protein structure and expression of mCLCA3 in asthmatic mouse lung, an N-terminal 269 amino acid peptide of mCLCA3 was expressed in E. coli, purified to homogeneity and rabbit polyclonal antibodies against this peptide were generated. Immunohistochemistry of asthmatic mouse lung using the antibody indicated exclusive mCLCA3 expression in mucin granules of goblet cells in airway surface and lumen. Immunoblot analysis of lavage fluid from asthmatic mouse lung revealed a single 90 kDa protein form of mClCA3. The results demonstrate that the 90 kDa N-terminal peptide, neither the full-length protein nor the reported N-terminal 35 kDa cleaved form of mClCA3 is the major functional form involved in the packaging and exocytosis of mucin granules in asthmatic goblet cells.

  14. Two distinct signaling pathways participate in auxin-induced swelling of pea epidermal protoplasts.

    Science.gov (United States)

    Yamagami, Mutsumi; Haga, Ken; Napier, Richard M; Iino, Moritoshi

    2004-02-01

    Protoplast swelling was used to investigate auxin signaling in the growth-limiting stem epidermis. The protoplasts of epidermal cells were isolated from elongating internodes of pea (Pisum sativum). These protoplasts swelled in response to auxin, providing the clearest evidence that the epidermis can directly perceive auxin. The swelling response to the natural auxin IAA showed a biphasic dose response curve but that to the synthetic auxin 1-naphthalene acetic acid (NAA) showed a simple bell-shaped dose response curve. The responses to IAA and NAA were further analyzed using antibodies raised against ABP1 (auxin-binding protein 1), and their dependency on extracellular ions was investigated. Two signaling pathways were resolved for IAA, an ABP1-dependent pathway and an ABP1-independent pathway that is much more sensitive to IAA than the former. The response by the ABP1 pathway was eliminated by anti-ABP1 antibodies, had a higher sensitivity to NAA, and did not depend on extracellular Ca(2+). In contrast, the response by the non-ABP1 pathway was not affected by anti-ABP1 antibodies, had no sensitivity to NAA, and depended on extracellular Ca(2+). The swelling by either pathway required extracellular K(+) and Cl(-). The auxin-induced growth of pea internode segments showed similar response patterns, including the occurrence of two peaks in the dose response curve for IAA and the difference in Ca(2+) requirements. It is suggested that two signaling pathways participate in auxin-induced internode growth and that the non-ABP1 pathway is more likely to be involved in the control of growth by constitutive concentrations of endogenous auxin. PMID:14764902

  15. 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. PMID:22842862

  16. 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.

  17. 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.

  18. 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

  19. Sensitization with 7S Globulins from Peanut, Hazelnut, Soy or Pea Induces IgE with Different Biological Activities Which Are Modified by Soy Tolerance

    DEFF Research Database (Denmark)

    Kroghsbo, Stine; Bøgh, Katrine Lindholm; Rigby, Neil M.;

    2011-01-01

    , such as stability to digestion, have also been suggested. 7S globulins from peanut, hazelnut, soy, and pea were studied to determine whether related proteins would induce a similar sensitization when removed from their ‘normal’ matrix. Methods: Brown Norway rats (soy tolerant or nontolerant) were immunized i.p. 3...... times with 100 μg purified peanut, hazelnut, soy, or pea 7S without adjuvant. Sera were analyzed for specific antibodies by different ELISAs (IgG1, IgG2a, and IgE), inhibition ELISA, and rat basophilic leukemia cell assay. Results: The 4 related 7S globulins induced a response with an almost identical...... level of specific antibodies, but peanut 7S induced IgE of higher avidity than hazelnut and pea 7S which, again, had a higher avidity than IgE induced by soy 7S. Soy tolerance reduced the functionality of IgE without influencing antibody titers. Conclusions: Although the 4 7S globulins are structurally...

  20. Predicting the expression of recombinant monoclonal antibodies in Chinese hamster ovary cells based on sequence features of the CDR3 domain.

    Science.gov (United States)

    Pybus, Leon P; James, David C; Dean, Greg; Slidel, Tim; Hardman, Colin; Smith, Andrew; Daramola, Olalekan; Field, Ray

    2014-01-01

    Despite the development of high-titer bioprocesses capable of producing >10 g L(-1) of recombinant monoclonal antibody (MAb), some so called "difficult-to-express" (DTE) MAbs only reach much lower process titers. For widely utilized "platform" processes the only discrete variable is the protein coding sequence of the recombinant product. However, there has been little systematic study to identify the sequence parameters that affect expression. This information is vital, as it would allow us to rationally design genetic sequence and engineering strategies for optimal bioprocessing. We have therefore developed a new computational tool that enables prediction of MAb titer in Chinese hamster ovary (CHO) cells based on the recombinant coding sequence of the expressed MAb. Model construction utilized a panel of MAbs, which following a 10-day fed-batch transient production process varied in titer 5.6-fold, allowing analysis of the sequence features that impact expression over a range of high and low MAb productivity. The model identified 18 light chain (LC)-specific sequence features within complementarity determining region 3 (CDR3) capable of predicting MAb titer with a root mean square error of 0.585 relative expression units. Furthermore, we identify that CDR3 variation influences the rate of LC-HC dimerization during MAb synthesis, which could be exploited to improve the production of DTE MAb variants via increasing the transfected LC:HC gene ratio. Taken together these data suggest that engineering intervention strategies to improve the expression of DTE recombinant products can be rationally implemented based on an identification of the sequence motifs that render a recombinant product DTE.

  1. Frequencies of the expression of main protein antigens from Helicobacter pylori isolates and production of specific serum antibodies in infected patients

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Ya-Fei Mao; Zhe-Xin Shao

    2005-01-01

    AIM: To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori(H pylori)isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori.METHODS: H pylori strains in biopsy specimens from 157patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected.The target recombinant proteins rUreB, rVacA, rCagA1,rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography.Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA,rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody,expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed.RESULTS: In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2%respectively. In the 125 serum samples from the H pyloriinfected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and Flab were

  2. Approaches to systems biology. Four methods to study single-cell gene expression, cell motility, antibody reactivity, and respiratory metabolism

    DEFF Research Database (Denmark)

    Hagedorn, Peter

    To understand how complex systems, such as cells, function, comprehensive Measurements of their constituent parts must be made. This can be achieved by combining methods that are each optimized to measure specific parts of the system. Four such methods,each covering a different area, are presented......: Transcript profiling of one cell type extracted from a complex tissue containing several cell types; observation and recording of cell motility; measurement of antibody reactivities using microarrays; and invivo measurement of free and bound NADH in mitochondria. Detailed statistical analysis of the data...... from such measurements allows models of the system to be developed and tested. For each of the methods, such analysis and modelling approaches have beenapplied and are presented: Differentially regulated genes are identified and classified according to function; cell-specfic motility models...

  3. Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

    Directory of Open Access Journals (Sweden)

    Roghayeh Rahimi

    2015-02-01

    Full Text Available Objective(s:Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4 expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. Materials and Methods: In this study, expression was induced in BL21 (DE3 E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture            medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. [AGA1] . Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. [AGA2] The final concentration of purified protein was 500 µg/ml.

  4. Testicular germ cell sensitivity to TRAIL-induced apoptosis is dependent upon p53 expression and is synergistically enhanced by DR5 agonistic antibody treatment.

    Science.gov (United States)

    McKee, Chad M; Ye, Yang; Richburg, John H

    2006-12-01

    The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 mug/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32 degrees C but not 37 degrees C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32 degrees C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL.

  5. 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

  6. 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

  7. Pea Island National Wildlife Refuge : Narrative Report : Fiscal Year 1974

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This annual narrative report for Pea Island National Wildlife Refuge summarizes Refuge activities during the 1974 fiscal year. The report begins with a summary of...

  8. [Narrative report : Pea Island Migratory Wildfowl Refuge : Year 1941

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This 1941 narrative report for Pea Island Migratory Wildfowl Refuge lists wildlife observed on the refuge and compares it with 1940 data. Water conditions,...

  9. Pea Island National Wildlife Refuge: Comprehensive Conservation Plan

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This Comprehensive Conservation Plan (CCP) was written to guide management on Pea Island NWR for the next 15 years. This plan outlines the Refuge vision and purpose...

  10. Narrative report : Pea Island Migratory Wildfowl Refuge : Year 1940

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — This 1940 narrative report for Pea Island Migratory Wildfowl Refuge provides a roster of army personnel and service personnel, a summary of education, information...

  11. Purification and properties of the cytoplasmic glucose-6-phosphate dehydrogenase from pea leaves.

    Science.gov (United States)

    Fickenscher, K; Scheibe, R

    1986-06-01

    A method involving affinity chromatography on the yellow dye Remazol Brilliant Gelb GL to highly purify the cytoplasmic isoenzyme of glucose-6-phosphate dehydrogenase from pea shoots is described. Purification is at least 6000-fold. The specific activity of the purified enzyme is 185 mumol NADP reduced/min per mg protein. The preparation was free from any contamination of chloroplastic isoenzyme. The purified enzyme retains its activity in the presence of reducing agents which, in contrast, inactivate the chloroplast enzyme. The state of activity of the cytoplasmic and the chloroplastic isoenzyme in illuminated or darkened pea leaves was investigated using specific antibodies. While upon illumination the chloroplastic isoenzyme was inactivated by 80 to 90%, we could not find any change in activity of the cytoplasmic glucose-6-phosphate dehydrogenase. ATP, ADP, NAD, NADH, and various sugar phosphates do not inhibit the enzyme activity. Only NADPH is a strong competitive inhibitor with respect to NADP, suggesting that the enzyme is regulated by feedback inhibition by one of its products. Mg2+ ions have no influence on the activity of the enzyme. The molecular weight has found to be 240,000 for the native enzyme and 60,000 for the subunit. Throughout the purification procedure the enzyme was very unstable unless NADP was present in the buffer. PMID:3717951

  12. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

    Directory of Open Access Journals (Sweden)

    Erika Assarsson

    Full Text Available Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA for improved high throughput detection of protein biomarkers. This was enabled by: (1 a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2 a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3 introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility. The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

  13. Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

    Science.gov (United States)

    Assarsson, Erika; Lundberg, Martin; Holmquist, Göran; Björkesten, Johan; Thorsen, Stine Bucht; Ekman, Daniel; Eriksson, Anna; Rennel Dickens, Emma; Ohlsson, Sandra; Edfeldt, Gabriella; Andersson, Ann-Catrin; Lindstedt, Patrik; Stenvang, Jan; Gullberg, Mats; Fredriksson, Simon

    2014-01-01

    Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

  14. Purification and on-column refolding of a single-chain antibody fragment against rabies virus glycoprotein expressed in Escherichia coli.

    Science.gov (United States)

    Xi, Hualong; Yuan, Ruosen; Chen, Xiaoxu; Gu, Tiejun; Cheng, Yue; Li, Zhuang; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2016-10-01

    An anti-rabies virus single-chain antibody fragment of an anti-glycoprotein with the VL-linker-VH orientation, designated scFv57RN, was successfully and conveniently prepared in this study. The scFv57RN protein was mainly expressed in inclusion bodies in Escherichia coli. After washing and purification, the inclusion bodies were finally obtained with an on-column refolding procedure. Further purification by gel exclusion chromatography was performed to remove inactive multimers. About 360 mg of final product was recovered from 1 L of bacterial culture. The final product showed a high neutralizing titer of 950 IU/mg to the CVS-11 strain as measured using the rapid fluorescent focus inhibition test. Our study demonstrated a highly efficient method to mass produce scFV57RN with activity from inclusion bodies, which may be applied in the purification of other insoluble proteins.

  15. Green Production of Anionic Surfactant Obtained from Pea Protein

    OpenAIRE

    Rondel, Caroline; Portet, Bénédicte; Alric, Isabelle; Mouloungui, Zephirin; Blanco, Jean-François; Silvestre, Françoise

    2011-01-01

    A pea protein isolate was hydrolyzed by a double enzyme treatment method in order to obtain short peptide sequences used as raw materials to produce lipopeptides-based surfactants. Pea protein hydrolysates were prepared using the combination of Alcalase and Flavourzyme. The influence of the process variables was studied to optimize the proteolytic degradation to high degrees of hydrolysis. The average peptide chain lengths were obtained at 3–5 amino acid units after a hydrolysis of 30 min wit...

  16. 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.

  17. Variation potential influence on photosynthetic cyclic electron flow in pea

    OpenAIRE

    Sukhov, Vladimir; Surova, Lyubov; Sherstneva, Oksana; Katicheva, Lyubov; Vodeneev, Vladimir

    2015-01-01

    Cyclic electron flow is an important component of the total photosynthetic electron flow and participates in adaptation to the action of stressors. Local leaf stimulation induces electrical signals, including variation potential (VP), which inactivate photosynthesis; however, their influence on cyclic electron flow has not been investigated. The aim of this study was to investigate VP's influence on cyclic electron flow in pea (Pisum sativum L.). VP was induced in pea seedling leaves by local...

  18. 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.

  19. 降钙素原的克隆表达及多克隆抗体制备%Cloning,expression and the polyclonal antibody preparation of procalcitonin

    Institute of Scientific and Technical Information of China (English)

    王春芳; 周新; 谢焱; 郑璇

    2011-01-01

    目的 克隆表达与纯化降钙素原(PCT),制备多克隆抗体,为其单抗制备及在临床上的应用研究提供有用的实验基础.方法 培养人甲状腺髓伴癌细胞株(TT细胞)提取总RNA进行RT-PCR获取PCT的全长基因,构建pET-PCT重组质粒,转化至DE3经IPTG诱导表达出融合蛋白,纯化并进行10%聚丙烯酰胺凝胶电泳(SDS-PAGE)、质谱测序,融合蛋白免疫兔制备多抗,采用ELISA和Western-blot进行鉴定.结果 成功获得PCT全长基因,10%SDS-PAGE电泳和质谱测序证明表达的融合蛋白为PCT,成功制备了PCT多抗.结论 成功获得PCT的全长基因,表达出融合蛋白并制备多抗,为研究PCT的病理、生理作用及功能奠定基础,为PCT的单抗制备及在临床应用研究提供了有用的实验材料.%Objective To construct Pet-PCT recombinant plasmid which contained the full length sequence of human PCT gene, express fusion protein,and produce polyclonal antibody production of PCT to serve as a useful experimental basis for preparing the procalcitonin monoclonal antibody and clinical application. Methods Full length Mrna of PCT was obtained from TT cells through RT-PCR,and inserted into Pet-32a( + ) expression vector,which was used to transfect E. Coli DE3 cells. Transfected DE3 cells were induced by IPTG to produce fusion PCT protein. The fusion protein with His tag was purified by Ni-NTA affinity chro-matography column, and verified by multiple techniques such as 10% sodium dodecy sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ,and MALDI-TOF mass spectrometry. The purified protein was used to immunize New Zealand rabbit to produce polyclonal antibody, which was verified by ELISA and Western blot. Results Recombinant vectors containing full length PCT Mrna insert were constructed,and were used to transfect the E. Coli DE3 cells for producing recombinant PCT protein. SDS-PAGE and MALDI-TOF mass spectrometry results showed the characteristics of the recombinant protein

  20. Strigolactones suppress adventitious rooting in Arabidopsis and pea.

    Science.gov (United States)

    Rasmussen, Amanda; Mason, Michael Glenn; De Cuyper, Carolien; Brewer, Philip B; Herold, Silvia; Agusti, Javier; Geelen, Danny; Greb, Thomas; Goormachtig, Sofie; Beeckman, Tom; Beveridge, Christine Anne

    2012-04-01

    Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation. PMID:22323776

  1. 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.

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

    International Nuclear Information System (INIS)

    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 (GA1) 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 [35S] 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

  3. An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes

    NARCIS (Netherlands)

    H. Bakker; G.J.A. Rouwendal; A.S. Karnoup; D.E.A. Florack; G.M. Stoopen; J.P.F.G. Helsper; R. van Ree; I. van Die; D. Bosch

    2006-01-01

    N-glycosylation of a mAb may have a major impact on its therapeutic merits. Here, we demonstrate that expression of a hybrid enzyme (called xylGaIT), consisting of the N-terminal domain of Arabidopsis thaliana xylosyltra nsf erase and the catalytic domain of human 0-1,4-galactosyltransf erase I (GaI

  4. Sequence and structural analysis of antibodies

    OpenAIRE

    Raghavan, A. K.

    2009-01-01

    The work presented in this thesis focusses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more \\humanlike" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practi...

  5. Anti-Phospholipase A2 Receptor (PLA2R Antibody and Glomerular PLA2R Expression in Japanese Patients with Membranous Nephropathy.

    Directory of Open Access Journals (Sweden)

    Kei Hihara

    Full Text Available The phospholipase A2 receptor (PLA2R is the major target antigen (Ag in idiopathic membranous nephropathy (IMN. Recently, several types of immunoassay systems for anti-PLA2R antibody (Ab have been developed. However, the correlation of serum anti-PLA2R Abs and glomerular expression of PLA2R Ag, and their association with clinicopathological characteristics have yet to be proven in Japanese patients. We examined serum anti-PLA2R Abs by both ELISA and cell-based indirect immunofluorescence assay (CIIFA, and glomerular PLA2R expression by immunofluorescence (IF in 59 biopsy-proven MN patients including IMN (n = 38 and secondary MN (SMN (n = 21. In this study, anti-PLA2R Abs were present in 50% of IMN patients, but was absent in SMN patients. The concordance rate between ELISA and CIIFA was 100%. Serum IgG levels were significantly lower in anti-PLA2R Ab-positive patients. Serum albumin levels correlated inversely with serum anti-PLA2R Ab titers. The prevalence and intensity of glomerular staining for IgG4 by IF were significantly higher in anti-PLA2R Ab-positive patients than in -negative patients. Glomerular PLA2 Ag expression evaluated by IF was positive in 52.6% of IMN patients, but was absent in SMN patients. The concordance rate between the prevalence of glomerular PLA2R Ag expression and anti-PLA2R Ab was 84.2%. The prevalence of anti-PLA2R Abs measured by ELISA/CIIFA was equivalent to previous Japanese studies evaluated using Western blotting. These analyses showed an excellent specificity for the diagnosis of IMN, and anti-PLA2R positivity was associated with some clinicopathological features, especially glomerular IgG4-dominant deposition.

  6. Anti-Phospholipase A2 Receptor (PLA2R) Antibody and Glomerular PLA2R Expression in Japanese Patients with Membranous Nephropathy.

    Science.gov (United States)

    Hihara, Kei; Iyoda, Masayuki; Tachibana, Shohei; Iseri, Ken; Saito, Tomohiro; Yamamoto, Yasutaka; Suzuki, Taihei; Wada, Yukihiro; Matsumoto, Kei; Shibata, Takanori

    2016-01-01

    The phospholipase A2 receptor (PLA2R) is the major target antigen (Ag) in idiopathic membranous nephropathy (IMN). Recently, several types of immunoassay systems for anti-PLA2R antibody (Ab) have been developed. However, the correlation of serum anti-PLA2R Abs and glomerular expression of PLA2R Ag, and their association with clinicopathological characteristics have yet to be proven in Japanese patients. We examined serum anti-PLA2R Abs by both ELISA and cell-based indirect immunofluorescence assay (CIIFA), and glomerular PLA2R expression by immunofluorescence (IF) in 59 biopsy-proven MN patients including IMN (n = 38) and secondary MN (SMN) (n = 21). In this study, anti-PLA2R Abs were present in 50% of IMN patients, but was absent in SMN patients. The concordance rate between ELISA and CIIFA was 100%. Serum IgG levels were significantly lower in anti-PLA2R Ab-positive patients. Serum albumin levels correlated inversely with serum anti-PLA2R Ab titers. The prevalence and intensity of glomerular staining for IgG4 by IF were significantly higher in anti-PLA2R Ab-positive patients than in -negative patients. Glomerular PLA2 Ag expression evaluated by IF was positive in 52.6% of IMN patients, but was absent in SMN patients. The concordance rate between the prevalence of glomerular PLA2R Ag expression and anti-PLA2R Ab was 84.2%. The prevalence of anti-PLA2R Abs measured by ELISA/CIIFA was equivalent to previous Japanese studies evaluated using Western blotting. These analyses showed an excellent specificity for the diagnosis of IMN, and anti-PLA2R positivity was associated with some clinicopathological features, especially glomerular IgG4-dominant deposition.

  7. Alternative-splicing-based bicistronic vectors for ratio-controlled protein expression and application to recombinant antibody production.

    OpenAIRE

    Fallot, Stéphanie; Ben Naya, Raouia; Hieblot, Corinne; Mondon, Philippe; Lacazette, Eric; Bouayadi, Khalil; Kharrat, Abdelhakim; Touriol, Christian; Prats, Hervé

    2009-01-01

    In the last decade polycistronic vectors have become essential tools for both basic science and gene therapy applications. In order to co-express heterologous polypeptides, different systems have been developed from Internal Ribosome Entry Site (IRES) based vectors to the use of the 2A peptide. Unfortunately, these methods are not fully suitable for the efficient and reproducible modulation of the ratio between the proteins of interest. Here we describe a novel bicistronic vector type based o...

  8. Soluble Expression, Purification and Characterization of Single-chain Fv Catalytic Antibody(sFv-2F3)

    Institute of Scientific and Technical Information of China (English)

    SUN Ye; LI Wei-jia; MA Ji-sheng; MU Ying; DU Xiu-bo; YAN Gang-lin; LUO Gui-min

    2004-01-01

    To find optimal conditions for expressing the soluble form of sFv-2F3 and to study the purification and property of its derivative Se-sFv-2F3, the preferred expression conditions were investigated by means of orthogonal design. These culture conditions included incubation temperature, inducer concentration, induction time and cell concentration. The evaluation of expression was accomplished by the analysis of whole cell lysates and the yield of soluble sFv-2F3 was calculated according to the analysis of Profinder(FTI-500,Pharmacia). The purification procedure was carried out via a two-step purification procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography(IMAC). The antioxidant efficacy of Se-sFv-2F3 was demonstrated by the determination of the content of the main product of lipid peroxidation, MDA, the viability of cells and the activity of LDH. We obtained the preferred culture conditions to grow the engineered bacteria and the procedure for preparing soluble sFv-2F3 and confirmed the antioxidant efficacy of Se-sFv-2F3.

  9. Red light regulation of ethylene biosynthesis and gravitropism in etiolated pea stems

    Science.gov (United States)

    Steed, C. L.; Taylor, L. K.; Harrison, M. A.

    2004-01-01

    During gravitropism, the accumulation of auxin in the lower side of the stem causes increased growth and the subsequent curvature, while the gaseous hormone ethylene plays a modulating role in regulating the kinetics of growth asymmetries. Light also contributes to the control of gravitropic curvature, potentially through its interaction with ethylene biosynthesis. In this study, red-light pulse treatment of etiolated pea epicotyls was evaluated for its effect on ethylene biosynthesis during gravitropic curvature. Ethylene biosynthesis analysis included measurements of ethylene; the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC); malonyl-conjugated ACC (MACC); and expression levels of pea ACC oxidase (Ps-ACO1) and ACC synthase (Ps-ACS1, Ps-ACS2) genes by reverse transcriptase-polymerase chain reaction analysis. Red-pulsed seedlings were given a 6 min pulse of 11 micromoles m-2 s-1 red-light 15 h prior to horizontal reorientation for consistency with the timeline of red-light inhibition of ethylene production. Red-pulse treatment significantly reduced ethylene production and MACC levels in epicotyl tissue. However, there was no effect of red-pulse treatment on ACC level, or expression of ACS or ACO genes. During gravitropic curvature, ethylene production increased from 60 to 120 min after horizontal placement in both control and red-pulsed epicotyls. In red-pulsed tissues, ACC levels increased by 120 min after horizontal reorientation, accompanied by decreased MACC levels in the lower portion of the epicotyl. Overall, our results demonstrate that ethylene production in etiolated epicotyls increases after the initiation of curvature. This ethylene increase may inhibit cell growth in the lower portion of the epicotyl and contribute to tip straightening and reduced overall curvature observed after the initial 60 min of curvature in etiolated pea epicotyls.

  10. Anti-CD20 antibody promotes cancer escape via enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L.

    Science.gov (United States)

    Bodogai, Monica; Lee Chang, Catalina; Wejksza, Katarzyna; Lai, Jinping; Merino, Maria; Wersto, Robert P; Gress, Ronald E; Chan, Andrew C; Hesdorffer, Charles; Biragyn, Arya

    2013-04-01

    The possible therapeutic benefits of B-cell depletion in combating tumoral immune escape have been debated. In support of this concept, metastasis of highly aggressive 4T1 breast cancer cells in mice can be abrogated by inactivation of tumor-evoked regulatory B cells (tBreg). Here, we report the unexpected finding that B-cell depletion by CD20 antibody will greatly enhance cancer progression and metastasis. Both murine and human tBregs express low levels of CD20 and, as such, anti-CD20 mostly enriches for these cells. In the 4T1 model of murine breast cancer, this effect of enriching for tBregs suggests that B-cell depletion by anti-CD20 may not be beneficial at all in some cancers. In contrast, we show that in vivo-targeted stimulation of B cells with CXCL13-coupled CpG oligonucleotides (CpG-ODN) can block cancer metastasis by inhibiting CD20(Low) tBregs. Mechanistic investigations suggested that CpG-ODN upregulates low surface levels of 4-1BBL on tBregs to elicit granzyme B-expressing cytolytic CD8(+) T cells, offering some explanative power for the effect. These findings underscore the immunotherapeutic importance of tBreg inactivation as a strategy to enhance cancer therapy by targeting both the regulatory and activating arms of the immune system in vivo. PMID:23365136

  11. The optimized capsid gene of porcine circovirus type 2 expressed in yeast forms virus-like particles and elicits antibody responses in mice fed with recombinant yeast extracts.

    Science.gov (United States)

    Bucarey, Sergio A; Noriega, Jorge; Reyes, Paulina; Tapia, Cecilia; Sáenz, Leonardo; Zuñiga, Alejandro; Tobar, Jaime A

    2009-09-25

    Porcine circovirus type 2 (PCV2)-associated diseases are considered to be the biggest problem for the worldwide swine industry. The PCV2 capsid protein (Cap) is an important antigen for development of vaccines. At present, most anti-PCV2 vaccines are produced as injectable formulations. Although effective, these vaccines have certain drawbacks, including stress with concomitant immunosuppresion, and involve laborious and time-consuming procedures. In this study, Saccharomyces cerevisiae was used as a vehicle to deliver PCV2 antigen in a preliminary attempt to develop an oral vaccine, and its immunogenic potential in mice was tested after oral gavage-mediated delivery. The cap gene with a yeast-optimized codon usage sequence (opt-cap) was chemically synthesized and cloned into Escherichia coli/Saccharomyces cerevisiae shuttle vector, pYES2, under the control of the Gal1 promoter. Intracellular expression of the Cap protein was confirmed by Western blot analysis and its antigenic properties were compared with those of baculovirus/insect cell-produced Cap protein derived from the native PCV2 cap gene. It was further demonstrated by electron micrography that the yeast-derived PCV2 Cap protein self-assembles into virus-like particles (VLPs) that are morphologically and antigenically similar to insect cell-derived VLPs. Feeding raw yeast extract containing Cap protein to mice elicited both serum- and fecal-specific antibodies against the antigen. These results show that it is feasible to use S. cerevisiae as a safe and simple system to produce PCV2 virus-like particles, and that oral yeast-mediated antigen delivery is an alternative strategy to efficiently induce anti-PCV2 antibodies in a mouse model, which is worthy of further investigation in swine. PMID:19664739

  12. Prokaryotic Expression of Rubber Elongation Factor Gene and Preparation of Its Polyclonal Antibody%橡胶延长因子基因的原核表达及其多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    陈章权; 吴坤鑫; 梁晓东; 黄震; 陆田田

    2008-01-01

    [Objective] The aim of this study was to prepare the recombination protein of rubber elongation factor and its polyclonal antibodies. [Method] The encoding gene of rubber elongation factor (REF) was amplified by RT-PCR, and cloned into the prokaryotic expression vector pDEST17 to transform into Escherichia coil BI2I-AI. The recombinant protein induced by L-Arabinose was purified by the affinity chromatography. As the immunogen, the recombination protein was used to immunize mice for preparing polyclonal antibodies, while ELISA and Western blot hybridization were used to detect the titers and specificity. [Result] The purified recombination protein of REF with high expression was used to immunize house mice for preparing polyclonal antibodies with high titer and specificity. The western blot hybridization showed that the antibody could recognize the natural REF from latex. [Conclusion] The recombination protein of REF was successfully obtained and the mouse anti REF antibody with high titer and specificity was prepared, which lays a basis for further studies on biological functions of rubber elongation factor and other membrane proteins in rubber particles.

  13. Olfactory cues from different plant species in host selection by female pea moths.

    Science.gov (United States)

    Thöming, Gunda; Norli, Hans Ragnar

    2015-03-01

    In herbivorous insects specialized on few plant species, attraction to host odor may be mediated by volatiles common to all host species, by specific compounds, or combinations of both. The pea moth Cydia nigricana is an important pest of the pea. Volatile signatures of four host plant species were studied to identify compounds involved in pea moth host selection and to improve previously reported attractive volatile blends. P. sativum and alternative Fabaceae host species were compared regarding female attraction, oviposition, and larval performance. Pea moth females were strongly attracted to the sweet pea Lathyrus odoratus, but larval performance on that species was moderate. Chemical analyses of sweet pea odor and electrophysiological responses of moth antennae led to identification of seven sweet-pea-specific compounds and ten compounds common to all tested host species. Blends of these specific and common cues were highly attractive to mated pea moth females in wind tunnel and field experiments.

  14. Quantitative PET of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Weibo; Chen, Kai; He, Lina; Cao, Qizhen; Chen, Xiaoyuan [Stanford University School of Medicine, The Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford, CA (United States); Koong, Albert [Stanford University School of Medicine, Department of Radiation Oncology, Stanford, CA (United States)

    2007-06-15

    Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) on the surface of cancer cells, was approved by the FDA to treat patients with metastatic colorectal cancer. It is currently also in advanced-stage development for the treatment of several other solid tumors. Here we report for the first time the quantitative positron emission tomography (PET) imaging of EGFR expression in xenograft-bearing mice using {sup 64}Cu-labeled cetuximab. We conjugated cetuximab with macrocyclic chelating agent 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA), labeled with {sup 64}Cu, and tested the resulting {sup 64}Cu-DOTA-cetuximab in seven xenograft tumor models. The tracer uptake measured by PET was correlated with the EGFR expression quantified by western blotting. The estimated human dosimetry based on the PET data in Sprague-Dawley rats was also calculated. MicroPET imaging showed that {sup 64}Cu-DOTA-cetuximab had increasing tumor activity accumulation over time in EGFR-positive tumors but relatively low uptake in EGFR-negative tumors at all times examined (<5%ID/g). There was a good correlation (R {sup 2} = 0.80) between the tracer uptake (measured by PET) and the EGFR expression level (measured by western blotting). Human dosimetry estimation indicated that the tracer may be safely administered to human patients for tumor diagnosis, with the dose-limiting organ being the liver. The success of EGFR-positive tumor imaging using {sup 64}Cu-DOTA-cetuximab can be translated into the clinic to characterize the pharmacokinetics, to select the right population of patients for EGFR-targeted therapy, to monitor the therapeutic efficacy of anti-EGFR treatment, and to optimize the dosage of either cetuximab alone or cetuximab in combination with other therapeutic agents. (orig.)

  15. Essential Role for the Lectin Pathway in Collagen Antibody-Induced Arthritis Revealed Through Use of Adenovirus Programming Complement Inhibitor MAp44 Expression

    Science.gov (United States)

    Banda, Nirmal K.; Mehta, Gaurav; Kjaer, Troels R.; Takahashi, Minoru; Schaack, Jerome; Morrison, Thomas E.; Thiel, Steffen; Arend, William P.; Holers, V. Michael

    2014-01-01

    Previous studies using mannose-binding lectin (MBL) and complement C4 deficient mice have suggested that the lectin pathway (LP) is not required for the development of inflammatory arthritis in the collagen antibody-induced arthritis (CAIA) model. MBL, ficolins and collectin-11 are key LP pattern recognition molecules that associate with three serine proteases, MASP-1, MASP-2 and MASP-3, and also with two MBL-associated proteins designated sMAP and MAp44. Recent studies have shown that MAp44, an alternatively spliced product of the MASP-1/3 gene, is a competitive inhibitor of the binding of the recognition molecules to all three MASPs. In these studies we examined the effect of treatment of mice with adenovirus (Ad) programmed to express human MAp44 (AdhMAp44) on the development of CAIA. AdhMAp44 and Ad programming Green fluorescent protein (AdGFP) expression were injected intraperitoneally in C57BL/6 wild-type mice prior to the induction of CAIA. AdhMAp44 significantly reduced the clinical disease activity score (CDA) by 81% compared to mice injected with AdGFP. Similarly, histopathologic injury scores for inflammation, pannus, cartilage and bone damage, as well as C3 deposition in the cartilage and synovium, were significantly reduced by AdhMAp44 pretreatment. Mice treated with AdmMAp44, programming expression of mouse MAp44, also showed significantly decreased CDA and histopathologic injury scores. Additionally, administration of AdhMAp44 significantly diminished the severity of Ross River Virus-induced arthritis, a LP-dependent model. Our study provides conclusive evidence that an intact complement LP is essential to initiate CAIA, and that MAp44 may be an appropriate treatment for inflammatory arthritis. PMID:25070856

  16. The influence of feeding GMO-peas on growth of animal models

    OpenAIRE

    Petr Mares; Tunde Jurikova Pokorna; Jiri Sochor; Ladislav Zeman; Mojmir Baron; Jiri Mlcek; Stefan Balla

    2014-01-01

    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 anim...

  17. Characterization of a rabbit-antiserum for detection of pea protein in foods

    OpenAIRE

    Lundholm, Linnéa

    2008-01-01

    Food allergy is an IgE-mediated immunological disease, which affects almost 4% of the adult population and up to 6% of children. Proteins from milk, egg, peanuts, soybean, wheat, fish and nuts are the main cause of food allergies. A less common allergen is pea protein. The National Food Administration analyses undeclared pea protein and contaminations of pea protein in foods using rocket immunoelectrophoresis and immunodiffusion. For both methods an antiserum against pea protein is needed. Th...

  18. 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.)

  19. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    Science.gov (United States)

    Levite, Mia

    2014-08-01

    pathological effects: they activate glutamate/AMPA receptors, kill neurons by 'Excitotoxicity', and/or by complement activation modulated by complement regulatory proteins, cause multiple brain damage, aggravate chemoconvulsant-induced seizures, and also induce behavioral/motor impairments. Some patients with 'Autoimmune Epilepsy' that have anti-AMPA-GluR3B antibodies respond well (although sometimes transiently) to immunotherapy, and thanks to that have reduced seizures and overall improved neurological functions. (2) Anti-NMDA-NR1 antibodies are present in patients with autoimmune 'Anti-NMDA-receptor Encephalitis'. In humans, in animal models and in vitro the anti-NMDA-NR1 antibodies can be very pathogenic since they can cause a pronounced decrease of surface NMDA receptors expressed in hippocampal neurons, and also decrease the cluster density and synaptic localization of the NMDA receptors. The anti-NMDA-NR1 antibodies induce these effects by crosslinking and internalization of the NMDA receptors. Such changes can impair glutamate signaling via the NMDA receptors and lead to various neuronal/behavior/cognitive/psychiatric abnormalities. Anti-NMDA-NR1 antibodies are frequently present in high levels in the CSF of the patients with 'Anti-NMDA-receptor encephalitis' due to their intrathecal production. Many patients with 'Anti-NMDA receptor Encephalitis' respond well to several modes of immunotherapy. (3) Anti-NMDA-NR2A/B antibodies are present in a substantial number of patients with Systemic Lupus Erythematosus (SLE) with or without neuropsychiatric problems. The exact percentage of SLE patients having anti-NMDA-NR2A/B antibodies varies in different studies from 14 to 35%, and in one study such antibodies were found in 81% of patients with diffuse 'Neuropshychiatric SLE', and in 44% of patients with focal 'Neuropshychiatric SLE'. Anti-NMDA-NR2A/B antibodies are also present in subpopulations of patients with Epilepsy of several types, Encephalitis of several types (e

  20. Glutamate receptor antibodies in neurological diseases: anti-AMPA-GluR3 antibodies, anti-NMDA-NR1 antibodies, anti-NMDA-NR2A/B antibodies, anti-mGluR1 antibodies or anti-mGluR5 antibodies are present in subpopulations of patients with either: epilepsy, encephalitis, cerebellar ataxia, systemic lupus erythematosus (SLE) and neuropsychiatric SLE, Sjogren's syndrome, schizophrenia, mania or stroke. These autoimmune anti-glutamate receptor antibodies can bind neurons in few brain regions, activate glutamate receptors, decrease glutamate receptor's expression, impair glutamate-induced signaling and function, activate blood brain barrier endothelial cells, kill neurons, damage the brain, induce behavioral/psychiatric/cognitive abnormalities and ataxia in animal models, and can be removed or silenced in some patients by immunotherapy.

    Science.gov (United States)

    Levite, Mia

    2014-08-01

    pathological effects: they activate glutamate/AMPA receptors, kill neurons by 'Excitotoxicity', and/or by complement activation modulated by complement regulatory proteins, cause multiple brain damage, aggravate chemoconvulsant-induced seizures, and also induce behavioral/motor impairments. Some patients with 'Autoimmune Epilepsy' that have anti-AMPA-GluR3B antibodies respond well (although sometimes transiently) to immunotherapy, and thanks to that have reduced seizures and overall improved neurological functions. (2) Anti-NMDA-NR1 antibodies are present in patients with autoimmune 'Anti-NMDA-receptor Encephalitis'. In humans, in animal models and in vitro the anti-NMDA-NR1 antibodies can be very pathogenic since they can cause a pronounced decrease of surface NMDA receptors expressed in hippocampal neurons, and also decrease the cluster density and synaptic localization of the NMDA receptors. The anti-NMDA-NR1 antibodies induce these effects by crosslinking and internalization of the NMDA receptors. Such changes can impair glutamate signaling via the NMDA receptors and lead to various neuronal/behavior/cognitive/psychiatric abnormalities. Anti-NMDA-NR1 antibodies are frequently present in high levels in the CSF of the patients with 'Anti-NMDA-receptor encephalitis' due to their intrathecal production. Many patients with 'Anti-NMDA receptor Encephalitis' respond well to several modes of immunotherapy. (3) Anti-NMDA-NR2A/B antibodies are present in a substantial number of patients with Systemic Lupus Erythematosus (SLE) with or without neuropsychiatric problems. The exact percentage of SLE patients having anti-NMDA-NR2A/B antibodies varies in different studies from 14 to 35%, and in one study such antibodies were found in 81% of patients with diffuse 'Neuropshychiatric SLE', and in 44% of patients with focal 'Neuropshychiatric SLE'. Anti-NMDA-NR2A/B antibodies are also present in subpopulations of patients with Epilepsy of several types, Encephalitis of several types (e

  1. The assay of thyrotropin receptor antibodies with human TSH/LH-CG chimeric receptor expressed on chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Yi, Ka Hee; Kim, Chang Min [Korea Cancer Center Hospital, Seoul (Korea, Republic of)

    1996-12-01

    TSH/LH-CG chimera cDNA is transfected to CHO-K1 cell to obtain the chimeric receptor expressed on the cell surface. The optimal conditions for TSAb and TSBAb measurements are determined using chimeric receptors and under these conditions activity of TSAb and TSBAb in the sera of the Graves` patients. The results obtained are compared to those of TSAb assays using FRTL5 cells CHO-TSHR cells which have wild type human TSH receptor. The transfection procedure of chimeric receptor gene to CHO-K1 cells are on going. The optimal conditions for TSAb and TSBAb measurement using chimeric receptor will be determined after success of transfection procedure. If this study is successfully completed, not only the heterogeneity of Graves. IgG but also pathogenesis of Graves` disease will be elucidated. (author). 25 refs.

  2. 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.

  3. Nodulin gene expression in the developing pea root nodule.

    NARCIS (Netherlands)

    Govers, F.

    1987-01-01

    Infection of leguminous plants with bacteria of the genus Rhizobium results in a symbiotic interaction which brings about the development of an entirely new organ on the plant, the root nodule. Within this organ about half of the plant cells are inhabited by bacteroids, the endosymblotic form

  4. PRESERVING QUALITY OF FROZEN GREEN PEAS DURING LONG TIME STORAGE

    Directory of Open Access Journals (Sweden)

    FELICIA DIMA

    2012-06-01

    Full Text Available The purpose of this study was to analyze the degree which fresh green peas maintain their technological properties and nutritional components during long-term storage, for a period of 24 months, in a defined condition of the air, at maximum -18°C. Peas were blanched, at four different durations of this operation and the same temperature of the blanching water, 95°C. The peas were frozen after end stored. In conditions of long-term storage of frozen peas, it has been found a considerable loss of the initial content of vitamin C and damage of chlorophyll (total, a and b, under the action of chlorophyllase, causing some gray compounds. The conditions of the storage in frozen stage produced a distortion of color and sensorial qualities, higher as the storage temperature was higher than prescribed. It was found that the best results in preserving quality of peas during long-term storage (24 months were obtained for the blanched samples at 4 minutes at 95°C. The reducing of the content of vitamin C, chlorophyll a and b, and sensorial qualities was the lowest, overall.

  5. 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.

  6. 76 FR 21712 - Notice of Availability for Final PEA and Draft FONSI

    Science.gov (United States)

    2011-04-18

    ... Department of the Navy Notice of Availability for Final PEA and Draft FONSI AGENCY: Department of the Navy... of the Navy announces the availability of, the Final Programmatic Environmental Assessment (PEA) and... appropriate. Dates and Addresses: The waiting period for the Final PEA and FONSI will end 30 days...

  7. 7 CFR 201.56-6 - Legume or pea family, Fabaceae (Leguminosae).

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Legume or pea family, Fabaceae (Leguminosae). 201.56-6...-6 Legume or pea family, Fabaceae (Leguminosae). Kinds of seed: Alfalfa, alyceclover, asparagusbean..., lupines, northern sweetvetch, peas, peanut, roughpea, sainfoin, sesbania, sourclover,...

  8. Characterization of alpha-toxin hla gene variants, alpha-toxin expression levels, and levels of antibody to alpha-toxin in hemodialysis and postsurgical patients with Staphylococcus aureus bacteremia.

    Science.gov (United States)

    Sharma-Kuinkel, Batu K; Wu, Yuling; Tabor, David E; Mok, Hoyin; Sellman, Bret R; Jenkins, Amy; Yu, Li; Jafri, Hasan S; Rude, Thomas H; Ruffin, Felicia; Schell, Wiley A; Park, Lawrence P; Yan, Qin; Thaden, Joshua T; Messina, Julia A; Fowler, Vance G; Esser, Mark T

    2015-01-01

    Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml; P toxin-expressing S. aureus isolates (P toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

  9. The branching gene RAMOSUS1 mediates interactions among two novel signals and auxin in pea.

    Science.gov (United States)

    Foo, Eloise; Bullier, Erika; Goussot, Magali; Foucher, Fabrice; Rameau, Catherine; Beveridge, Christine Anne

    2005-02-01

    In Pisum sativum, the RAMOSUS genes RMS1, RMS2, and RMS5 regulate shoot branching via physiologically defined mobile signals. RMS1 is most likely a carotenoid cleavage enzyme and acts with RMS5 to control levels of an as yet unidentified mobile branching inhibitor required for auxin inhibition of branching. Our work provides molecular, genetic, and physiological evidence that RMS1 plays a central role in a shoot-to-root-to-shoot feedback system that regulates shoot branching in pea. Indole-3-acetic acid (IAA) positively regulates RMS1 transcript level, a potentially important mechanism for regulation of shoot branching by IAA. In addition, RMS1 transcript levels are dramatically elevated in rms3, rms4, and rms5 plants, which do not contain elevated IAA levels. This degree of upregulation of RMS1 expression cannot be achieved in wild-type plants by exogenous IAA application. Grafting studies indicate that an IAA-independent mobile feedback signal contributes to the elevated RMS1 transcript levels in rms4 plants. Therefore, the long-distance signaling network controlling branching in pea involves IAA, the RMS1 inhibitor, and an IAA-independent feedback signal. Consistent with physiological studies that predict an interaction between RMS2 and RMS1, rms2 mutations appear to disrupt this IAA-independent regulation of RMS1 expression. PMID:15659639

  10. Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

    International Nuclear Information System (INIS)

    The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5' regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS, LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5' end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV

  11. [Graviresponse in higher plants and its regulation in molecular bases: relevance to growth and development, and auxin polar transport in etiolated pea seedlings].

    Science.gov (United States)

    Ueda, Junichi; Miyamoto, Kensuke

    2003-08-01

    We review the graviresponse under true and simulated microgravity conditions on a clinostat in higher plants, and its regulation in molecular bases, especially on the aspect of auxin polar transport in etiolated pea (Pisum sativum L. cv. Alaska) seedlings which were the plant materials subjected to STS-95 space experiments. True and simulated microgravity conditions substantially affected growth and development in etiolated pea seedlings, especially the direction of growth of stems and roots, resulting in automorphosis. In etiolated pea seedlings grown in space, epicotyls were the most oriented toward the direction far from the cotyledons, and roots grew toward the aerial space of Plant Growth Chamber. Automorphosis observed in space were well simulated by a clinorotation on a 3-dimensional clinostat and also phenocopied by the application of auxin polar transport inhibitors of 2,3,5-triiodobenzoic acid, N-(1-naphtyl)phthalamic acid and 9-hydroxyfluorene-9-carboxylic acid. Judging from the results described above together with the fact that activities of auxin polar transport in epicotyls of etiolated pea seedlings grown in space substantially were reduced, auxin polar transport seems to be closely related to automorphosis. Strenuous efforts to learn in molecular levels how gravity contributes to the auxin polar transport in etiolated pea epicotyls resulted in successful identification of PsPIN2 and PsAUX1 genes located in plasma membrane which products are considered to be putative efflux and influx carriers of auxin, respectively. Based on the results of expression of PsPIN2 and PsAUX1 genes under various gravistimulations, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed. PMID:14555809

  12. Early embryo invasion as a determinant in pea of the seed transmission of pea seed-borne mosaic virus.

    Science.gov (United States)

    Wang, D; Maule, A J

    1992-07-01

    Seed transmission of an isolate of pea seed-borne mosaic virus (PSbMV) in several pea genotypes has been studied. Cross-pollination experiments showed that pollen transmission of PSbMV did not occur and accordingly, virus was not detected in pollen grains by ELISA or electron microscopy. Comparative studies between two pea cultivars, one with a high incidence of seed transmission and one with none, showed that PSbMV infected the floral tissues (sepals, petals, anther and carpel) of both cultivars, but was not detected in ovules prior to fertilization. Virus was detected equally well in seed coats of the progeny in both cultivars. Analysis of virus incidence and concentration in pea seeds of different developmental stages demonstrated that in the cultivar with a high incidence of seed transmission, PSbMV directly invaded immature embryos, multiplied in the embryonic tissues and persisted during seed maturation. In contrast, the cultivar without seed transmission did not show invasion of immature embryos by the virus; there was no evidence for virus multiplication or persistence during embryo development and seed maturation. Hence seed transmission of PSbMV resulted from direct invasion of immature pea embryos by the virus and the block to seed transmission in the non-permissive cultivar probably occurred at this step.

  13. 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

    production decreased from a maximum of 1.34 to as low as 0.85 with increased fertilizer N supply. The study suggests that pea–wheat intercropping is a cropping strategy that use N sources efficiently due to its spatial self-regulating dynamics where pea improve its interspecific competitive ability in areas......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 natural abundance techniques were used to determine N accumulation in the crops from the soil, fertilizer and symbiotic N2 fixation. Intercrops of pea and wheat showed maximum productivity without the supply of N fertilizer. Intercropping increased total dry matter (DM) and N yield, grain DM and N yield...

  14. Genome sequence of the pea aphid Acyrthosiphon pisum

    DEFF Research Database (Denmark)

    Richards, S.; Gibbs, R. A.; Gerardo, N. M.;

    2010-01-01

    Aphids are important agricultural pests and also biological models for studies of insect-plant interactions, symbiosis, virus vectoring, and the developmental causes of extreme phenotypic plasticity. Here we present the 464 Mb draft genome assembly of the pea aphid Acyrthosiphon pisum. This first...... published whole genome sequence of a basal hemimetabolous insect provides an outgroup to the multiple published genomes of holometabolous insects. Pea aphids are host-plant specialists, they can reproduce both sexually and asexually, and they have coevolved with an obligate bacterial symbiont. Here we...... include genes involved in chromatin modification, miRNA synthesis, and sugar transport. Gene losses include genes central to the IMD immune pathway, selenoprotein utilization, purine salvage, and the entire urea cycle. The pea aphid genome reveals that only a limited number of genes have been acquired...

  15. 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

    with homologous FMDV, positive reactions were obtained in all but one case. In some of these cattle the antibody response was detected late in comparison to the non-vaccinated infected cattle. The test gave results that compared favourably with two commercial ELISA's when used to test sera from cattle, pigs......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...... antibody. Sera from naive, vaccinated and infected cattle, sheep and pigs were examined. The specificity of the test was high. Non-specific reactions observed in particular in sera of cattle and sheep could be removed by filtration and inactivation. Positive reactions were obtained for sera from cattle...

  16. Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv).

    Science.gov (United States)

    Ferreira, A R; Ataíde, F; von Stosch, M; Dias, J M L; Clemente, J J; Cunha, A E; Oliveira, R

    2012-11-01

    In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75 h, typically between 140 and 160 g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5 % of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0 % boundary for longer periods of time when compared to a traditional proportional-integral-derivative algorithm, which tends to destabilize with increasing cell density. PMID:22610694

  17. Bispecific Antibody Conjugated Manganese-Based Magnetic Engineered Iron Oxide for Imaging of HER2/neu- and EGFR-Expressing Tumors

    Science.gov (United States)

    Wu, Shou-Cheng; Chen, Yu-Jen; Wang, Hsiang-Ching; Chou, Min-Yuan; Chang, Teng-Yuan; Yuan, Shyng-Shiou; Chen, Chiao-Yun; Hou, Ming-Feng; Hsu, John Tsu-An; Wang, Yun-Ming

    2016-01-01

    The overexpression of HER2/neu and EGFR receptors plays important roles in tumorigenesis and tumor progression. Targeting these two receptors simultaneously can have a more widespread application in early diagnosis of cancers. In this study, a new multifunctional nanoparticles (MnMEIO-CyTE777-(Bis)-mPEG NPs) comprising a manganese-doped iron oxide nanoparticle core (MnMEIO), a silane-amino functionalized poly(ethylene glycol) copolymer shell, a near infrared fluorescence dye (CyTE777), and a covalently conjugated anti-HER2/neu and anti-EGFR receptors bispecific antibody (Bis) were successfully developed. In vitro T2-weighted MR imaging studies in SKBR-3 and A431 tumor cells incubated with MnMEIO-CyTE777-(Bis)-mPEG NPs showed - 94.8 ± 3.8 and - 84.1 ± 2.8% negative contrast enhancement, respectively. Pharmacokinetics study showed that MnMEIO-CyTE777-(Bis)-mPEG NPs were eliminated from serum with the half-life of 21.3 mins. In vivo MR imaging showed that MnMEIO-CyTE777-(Bis)-mPEG NPs could specifically and effectively target to HER2/neu- and EGFR-expressing tumors in mice; the relative contrast enhancements were 11.8 (at 2 hrs post-injection) and 61.5 (at 24 hrs post-injection) fold higher in SKBR-3 tumors as compared to Colo-205 tumors. T2-weighted MR and optical imaging studies revealed that the new contrast agent (MnMEIO-CyTE777-(Bis)-mPEG NPs) could specifically and effectively target to HER2/neu- and/or EGFR-expressing tumors. Our results demonstrate that MnMEIO-CyTE777-(Bis)-mPEG NPs are able to recognize the tumors expressing both HER2/neu and/or EGFR, and may provide a novel molecular imaging tool for early diagnosis of cancers expressing HER2/neu and/or EGFR. PMID:26722378

  18. 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.

  19. Characterization of DNA-binding proteins from pea mitochondria

    DEFF Research Database (Denmark)

    Hatzack, F.A.; Dombrowski, S.; Brennicke, A.;

    1998-01-01

    unknown. Proteins binding to double-stranded oligonucleotides representing different parts of the pea (Pisum sativum) mitochondrial atp9 were analyzed by denaturation-renaturation chromatography and mobility-shift experiments. Two DNA-protein complexes were detected, which appeared to be sequence specific...... 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...

  20. Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

    Science.gov (United States)

    Tamura, Masaaki; Yan, Heping; Zegarra-Moro, Ofelia; Edl, Jennifer; Oursler, Stephanie; Chard-Bergstrom, Cindy; Andrews, Gordon; Kanehira, Tsutomu; Takekoshi, Susumu; Mernaugh, Ray

    2008-08-01

    To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

  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...

  2. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    Science.gov (United States)

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

  3. Binding of estrogen receptors to switch sites and regulatory elements in the immunoglobulin heavy chain locus of activated B cells suggests a direct influence of estrogen on antibody expression.

    Science.gov (United States)

    Jones, Bart G; Penkert, Rhiannon R; Xu, Beisi; Fan, Yiping; Neale, Geoff; Gearhart, Patricia J; Hurwitz, Julia L

    2016-09-01

    Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease. PMID:27494228

  4. Cloning and analysis of a cDNA encoding acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Using cDNA representational difference analysis (cDNA RDA) method, we have successfully isolated a gene fragment whose expression was specifically induced by external GA3 application. Screening a G2 pea cDNA library using this fragment as a probe, we obtained a 2036 bp full-length cDNA. It contains a 1746 bp open reading frame and encodes a protein of 581 amino acids with a theoretical molecular weight of 64 ku. It shares high-level sequence identity with AAIR genes from other plant species. This cDNA was cloned into expression vector and recombinant E. coli DH5α cells with remarkable AAIR enzyme activity were obtained.

  5. Parkinson-like phenotype in insulin-resistant PED/PEA-15 transgenic mice

    Science.gov (United States)

    Perruolo, Giuseppe; Viggiano, Davide; Fiory, Francesca; Cassese, Angela; Nigro, Cecilia; Liotti, Antonietta; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2016-01-01

    Neurological abnormalities, such as Parkinson-like disorders (PlD), are often co-morbidities of Type 2 Diabetic (T2D) patients, although the epidemiological link between these two disorders remains controversial. The PED/PEA-15 protein represents a possible candidate linking T2D and PD, because it is increased in subjects with T2D and is highly expressed in the brain. To test this hypothesis, we have analyzed the neurological and neurochemical phenotype of transgenic mice overexpressing PED/PEA-15 (tgPED). These mice develop impaired glucose tolerance and insulin resistance, accompanied by neurological features resembling PlD: feet clasping, slow and delayed locomotor movements in different behavioral tests in absence of clear cognitive deficits, ataxia or anxiety. Morphological analysis of the brains showed selective modifications of metabolic activity in the striatal region. In the same region, we have observed 26% decrease of dopamine fibers, confirmed by immunohistochemistry and Western Blot for tyrosine hydroxylase. Moreover, they also showed 48% reduction of dopamine levels in the striatum. Thus the tgPED mice may represent a genetic animal model of neurological disease linked to T2D. PMID:27426254

  6. 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.

  7. 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.

  8. Isolation and transcript analysis of gibberellin 20-oxidase genes in pea and bean in relation to fruit development.

    Science.gov (United States)

    García-Martínez, J L; López-Diaz, I; Sánchez-Beltrán, M J; Phillips, A L; Ward, D A; Gaskin, P; Hedden, P

    1997-04-01

    PCR was used with degenerate primers based on conserved amino acid sequences in gibberellin (GA) 20-oxidases to isolate cDNA clones for these enzymes from young seeds of pea (Pisum sativum) and developing embryos of French bean (Phaseolus vulgaris). One GA 20-oxidase cDNA (Ps27-12) was obtained from pea and three (Pv 15-11, Pv73-1 and Pv85-26) from bean. Their identities were confirmed by demonstrating that fusion proteins expressed in Escherichia coli exhibited GA 20-oxidase activity, converting [14C]GA12 to [14C]GA9. The intermediates in this three-step reaction, GA15 and GA24, were also identified as products. The expression proteins from three of the clones (Ps27-12, Pv15-11 and Pv73-1) were also shown to convert GA53 to GA20, as effectively as they did GA12. On the basis of transcript levels measured by northern blot analysis, the pea GA 20-oxidase gene is most highly expressed in young leaves, fully expanded internodes, very young seeds (until 4 days after anthesis) and expanding pods (from 3 days after anthesis at least until day 6). Expression in pods from 3-day-old unpollinated ovaries is higher than in those from pollinated ovaries. Treatment of unpollinated ovaries with GA3 to induce parthenocarpic fruit-set severely reduced the amount of GA 20-oxidase mRNA, whereas treatment with 2,4-D, although inducing fruit-set, did not reduce the levels of these transcripts. Plant decapitation above an unpollinated ovary resulted in very high levels of GA 20-oxidase mRNA in the pod. The three GA 20-oxidase genes from French bean showed very different patterns of expression: Pv 15-1 was expressed in the roots, young leaves, and developing seeds, but most highly in immature cotyledons, while Pv73-1 has a similar expression pattern to Ps27-12, with transcripts found only in young seeds and young leaves, where it was particularly abundant. Transcripts corresponding to Pv85-26 were detected in developing seeds, and just traces in the young leaves. Southern blot analysis

  9. Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides.

    Science.gov (United States)

    Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

    2010-09-01

    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli. PMID:20592135

  10. 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

  11. Associations of wheat with pea can reduce aphid infestations.

    Science.gov (United States)

    Lopes, T; Bodson, B; Francis, F

    2015-06-01

    Increasing plant diversity within crops can be beneficial for pest control. In this field study, the effects of two wheat and pea associations (mixed cropping and strip cropping) on aphid populations were compared with pure stands of both crops by observations on tillers and plants. Pea was more susceptible to infestations than wheat. As expected, the density of aphid colonies was significantly higher in pure stands during the main occurrence periods, compared with associations. Additionally, flying beneficials, such as not only aphidophagous adult ladybirds but also parasitoid, hoverfly and lacewing species that feed on aphids at the larval stage, were monitored using yellow pan traps. At specific times of the sampling season, ladybirds and hoverflies were significantly more abundant in the pure stand of pea and wheat, respectively, compared with associations. Few parasitoids and lacewings were trapped. This study showed that increasing plant diversity within crops by associating cultivated species can reduce aphid infestations, since host plants are more difficult to locate. However, additional methods are needed to attract more efficiently adult beneficials into wheat and pea associations. PMID:26013274

  12. 78 FR 68410 - United States Standards for Whole Dry Peas

    Science.gov (United States)

    2013-11-14

    ... marketing environment. According to information received by GIPSA from the USADPLC and USPLTA, some... facilitate the marketing of the class, Smooth Yellow Dry Peas and help ensure the purity of classes for Whole... Section 203(c) of the Agricultural Marketing Act of 1946, as amended (AMA) (7 U.S.C. 1622(c)), directs...

  13. Pea (Pisum sp.) genetic resources, its analysis and exploration

    Science.gov (United States)

    Pea is important temperate region pulse, with feed, fodder and vegetable uses. Originated and domesticated in Middle East and Mediterranean, it formed important dietary components of early civilizations. Although Pisum is a small genus with two or three species, it is very diverse and structured, r...

  14. Ly$\\alpha$ and UV Sizes of Green Pea Galaxies

    CERN Document Server

    Yang, Huan; Rhoads, James E; Leitherer, Claus; Wofford, Aida; Jiang, Tianxing; Wang, Junxian

    2016-01-01

    Green Peas are nearby analogs of high-redshift Ly$\\alpha$-emitting galaxies. To probe their Ly$\\alpha$ escape, we study the spatial profiles of Ly$\\alpha$ and UV continuum emission of 24 Green Pea galaxies using the Cosmic Origins Spectrograph (COS) on Hubble Space Telescope (HST). We extract the spatial profiles of Ly$\\alpha$ emission from their 2D COS spectra, and of UV continuum from both the 2D spectra and NUV images. The Ly$\\alpha$ emission shows more extended spatial profiles than the UV continuum in most Green Peas. The deconvolved Full Width Half Maximum (FWHM) of the Ly$\\alpha$ spatial profile is about 2 to 4 times that of the UV continuum in most cases. The Ly$\\alpha$ light shows significant offsets from the UV continuum in four galaxies and central absorption in one galaxy. We also compare the spatial profiles of Ly$\\alpha$ photons at blueshifted and redshifted velocities in eight Green Peas with sufficient data quality, and find the blue wing of the Ly$\\alpha$ line has a larger spatial extent than...

  15. Function-structure relationships of acetylated pea starches

    NARCIS (Netherlands)

    Huang, J.

    2006-01-01

    Cowpea, chickpea and yellow pea starches were studied and the results showed that their properties were strongly related to the chemical fine structures of the starches. Furthermore, granular starches were modified using two types of chemical acetylation reagents and then separated into different si

  16. 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 m

  17. Extracellular proteins in pea root tip and border cell exudates.

    Science.gov (United States)

    Wen, Fushi; VanEtten, Hans D; Tsaprailis, George; Hawes, Martha C

    2007-02-01

    Newly generated plant tissue is inherently sensitive to infection. Yet, when pea (Pisum sativum) roots are inoculated with the pea pathogen, Nectria haematococca, most newly generated root tips remain uninfected even though most roots develop lesions just behind the tip in the region of elongation. The resistance mechanism is unknown but is correlated spatially with the presence of border cells on the cap periphery. Previously, an array of >100 extracellular proteins was found to be released while border cell separation proceeds. Here we report that protein secretion from pea root caps is induced in correlation with border cell separation. When this root cap secretome was proteolytically degraded during inoculation of pea roots with N. haematococca, the percentage of infected root tips increased from 4% +/- 3% to 100%. In control experiments, protease treatment of conidia or roots had no effect on growth and development of the fungus or the plant. A complex of >100 extracellular proteins was confirmed, by multidimensional protein identification technology, to comprise the root cap secretome. In addition to defense-related and signaling enzymes known to be present in the plant apoplast were ribosomal proteins, 14-3-3 proteins, and others typically associated with intracellular localization but recently shown to be extracellular components of microbial biofilms. We conclude that the root cap, long known to release a high molecular weight polysaccharide mucilage and thousands of living cells into the incipient rhizosphere, also secretes a complex mixture of proteins that appear to function in protection of the root tip from infection.

  18. 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 Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION CANNED VEGETABLES Requirements for Specific Standardized Canned Vegetables § 155.172 Canned...

  19. Siiliga seotud kahjud on pea kõik heastatud

    Index Scriptorium Estoniae

    2015-01-01

    Kaitseväe peastaabi pressiesindaja sõnul on kaitsevägi praeguseks lõpetanud pea kõikide kevadel aset leidnud suurõppuse Siil ajal tekitatud kahjustuste kõrvaldamise. Ligi 60 registreeritud juhtumist moodustasid suurema osa pinnase ning kruusateede kahjustused ning neid parandas kaitseväe pioneeripataljon

  20. Effect of cooling on Clostridium perfringens in pea soup

    NARCIS (Netherlands)

    Jong, de A.E.I.; Rombouts, F.M.; Beumer, R.R.

    2004-01-01

    Foods associated with Clostridium perfringens outbreaks are usually abused after cooking. Because of their short generation times, C. perfringens spores and cells can grow out to high levels during improper cooling. Therefore, the potential of C. perfringens to multiply in Dutch pea soup during diff

  1. 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

    OpenAIRE

    Nielsen, Morten A.; Mafalda Resende; de Jongh, Willem A.; Ditlev, Sisse B.; Benjamin Mordmüller; Sophie Houard; Nicaise Tuikue Ndam; Mette Ø Agerbæk; Mette Hamborg; Achille Massougbodji; Saddou Issifou; Anette Strøbæk; Lars Poulsen; Odile Leroy; Kremsner, Peter G

    2015-01-01

    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...

  2. Tissue-specific and pathogen-inducible expression of a fusion protein containing a Fusarium-specific antibody and a fungal chitinase protects wheat against Fusarium pathogens and mycotoxins.

    Science.gov (United States)

    Cheng, Wei; Li, He-Ping; Zhang, Jing-Bo; Du, Hong-Jie; Wei, Qi-Yong; Huang, Tao; Yang, Peng; Kong, Xian-Wei; Liao, Yu-Cai

    2015-06-01

    Fusarium head blight (FHB) in wheat and other small grain cereals is a globally devastating disease caused by toxigenic Fusarium pathogens. Controlling FHB is a challenge because germplasm that is naturally resistant against these pathogens is inadequate. Current control measures rely on fungicides. Here, an antibody fusion comprised of the Fusarium spp.-specific recombinant antibody gene CWP2 derived from chicken, and the endochitinase gene Ech42 from the biocontrol fungus Trichoderma atroviride was introduced into the elite wheat cultivar Zhengmai9023 by particle bombardment. Expression of this fusion gene was regulated by the lemma/palea-specific promoter Lem2 derived from barley; its expression was confirmed as lemma/palea-specific in transgenic wheat. Single-floret inoculation of independent transgenic wheat lines of the T3 to T6 generations revealed significant resistance (type II) to fungal spreading, and natural infection assays in the field showed significant resistance (type I) to initial infection. Gas chromatography-mass spectrometry analysis revealed marked reduction of mycotoxins in the grains of the transgenic wheat lines. Progenies of crosses between the transgenic lines and the FHB-susceptible cultivar Huamai13 also showed significantly enhanced FHB resistance. Quantitative real-time PCR analysis revealed that the tissue-specific expression of the antibody fusion was induced by salicylic acid drenching and induced to a greater extent by F. graminearum infection. Histochemical analysis showed substantial restriction of mycelial growth in the lemma tissues of the transgenic plants. Thus, the combined tissue-specific and pathogen-inducible expression of this Fusarium-specific antibody fusion can effectively protect wheat against Fusarium pathogens and reduce mycotoxin content in grain.

  3. Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

    Science.gov (United States)

    Burgess, D; Penton, A; Dunsmuir, P; Dooner, H

    1997-02-01

    Three ADP-glucose pyrophosphorylase (ADPG-PPase) cDNA clones have been isolated and characterized from a pea cotyledon cDNA library. Two of these clones (Psagps1 and Psagps2) encode the small subunit of ADPG-PPase. The deduced amino acid sequences for these two clones are 95% identical. Expression of these two genes differs in that the Psagps2 gene shows comparatively higher expression in seeds relative to its expression in other tissues. Psagps2 expression also peaks midway through seed development at a time in which Psagps1 transcripts are still accumulating. The third cDNA isolated (Psagp11) encodes the large subunit of ADPG-PPase. It shows greater selectivity in expression than either of the small subunit clones. It is highly expressed in sink organs (seed, pod, and seed coat) and undetectable in leaves.

  4. Studies on the relationship between cyanide-resistant respi-ration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide

    Institute of Scientific and Technical Information of China (English)

    LI; Chijun; (

    2001-01-01

    [1]Liang, Z., Liang, H. G., The respiratory metabolism of plants, in Plant Physiology and Molecular Biology (eds. Yu, S. W., Tang, Z. C.) (in Chinese), 2nd ed., Beijing: Science Press, 1998, 344-365.[2]Lü, C. S., Liang, H. G., Induced respiration in melon fruits, Scientia Sinica, 1963, 12(4): 616.[3]Liang, H. G., Lü, C. S., A comparative study of CN-resistant respiration in different cultures of tobacco callus, Plant Physiol., 1984, 75: 876.[4]Elthon, T. E., McIntosh, L., Identification of the alternative terminal oxidase of higher plant mitochondria, Proc. Natl. Acad. Sci. USA, 1987, 84: 8399.[5]Elthon, T. E., Nickels, R. L., McIntosh, L., Monoclonal antibodies to the alternative oxidase of higher plant mitochondria, Plant Physiol., 1989, 89: 1311.[6]Liang, W. S., Liang, H. G., Progress of the alternative oxidase, Chinese Bulletin of Botany (in Chinese), 1997, 14(2): 9.[7]Liang, W. S., Liang, H. G., Induction of alternative oxidase expression by endogenous ethylene in aging potato slices, Acta Phytophysiol. Sin. (in Chinese), 1999, 25(2): 205.[8]He, J. X., Wei, Z. Q., Liang, H. G., Effects of water stress on development, and operation and gene expression of cyanide-resistant respiratory pathway in wheat, Science in China, Ser. C, 1999, 42(3): 300.[9]McIntosh, L., Molecular biology of the alternative oxidase, Plant Physiol., 1994, 105: 781.[10] Wang, J., Zhang, L. X., Liu, Z. L. et al., A possible calcium binding site in D1 protein: A fluorescence and FTIR study of the interaction between lanthanides and a synthetic peptide, Photosynthesis Research, 1995, 44: 297.[11] Li, X. P., Du, L. F., Liang, H. G. et al., Preparation and identification of antidodecapeptide of polypeptide D1 or photosys-tem II reaction center, Prog. Biochem. Biophys. (in Chinese), 1997, 24(3): 283.[12] Wen, J. Q., Liang, H. G., Studies on energy status and mitochondria respiration during growth and senescence of mung bean cotyledons, Physiol

  5. Expression and purification of the nucleocapsid protein of Schmallenberg virus, and preparation and characterization of a monoclonal antibody against this protein.

    Science.gov (United States)

    Zhang, Yongning; Wu, Shaoqiang; Wang, Jianchang; Wernike, Kerstin; Lv, Jizhou; Feng, Chunyan; Zhang, Jihong; Wang, Caixia; Deng, Junhua; Yuan, Xiangfen; Lin, Xiangmei

    2013-11-01

    Schmallenberg virus (SBV) is a novel orthobunyavirus that primarily infects ruminants such as cattle, sheep and goats. The nucleocapsid (N) protein of SBV has been shown to be an ideal target antigen for serological detection. To prepare a monoclonal antibody (mAb) against the N protein, the full-length coding sequence of the SBV N gene was cloned into pET-28a-c(+) and pMAL-c5X vectors to generate two recombinant plasmids, which were expressed in Escherichia coli BL21 as histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins, respectively. After affinity purification of His-SBV-N with Ni-NTA agarose and MBP-SBV-N with amylose resin, His-SBV-N was used to immunize BALB/c mice, while MBP-SBV-N was utilized to screen for mAb-secreting hybridomas. Six hybridoma cell lines stably secreting mAbs against N were obtained. Clone 2C8 was selected for further study because of its rapid growth characteristics in vitro and good reactivity with recombinant SBV N proteins in enzyme-linked immunosorbent assays. The epitope recognized by 2C8 is located at amino acids 51-76 of the SBV N protein. Western blot analyses showed that 2C8 reacts with both recombinant SBV N proteins and SBV isolates. It is also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein. The successful preparation of recombinant N proteins and mAbs provides valuable materials that can be used in the serological diagnosis of SBV. PMID:23988909

  6. Molecular polygamy: The promiscuity of l-phenylalanyl-tRNA-synthetase triggers misincorporation of meta- and ortho-tyrosine in monoclonal antibodies expressed by Chinese hamster ovary cells.

    Science.gov (United States)

    Popp, Oliver; Larraillet, Vincent; Kettenberger, Hubert; Gorr, Ingo H; Hilger, Maximiliane; Lipsmeier, Florian; Zeck, Anne; Beaucamp, Nicola

    2015-06-01

    In-depth analytical characterization of biotherapeutics originating from different production batches is mandatory to ensure product safety and consistent molecule efficacy. Previously, we have shown unintended incorporation of tyrosine (Tyr) and leucine/isoleucine (Leu/Ile) at phenylalanine (Phe) positions in a recombinant produced monoclonal antibody (mAb) using an orthogonal MASCOT/SIEVE based approach for mass spectrometry data analysis. The misincorporation could be avoided by sufficient supply of phenylalanine throughout the process. Several non-annotated signals in the primarily chromatographic peptide separation step for apparently single Phe→Tyr sequence variants (SVs) suggest a role for isobar tyrosine isoforms. Meta- and ortho-Tyr are spontaneously generated during aerobic fed-batch production processes using Chinese hamster ovary (CHO) cell lines. Process induced meta- and ortho-Tyr but not proteinogenic para-Tyr are incorporated at Phe locations in Phe-starved CHO cultures expressing a recombinant mAb. Furthermore, meta- and ortho-Tyr are preferably misincorporated over Leu. Structural modeling of the l-phenylalanyl-tRNA-synthetase (PheRS) substrate activation site indicates a possible fit of non-cognate ortho-Tyr and meta-Tyr substrates. Dose-dependent misincorporations of Tyr isoforms support the hypothesis that meta- and ortho-Tyr are competing, alternative substrates for PheRS in CHO processes. Finally, easily accessible at-line surrogate markers for Phe→Tyr SV formation in biotherapeutic production were defined by the calculation of critical ratios for meta-Tyr/Phe and ortho-Tyr/Phe to support early prediction of SV probability, and finally, to allow for immediate process controlled Phe→Tyr SV prevention.

  7. Involvement of diamine oxidase and peroxidase in insolubilization of the extracellular matrix: implications for pea nodule initiation by Rhizobium leguminosarum.

    Science.gov (United States)

    Wisniewski, J P; Rathbun, E A; Knox, J P; Brewin, N J

    2000-04-01

    Rhizobium leguminosarum colonizes host cells and tissues through infection threads, which are tubular in-growths of the plant cell wall. Monoclonal antibody MAC265 recognizes a plant matrix glycoprotein (MGP) associated with the lumen of these infection threads. This glycoprotein is also released in soluble form from the root tips of pea seedlings. In the presence of hydrogen peroxide, release of glycoprotein from root tips was not observed. Extractability from root tips was therefore used as the basis for investigating the peroxide-driven insolubilization of MGP and the possible involvement of two extracellular enzymes, peroxidase (POD) and diamine oxidase (DAO), was investigated. Release of MGP from root tips was enhanced by application of POD and DAO inhibitors (salicylhydroxamic acid and o-phenanthroline, respectively). Furthermore, release of MGP was inhibited by pretreatment of roots with putrescine (the substrate of DAO) and also by application of a partially purified extract of DAO from pea shoots. Following inoculation of pea roots with R. leguminosarum, elevated levels of DAO transcript were observed by reverse transcriptase-polymerase chain reaction (RT-PCR), but these then dropped to a low level from 4 to 10 days post inoculation, rising again in more mature nodules. In situ hybridization studies indicated that the bulk of the transcription was associated with the infected tissue in the center of the nodule. On the basis of these observations, we postulate that DAO may be involved in the peroxide-driven hardening of MGP in the lumen of infection threads and in the intercellular matrix.

  8. 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. PMID:24291196

  9. 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.

  10. 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.

    Science.gov (United States)

    Satoh, Nozomi; Kon, Tatsuya; Yamagishi, Noriko; Takahashi, Tsubasa; Natsuaki, Tomohide; Yoshikawa, Nobuyuki

    2014-11-01

    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. PMID:25386843

  11. 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.

  12. 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

  13. Optimization of Agroinfiltration in Pisum sativum Provides a New Tool for Studying the Salivary Protein Functions in the Pea Aphid Complex.

    Science.gov (United States)

    Guy, Endrick; Boulain, Hélène; Aigu, Yoann; Le Pennec, Charlotte; Chawki, Khaoula; Morlière, Stéphanie; Schädel, Kristina; Kunert, Grit; Simon, Jean-Christophe; Sugio, Akiko

    2016-01-01

    Aphids are piercing-sucking insect pests and feed on phloem sap. During feeding, aphids inject a battery of salivary proteins into host plant. Some of these proteins function like effectors of microbial pathogens and influence the outcome of plant-aphid interactions. The pea aphid (Acyrthosiphon pisum) is the model aphid and encompasses multiple biotypes each specialized to one or a few legume species, providing an opportunity to investigate the underlying mechanisms of the compatibility between plants and aphid biotypes. We aim to identify the aphid factors that determine the compatibility with host plants, hence involved in the host plant specialization process, and hypothesize that salivary proteins are one of those factors. Agrobacterium-mediated transient gene expression is a powerful tool to perform functional analyses of effector (salivary) proteins in plants. However, the tool was not established for the legume species that A. pisum feeds on. Thus, we decided to optimize the method for legume plants to facilitate the functional analyses of A. pisum salivary proteins. We screened a range of cultivars of pea (Pisum sativum) and alfalfa (Medicago sativa). None of the M. sativa cultivars was suitable for agroinfiltration under the tested conditions; however, we established a protocol for efficient transient gene expression in two cultivars of P. sativum, ZP1109 and ZP1130, using A. tumefaciens AGL-1 strain and the pEAQ-HT-DEST1 vector. We confirmed that the genes are expressed from 3 to 10 days post-infiltration and that aphid lines of the pea adapted biotype fed and reproduced on these two cultivars while lines of alfalfa and clover biotypes did not. Thus, the pea biotype recognizes these two cultivars as typical pea plants. By using a combination of ZP1109 and an A. pisum line, we defined an agroinfiltration procedure to examine the effect of in planta expression of selected salivary proteins on A. pisum fitness and demonstrated that transient expression of

  14. A simple vector system to improve performance and utilisation of recombinant antibodies

    OpenAIRE

    Vincent Karen J; Mitchell Joanne N; Rojas Gertrudis; Martin Cecile D; Wu Jiahua; McCafferty John; Schofield Darren J

    2006-01-01

    Abstract Background Isolation of recombinant antibody fragments from antibody libraries is well established using technologies such as phage display. Phage display vectors are ideal for efficient display of antibody fragments on the surface of bacteriophage particles. However, they are often inefficient for expression of soluble antibody fragments, and sub-cloning of selected antibody populations into dedicated soluble antibody fragment expression vectors can enhance expression. Results We ha...

  15. Medicago truncatula, an intergenomic vehicle for the map-based cloning of pea (Pisum sativum) genes. Comparative structural genomic studies of the pea Sym2-Nod3 region

    NARCIS (Netherlands)

    Gualtieri González-Latorre, G.S.

    2001-01-01

    To determine the usefulness of M. truncatula as intergenomic vehicle for the positional cloning of pea genes it was studied whether these legumes are microsyntenic. These studies were focused on the pea Sym2 and Nod3 genomic regions. The M. truncatula orthologous genomic regions have been cloned and

  16. Construction of prokaryotic expression system of 2 148-bp fragment from cagA gene and detection of cagA gene, CagA protein in Helicobacter pyloriisolates and its antibody in sera of patients

    Institute of Scientific and Technical Information of China (English)

    Jie Yan; Yuan Wang; Shi-He Shao; Ya-Fei Mao; Hua-Wen Li; Yi-Hui Luo

    2004-01-01

    AIM: To construct a prokaryotic expression system of a Helicobacter pylori ( H pylori) cagA gene fragment and establish enzyme-linked immunosorbent assays (ELISA) for detecting CagA and its antibody, so as to understand the manner in which the infection of CagA-expressing H pylori (CagA+ H pylori) isolates cause diseases.METHODS: H pylori strains in gastric biopsy specimens from 156 patients with positive results in rapid urease test were isolated. PCR was used to detect the frequency of cagA gene in the 109 H pylori isolates and to amplify a 2 148-bp fragment (cagA1) of cagA gene from a clinical strain Y06. A prokaryotic expression system of cagA1 gene was constructed,and the expression of the target recombinant protein (rCagA1) was examined by SDS-PAGE. Western blotting and immunodiffusion assay were employed to determine the immunoreactivity and antigenicity of rCagA1, respectively.Two ELISAs were established to detect CagA expression in 109 H pylori isolates and the presence of CagA antibody in the corresponding patients′ sera, and the correlations between infection with CagA+ H pylori and gastritis as well as peptic ulcer were analyzed.RESULTS: Of all the clinical specimens obtained, 80.8%(126/156) were found to have H pylori isolates and 97.2%of the isolates (106/109) were positive for caaA gene. In comparison with the reported data, the cloned cagA1fragment possessed 94.83% and 93.30% homologies with the nucleotide and putative amino acid sequences,respectively. The output of rCagA1 produced by the constructed recombinant prokaryotic expression system was approximately 30% of the total bacterial protein, rCagA1was able to bind to the commercial antibody against the whole-cells of H pylori and to induce the immunized rabbits to produce antibody with an immunodiffusion titer of 1:4. A proportion as high as 92.6% of the H pylori isolates (101/109)expressed CagA and 88.1% of the patients′ serum samples (96/109) were CagA antibody-positive. The percentage of

  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

    a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave, positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.......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...

  18. Graviresponse and its regulation from the aspect of molecular levels in higher plants: growth and development, and auxin polar transport in etiolated pea seedlings under microgravity.

    Science.gov (United States)

    Miyamoto, Kensuke; Hoshino, Tomoki; Hitotsubashi, Reiko; Tanimoto, Eiichi; Ueda, Junichi

    2003-10-01

    In STS-95 space experiments we have demonstrated that microgravity conditions resulted in automorphosis in etiolated pea (Pisum sativum L. cv. Alaska) seedlings (Ueda et al. 1999). Automorphosis-like growth and development in etiolated pea seedlings were also induced under simulated microgravity conditions on a 3-dimensional (3-D) clinostat, epicotyls being the most oriented toward the direction far from the cotyledons. Detail analysis of epicotyl bending revealed that within 36 h after watering, no significant difference in growth direction of epicotyls was observed in between seedlings grown on the 3-D clinostat and under 1 g conditions, differential growth near the cotyledonary node resulting in epicotyl bending of ca. 45 degrees toward the direction far from the cotyledons. Thereafter epicotyls continued to grow almost straightly keeping this orientation on the 3-D clinostat. On the other hand, the growth direction in etiolated seedlings changed to antigravity direction by negative gravitropic response under 1 g conditions. Automorphological epicotyl bending was also phenocopied by the application of auxin polar transport inhibitors such as 9-hydroxyfluorene-9-carboxylic acid, N-(1-naphtyl)phthalamic acid and 2,3,5-triiodobenzoic acid. These results together with the fact that auxin polar transport activity in etiolated pea epicotyls was substantially reduced in space suggested that reduced auxin polar transport is closely related to automorphosis. Strenuous efforts to learn how gravity contributes to the auxin polar transport in etiolated pea epicotyls in molecular bases resulted in successful identification of PsPIN2 and PsAUX1 encoding putative auxin-efflux and influx carrier proteins, respectively. Based on the results of these gene expression under simulated microgravity conditions, a possible role of PsPIN2 and PsAUX1 genes for auxin polar transport in etiolated pea seedlings will be discussed. PMID:14676393

  19. 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.

  20. Detection of antibody against antigen expressed by molecularly cloned hepatitis C virus cDNA: Application to diagnosis and blood screening for posttransfusion hepatitis

    International Nuclear Information System (INIS)

    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. 登革病毒4型E蛋白的表达与多克隆抗体的制备%Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

    Institute of Scientific and Technical Information of China (English)

    李竹石; 俞永新; 李玉华; 林华; 杨健; 杨会强; 曾献武; 赵宇; 刘俐; 刘莉娜; 康庄

    2012-01-01

    目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白Ⅲ区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体.方法 将登革病毒4型E蛋白与E蛋白Ⅲ区编码序列克隆到pET-32a(+)质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测.分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白Ⅲ区的多克隆抗体,并对其进行Western blot鉴定.结果 登革病毒4型E蛋白与E蛋白Ⅲ区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带.结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础.%Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a ( + ) plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody formed bands as expected when reacting with the dengue virus. Conclusions The polyclonal antibody is prepared and may be used in the detection of dengue virus.

  2. 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.

  3. DEVELOPMENT AND PERFORMANCE EVALUATION OF TRACTOR FRONT MOUNTED PIGEON PEA STEM CUTTER

    OpenAIRE

    Atul R. Dange; S.K.Thakare

    2010-01-01

    Pigeon pea or tur (Cajanus cajan L. Mills.) is one of the important pulse crops of India and ranks second to chickpea in area and production. Traditionally the harvesting of pigeon pea is done manually by sickle, which demands considerable amount of labour, drudgery, time and cost to harvest, which reflects on total production cost of the crop. In view of this a tractor operated front mounted pigeon pea stem cutter was developed and being front mounted implement it facilitated better visibil...

  4. Towards genome-wide breeding for yield stability in spring pea

    OpenAIRE

    Klein, Anthony; Tayeh, Nadim; Siol, Mathieu; Herbommez, J.F.; Pichon, Jean-Philippe; Duarte, Jorge; Duborjal, H.; Houtin, Hervé; Blanc, Norbert; Valdrini, Jean-Marc; Walczak, Patrice; Bleriot, Olivier; Hanocq, Eric; Chassin, Alain; Bidon, Marc

    2014-01-01

    Field pea (Pisum sativum L.) is an attractive crop for human and livestock nutrition and an important contributor to low-input farming systems. Multiple environmental challenges face field pea production and penalize yield regularity. The work-package 1 of the French National ANR project PeaMUST aims at identifying efficient gene combinations for yield stability in low-input cropping systems through genomic selection. Genomic selection is a new breeding method that uses increasingly abundant ...

  5. Prokaryotic Expression and Polyclonal Antibody Preparation of SDG711 C-terminal from Rice%水稻SDG711蛋白C末端原核表达及多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    张志刚; 李超; 林欣欣; 巫光宏; 杜平州; 王玉琪

    2013-01-01

    [目的]对水稻SDG711蛋白C末端进行原核表达,并制备其多克隆抗体.[方法]选取水稻SDG711蛋白抗原决定簇较密集的C末端进行原核表达,通过构建原核表达载体pET28a-711C,转化E.coli BL21(DE3)感受态细胞,IPTG诱导表达融合蛋白后进行纯化,再以纯化的融合蛋白为抗原免疫新西兰白兔,制备多克隆抗体,并对其进行Western-blot分析.[结果]试验制备的多克隆抗体能有效地检测抗原的表达.[结论]该研究为进一步深入研究SDG711蛋白的功能奠定了基础.%[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and prepare its polyclonal antibody. [ Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherkhia coli BL21 (DE3) competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained for Western-blot analysis. [Result] The polyclonal antibody prepared could efficiently detect the antigen expression. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein.

  6. Sequence and structual analysis of antibodies.

    OpenAIRE

    Raghavan, A. K.

    2008-01-01

    The work presented in this thesis focuses on the sequence and structural analysis of antibodies and has fallen into three main areas. First I developed a method to assess how typical an antibody sequence is of the expressed human antibody repertoire. My hypothesis was that the more "human like" an antibody sequence is (in other words how typical it is of the expressed human repertoire), the less likely it is to elicit an immune response when used in vivo in humans. In practice, I found that, ...

  7. Clinorotation affects mesophyll photosynthetic cells in leaves of pea seedlings.

    Science.gov (United States)

    Adamchuk, N I

    1998-07-01

    Experiments with autotrophs in altered gravity condition have a grate significant for development of space biology. The main results of investigation in the photosynthetic apparatus state under microgravity condition have based on the experiments with maturity plants and their differentiated cells. The structural and functional organization of photosynthetic cells in seedlings is poor understandable still. Along with chloroplasts preserving a native membrane system in palisade parenchyma cells of the 29-day pea plant leaves in microgravity, chloroplasts with fribly packed or damaged granae, whose thylakoids appeared as vesicles with an electrontransparent content, were also observed. The investigation of preceding process induced these effects have a sense. That is why, the goal of our experiments was to perform the study of a structural organization of the photosynthetic cells of 3-d pair of pea seedlings leaves under the influence of clinorotation.

  8. 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.

  9. 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.

  10. The QE numerical simulation of PEA semiconductor photocathode

    CERN Document Server

    Li, Xudong; Zhang, Meng; Zhao, Minghua

    2011-01-01

    Several kinds of models have already been proposed for explaining the photoemission process. The exact photoemission theory of semiconductor photocathode was not well established after decades of research. In this paper an integral equation of quantum efficiency (QE) is constructed to describe the photoemission of positive electron affinity (PEA) semiconductor photocathode based on three-step photoemission model. The influences of forbidden gap, electron affinity, photon energy, incident angle, degree of polarization, refractive index, extinction coefficient, initial/final electron energy, relaxation time and external electric field on the QE of PEA semiconductor photocathode are taken into account. In addition, a computer code is also programmed to calculate the QE of K2CsSb photocathode theoretically at 532nm wavelength, the result is in line with the experimental value by and large. What are the reasons caused to the distinction between the experimental measuring and theoretical QE are discussed.

  11. Pulsed electro-acoustic (PEA) measurements of embedded charge distributions

    Science.gov (United States)

    Dennison, J. R.; Pearson, Lee H.

    2013-09-01

    Knowledge of the spatial distribution and evolution of embedded charge in thin dielectric materials has important applications in semiconductor, high-power electronic device, high-voltage DC power cable insulation, high-energy and plasma physics apparatus, and spacecraft industries. Knowing how, where, and how much charge accumulates and how it redistributes and dissipates can predict destructive charging effects. Pulsed Electro-acoustic (PEA) measurements— and two closely related methods, Pressure Wave Propagation (PWP) and Laser Intensity Modulation (LIMM)— nondestructively probe such internal charge distributions. We review the instrumentation, methods, theory and signal processing of simple PEA experiments, as well as the related PPW and LIMM methods. We emphasize system improvements required to achieve high spatial resolution for in vacuo measurements of thin dielectrics charged using electron beam injection.

  12. Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours.

    Science.gov (United States)

    Aluko, Rotimi E; Mofolasayo, Olawunmi A; Watts, Beverley M

    2009-10-28

    Commercial yellow pea seed flours prepared by a patented wet-milling process and pea protein isolate (PPI) were analyzed for emulsifying and foaming properties at pH 3.0, 5.0, and 7.0 and compared to soybean protein isolate (SPI). PPI and SPI formed emulsions with significantly smaller (p pea starch into SPI emulsions produced a synergistic effect that led to significant increases (p pea starch could be used to improve the quality of SPI-stabilized food emulsions. PMID:20560631

  13. Expression and purification of HIV-1 subtype C Gp120, and its antibodies preparation%HIV-1 C亚型Gp120蛋白表达、纯化及其抗体制备的研究

    Institute of Scientific and Technical Information of China (English)

    杨海儒; 冯霞; 余双庆; 张晓光; 张晓梅; 陈国敏; 李泽琳; 曾毅

    2009-01-01

    目的 制备HIV-1 C亚型Gp120蛋白及其抗体.方法 用PCR技术从表达C亚型HIV-1全长gp16O基因的质粒上扩增gP120基因羧基末端部分片段,其长度为612个核苷酸,编码204个氨基酸.将测序鉴定正确的gp120基因片段克隆到原核表达载体pET-30a上,以包涵体的形式在大肠埃希菌BL21(DE3)中高效表达,Gp120蛋白C端融合6×His标签便于纯化.将纯化的蛋白免疫新西兰大白兔制备C亚型Gp120特异性兔多克隆抗体,用间接ELISA法测定抗体滴度,并用Western Blot方法验证该抗体是否可识别在哺乳动物细胞内表达的C亚型Gp160蛋白.结果 成功地获得高纯度的C亚型Gp120融合蛋白,用其制备的多克隆抗体滴度可达1:204 800,该抗体可特异性识别在COS-1细胞中瞬时表达的C亚型Gp160蛋白.结论 获得了高纯度的C亚型Gp120融合蛋白及其高效价的抗体.%Objective To prepare H1V-1 subtype C Gp120 protein and to produce its polyelonal antibodies.Methods A C-terminal fragment of gp120 gene was amplified by PCR from a plasmid expressing full- length HIV-1 subtype C gp160 gene. The length of the subtype C gp120 fragment was 612 nt and it encodes 204 amino acid residues. The resulting DNA construct was cloned into a prokaryotic expression vector (pET-30a) and recombinant pET-3Oa-gp120 was expressed in Escherichia coli BL21 (DE3) as an insoluble protein. The vector also contained a six-histidine (His6) tag at the C-terminus for convenient purification. To produce subtype CGp120-specific polyelonal antibodies, New-Zealand rabbit was immunized with the purified Gp120 protein. Serum samples were tested by enzyme-linked immunosorbont assays (ELISA) to determine the level of antibodies. And Western blotting was used to further verify whether the polyclonal antibodies could specifically recognize subtype C Gp160 protein expressed in mammalian cells.Results HIV-1 subtype C Gp120 protein was successfully acquired and the titer of its polyclonal

  14. Extrusion-Cooking of Pea Flour: Structural and Immunocytochemical Aspects

    OpenAIRE

    Ben-Hdech, Hassane; Gallant, Daniel J.; Bouchet, Brigitte; Gueguen, Jacques; Melcion, Jean-Pierre

    1991-01-01

    Pea flour was submitted to extrusion-cooking under various conditions. The progressive structural transformation was investigated by light microscopy and immuno- gold transmission electron microscopy. Each of the three major compounds, i.e., starch granules, protein bodies, and cell wall fragments, develop a specific, independent structure. Protein bodies aggregate and fuse giving a protein matrix. Starch granules swell, deform, come into contact with each other, and ultimately also fuse toge...

  15. Function-structure relationships of acetylated pea starches

    OpenAIRE

    J. Huang

    2006-01-01

    Cowpea, chickpea and yellow pea starches were studied and the results showed that their properties were strongly related to the chemical fine structures of the starches. Furthermore, granular starches were modified using two types of chemical acetylation reagents and then separated into different size fractions. The amount of introduced acetyl groups was found to depend on the size of the granules for the reaction with rapidly reacting reagent acetic acid anhydride, whereas the amount of intr...

  16. Distribution of N within Pea, Lupin, and Soybean Nodules.

    Science.gov (United States)

    Kohl, D H; Reynolds, P H; Shearer, G

    1989-06-01

    The (15)N abundance of some, but not all, legume root nodules is significantly elevated compared to that of the whole plant. It seems probable that differences in (15)N enrichment reflect differences in the assimilatory pathway of fixed N. In that context, we have determined the distribution of naturally occurring (15)N in structural fractions of nodules from soybean (Glycine max L. Merr.), yellow lupin (Lupinus luteus), and pea (Pisum sativum) nodules and in chemical components from soybean nodules and to a lesser extent, pea and lupin nodules. None of the fractions of pea nodules (cortex, bacteriod, or host plant cytoplasm) was enriched in (15)N. The differences among bacteriods, cortex, and plant cytoplasm were smaller in lupin than in soybean nodules, but in both, bacteriods had the highest (15)N enrichment. In soybean nodules, the (15)N abundance of bacteriods and cortex was higher than plant cytoplasm, but all three fractions were more enriched in (15)N than the entire plant. Plant cytoplasm from soybean nodules was fractionated into protein-rich material, nonprotein alcohol precipitable material (NA), and a low molecular weight fraction. The N of the latter was further separated into N of ureides, nucleotides and free amino acids. Most of these components were either similar to or lower in (15)N abundance than the plant cytoplasm as a whole, but the NA fraction showed unusual (15)N enrichment. However, the percentage of nodule N in this fraction was small. NA fractions from yellow lupin and pea nodules and from soybean leaves were not enriched in (15)N. Nor was the NA fraction in ruptured bacteriods and cortical tissue of soybean nodules. Variation among soybean nodule fractions in the preponderance in protein of different amino acids was not large enough to explain the differences in (15)N abundances among them. A hypothesis, consistent with all known data, concerning the mechanism leading to the observed excess (15)N of lupin and soybean bacteriods is

  17. Sort sourceof pea in Ukraine and in the world

    OpenAIRE

    ШЕВЧЕНКО А.М.

    2006-01-01

    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.

  18. 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

  19. 抗人CD25嵌合抗体基因的构建及其瞬时表达研究%Study on construction and transient expression of human-mouse chimeric antibody gene against human CD25

    Institute of Scientific and Technical Information of China (English)

    胡迪超; 张爱华; 潘勇兵; 詹珊珊; 杨晓明

    2011-01-01

    目的:构建抗人CD25嵌合抗体基因并在哺乳动物细胞中进行瞬时表达和初步鉴定.方法:采用RLM-RACE法克隆WuTac抗体可变区和信号肽序列,并利用基因拼接法构建嵌合抗体基因.用脂质体法瞬时转染三种哺乳动物细胞,并使用ELISA、FCM、WB、Dot blot和免疫荧光法进行检测.结果:成功克隆WuTac抗体可变区和信号肽序列,并构建了抗人CD25嵌合抗体表达质粒.瞬时转染结果表明所表达的嵌合抗体保留了亲本抗体WuTac的抗原结合力.结论:成功构建了抗人CD25嵌合抗体基因,为其进一步研究打下基础.%Objective:To construct chimeric antibody gene against human an CD25 angigen,and prelin inarily identify the expressed prod-ucts produced from transiently transfected mammalian cells in order to facilitate the further study of stable expression.Methods:The RLM-RACE was employed to clone variable region genes and leader sequences,and the Overlap PVR method was used to construct the chimeric anti-body gene.After transiently transfected in three mammalian cells with liposome method, the expressed products were determined by ELISA,FCM,W B,Dotblot and immunofluorescence assay.Results:The variable region genes and leader sequences were successfully amplified,and the eukayotic expression plasmids were constructed.The results from transient transfection indicate the expressed products retain the antigen binding capacity of parental antibody WuTac.Conclusion:The successfully constructed chimeric antibody gene against human CD25 lays sound foun-dation for further study.

  20. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on antibody-mediated immune response and gene expression of pattern recognition receptors and cytokines in broiler chickens immunized with T-cell dependent antigens.

    Science.gov (United States)

    Alizadeh, M; Rodriguez-Lecompte, J C; Echeverry, H; Crow, G H; Slominski, B A

    2016-04-01

    This study evaluated the effect of yeast-derived products on innate and antibody mediated immune response in broiler chickens following immunization with sheep red blood cells (SRBC) and bovine serum albumin (BSA). One-day-old male broiler chickens (Ross-308) were randomly assigned to 6 dietary treatments of 9 replicate cages of 5 birds each per treatment. Dietary treatments consisted of a Control diet without antibiotic, and diets containing 11 mg/kg of virginiamycin, 0.25% of yeast cell wall (YCW), 0.2% of a commercial product Maxi-Gen Plus containing processed yeast and nucleotides, 0.05% of nucleotides, or a diet containing 10% of DDGS. On days 21 and 28 post-hatching, 5 birds per treatment were immunized intramuscularly with both SRBC and BSA. One week after each immunization, blood samples were collected. Serum samples were analyzed by hemagglutination test for antibody response to SRBC, and by ELISA for serum IgM and IgG response to BSA. On d 35, 5 birds per treatment were euthanized and the tissue samples from the cecal tonsils were collected to assess the gene expression of toll-like receptors TLR2b, TLR4, and TLR21, monocyte mannose receptor (MMR), and cytokines IL-10, IL-13, IL-4, IL-12p35, and IFN-γ. The results for gene expression analysis demonstrated that the diet supplemented with YCW increased the expression of TLR2b and T-helper type 2 cytokines IL-10, IL-4, and IL-13 relative to the Control; and the expression of TLR4 and IL-13 was upregulated in the nucleotide-containing diet. However, the diets containing antibiotics or Maxi-Gen Plus downregulated the expression of IFN-γ compared to the control. The primary antibody response to SRBC was not affected by diets. However, the diet containing YCW increased the secondary antibody response to SRBC compared to the antibiotic treatment. Neither primary nor secondary IgG and IgM response against BSA were affected by diets. In conclusion, supplementation of the diet with YCW stimulated Th2 cell

  1. 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.

  2. Auxin biosynthesis in pea: characterization of the tryptamine pathway.

    Science.gov (United States)

    Quittenden, Laura J; Davies, Noel W; Smith, Jason A; Molesworth, Peter P; Tivendale, Nathan D; Ross, John J

    2009-11-01

    One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots. PMID:19710233

  3. Green Pea Galaxies Reveal Secrets of Ly$\\alpha$ Escape

    CERN Document Server

    Yang, Huan; Gronke, Max; Rhoads, James E; Jaskot, Anne; Zheng, Zhenya; Dijkstra, Mark

    2015-01-01

    Star-formation in galaxies generates a lot of Ly$\\alpha$ photons. Understanding the escape of Ly$\\alpha$ photons from galaxies is a key issue in studying high redshift galaxies and probing cosmic reionization with Ly$\\alpha$. To understand Ly$\\alpha$ escape, it is valuable to study analogs of high redshift Ly$\\alpha$ emitters in nearby universe. However, most nearby analogs have too small a Ly$\\alpha$ equivalent width and escape fraction compared to high redshift Ly$\\alpha$ emitters. One different group of nearby analogs are "Green Pea" galaxies, selected by their high equivalent width optical emission lines. Here we show that Green Pea galaxies have strong Ly$\\alpha$ emission lines and high Ly$\\alpha$ escape fraction (see also Henry et al. 2015), providing an opportunity to solve Ly$\\alpha$ escape problem. Green Peas have a Ly$\\alpha$ equivalent width distribution similar to high redshift Ly$\\alpha$ emitters. The Ly$\\alpha$ escape fraction correlates with many quantities of Ly$\\alpha$ profile, especially the...

  4. Green Pea Galaxies Reveal Secrets of Lyα Escape

    Science.gov (United States)

    Yang, Huan; Malhotra, Sangeeta; Gronke, Max; Rhoads, James E.; Dijkstra, Mark; Jaskot, Anne; Zheng, Zhenya; Wang, Junxian

    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. Effective stabilization of CLA by microencapsulation in pea protein.

    Science.gov (United States)

    Costa, A M M; Nunes, J C; Lima, B N B; Pedrosa, C; Calado, V; Torres, A G; Pierucci, A P T R

    2015-02-01

    CLA was microencapsulated by spray drying in ten varied wall systems (WS) consisting of pea protein isolate or pea protein concentrate (PPC) alone at varied core:WS ratios (1:2; 1:3 and 1:4), or blended with maltodextrin (M) and carboxymethylcellulose at a pea protein:carbohydrate ratio of 3:1. The physical-chemical properties of the CLA microparticles were characterised by core retention, microencapsulation efficiency (ME), particle size and moisture. CLA:M:PPC (1:1:3) showed the most promising results, thus we evaluated the effect of M addition in the WS on other physical-chemical characteristics and oxidative stability (CLA isomer profile, quantification of CLA and volatile compounds by SPME coupled with CG-MS) during two months of storage at room temperature, CLA:PPC (1:4) was selected for comparisons. CLA:M:PPC (1:1:3) microparticles demonstrated better morphology, solubility, dispersibility and higher glass-transition temperature values. M addition did not influence the oxidative stability of CLA, however its presence improved physical-chemical characteristics necessary for food applications.

  6. 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...

  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 that...

  8. CELL TYPE-DEPENDENT AND DIFFERENTIATION STAGE-DEPENDENT EXPRESSION OF PML DOMAINS IN RAT, DETECTED BY MONOCLONAL-ANTIBODY HIS55

    NARCIS (Netherlands)

    LAM, YW; AMMERLAAN, W; O, WS; KROESE, F; OPSTELTEN, D

    1995-01-01

    Using mouse monoclonal antibody (mAb) HIS55, we identified a nuclear antigen (ag) that exhibited a staining pattern of discrete foci. Such foci could be detected in cells of many mammalian species. These nuclear foci were not associated with nuclear membrane, nucleoli, or mitotic chromosomes. In iso

  9. Expression, Purification of E.coli NrfA Protein and Preparation of Polyclonal Antibody Against NrfA%大肠杆菌NrfA蛋白表达、纯化及多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    何婷婷; 龚钢明; 高然

    2012-01-01

    [ Objective] This study aimed to clone the E. Coli NrfA gene and construct the pET-28a( + )-NrfA prokaryotic expression vector for preparation of polyclonal antibody against E. Coli NrfA. [ Method] E. Coli NrfA gene was cloned from the E. Coli genome DNA by PCR and inserted into the vector pET-28a( + )to construct prokaryotic expression vector pET-28a( + )-NrfA. E. Coli NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by immunizing rabbit with routine method. The specificity and titer of polyclonal antibody were confirmed by ELISA and Western Blotting. [ Result] The constructed prokaryotic expression vector pET-28a( + )-NrfA was induced by IPTG, the recombinant NrfA protein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained after immunization and purification was about 1 =204 900. Western Blotting analysis indicated that the obtained polyclonal antibody against E. Coli NrfA protein had high titer and high specificity. [Conclusion] E. Coli NrfA gene was cloned and the prokaryotic expression vector pET-28a( + )-NrfA was constructed successfully, and the polyclonal antibody with high titer and high specificity was prepared, which had laid the foundation for the study of NrfA in different strains of bacteria.%[目的]克隆大肠杆菌NrfA基因,构建pET-28a(+ )-NrfA表达载体,制备相应的多克隆抗体并对其进行鉴定.[方法]以大肠杆菌基因组DNA为模板,PCR扩增得到NrfA基因编码区,构建pET-28a(+ )-NrfA表达载体;经IPTG诱导表达并纯化重组蛋白;再免疫新西兰雄兔,制备多克隆抗体;用ELISA方法检测抗体的效价,Western Blotting检测抗体的特异性.[结果]构建的表达载体pET-28a(+)-NrfA在大肠杆菌中诱导后可高效表达NrfA蛋白;免疫获得的多克隆抗体用ELISA检测,其效价为1∶204 900;经Western Blotting分析,抗体的特异性较好.[结论]成功克隆大肠杆菌的NrfA基

  10. [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

  11. Active treatment of murine tumors with a highly attenuated vaccinia virus expressing the tumor associated antigen 5T4 (TroVax) is CD4+ T cell dependent and antibody mediated.

    Science.gov (United States)

    Harrop, Richard; Ryan, Matthew G; Myers, Kevin A; Redchenko, Irina; Kingsman, Susan M; Carroll, Miles W

    2006-09-01

    5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26-h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8+ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4+ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4+ and CD8+ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26-h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4+ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model. PMID:16311730

  12. Isoform-specific anti-MeCP2 antibodies confirm that expression of the e1 isoform strongly predominates in the brain [v1; ref status: indexed, http://f1000r.es/1mg

    Directory of Open Access Journals (Sweden)

    Lara Kaddoum

    2013-10-01

    Full Text Available Rett syndrome is a neurological disorder caused by mutations in the MECP2 gene.  MeCP2 transcripts are alternatively spliced to generate two protein isoforms (MeCP2_e1 and MeCP2_e2 that differ at their N-termini. Whilst mRNAs for both forms are expressed ubiquitously, the one for MeCP2_e1 is more abundant than for MeCP2_e2 in the central nervous system. In transfected cells, both protein isoforms are nuclear and colocalize with densely methylated heterochromatic foci. With a view to understanding the physiological contribution of each isoform, and their respective roles in the pathogenesis of Rett syndrome, we set out to generate isoform-specific anti-MeCP2 antibodies. To this end, we immunized rabbits against the peptides corresponding to the short amino-terminal portions that are different between the two isoforms. The polyclonal antibodies thus obtained specifically detected their respective isoforms of MeCP2 in Neuro2a (N2A cells transfected to express either form. Both antisera showed comparable sensitivities when used for Western blot or immunofluorescence, and were highly specific for their respective isoform. When those antibodies were used on mouse tissues, specific signals were easily detected for Mecp2_e1, whilst Mecp2_e2 was very difficult to detect by Western blot, and even more so by immunofluorescence. Our results thus suggest that brain cells express low amounts of the Mecp2-e2 isoform. Our findings are compatible with recent reports showing that MeCP2_e2 is dispensable for healthy brain function, and that it may be involved in the regulation of neuronal apoptosis and embryonic development.

  13. Medicago truncatula, an intergenomic vehicle for the map-based cloning of pea (Pisum sativum) genes. Comparative structural genomic studies of the pea Sym2-Nod3 region

    OpenAIRE

    Gualtieri González-Latorre, G.S.

    2001-01-01

    To determine the usefulness of M. truncatula as intergenomic vehicle for the positional cloning of pea genes it was studied whether these legumes are microsyntenic. These studies were focused on the pea Sym2 and Nod3 genomic regions. The M. truncatula orthologous genomic regions have been cloned and it was shown that these regions of the two legumes are microsyntenic. Both Sym2 and Nod3 play a key role in the pea- Rhizobium symbiosis, controlling Nod factor-structure dependent infection and a...

  14. Expression of mouse calreticuin protein and preparation of its polyclonal antibodies%小鼠钙网蛋白的原核表达和多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    汪龚泽; 刘朝奇; 杨建林; 聂纪芹

    2011-01-01

    In order to investigate the biological properties of calreticulin(CRT), a protein present in the mammalian cells with highly conservative and multiple biological properties, a short fragment of mouse CRT was amplified through RT-PCR method and then was cloned into prokaryotic expression vector pDET28a( + ). The recombinant protein CRT was induced by IPTG in E.coli BL2KDE3) and the expressed protein was detected by Western blotting. The purified protein was used to immune mouse to prepare polyclonal antibody and the specificity and titer of the antibody were detected by ELISA, Western blotting and FCM. It was demonstrated that the recombinant prokaryotic expression plasmid pET28a( + ) /CRT was successfully con structed and the CRT protein was expressed in E. Coli BL2KDE3) with high efficiency. Western blot assay showed that this re combinant protein was characterized with its antibody. Mouse immunized with the purified protein produced high titer of anti body. Western blot assay displayed that the CRT protein was highly expressed in some of eukaryotic cells and could specifically combine with the antibody. FCM assay displayed that the antibody could also specifically combine with the membrane extracel lular region of CRT. It is evident that the preparation of recombinant CRT and its polyclonal antibodies have a strong specificity to match with the CRT protein from mouse and human.%钙网蛋白(calreticulin,CRT)是存在于哺乳动物细胞具有高度保守性和多种生物学活性的蛋白,为了更好的研究其生物学活性,本课题组通过PCR方法扩增CRT截短基因,将其克隆到原核表达载体PET-28a(+)中,经大肠杆菌表达并纯化CRT蛋白.以纯化的CRT蛋白抗原免疫BALB/c小鼠,制备多克隆的CRT血清.进一步采用Western blot、ELISA、流式细胞术等技术对制备的抗体进行初步鉴定.结果显示:原核表达重组质粒在大肠杆菌中能高效表达CRT蛋白;获得多抗血清应用Western blot鉴定几种

  15. Prokaryotic expression of BTBD10 and preparation of its polyclonal antibody%BTBD10基因的原核表达和多克隆抗体制备

    Institute of Scientific and Technical Information of China (English)

    刘瑜; 谷昭艳; 李春霖; 陈凤玲; 胡仁明

    2011-01-01

    Objective To clone and express the BTBD10, and prepare the rabbit polyclonal antibody against BTBD10. Methods Full length BTBD10 gene was amplified by PCR and cloned into the expression vector pET32a to construct recombinant expression plasmid pET32a- BTBD10. The recombinant plasmid was transformed into E. Coli ROSSET and induced to express recombinant protein with IPTG. The fusion protein was further purified by affinity chromatography. A rabbit was immunized with the purified BTBD10 fusion protein to produce polyclonal antibody, and the production of antibody was identified by Western blot. Results Restriction enzyme digestion and DNA sequencing analysis suggested that the recombinant expression plasmid contained the correct BTBD10 code reading frame. SDS-PAGE demonstrated that the induced IPTG expressed the fused protein at 85kU, which was consistent the expected molecular weight. Western blot showed that the antibody showed a good specificity and a high titer at S6kU on the target band. Conclusion The BTBD10 prokaryotic expression plasmid has been successfully constructed, with highly-purified recombinant BTBD10 protein and rabbit polyclonal antibody against BTBD10 obtained.%目的 克隆和表达BTBD10基因,为进一步研究BTBD 10与胰岛细胞增殖功能提供工具.方法 制备兔抗BTBD10多克隆抗体,利用PCR方法扩增BTBD10全长,经Bam H I和Sal I酶切后连接入pET32a原核表达载体,将重组表达质粒pET32a-BTBD 10转化大肠杆菌ROSSET,经IPTG诱导表达蛋白,并以亲和层析的方法进行纯化,以纯化的BTBD10蛋白免疫新两兰大白兔,制备兔抗BTBD10的多克隆抗体,利用Western blot方法分析鉴定.结果 经双酶切和核酸序列分析证实重组质粒包含有正确编码的BTBD10读码框.SDS-PAGE电泳分析显示IPTG诱导后表达约85kU的融合蛋白,与预期结果相符.将纯化的融合蛋白免疫家兔,获得兔抗BTBD10多克隆抗体血清,Western blot结果显示该抗体特异性

  16. Monoclonal antibodies

    International Nuclear Information System (INIS)

    Monoclonal antibodies (MAbs) are antibodies having single specificity for a given antigen site (epitope). The development of hybridoma technology and the relative ease by which MAbs can be prepared has revolutionized many aspects of serological applications in diagnosis and differentiation of disease producing agents. The property of monospecificity offers advantages in diagnostic applications over polyclonal sera in that tests can be defined exactly with regard to the antigen detected and the affinity of reaction between the given antigenic site and the monoclonal reagent. In addition, MAbs offer better possibilities for test standardization, because the same reagent can be used in different laboratories. Such an MAb can be supplied by a central laboratory or 'grown' from hybridoma cells, ensuring that the resultant product is identical from laboratory to laboratory and that the part of the test involving the MAb reaction is the same. The methodologies for inoculation regimes, mice, cloning methods, selection of fusion partners, etc., have been validated extensively in developed country laboratories. The decision to establish a MAb production facility must be examined on a strict cost-benefit basis, since it is still expensive to produce a product. There are many MAbs available that should be sought to allow exploitation in developing tests. If a production facility is envisaged, it should produce reagents for national needs, i.e. there should be a clear problem oriented approach whereby exact needs are defined. In the field of veterinary applications, MAbs are the central reagent in many immunoassays based on the enzyme linked immunosorbent assay (ELISA). The development of specific tests for diagnosing diseases is dominated by MAbs and has been fuelled by a strong research base, mainly in developed countries allied to developing countries through the study of related diseases. Thus, there are very many assays dependent on MAbs, some of which form the basis of

  17. Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?

    Science.gov (United States)

    Burrai, G P; Tanca, A; De Miglio, M R; Abbondio, M; Pisanu, S; Polinas, M; Pirino, S; Mohammed, S I; Uzzau, S; Addis, M F; Antuofermo, E

    2015-11-01

    Canine mammary tumors (CMTs) share many features with human breast cancer (HBC), specifically concerning cancer-related pathways. Although the human epidermal growth factor receptor 2 (HER2) plays a significant role as a therapeutic and prognostic biomarker in HBC, its relevance in the pathogenesis and prognosis of CMT is still controversial. The aim of this study was to investigate HER2 expression in canine mammary hyperplasic and neoplastic tissues as well as to evaluate the specificity of the most commonly used polyclonal anti HER2 antibody by multiple molecular approaches. HER2 protein and RNA expression were determined by immunohistochemistry (IHC) and by quantitative real-time (qRT) PCR. A strong cell membrane associated with non-specific cytoplasmic staining was observed in 22% of carcinomas by IHC. Adenomas and carcinomas exhibited a significantly higher HER2 mRNA expression when compared to normal mammary glands, although no significant difference between benign and malignant tumors was noticed by qRT-PCR. The IHC results suggest a lack of specificity of the FDA-approved antibody in CMT samples as further demonstrated by Western immunoblotting (WB) and reverse phase protein arrays (RPPA). Furthemore, HER2 was not detected by mass spectrometry (MS) in a protein-expressing carcinoma at the IHC investigation. This study highlights that caution needs to be used when trying to translate from human to veterinary medicine information concerning cancer-related biomarkers and pathways. Further investigations are necessary to carefully assess the diagnostic and biological role specifically exerted by HER2 in CMTs and the use of canine mammary tumors as a model of HER2 over-expressing breast cancer. PMID:26088453

  18. PsaA Antigen Expression of Y.pestis and Its Antibody Development and Application%鼠疫菌PsaA抗原的表达及抗体制备和应用

    Institute of Scientific and Technical Information of China (English)

    洪文艳; 王浩然; 王津; 郭兆彪; 周蕾; 杨瑞馥

    2011-01-01

    Objective: To develop the PsaA antigen of Y.Pestis and its antibody, and to develop the fast detection assays, and to detect the positive ratio of PsaA antibody in 18 plague monkey serum samples. Methods: The PsaA gene was amplificated by PCR method. The production was cloned and expressed using an E. coli expression system, purified with the Ni-NTA affinity chromatography,and renatured the inclusion body expressed proteins using a urea gradient dialysis method. The corresponding polyclonal antibody was prepared by immunizing rabbits using the conventional method. Bloods were obtained from rabbits for collection of sera and purification of specific immunoglobulin G (IgG) with a caprylic acid-saturated ammonium sulfate precipitation method. Then both indirect ELISA method and Up-converting Phospher Technology (UPT) lateral flow assay were developed to detect PsaA antibodies of samples. Lastly,18 monkey serum samples were tested. Results: The data showed that the positive ratio of the PsaA antibody in plague monkey serum samples was 62%(8/13). Conclusion: The fast detection assays were successfully developed for the PsaA antibody test. And the positive ratio of the antibody in plague monkey serum samples was 62%.%目的:制备鼠疫菌PsaA抗原及抗体,建立针对PsaA抗体的快速检测方法,并检测鼠疫感染猴血清标本中的PsaA抗体的阳性率.方法:利用PCR方法扩增出PsaA蛋白基因片段,在大肠杆菌原核表达系统中表达出重组PsaA抗原,以镍柱亲和层析纯化包涵体形式的表达蛋白,以尿素梯度透析复性成可溶蛋白.再以表达蛋白为免疫原,常规免疫家兔,收集兔血清制备多抗,并以正辛酸-硫酸铵法提纯获得PsaA抗体IgG.利用得到的PsaA抗原和抗体为材料,建立两种检测PsaA抗体的快速检测方法,即间接ELISA法和上转换发光(Up-converting Phospher Technology,UPT)免疫层析试纸条法.最后利用这两种方法检测18份猴血清标本中的PsaA抗体.

  19. Prokaryotic expression, purification and antibody preparation of human fibronectin 1%纤维连接蛋白1的原核表达、纯化及抗体的制备

    Institute of Scientific and Technical Information of China (English)

    王菲菲; 苏畅; 吴海东; 王萌; 孔鹏洲; 刘民; 李欣; 汤华

    2010-01-01

    Objective To express human fibronectin 1 and generate specific human fibronectin 1 antibody in rabbit. Methods A 450 bp length fragment of human FN1 gene, containing the ISO amino acids of the C-terminal, was amplified by RT-PCR, and was cloned to pRSETA2 vector. The expression of FN1 was in BL21 (DE3) and the protein was purified by the Ni2 + -NTA resin. Purified FN1 was injected to the rabbit to raise antibody, and purified the antibody by affinity method. The specification of the antibody was detected through Western blot and immunohistochemistry. Results The pRSETA2 -FN1 vector was confirmed correctly through restriction enzyme digestion and sequencing. The fusion protein with 20 000 (FN1-His tag) was purified by Ni~(2+) -NTA column. The titer of generated FN1 antibody in rabbit was about 1: 10 000 by ELISA. The purified FN1 antibody by affinity was used to detect the FN1 in lung and lung cancer tissue by immunohistochemistry , and to detect FN1 in human plasma and HepG2 cell lysate by western blot. Conclusion The C-terminal of human FN1 was successfully prokaryotically expressed in this study. A specific FN1 antibody was generated by this recombinant FN1 protein.%目的 构建人纤维连接蛋白1(FN1)基因的原核表达载体,诱导其表达并纯化该蛋白,制备特异性抗体.方法 利用RT-PCR方法 扩增人FN1编码序列450 bp的片段,包含FN1羧基端的150个氨基酸.构建原核表达质粒pRSETA2-FN1,转化大肠杆菌BL21(DE3),FN1蛋白经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达.融合蛋白通过Ni~(2+)-NTA树脂亲和纯化后免疫兔制备抗体血清.蛋白质免疫印迹(Western blot)和免疫组化检测其特异性.结果成功构建重组质粒pRSETA2-FN1,限制性内切酶酶切鉴定及DNA测序均显示插入片段正确.SDS-PAGE凝胶显示表达的融合蛋白相对分子质量约为20 000(FN1-His标签),与预期结果一致.酶联免疫吸附试验(ELISA)检测抗体效价约为1:10 000, Western blot显示可

  20. 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.

  1. Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae.

    Science.gov (United States)

    Piekarowicz, Andrzej; Kłyż, Aneta; Majchrzak, Michał; Stein, Daniel C

    2016-01-01

    All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development. PMID:26939573

  2. Characterization of regenerated butterfly pea (Clitoria ternatea L.) accessions for morphological, phenology, reproductive and potential nutraceutical, pharmaceutical trait utilization.

    Science.gov (United States)

    Butterfly pea, Clitoria ternatea, has been used in Africa as a companion crop and in the United States as an ornamental. The USDA, ARS, PGRCU curates 28 butterfly pea accessions. Butterfly pea accessions were transplanted from about 30-day-old seedlings to the field in Griffin, GA, around 01 June ...

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

    OpenAIRE

    Clementi Massimo; Burioni Roberto; Liu Gerald; Prabhu Ramesh; Gunduz Feyza; Poat Bret; Hazari Sidhartha; Chandra Partha K; Garry Robert F; Dash Srikanta

    2010-01-01

    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...

  4. 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 albumin

  5. 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...

  6. 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 (Pseudom

  7. Genetic Diversity of Chinese and Global Pea (Pisum sativum L.) Collections.

    Science.gov (United States)

    Pea (Pisum sativum L.) is an important food and feed legume grown across many temperate regions of the world, especially from Asia to Europe and North America. The goal of this study was to use 30 informative pea microsatellite markers to compare genetic diversity in a global core from the USDA and ...

  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. 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

  10. 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...

  11. Monoclonal antibodies.

    Science.gov (United States)

    2009-01-01

    The ability to produce and exploit monoclonal antibodies (mAbs) has revolutionized many areas of biological sciences. The unique property of an mAb is that it is a single species of immunoglobulin (IG) molecule. This means that the specificity of the interaction of the paratopes on the IG, with the epitopes on an antigenic target, is the same on every molecule. This property can be used to great benefit in immunoassays to provide tests of defined specificity and sensitivity, which improve the possibilities of standardization. The performance of assays can often be determined relating the actual weight of antibody (hence the number of molecules) to the activity. Often the production of an mAb against a specific epitope is the only way that biological entities can be differentiated. This chapter outlines the areas involving the development of assays based on mAbs. The problems involved address include the physical aspects of mAbs and how they may affect assay design and also the implications of results based on monospecific reagents. Often these are not fully understood, leading to assays that are less than satisfactory, which does not justify the relatively high cost of preparing and screening of mAbs. There are many textbooks and reviews dealing with the preparation of mAbs, the principles involved, and various purification and manipulative methods for the preparation of fragments and conjugation. There has been little general information attempting to summarize the best approaches to assay design using mAbs. Much time can be wasted through bad planning, and this is particularly relevant to mAbs. A proper understanding of some basic principles is essential. It is beyond the scope of this chapter to discuss all aspects, but major areas are highlighted. PMID:19219589

  12. Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

    Science.gov (United States)

    Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

    1996-01-01

    A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

  13. Cloning and expression of MICB gene and its application for the detection of anti-MICB antibodies%MICB基因的克隆表达及其在抗体测定中的初步应用

    Institute of Scientific and Technical Information of China (English)

    应燕玲; 和艳敏; 陶苏丹; 何吉; 朱发明; 吕杭军

    2015-01-01

    目的:构建可行的MICB ( major histocompatibility complex class Ⅰ chain-related gene B)基因原核表达系统及蛋白质纯化体系,利用表达纯化蛋白建立ELISA方法初步应用于肾移植患者中MICB抗体的检测。方法外周血RNA经RT-PCR和特异性引物扩增获得MICB基因cDNA, MICB cDNA和原核表达载体pET-28a分别双酶切后连接构建重组表达载体,转染宿主菌E. coli BL21 DE3,IPTG诱导表达。亲和层析的Ni-NTA Spin纯化蛋白质,并通过Western blot鉴定。利用纯化蛋白建立ELISA法检测肾移植患者中MICB抗体。结果构建3个MICB常见等位基因2、3外显子的pET-28a-MICB重组表达体系,经Ni-NTA Spin纯化获得重组的MICB蛋白,Western blot鉴定。利用3个不同MICB蛋白质成功构建了ELISA方法,初步检测24份肾移植患者标本MICB抗体,显示不同的重组蛋白敏感性存在差异。结论本研究建立MICB基因克隆与体外表达技术,鉴定并纯化具有生物活性的蛋白质,同时建立MICB抗体检测的ELISA方法,为进一步研究MICB与移植免疫的关系提供基础资料。%Objective To construct a prokaryotic expression system for major histocompatibility complex class Ⅰ chain-related gene B ( MICB) and to establish an ELISA method for the detection of anti-MICB antibodies in patients with kidney transplantation. Methods The MICB cDNA fragments were ob-tained by RT-PCR with a pair of specific primers. The MICB cDNA and the prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct the recombinant expression plas-mid pET-28a-MICB. The transformed E. coli BL21 DE3 strains carrying recombinant expression plasmid were induced by IPTG to express MICB protein. The expressed recombinant proteins were identified by Western blot assay and purified by Ni-NTA Spin column. The purified proteins were coupled to ELISA for the detection of anti-MICB antibodies in patients with kidney transplantation. Results

  14. Formation of electric dipoles in pea stem tissue due to an electric field

    Science.gov (United States)

    Ahmadi, Fatemeh; Farahani, Elham

    2016-07-01

    For examining the effect of an electrical field (DC) on pea seed, we exposed the pea seeds to electric fields with intensities 1, 4 and 7 kV/cm for 30, 230, 430 and 630 seconds. The tests were repeated three times, and each iteration had 5 seeds. Then, the seeds were moved to packaged plates. Finally, microscopic observation of the pea stem tissue showed that the application of a DC electrical field caused a deformation in the pea stem tissue. The results led us to examine the deformation of the tissue theoretically and to address that deformation as an electrostatic problem. In this regard, we modeled the pea stem based on the formation of electric dipoles. Then, theoretically, we calculated the force acting on each xylem section by coding, and the results were consistent with the experimental data.

  15. Isolation of pea matrix attachment region and study on its function in transgenic tobaccos

    Institute of Scientific and Technical Information of China (English)

    李旭刚; 朱祯; 徐军望; 吴茜; 徐鸿林

    2001-01-01

    A DNA fragment containing consensus sequence of matrix attachment region (MAR) has been isolated from pea genome. Compared with original DNA sequence, one 115 bp-long repeat sequence is deleted in the obtained DNA sequence. DNA fragments located upstream and down-stream of repeat DNA sequence respectively share 84% and 93% homology to the corresponding original sequence, and contain A-box or T-box and TATAA sequence, which is characteristics short sequence of MARs. To test the function of the DNA sequence, the plant expression vectors in which b-glucuronidase gene (GUS, uidA) was used as reporter gene were constructed and transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assay showed that the average level of uidA expression was increased twofold for the presence of MAR, and the highest level of GUS activity of transgenic plants could be increased six times. The results cited above suggest that the isolated DNA sequence contains consensus sequence of MARs and has capability to increase expression level of gene in transgenic plants.

  16. Biomass production and nitrogen accumulation in pea, oat, and vetch green manure mixtures

    International Nuclear Information System (INIS)

    Interest in the use of green manures has revived because of their role in improving soil quality and their beneficial N and non-N rotation effects. This study evaluated biomass production, N content, radiation interception (RI), and radiation use efficiency (RUE) of pea (Pisum sativum L.), oat (Avena sativa L.), and hairy vetch (Vicia villosa Roth) mixtures. Treatments were a three-way factorial of pea genotype ('Century' vs 'Tipu'), pea planting density (90 vs 224 kg ha-1), and cropping mixture (solecropped pea vs pea planted with a mixture of oat and hairy vetch). A mixture of oat and vetch without pea was also planted. Treatments were planted in early June on a Caribou gravelly loam (coarse-loamy, mixed, frigid Typic Haplorthods) in Presque Isle, ME, in 1993 and 1994. Biomass production and radiation interception were measured by repeated sampling. Mixture biomass was affected by a year x pea density interaction: respective yields for mixtures containing low-density and high-density pea were 770 and 880 g m-2 in 1993 vs 820 and 730 g m-2 in 1994. Mixture N content paralleled biomass production and averaged 209 g m-2 across all treatments. While pea sole crops did not consistently produce biomass or N equal to three-species mixtures the two-species mixture of oat and vetch did, yielding 820 g m-2 of biomass and 21.7 g m-2 of N, averaged over the 2 yr. Multiple regression showed that 61% of the variability in mixture biomass production was accounted for by a combination of early-season pea RI and midseason total mixture RUE. Economic analyses showed that rotation including these green manures may be economically competitive with a conventional rotation of barley (Hordeum vulgare L.) undersown with clover (Trifolium spp.) in a potato (Solanum tuberosum L.) production system

  17. 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; Nakahara, Kenji S; Atsumi, Go; Shimada, Ryoko; Hisa, Yusuke; Naito, Satoshi; Uyeda, Ichiro

    2013-07-01

    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

  18. 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.

  19. Induction of mutation in pea by gamma irradiation

    International Nuclear Information System (INIS)

    A study on gamma rays induced mutations in the variety Bonneville of pea was aimed at determining the dose-event relationship using various indices in the M1 and M2 generations and isolation of mutants of economic value. Frequency of chlorophyll mutations followed a linear relationship with dose level. A wide spectrum of both lethal and viable mutations was observed in the effective dose range. Among the viable a few mutants affecting the maturity period, grain type and pod number appear to be of practical value. (author)

  20. Inheritance of enlarged leaf mutations in pea (Pisum sativum L.)

    International Nuclear Information System (INIS)

    In the present study, two pea (Pisum sativum L.) mutants experimentally obtained after gamma irradiation of dry seeds are described. Both mutants were characterized by enlarged leaf size. The mutant 2/462 was shown to have semi-dominant inheritance pattern; 2/927 was sterile and inherited monogenic recessive. The induced mutations have pleiotropic effect, affecting morphological and reproductive traits. New mutants had similar phenotypes to previously named mutants latifolium (lat) and cabbage leaf (calf), but no allelism tests were made between the new and the previously reported mutants. Both mutant lines may be useful plant material for research on leaf development