Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

NASA Technical Reports Server (NTRS)

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.

The major nucleoside triphosphatase in pea (Pisum sativum L.) nuclei and in rat liver nuclei share common epitopes also present in nuclear lamins

NASA Technical Reports Server (NTRS)

Tong, C. G.; Dauwalder, M.; Clawson, G. A.; Hatem, C. L.; Roux, S. J.

1993-01-01

The major nucleoside triphosphatase (NTPase) activities in mammalian and pea (Pisum sativum L.) nuclei are associated with enzymes that are very similar both biochemically and immunochemically. The major NTPase from rat liver nuclei appears to be a 46-kD enzyme that represents the N-terminal portion of lamins A and C, two lamina proteins that apparently arise from the same gene by alternate splicing. Monoclonal antibody (MAb) G2, raised to human lamin C, both immunoprecipitates the major (47 kD) NTPase in pea nuclei and recognizes it in western blot analyses. A polyclonal antibody preparation raised to the 47-kD pea NTPase (pc480) reacts with the same lamin bands that are recognized by MAb G2 in mammalian nuclei. The pc480 antibodies also bind to the same lamin-like bands in pea nuclear envelope-matrix preparations that are recognized by G2 and three other MAbs known to bind to mammalian lamins. In immunofluorescence assays, pc480 and anti-lamin antibodies stain both cytoplasmic and nuclear antigens in plant cells, with slightly enhanced staining along the periphery of the nuclei. These results indicate that the pea and rat liver NTPases are structurally similar and that, in pea nuclei as in rat liver nuclei, the major NTPase is probably derived from a lamin precursor by proteolysis.

Expression of small heat shock proteins from pea seedlings under gravity altered conditions

NASA Astrophysics Data System (ADS)

Talalaev, Alexandr S.

2005-08-01

A goal of our study was to evaluate the stress gene expression in Pisum sativum seedlings exposed to altered gravity and temperature elevation. We investigate message for the two inducible forms of the cytosolic small heat shock proteins (sHsp), sHsp 17.7 and sHsp 18.1. Both proteins are able to enhance the refolding of chemically denatured proteins in an ATP- independent manner, in other words they can function as molecular chaperones. We studied sHsps expression in pea seedlings cells by Western blotting. Temperature elevation, as the positive control, significantly increased PsHsp 17.7 and PsHsp 18.1 expression. Expression of the housekeeping protein, actin was constant and comparable to unstressed controls for all treatments. We concluded that gravitational perturbations incurred by clinorotation did not change sHsp genes expression.

Requirement of PEA3 for Transcriptional Activation of FAK Gene in Tumor Metastasis

PubMed Central

Li, Shufeng; Huang, Xiaofeng; Zhang, Dapeng; Huang, Qilai; Pei, Guoshun; Wang, Lixiang; Jiang, Wenhui; Hu, Qingang; Tan, Renxiang; Hua, Zi-Chun

2013-01-01

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides −170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis. PMID:24260201

Immunological and biochemical evidence for nuclear localization of annexin in peas

NASA Technical Reports Server (NTRS)

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

1998-01-01

Immunofluorescent localization of annexins using an anti-pea annexin polyclonal antibody (anti-p35) in pea (Pisum sativum) leaf and stem epidermal peels showed staining of the nuclei and the cell periphery. Nuclear staining was also seen in cell teases prepared from pea plumules. The amount of nuclear stain was reduced both by fixation time and by dehydration and organic solvent treatment. Observation with confocal microscopy demonstrated that the anti-p35 stain was diffusely distributed throughout the nuclear structure. Immunoblots of purified nuclei, nuclear envelope matrix, nucleolar, and chromatin fractions showed a cross-reactive protein band of 35 kDa. These data are the first to show annexins localized in plant cell nuclei where they may play a role in nuclear function.

Gibberellin 3-oxidase gene expression patterns influence gibberellin biosynthesis, growth, and development in pea.

PubMed

Reinecke, Dennis M; Wickramarathna, Aruna D; Ozga, Jocelyn A; Kurepin, Leonid V; Jin, Alena L; Good, Allen G; Pharis, Richard P

2013-10-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.

Expression of Cry2Aa, a Bacillus thuringiensis insecticidal protein in transgenic pigeon pea confers resistance to gram pod borer, Helicoverpa armigera.

PubMed

Singh, Shweta; Kumar, Nikhil Ram; Maniraj, R; Lakshmikanth, R; Rao, K Y S; Muralimohan, N; Arulprakash, T; Karthik, K; Shashibhushan, N B; Vinutha, T; Pattanayak, Debasis; Dash, Prasanta K; Kumar, P Ananda; Sreevathsa, Rohini

2018-06-11

Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T 1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T 3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80-100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25-80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.

Cloning and expression of a nuclear encoded plastid specific 33 kDa ribonucleoprotein gene (33RNP) from pea that is light stimulated.

PubMed

Reddy, M K; Nair, S; Singh, B N; Mudgil, Y; Tewari, K K; Sopory, S K

2001-01-24

We report the cloning and sequencing of both cDNA and genomic DNA of a 33 kDa chloroplast ribonucleoprotein (33RNP) from pea. The analysis of the predicted amino acid sequence of the cDNA clone revealed that the encoded protein contains two RNA binding domains, including the conserved consensus ribonucleoprotein sequences CS-RNP1 and CS-RNP2, on the C-terminus half and the presence of a putative transit peptide sequence in the N-terminus region. The phylogenetic and multiple sequence alignment analysis of pea chloroplast RNP along with RNPs reported from the other plant sources revealed that the pea 33RNP is very closely related to Nicotiana sylvestris 31RNP and 28RNP and also to 31RNP and 28RNP of Arabidopsis and spinach, respectively. The pea 33RNP was expressed in Escherichia coli and purified to homogeneity. The in vitro import of precursor protein into chloroplasts confirmed that the N-terminus putative transit peptide is a bona fide transit peptide and 33RNP is localized in the chloroplast. The nucleic acid-binding properties of the recombinant protein, as revealed by South-Western analysis, showed that 33RNP has higher binding affinity for poly (U) and oligo dT than for ssDNA and dsDNA. The steady state transcript level was higher in leaves than in roots and the expression of this gene is light stimulated. Sequence analysis of the genomic clone revealed that the gene contains four exons and three introns. We have also isolated and analyzed the 5' flanking region of the pea 33RNP gene.

Bean α-amylase inhibitor 1 in transgenic peas (Pisum sativum) provides complete protection from pea weevil (Bruchus pisorum) under field conditions

PubMed Central

Morton, Roger L.; Schroeder, Hart E.; Bateman, Kaye S.; Chrispeels, Maarten J.; Armstrong, Eric; Higgins, Thomas J. V.

2000-01-01

Two α-amylase inhibitors, called αAI-1 and αAI-2, that share 78% amino acid sequence identity and have a differential specificity toward mammalian and insect α-amylases are present in different accessions of the common bean (Phaseolus vulgaris). Using greenhouse-grown transgenic peas (Pisum sativum), we have shown previously that expression of αAI-1 in pea seeds can provide complete protection against the pea weevil (Bruchus pisorum). Here, we report that αAI-1 also protects peas from the weevil under field conditions. The high degree of protection is explained by our finding that αAI-1 inhibits pea bruchid α-amylase by 80% over a broad pH range (pH 4.5–6.5). αAI-2, on the other hand, is a much less effective inhibitor of pea bruchid α-amylase, inhibiting the enzyme by only 40%, and only in the pH 4.0–4.5 range. Nevertheless, this inhibitor was still partially effective in protecting field-grown transgenic peas against pea weevils. The primary effect of αAI-2 appeared to be a delay in the maturation of the larvae. This contrasts with the effect of αAI-1, which results in larval mortality at the first or second instar. These results are discussed in relationship to the use of amylase inhibitors with different specificities to bring about protection of crops from their insect pests or to decrease insect pest populations below the economic injury level. PMID:10759552

Cloning and characterization of a cDNA encoding topoisomerase II in pea and analysis of its expression in relation to cell proliferation.

PubMed

Reddy, M K; Nair, S; Tewari, K K; Mudgil, Y; Yadav, B S; Sopory, S K

1999-09-01

We have isolated and sequenced four overlapping cDNA clones to identify the full-length cDNA for topoisomerase II (PsTopII) from pea. Using degenerate primers, based on the conserved amino acid sequences of other eukaryotic type II topoisomerases, a 680 bp fragment was PCR-amplified with pea cDNA as template. This fragment was used as a probe to screen an oligo-dT-primed pea cDNA library. A partial cDNA clone was isolated that was truncated at the 3' end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of PsTopII is 4639 bp with an open reading frame of 4392 bp. The deduced amino acid sequence shows a strong homology to other eukaryotic topoisomerase II (topo II) at the N-terminus end. The topo II transcript was abundant in proliferative tissues. We also show that the level of topo II transcripts could be stimulated by exogenous application of growth factors that induced proliferation in vitro cultures. Light irradiation to etiolated tissue strongly stimulated the expression of topo II. These results suggest that topo II gene expression is up-regulated in response to light and hormones and correlates with cell proliferation. Besides, we have also isolated and analysed the 5'-flanking region of the pea TopII gene. This is first report on the isolation of a putative promoter for topoisomerase II from plants.

Immunolocalization of pectic polysaccharides during abscission in pea seeds (Pisum sativum L.) and in abscission less def pea mutant seeds.

PubMed

Lee, YeonKyeong; Ayeh, Kwadwo Owusu; Ambrose, Mike; Hvoslef-Eide, Anne Kathrine

2016-08-31

In pea seeds (Pisum sativum L.), the presence of the Def locus determines abscission event between its funicle and the seed coat. Cell wall remodeling is a necessary condition for abscission of pea seed. The changes in cell wall components in wild type (WT) pea seed with Def loci showing seed abscission and in abscission less def mutant peas were studied to identify the factors determining abscission and non-abscission event. Changes in pectic polysaccharides components were investigated in WT and def mutant pea seeds using immunolabeling techniques. Pectic monoclonal antibodies (1 → 4)-β-D-galactan (LM5), (1 → 5)-α-L-arabinan(LM6), partially de-methyl esterified homogalacturonan (HG) (JIM5) and methyl esterified HG (JIM7) were used for this study. Prior to abscission zone (AZ) development, galactan and arabinan reduced in the predestined AZ of the pea seed and disappeared during the abscission process. The AZ cells had partially de-methyl esterified HG while other areas had highly methyl esterified HG. A strong JIM5 labeling in the def mutant may be related to cell wall rigidity in the mature def mutants. In addition, the appearance of pectic epitopes in two F3 populations resulting from cross between WT and def mutant parents was studied. As a result, we identified that homozygous dominant lines (Def/Def) showing abscission and homozygous recessive lines (def/def) showing non-abscission had similar immunolabeling pattern to their parents. However, the heterogeneous lines (Def/def) showed various immunolabeling pattern and the segregation pattern of the Def locus. Through the study of the complexity and variability of pectins in plant cell walls as well as understanding the segregation patterns of the Def locus using immunolabeling techniques, we conclude that cell wall remodeling occurs in the abscission process and de-methyl esterification may play a role in the non-abscission event in def mutant. Overall, this study contributes new insights into

Effect of roasted pea flour/starch and encapsulated pea starch incorporation on the in vitro starch digestibility of pea breads.

PubMed

Lu, Zhan-Hui; Donner, Elizabeth; Liu, Qiang

2018-04-15

Oven or microwave roasting and alginate encapsulation of pea flour and starch to produce novel pea ingredients for enrichment of slowly digestible starch (SDS) and resistant starch (RS) content in pea bread were investigated. Pea flour treated either by oven roasting (160°C, 30min) or by microwave roasting (1.1kW, 6min) effectively retained its low starch digestibility similar to its native form (∼25% SDS; ∼60% RS). When oven roasting was applied to pea starch, SDS content increased triply compared to the fully boiled counterpart. Alginate encapsulation effectively controlled carbohydrate release to simulated gastric, intestinal and colonic fluids, and thus largely enriched the SDS and RS fractions in starch. Pea bread containing up to 37.5% of encapsulated roasted MPS pea starch not only provided high SDS and RS fractions (23.9% SDS and 30.2% RS) compared to a white bread control (0.2% SDS and 2.5% RS), but also provided an acceptable palatability. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Characterization by enzyme-linked immunosorbent assay of monoclonal antibodies to pisum and Avena phytochrome.

PubMed

Cordonnier, M M; Greppin, H; Pratt, L H

1984-01-01

Nine monoclonal antibodies to pea (Pisum sativum L.) and 16 to oat (Avena sativa L.) phytochrome are characterized by enzyme-linked immunosorbent assay against phytochrome from six different sources: pea, zucchini (Cucurbita pepo L.), lettuce (Lactuca sativa L.), oat, rye (Secale cereale L.), and barley (Hordeum vulgare L.). All antibodies were raised against phytochrome with a monomer size near 120,000 daltons. Nevertheless, none of them discriminated qualitatively between 118/114-kilodalton oat phytochrome and a photoreversible, 60-kilodalton proteolytic degradation product derived from it. In addition, none of the 23 antibodies tested discriminated substantially between phytochrome-red-absorbing form and phytochrome-far red-absorbing form. Two antibodies to pea and six to oat phytochrome also bound strongly to phytochrome from the other species, even though these two plants are evolutionarily widely divergent. Of these eight antibodies, two bound significantly to all of the six phytochrome preparations tested, indicating that these two may recognize highly conserved regions of the chromoprotein. Since the molecular function of phytochrome is unknown, these two antibodies may serve as unique probes for regions of this pigment that are important to its mode of action.

Nodulation, Nitrogen Fixation, and Hydrogen Oxidation by Pigeon Pea Bradyrhizobium spp. in Symbiotic Association with Pigeon Pea, Cowpea, and Soybean †

PubMed Central

Nautiyal, C. S.; Hegde, S. V.; van Berkum, P.

1988-01-01

The pigeon pea strains of Bradyrhizobium CC-1, CC-8, UASGR(S), and F4 were evaluated for nodulation, effectiveness for N2 fixation, and H2 oxidation with homologous and nonhomologous host plants. Strain CC-1 nodulated Macroptilium atropurpureum, Vigna unguiculata, Glycine max, and G. soja but did not nodulate Pisum sativum, Phaseolus vulgaris, Trigonella foenum-graecum, and Trifolium repens. Strain F4 nodulated G. max cv. Peking and PI 434937 (Malayan), but the symbioses formed were poor. Similarly, G. max cv. Peking, cv. Bragg, PI 434937, PR 13-28-2-8-7, and HM-1 were nodulated by strain CC-1, and symbioses were also poor. G. max cv. Williams and cv. Clark were not nodulated. H2 uptake activity was expressed with pigeon pea and cowpea, but not with soybean. G. max cv. Bragg grown in Bangalore, India, in local soil not previously exposed to Bradyrhizobium japonicum formed nodules with indigenous Bradyrhizobium spp. Six randomly chosen isolates, each originating from a different nodule, formed effective symbioses with pigeon pea host ICPL-407, nodulated PR 13-28-2-8-7 soybean forming moderately effective symbioses, and did not nodulate Williams soybean. These results indicate the six isolates to be pigeon pea strains although they originated from soybean nodules. Host-determined nodulation of soybean by pigeon pea Bradyrhizobium spp. may depend upon the ancestral backgrounds of the cultivars. The poor symbioses formed by the pigeon pea strains with soybean indicate that this crop should be inoculated with B. japonicum for its cultivation in soils containing only pigeon pea Bradyrhizobium spp. PMID:16347542

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea.

PubMed

Legrand, Sylvain; Marque, Gilles; Blassiau, Christelle; Bluteau, Aurélie; Canoy, Anne-Sophie; Fontaine, Véronique; Jaminon, Odile; Bahrman, Nasser; Mautord, Julie; Morin, Julie; Petit, Aurélie; Baranger, Alain; Rivière, Nathalie; Wilmer, Jeroen; Delbreil, Bruno; Lejeune-Hénaut, Isabelle

2013-09-01

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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.

Genetically modified α-amylase inhibitor peas are not specifically allergenic in mice.

PubMed

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

2013-01-01

Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice.

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

PubMed Central

Dekan, Gerhard; Moore, Andrew E.; Higgins, T. J. V.; Epstein, Michelle M.

2013-01-01

Weevils can devastate food legumes in developing countries, but genetically modified peas (Pisum sativum), chickpeas and cowpeas expressing the gene for alpha-amylase inhibitor-1 (αAI) from the common bean (Phaseolus vulgaris) are completely protected from weevil destruction. αAI is seed-specific, accumulated at high levels and undergoes post-translational modification as it traverses the seed endomembrane system. This modification was thought to be responsible for the reported allergenicity in mice of the transgenic pea but not the bean. Here, we observed that transgenic αAI peas, chickpeas and cowpeas as well as non-transgenic beans were all allergenic in BALB/c mice. Even consuming non-transgenic peas lacking αAI led to an anti-αAI response due to a cross-reactive response to pea lectin. Our data demonstrate that αAI transgenic peas are not more allergenic than beans or non-transgenic peas in mice. This study illustrates the importance of repeat experiments in independent laboratories and the potential for unexpected cross-reactive allergic responses upon consumption of plant products in mice. PMID:23326368

Expression and assembly of a fully active antibody in algae

NASA Astrophysics Data System (ADS)

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.

Functional Conservation of PISTILLATA Activity in a Pea Homolog Lacking the PI Motif1

PubMed Central

Berbel, Ana; Navarro, Cristina; Ferrándiz, Cristina; Cañas, Luis Antonio; Beltrán, José-Pío; Madueño, Francisco

2005-01-01

Current understanding of floral development is mainly based on what we know from Arabidopsis (Arabidopsis thaliana) and Antirrhinum majus. However, we can learn more by comparing developmental mechanisms that may explain morphological differences between species. A good example comes from the analysis of genes controlling flower development in pea (Pisum sativum), a plant with more complex leaves and inflorescences than Arabidopsis and Antirrhinum, and a different floral ontogeny. The analysis of UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), the pea orthologs of LEAFY and UNUSUAL FLORAL ORGANS, has revealed a common link in the regulation of flower and leaf development not apparent in Arabidopsis. While the Arabidopsis genes mainly behave as key regulators of flower development, where they control the expression of B-function genes, UNI and STP also contribute to the development of the pea compound leaf. Here, we describe the characterization of P. sativum PISTILLATA (PsPI), a pea MADS-box gene homologous to B-function genes like PI and GLOBOSA (GLO), from Arabidopsis and Antirrhinum, respectively. PsPI encodes for an atypical PI-type polypeptide that lacks the highly conserved C-terminal PI motif. Nevertheless, constitutive expression of PsPI in tobacco (Nicotiana tabacum) and Arabidopsis shows that it can specifically replace the function of PI, being able to complement the strong pi-1 mutant. Accordingly, PsPI expression in pea flowers, which is dependent on STP, is identical to PI and GLO. Interestingly, PsPI is also transiently expressed in young leaves, suggesting a role of PsPI in pea leaf development, a possibility that fits with the established role of UNI and STP in the control of this process. PMID:16113230

Sensitization with 7S globulins from peanut, hazelnut, soy or pea induces IgE with different biological activities which are modified by soy tolerance.

PubMed

Kroghsbo, Stine; Bøgh, Katrine L; Rigby, Neil M; Mills, E N Clare; Rogers, Adrian; Madsen, Charlotte B

2011-01-01

It is not known why some foods sensitizing via the gastrointestinal tract are prevalent allergenic foods and others are not. Eating habits, processing, and the food matrix have been suggested to influence the allergenicity of a given food. Factors related to protein structure, 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. 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. 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. Although the 4 7S globulins are structurally related allergens, they induce antibodies with different antigen-binding characteristics. Peanut 7S induces IgE of a higher avidity than hazelnut and pea 7S which, again, has a higher avidity than IgE induced by soy 7S. We also show that soy tolerance influences the function of antibodies to peanut 7S. These findings may help explain how antibodies of different clinical significances can develop in different individuals sensitized to the same allergen. Copyright © 2011 S. Karger AG, Basel.

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells

PubMed Central

Melidoni, Anna N.; Dyson, Michael R.; Wormald, Sam; McCafferty, John

2013-01-01

Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by introducing and expressing antibody gene populations in ES cells. Following induced antibody expression and secretion, changes in differentiation outcomes of individual antibody-expressing ES clones are monitored using lineage-specific gene expression to identify clones that encode and express signal-modifying antibodies. This in-cell expression and reporting system was exemplified by generating blocking antibodies to FGF4 and its receptor FGFR1β, identified through delayed onset of ES cell differentiation. Functionality of the selected antibodies was confirmed by addition of exogenous antibodies to three different ES reporter cell lines, where retained expression of pluripotency markers Oct4, Nanog, and Rex1 was observed. This work demonstrates the potential for discovery and utility of functional antibodies in stem cell differentiation. This work is also unique in constituting an example of ES cells carrying an inducible antibody that causes a functional protein “knock-down” and allows temporal control of stable signaling components at the protein level. PMID:24082130

Characterization of proanthocyanidin metabolism in pea (Pisum sativum) seeds.

PubMed

Ferraro, Kiva; Jin, Alena L; Nguyen, Trinh-Don; Reinecke, Dennis M; Ozga, Jocelyn A; Ro, Dae-Kyun

2014-09-16

Proanthocyanidins (PAs) accumulate in the seeds, fruits and leaves of various plant species including the seed coats of pea (Pisum sativum), an important food crop. PAs have been implicated in human health, but molecular and biochemical characterization of pea PA biosynthesis has not been established to date, and detailed pea PA chemical composition has not been extensively studied. PAs were localized to the ground parenchyma and epidermal cells of pea seed coats. Chemical analyses of PAs from seeds of three pea cultivars demonstrated cultivar variation in PA composition. 'Courier' and 'Solido' PAs were primarily prodelphinidin-types, whereas the PAs from 'LAN3017' were mainly the procyanidin-type. The mean degree of polymerization of 'LAN3017' PAs was also higher than those from 'Courier' and 'Solido'. Next-generation sequencing of 'Courier' seed coat cDNA produced a seed coat-specific transcriptome. Three cDNAs encoding anthocyanidin reductase (PsANR), leucoanthocyanidin reductase (PsLAR), and dihydroflavonol reductase (PsDFR) were isolated. PsANR and PsLAR transcripts were most abundant earlier in seed coat development. This was followed by maximum PA accumulation in the seed coat. Recombinant PsANR enzyme efficiently synthesized all three cis-flavan-3-ols (gallocatechin, catechin, and afzalechin) with satisfactory kinetic properties. The synthesis rate of trans-flavan-3-ol by co-incubation of PsLAR and PsDFR was comparable to cis-flavan-3-ol synthesis rate by PsANR. Despite the competent PsLAR activity in vitro, expression of PsLAR driven by the Arabidopsis ANR promoter in wild-type and anr knock-out Arabidopsis backgrounds did not result in PA synthesis. Significant variation in seed coat PA composition was found within the pea cultivars, making pea an ideal system to explore PA biosynthesis. PsANR and PsLAR transcript profiles, PA localization, and PA accumulation patterns suggest that a pool of PA subunits are produced in specific seed coat cells early in

Gibberellin 3-oxidase Gene Expression Patterns Influence Gibberellin Biosynthesis, Growth, and Development in Pea1[W][OPEN

PubMed Central

Reinecke, Dennis M.; Wickramarathna, Aruna D.; Ozga, Jocelyn A.; Kurepin, Leonid V.; Jin, Alena L.; Good, Allen G.; Pharis, Richard P.

2013-01-01

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE) encodes a GA 3β-hydroxylase that catalyzes the conversion of GA20 to biologically active GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth and development, we generated transgenic pea lines (in a lele background) with cauliflower mosaic virus-35S-driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1 concentrations in a tissue-specific and development-specific manner, altering GA biosynthesis and catabolism gene expression and plant phenotype. PsGA3ox1 transgenic plants had longer internodes, tendrils, and fruits, larger stipules, and displayed delayed flowering, increased apical meristem life, and altered vascular development relative to the null controls. Transgenic PsGA3ox1 overexpression lines were then compared with lines where endogenous PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background (BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous promoter was coordinated within the internode tissue to avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These studies further our fundamental understanding of the regulation of GA biosynthesis and catabolism at the tissue and organ level and demonstrate that the timing/localization of GA3ox expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth. PMID:23979969

The pea stem

PubMed Central

Karahara, Ichirou

2012-01-01

The Casparian strip is commonly observed in the endodermis of roots of vascular plants and, in some cases, also in the stems. Pea stems develop the Casparian strip, and its development has been reported to be regulated by blue light. In addition, for the purpose of photobiological studies, pea stems provide a unique experimental system for other physiological studies of the development of the Casparian strip. In this article, I have briefly summarized (1) the effects of environmental factors on the development of the Casparian strip, (2) the advantage of using pea stems for physiological studies of the development of the Casparian strip, and (3) cellular events indicated to be involved in the development of the Casparian strip, focusing on the studies using pea stems as well as other recent studies. PMID:22899074

How does pea architecture influence light sharing in virtual wheat–pea mixtures? A simulation study based on pea genotypes with contrasting architectures

PubMed Central

Barillot, Romain; Combes, Didier; Chevalier, Valérie; Fournier, Christian; Escobar-Gutiérrez, Abraham J.

2012-01-01

Background and aims Light interception is a key factor driving the functioning of wheat–pea intercrops. The sharing of light is related to the canopy structure, which results from the architectural parameters of the mixed species. In the present study, we characterized six contrasting pea genotypes and identified architectural parameters whose range of variability leads to various levels of light sharing within virtual wheat–pea mixtures. Methodology Virtual plants were derived from magnetic digitizations performed during the growing cycle in a greenhouse experiment. Plant mock-ups were used as inputs of a radiative transfer model in order to estimate light interception in virtual wheat–pea mixtures. The turbid medium approach, extended to well-mixed canopies, was used as a framework for assessing the effects of leaf area index (LAI) and mean leaf inclination on light sharing. Principal results Three groups of pea genotypes were distinguished: (i) early and leafy cultivars, (ii) late semi-leafless cultivars and (iii) low-development semi-leafless cultivars. Within open canopies, light sharing was well described by the turbid medium approach and was therefore determined by the architectural parameters that composed LAI and foliage inclination. When canopy closure started, the turbid medium approach was unable to properly infer light partitioning because of the vertical structure of the canopy. This was related to the architectural parameters that determine the height of pea genotypes. Light capture was therefore affected by the development of leaflets, number of branches and phytomers, as well as internode length. Conclusions This study provides information on pea architecture and identifies parameters whose variability can be used to drive light sharing within wheat–pea mixtures. These results could be used to build up the architecture of pea ideotypes adapted to multi-specific stands towards light competition. PMID:23240074

Replication of pea enation mosaic virus RNA in isolated pea nuclei

PubMed Central

Powell, C. A.; Zoeten, G. A. de

1977-01-01

Isolated nuclei from healthy pea plants were primed with pea enation mosaic virus (PEMV), southern bean mosaic virus (SBMV), radish mosaic virus (RdMV), tobacco mosaic virus (TMV), PEMV RNA, SBMV RNA, RdMV RNA, or TMV RNA. RNA replication occurred only with PEMV RNA and not with intact PEMV or any of the other viruses or RNAs, as judged by ensuing actinomycin D-insensitive polymerase activity. Molecular hybridization experiments showed that some of the product of the polymerase was PEMV-specific (-)RNA. The substrate and ionic requirements of this polymerase were the same as those for the RNA-dependent RNA polymerase present in nuclei isolated from PEMV-infected pea plants. No virus particles could be recovered from nuclei primed with PEMV RNA. These results are discussed in relation to the possible mechanism for in vivo infection of pea cells. PMID:16592421

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

PubMed Central

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

Quantitating antibody uptake in vivo: conditional dependence on antigen expression levels.

PubMed

Thurber, Greg M; Weissleder, Ralph

2011-08-01

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. Using a cell line with high epithelial cell adhesion molecule expression and moderate epidermal growth factor receptor 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. 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. 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.

Discrete forms of amylose are synthesized by isoforms of GBSSI in pea.

PubMed

Edwards, Anne; Vincken, Jean-Paul; Suurs, Luc C J M; Visser, Richard G F; Zeeman, Sam; Smith, Alison; Martin, Cathie

2002-08-01

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2017-08-24

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

Differential expression and functional analysis of three calmodulin isoforms in germinating pea (Pisum sativum L.) seeds.

PubMed

Duval, Frédéric D; Renard, Michelle; Jaquinod, Michel; Biou, Valérie; Montrichard, Françoise; Macherel, David

2002-11-01

Implication of the ubiquitous, highly conserved, Ca2+ sensor calmodulin (CaM) in pea seed germination has been investigated. Mass spectrometry analysis of purified CaM revealed the coexistence in seeds of three protein isoforms, diverging from each other by single amino acid substitution in the N-terminal alpha-helix. CaM was shown to be encoded by a small multigenic family, and full-length cDNAs of the three isoforms (PsCaM1, 2 and 3) were isolated to allow the design of specific primers in more divergent 5' and 3' untranslated regions. Expression studies, performed by semiquantitative RT-PCR, demonstrated differential expression patterns of the three transcripts during germination. PsCaM1 and 2 were detected at different levels in dry axes and cotyledons, and they accumulated during imbibition and prior to radicle protrusion. In contrast, PsCaM3 appeared only upon radicle protrusion, then gradually increased in both tissues. To characterise the biochemical properties of the CaM isoforms, functional analyses were conducted in vitro using recombinant Strep-tagged proteins (CaM1-ST, CaM2-ST and CaM3-ST) expressed in Escherichia coli. Gel mobility shift assays revealed that CaM1-ST exhibited a stoichiometric binding of a synthetic amphiphilic CaM kinase II peptide while CaM2-ST and CaM3-ST affinities for the same peptide were reduced. Affinity differences were also observed for CaM isoform binding to Trp-3, an idealised helical CaM-binding peptide. However, the three proteins activated in the same way the CaM-dependent pea NAD kinase. Finally, the significance of the single substitutions upon CaM interaction with its targets is discussed in a structural context.

Influences of pea morphology and interacting factors on pea aphid (Homoptera: Aphididae) reproduction.

PubMed

Buchman, N; Cuddington, K

2009-08-01

It has been claimed that plant architecture can alter aphid reproductive rates, but the mechanism driving this effect has not been identified. We studied interactions between plant architecture, aphid density, environmental conditions, and nutrient availability on the reproduction of pea aphids [Acyrthosiphon pisum (Harris)] using four near-isogenic peas (Pisum sativum L.) that differ in morphology. Manipulations of aphid density (1, 5, and 10 adults per plant) allowed us to examine any effects of plant morphology on crowding and consequently reproduction. Pea morphology per se did not alter pea aphid crowding, as measured by mean nearest neighbor distance, and there was no effect on reproduction. In addition, reproduction increased with increasing adult density, indicating positive density dependence. In a separate experiment, peas were fertilized to determine whether differences between nutrient availability of the four different morphologies might drive any observed differences in aphid reproduction. Although plant nitrogen content was altered by fertilization treatments, this did not have an impact on aphid reproduction. Greenhouse experiments, however, suggested that pea morphology can interact with environmental conditions to reduce aphid reproduction under some conditions. We conclude that plant morphology only influences aphid reproduction when environmental conditions are less than optimal.

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

PubMed Central

2012-01-01

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, 277 (9.8%) pea ESTs

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

PubMed

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

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

Understanding the role of H(2)O(2) during pea seed germination: a combined proteomic and hormone profiling approach.

PubMed

Barba-Espín, Gregorio; Diaz-Vivancos, Pedro; Job, Dominique; Belghazi, Maya; Job, Claudette; Hernández, José Antonio

2011-11-01

In a previous publication, we showed that the treatment of pea seeds in the presence of hydrogen peroxide (H(2)O(2)) increased germination performance as well as seedling growth. To gain insight into the mechanisms responsible for this behaviour, we have analysed the effect of treating mature pea seeds in the presence of 20 mm H(2)O(2) on several oxidative features such as protein carbonylation, endogenous H(2)O(2) and lipid peroxidation levels. We report that H(2)O(2) treatment of the pea seeds increased their endogenous H(2)O(2) content and caused carbonylation of storage proteins and of several metabolic enzymes. Under the same conditions, we also monitored the expression of two MAPK genes known to be activated by H(2)O(2) in adult pea plants. The expression of one of them, PsMAPK2, largely increased upon pea seed imbibition in H(2)O(2) , whereas no change could be observed in expression of the other, PsMAPK3. The levels of several phytohormones such as 1-aminocyclopropane carboxylic acid, indole-3-acetic acid and zeatin appeared to correlate with the measured oxidative indicators and with the expression of PsMAPK2. Globally, our results suggest a key role of H(2)O(2) in the coordination of pea seed germination, acting as a priming factor that involves specific changes at the proteome, transcriptome and hormonal levels. © 2011 Blackwell Publishing Ltd.

Bowman-Birk inhibitor-like protein is secreted by sprouted pea seeds in response to induced colonization by enteropathogenic Escherichia coli.

PubMed

Anuradha, Ravi; Raveendran, Muthuraj; Babu, Subramanian

2013-11-01

The interaction between the clinical isolate of enteropathogenic Escherichia coli (EPEC) SBANU8 and pea sprouts was compared with avirulent K 12. E. coli. This was carried out by repeated co-incubation with pea sprouts for 5 days, and the protein profile of the culture supernatant was analyzed by single and two-dimensional electrophoresis. Mass spectrometry analysis led to the identification of two serine protease inhibitors including a Bowman-Birk-type protein secreted by pea sprouts in response to clinical isolate. Expression of the E. coli intimin gene involved in animal host colonization and virulence was studied by reverse transcription polymerase chain reaction. Expression of this gene was high in SBANU8 when co-incubated with pea sprouts. The present study gives baseline data on the molecular level interactions of EPEC and pea sprouts, which are needed to design the outbreak control strategies.

Discrete Forms of Amylose Are Synthesized by Isoforms of GBSSI in PeaW⃞

PubMed Central

Edwards, Anne; Vincken, Jean-Paul; Suurs, Luc C. J. M.; Visser, Richard G. F.; Zeeman, Sam; Smith, Alison; Martin, Cathie

2002-01-01

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses. PMID:12172021

[Prokaryotic expression of Nanog gene and preparation of anti-Nanog antibody].

PubMed

Li, Jun; Wang, Xiao-min; Dou, Zhong-ying; Li, Yong

2012-07-01

To express Nanog fusion protein in Escherichia coli ( E.coli), and to prepare rabbit anti-mouse polyclonal antibodies to the Nanog fusion protein. Mouse Nanog gene was amplified from the pNA992 recombinant plasmid and inserted into pET-32a vector to construct a recombinant expression vector pET-32a-Nanog. The recombinant vector was transfected into E.coli BL21 and induced by IPTG to express in them. The acquired Nanog fusion protein was purified with HisTrap affinity column and injected as an antigen into rabbits for preparing polyclonal antibodies. At last, the titer and specificity of the polyclonal antibodies were analyzed with indirect ELISA, Western blotting and immunocytochemical staining, respectively. The recombinant expression vector pET-32a-Nanog was successfully prepared, transfected and induced to obtain the high expression of the Nanog fusion protein in a form of inclusion bodies in E.coli. After purification, its purity was up to 97%. The titer of anti-Nanog antibodies was 1:32 000 in the immunized rabbit serum, and exhibited a high specificity to Nanog protein. The rabbit anti-mouse polyclonal antibodies have been prepared successfully with a high titer and specificity to the Nanog fusion protein.

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

PubMed Central

Wong, Chui E; Bhalla, Prem L; Ottenhof, Harald; Singh, Mohan B

2008-01-01

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, epigenetic regulation, auxin

Cloning, bacterial expression and crystallization of Fv antibody fragments

NASA Astrophysics Data System (ADS)

E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

1992-08-01

The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

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

PubMed Central

Nikolova, Ivelina Mitkova

2016-01-01

A study was conducted to determine the response of five pea varieties (Pisum sativum L.) to damage degree of Bruchus pisorum: Glyans, Modus, Kamerton, and Svit (Ukrainian cultivars) and Pleven 4 (Bulgarian cultivar). The seeds were classified into three types: healthy seeds (type 1), damaged seeds with parasitoid emergence hole (type 2), and damaged seeds with bruchid emergence hole (type 3) and they were sown. It was found that the weight of 1000 seeds did not affect the field germination of the pea varieties. Healthy and damaged seeds with parasitoid emergence holes (first and second seed types) provide a very good opportunity for growth and development while plants from damaged seeds with bruchid emergence holes had poor germination and vigor and low productivity. These seeds cannot provide the creation of well-garnished seeding and stable crop yields. Among tested varieties, the Ukrainian variety Glyans had considerably higher seed weight, field germination, and index germination and weak egg-laying activity of B. pisorum compared to others. Use of spring pea cultivars that are weakly preferred by the pea weevil in breeding programs would reduce losses due to pea weevil and provide an environmentally safer option to its control. PMID:27042379

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

PubMed

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.

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

Federal Register 2010, 2011, 2012, 2013, 2014

2013-10-23

... DEPARTMENT OF AGRICULTURE Grain Inspection, Packers and Stockyards Administration United States... Administration, USDA ACTION: Notice and request for comments. SUMMARY: The Department of Agriculture's (USDA... Standards for Feed Peas, Split Peas, and Lentils under the Agriculture Marketing Act (AMA) of 1946. To...

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

NASA Technical Reports Server (NTRS)

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

1992-01-01

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

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

PubMed

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.

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

PubMed

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

2017-01-01

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

Immunofluorescence detection of pea protein in meat products.

PubMed

Petrášová, Michaela; Pospiech, Matej; Tremlová, Bohuslava; Javůrková, Zdeňka

2016-08-01

In this study we developed an immunofluorescence method to detect pea protein in meat products. Pea protein has a high nutritional value but in sensitive individuals it may be responsible for causing allergic reactions. We produced model meat products with various additions of pea protein and flour; the detection limit (LOD) of the method for pea flour was 0.5% addition, and for pea protein it was 0.001% addition. The repeatabilities and reproducibilities for samples both positive and negative for pea protein were all 100%. In a blind test with model products and commercial samples, there was no statistically significant difference (p > 0.05) between the declared concentrations of pea protein and flour and the immunofluorescence method results. Sensitivity was 1.06 and specificity was 1.00. These results show that the immunofluorescence method is suitable for the detection of pea protein in meat products.

[Expression of human-mouse chimeric antibody directed against Chikungunya virus with site-specific integration system].

PubMed

Li, Jian-min; Chen, Wei; Jia, Xiu-jie; An, Xiao-ping; Li, Bing; Fan, Ying-ru; Tong, Yi-gang

2005-05-01

To obtain CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site and to express human-mouse chimeric antibody directed against Chikungunya Virus by using the cell line. The fusion gene of FRT and HBsAg was constructed by PCR and cloned into the MCS of pCI-neo to construct pCI-FRT-HBsAg. The pCI-FRT-HBsAg was transfected into CHO/dhfr(-) cells and cell clones with high expression of HBsAg were screened by detecting the amount of HBsAg with ELISA. A CHO cell clone with the highest expression was chosen and named as CHO/dhfr(-) FRT(+). pAFRT HFLF, a expression plasmid of chimeric antibody with RFT sequence was transfected into CHO/dhfr(-) FRT(+) cells and cell clones with high expression of the chimeric antibody were screened by increasing concentration of MTX. A CHO cell clone with high expression of the chimeric antibody was cultured in large scale and supernatant was collected from which the chimeric antibody was purified. The purified chimeric antibody was analyzed by SDS-PAGE, Western blot and IFA. A CHO/dhfr(-) cells line with integrated FRT sequence in the chromosome transcription active site was obtained successfully. A cell clone with yield of 5 mg/L of chimeric antibody was obtained, as compared with routine CHO cell expression system with a yield of 2 mg/L. A cell line with integrated FRT sequence in the chromosome transcription active site was obtained and with it human-mouse chimeric antibody directed against Chikungunya virus was expressed. This system lays a solid foundation which can be used for expressing antibodies and other proteins.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

PubMed

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana

PubMed Central

Balfanz, Sabine

2017-01-01

The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]i) whereas type 2 tyramine receptors can mediate Ca2+ signals or both Ca2+ signals and effects on [cAMP]i. Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine. PMID:29084141

PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana.

PubMed

Blenau, Wolfgang; Balfanz, Sabine; Baumann, Arnd

2017-10-30

The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP] i ) whereas type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . Here; we report that the American cockroach ( Periplaneta americana ) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP] i . Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana ; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

In Vitro Trypanocidal Activity of Antibodies to Bacterially Expressed Trypanosoma brucei Tubulin

PubMed Central

Kateete, DP; Alezuyo, C; Nanteza, A; Asiimwe, C; Lubega, GW

2012-01-01

Background There are only four drugs for treating African trypanosomiasis, a devastating disease in sub-Saharan Africa. With slow discovery of better drugs, vaccination is viewed as the best method of control. We previously showed that antibodies to native Trypanosoma brucei brucei tubulin inhibit the growth of trypanosomes in culture. Here, we aimed to determine the effect of antibodies to bacterially expressed trypanosome tubulin on T. brucei brucei growth. Methods T. brucei brucei alpha and beta tubulin genes were individually expressed in Escherichia coli under the tryptophan promoter. Monoclonal tubulin antibodies reacted specifically with the expressed tubulins with no cross-reaction with the opposite tubulin. Rabbits were immunized with 450µg each of the concentrated recombinant tubulin, and production of antibodies assessed by ELISA and Western blotting. The effect of polyclonal antibodies on trypanosome growth was determined by culturing bloodstream T. brucei brucei in up to 25% of antisera. Results Low antisera dilutions (25%) from the immunized rabbits inhibited trypanosome growth. The most cytotoxic antisera were from one rabbit immunized with a mixture of both alpha and beta tubulins. However, the result was not reproduced in other rabbits and there was no apparent effect on growth at higher antisera dilutions. Conclusion Antibodies to bacterially expressed trypanosome tubulin are not effective at killing cultured bloodstream trypanosomes. PMID:23109963

Pea weevil damage and chemical characteristics of pea cultivars determining their resistance to Bruchus pisorum L.

PubMed

Nikolova, I

2016-04-01

Bruchus pisorum (L.) is one of the most intractable pest problems of cultivated pea in Europe. Development of resistant cultivars is very important to environmental protection and would solve this problem to a great extent. Therefore, the resistance of five spring pea cultivars was studied to B. pisorum: Glyans, Modus; Kamerton and Svit and Pleven 4 based on the weevil damage and chemical composition of seeds. The seeds were classified as three types: healthy seeds (type one), damaged seeds with parasitoid emergence holes (type two) and damaged seeds with bruchid emergence holes (type three). From visibly damaged pea seeds by pea weevil B. pisorum was isolated the parasitoid Triaspis thoracica Curtis (Hymenoptera, Braconidae). Modus, followed by Glyans was outlined as resistant cultivars against the pea weevil. They had the lowest total damaged seed degree, loss in weight of damaged seeds (type two and type three) and values of susceptibility coefficients. A strong negative relationship (r = -0.838) between the weight of type one seeds and the proportion of type three seeds was found. Cultivars with lower protein and phosphorus (P) content had a lower level of damage. The crude protein, crude fiber and P content in damaged seeds significantly or no significantly were increased as compared with the healthy seeds due to weevil damage. The P content had the highest significant influence on pea weevil infestation. Use of chemical markers for resistance to the creation of new pea cultivars can be effective method for defense and control against B. pisorum.

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

PubMed

Fiory, Francesca; Parrillo, Luca; Raciti, Gregory Alexander; Zatterale, Federica; Nigro, Cecilia; Mirra, Paola; Falco, Roberta; Ulianich, Luca; Di Jeso, Bruno; Formisano, Pietro; Miele, Claudia; Beguinot, Francesco

2014-01-01

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.

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

PubMed Central

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

2015-01-01

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. PMID:25804541

Pea3 transcription factor promotes neurite outgrowth

PubMed Central

Kandemir, Basak; Caglayan, Berrak; Hausott, Barbara; Erdogan, Burcu; Dag, Ugur; Demir, Ozlem; Sogut, Melis S.; Klimaschewski, Lars; Kurnaz, Isil A.

2014-01-01

Pea3 subfamily of E–twenty six transcription factors consist of three major -exhibit branching morphogenesis, the function of Pea3 family in nervous system development and regeneration is only beginning to unfold. In this study, we provide evidence that Pea3 can directs neurite extension and axonal outgrowth in different model systems, and that Serine 90 is important for this function. We have also identified neurofilament-L and neurofilament-M as two putative novel targets for Pea3. PMID:25018694

Multiple, Distinct Isoforms of Sucrose Synthase in Pea1

PubMed Central

Barratt, D.H. Paul; Barber, Lorraine; Kruger, Nicholas J.; Smith, Alison M.; Wang, Trevor L.; Martin, Cathie

2001-01-01

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell. PMID:11598239

Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

PubMed Central

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-01-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

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

PubMed Central

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

2016-01-01

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 light and heavy chain sequences. No strictly conserved target sequence was evident, although the peptide bonds that were susceptible to proteolysis were predominantly and consistently located within or near to the interdomain or solvent-exposed regions in the antibody structure. Amino acids surrounding identified cleavage sites were mutated in an attempt to increase resistance. Different Guy’s 13 antibody heavy and light chain mutant combinations were expressed transiently in N. tabacum and demonstrated intensity shifts in the fragmentation pattern, resulting in alterations to the full-length antibody-to-fragment ratio. The work strengthens the understanding of proteolytic cleavage of antibodies expressed in plants and presents a novel approach to stabilize full-length antibody by site-directed mutagenesis.—Hehle, V. K., Paul, M. J., Roberts, V. A., van Dolleweerd, C. J., Ma, J. K.-C. Site-targeted mutagenesis for stabilization of recombinant monoclonal antibody expressed in tobacco (Nicotiana tabacum) plants. PMID:26712217

Effect of the combinations between pea proteins and soluble fibres on cholesterolaemia and cholesterol metabolism in rats.

PubMed

Parolini, Cinzia; Manzini, Stefano; Busnelli, Marco; Rigamonti, Elena; Marchesi, Marta; Diani, Erika; Sirtori, Cesare R; Chiesa, Giulia

2013-10-01

Many functional foods and dietary supplements have been reported to be beneficial for the management of dyslipidaemia, one of the major risk factors for CVD. Soluble fibres and legume proteins are known to be a safe and practical approach for cholesterol reduction. The present study aimed at investigating the hypocholesterolaemic effect of the combinations of these bioactive vegetable ingredients and their possible effects on the expression of genes regulating cholesterol homeostasis. A total of six groups of twelve rats each were fed, for 28 d, Nath's hypercholesterolaemic diets, differing in protein and fibre sources, being, respectively, casein and cellulose (control), pea proteins and cellulose (pea), casein and oat fibres (oat), casein and apple pectin (pectin), pea proteins and oat fibres (pea+oat) and pea proteins and apple pectin (pea+pectin). Administration of each vegetable-containing diet was associated with lower total cholesterol concentrations compared with the control. The combinations (pea+oat and pea+pectin) were more efficacious than fibres alone in modulating cholesterolaemia ( - 53 and - 54%, respectively, at 28 d; P< 0·005). In rats fed the diets containing oat fibres or apple pectin, alone or in combination with pea proteins, a lower hepatic cholesterol content (P< 0·005) and higher hepatic mRNA concentrations of CYP7A1 and NTCP were found when compared with the control rats (P< 0·05). In summary, the dietary combinations of pea proteins and oat fibres or apple pectin are extremely effective in lowering plasma cholesterol concentrations in rats and affect cellular cholesterol homeostasis by up-regulating genes involved in hepatic cholesterol turnover.

Domestication of Pea (Pisum sativum L.): The Case of the Abyssinian Pea

PubMed Central

Weeden, Norman F.

2018-01-01

Phylogenetic relationships of the Abyssinian pea (Pisum sativum ssp. abyssinicum) to other subspecies and species in the genus were investigated to test between different hypotheses regarding its origin and domestication. An extensive sample of the Pisum sativum ssp. sativum germplasm was investigated, including groups a-1, a-2, b, c, and d as identified by Kwon et al. (2012). A broad sample of P. fulvum but relatively few P. s. ssp. elatius accessions were analyzed. Partial sequences of 18 genes were compared and these results combined with comparisons of additional genes done by others and available in the literature. In total, 54 genes or gene fragment sequences were involved in the study. The observed affinities between alleles in P. ssp. sativum, P. s. ssp. abyssinicum, P. s. ssp. elatius, and P. fulvum clearly demonstrated a close relationship among the three P. sativum subspecies and rejected the hypothesis that the Abyssinian pea was formed by hybridization between one of the P. sativum subspecies and P. fulvum. If hybridization were involved in the generation of the Abyssinian pea, it must have been between P. s. ssp. sativum and P. s. ssp. elatius, although the Abyssinian pea possesses a considerable number of highly unique alleles, implying that the actual P. s. ssp. elatius germplasm involved in such a hybridization has yet to be tested or that the hybridization occurred much longer ago than the postulated 4000 years bp. Analysis of the P. s. ssp. abyssinicum alleles in genomic regions thought to contain genes critical for domestication indicated that the indehiscent pod trait was independently developed in the Abyssinian pea, whereas the loss of seed dormancy was either derived from P. s. ssp. sativum or at least partially developed before the P. s. ssp. abyssinicum lineage diverged from that leading to P. s. ssp. sativum. PMID:29720994

Expression of Recombinant Antibodies

PubMed Central

Frenzel, André; Hust, Michael; Schirrmann, Thomas

2013-01-01

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

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

PubMed

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. © 2015 American Society of Plant Biologists. All rights reserved.

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

PubMed

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

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

PubMed

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. Copyright © 2012 Elsevier Ltd. All rights reserved.

Profile and functional properties of seed proteins from six pea (Pisum sativum) genotypes.

PubMed

Barac, Miroljub; Cabrilo, Slavica; Pesic, Mirjana; Stanojevic, Sladjana; Zilic, Sladjana; Macej, Ognjen; Ristic, Nikola

2010-01-01

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

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

PubMed

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

2016-04-30

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 important allergenic specie of the legume family. This work is aimed at isolation of a novel LTP from pea seeds and characterization of its structural, functional, and allergenic properties. Three novel lipid transfer proteins, designated as Ps-LTP1-3, were found in the garden pea Pisum sativum, their cDNA sequences were determined, and mRNA expression levels of all the three proteins were measured at different pea organs. Ps-LTP1 was isolated for the first time from the pea seeds, and its complete amino acid sequence was determined. The protein exhibits antifungal activity and is a membrane-active compound that causes a leakage from artificial liposomes. The protein binds various lipids including bioactive jasmonic acid. Spatial structure of the recombinant uniformly (13)C,(15)N-labelled Ps-LTP1 was solved by heteronuclear NMR spectroscopy. In solution the unliganded protein represents the mixture of two conformers (relative populations ~ 85:15) which are interconnected by exchange process with characteristic time ~ 100 ms. Hydrophobic residues of major conformer form a relatively large internal tunnel-like lipid-binding cavity (van der Waals volume comes up to ~1000 Å(3)). The minor conformer probably corresponds to the protein with the partially collapsed internal cavity. For the first time conformational heterogeneity in solution was shown for an unliganded plant lipid transfer protein. Heat denaturation profile and simulated gastrointestinal digestion assay showed that Ps

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

PubMed

Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Kralicek, Andrew V; Shaw, Martin L; Lewis, John G; Conner, Anthony J; Barrell, Philippa J

2014-11-04

The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

Remediation of cadmium toxicity in field peas (Pisum sativum L.) through exogenous silicon.

PubMed

Rahman, Mohammad Farhadur; Ghosal, Anubrata; Alam, Mohammad Firoz; Kabir, Ahmad Humayan

2017-01-01

Cadmium (Cd) is an important phytotoxic element causing health hazards. This work investigates whether and how silicon (Si) influences the alleviation of Cd toxicity in field peas at biochemical and molecular level. The addition of Si in Cd-stressed plants noticeably increased growth and development as well as total protein and membrane stability of Cd-stressed plants, suggesting that Si does have critical roles in Cd detoxification in peas. Furthermore, Si supplementation in Cd-stressed plants showed simultaneous significant increase and decrease of Cd and Fe in roots and shoots, respectively, compared with Cd-stressed plants. At molecular level, GSH1 (phytochelatin precursor) and MT A (metallothionein) transcripts predominantly expressed in roots and strongly induced due to Si supplementation in Cd-stressed plants compared with Cd-free conditions, suggesting that these chelating agents may bind to Cd leading to vacuolar sequestration in roots. Furthermore, pea Fe transporter (RIT1) showed downregulation in shoots when plants were treated with Si along with Cd compared with Cd-treated conditions. It is consistent with the physiological observations and supports the conclusion that alleviation of Cd toxicity in pea plants might be associated with Cd sequestration in roots and reduced Cd translocation in shoots through the regulation of Fe transport. Furthermore, increased CAT, POD, SOD and GR activity along with elevated S-metabolites (cysteine, methionine, glutathione) implies the active involvement of ROS scavenging and plays, at least in part, to the Si-mediated alleviation of Cd toxicity in pea. The study provides first mechanistic evidence on the beneficial effect of Si on Cd toxicity in pea plants. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of feeding soybean meal and differently processed peas on the gut mucosal immune system of broilers.

PubMed

Röhe, I; Göbel, T W; Goodarzi Boroojeni, F; Zentek, J

2017-07-01

Peas are traditionally used as a protein source for poultry. However, peas contain antinutritional factors (ANF), which are associated with the initiation of local and systemic immune reactions. The current study examined the effect of feeding raw or differently processed peas in comparison with feeding a soybean meal (SBM) based control diet (C) on the gut mucosal immune system of broilers in a 35 day feeding trial. In six replicates, a total of 360 one-day-old male broilers were randomly allocated to four different groups receiving C, or three treatment diets containing raw, fermented, and enzymatically pre-digested peas, each supplying 30% of required crude protein. After slaughtering, jejunal samples were taken for immunohistochemical, flow cytometric, and gene expression analyses. Investigations were focused on the topological distribution of intraepithelial leukocytes (villus tip, villus mid, and crypt region) as well as on the further characterization of the different intraepithelial lymphocytes (IEL) and concomitant pro- and anti-inflammatory cytokines. Broilers receiving the raw or processed pea diets had higher numbers of intraepithelial CD45+ leukocytes in the tip (P = 0.004) and mid region (P < 0.001) of villi than birds fed C. Higher numbers of intraepithelial CD3+ lymphocytes were found in the villus tip (P = 0.002) and mid region (P = 0.003) of birds fed raw or processed pea containing diets in comparison with those fed C. The flow cytometric phenotyping showed a similar relative distribution of IEL among the feeding groups. The expression of intestinal pro- and anti-inflammatory cytokines was affected by feeding the different diets only to a minor extent. To conclude, feeding of diets formulated with raw and processed peas in comparison with feeding a SBM control diet initiated mucosal immune responses in the jejunum of broilers indicated by a quantitative increase of intraepithelial T cells. Further research is needed in order to ascertain the

Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from a dual-host phage display vector.

PubMed

Shang, Yonglei; Tesar, Devin; Hötzel, Isidro

2015-10-01

A recently described dual-host phage display vector that allows expression of immunoglobulin G (IgG) in mammalian cells bypasses the need for subcloning of phage display clone inserts to mammalian vectors for IgG expression in large antibody discovery and optimization campaigns. However, antibody discovery and optimization campaigns usually need different antibody formats for screening, requiring reformatting of the clones in the dual-host phage display vector to an alternative vector. We developed a modular protein expression system mediated by RNA trans-splicing to enable the expression of different antibody formats from the same phage display vector. The heavy-chain region encoded by the phage display vector is directly and precisely fused to different downstream heavy-chain sequences encoded by complementing plasmids simply by joining exons in different pre-mRNAs by trans-splicing. The modular expression system can be used to efficiently express structurally correct IgG and Fab fragments or other antibody formats from the same phage display clone in mammalian cells without clone reformatting. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

PubMed

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.

Genomic Tools in Pea Breeding Programs: Status and Perspectives

PubMed Central

Tayeh, Nadim; Aubert, Grégoire; Pilet-Nayel, Marie-Laure; Lejeune-Hénaut, Isabelle; Warkentin, Thomas D.; Burstin, Judith

2015-01-01

Pea (Pisum sativum L.) is an annual cool-season legume and one of the oldest domesticated crops. Dry pea seeds contain 22–25% protein, complex starch and fiber 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 tons 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 toward deciphering the pea genome. PMID:26640470

Yield potential of pigeon pea cultivars

USDA-ARS?s Scientific Manuscript database

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...

The pea GIGAS gene is a FLOWERING LOCUS T homolog necessary for graft-transmissible specification of flowering but not for responsiveness to photoperiod.

PubMed

Hecht, Valérie; Laurie, Rebecca E; Vander Schoor, Jacqueline K; Ridge, Stephen; Knowles, Claire L; Liew, Lim Chee; Sussmilch, Frances C; Murfet, Ian C; Macknight, Richard C; Weller, James L

2011-01-01

Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein.

PubMed

Escobar Galvis, M L; Marttila, S; Håkansson, G; Forsberg, J; Knorpp, C

2001-05-01

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein.

21 CFR 155.170 - Canned peas.

Code of Federal Regulations, 2014 CFR

2014-04-01

... the pea plant of the species Pisum sativum L. but excluding the subspecies macrocarpum. Only sweet... any combination of two or more of the dry or liquid forms of sugar, invert sugar sirup, dextrose... characteristics. Where the peas are of sweet green wrinkled varieties or hybrids having similar characteristics...

21 CFR 155.170 - Canned peas.

Code of Federal Regulations, 2012 CFR

2012-04-01

... the pea plant of the species Pisum sativum L. but excluding the subspecies macrocarpum. Only sweet... any combination of two or more of the dry or liquid forms of sugar, invert sugar sirup, dextrose... characteristics. Where the peas are of sweet green wrinkled varieties or hybrids having similar characteristics...

21 CFR 155.170 - Canned peas.

Code of Federal Regulations, 2013 CFR

2013-04-01

... the pea plant of the species Pisum sativum L. but excluding the subspecies macrocarpum. Only sweet... any combination of two or more of the dry or liquid forms of sugar, invert sugar sirup, dextrose... characteristics. Where the peas are of sweet green wrinkled varieties or hybrids having similar characteristics...

The pea aphid uses a version of the terminal system during oviparous, but not viviparous, development

PubMed Central

2013-01-01

Background In most species of aphid, female nymphs develop into either sexual or asexual adults depending on the length of the photoperiod to which their mothers were exposed. The progeny of these sexual and asexual females, in turn, develop in dramatically different ways. The fertilized oocytes of sexual females begin embryogenesis after being deposited on leaves (oviparous development) while the oocytes of asexual females complete embryogenesis within the mother (viviparous development). Compared with oviparous development, viviparous development involves a smaller transient oocyte surrounded by fewer somatic epithelial cells and a smaller early embryo that comprises fewer cells. To investigate whether patterning mechanisms differ between the earliest stages of the oviparous and viviparous modes of pea aphid development, we examined the expression of pea aphid orthologs of genes known to specify embryonic termini in other insects. Results Here we show that pea aphid oviparous ovaries express torso-like in somatic posterior follicle cells and activate ERK MAP kinase at the posterior of the oocyte. In addition to suggesting that some posterior features of the terminal system are evolutionarily conserved, our detection of activated ERK in the oocyte, rather than in the embryo, suggests that pea aphids may transduce the terminal signal using a mechanism distinct from the one used in Drosophila. In contrast with oviparous development, the pea aphid version of the terminal system does not appear to be used during viviparous development, since we did not detect expression of torso-like in the somatic epithelial cells that surround either the oocyte or the blastoderm embryo and we did not observe restricted activated ERK in the oocyte. Conclusions We suggest that while oviparous oocytes and embryos may specify posterior fate through an aphid terminal system, viviparous oocytes and embryos employ a different mechanism, perhaps one that does not rely on an interaction

Vicilin and convicilin are potential major allergens from pea.

PubMed

Sanchez-Monge, R; Lopez-Torrejón, G; Pascual, C Y; Varela, J; Martin-Esteban, M; Salcedo, G

2004-11-01

Allergic reactions to pea (Pisum sativum) ingestion are frequently associated with lentil allergy in the Spanish population. Vicilin have been described as a major lentil allergen. To identify the main IgE binding components from pea seeds and to study their potential cross-reactivity with lentil vicilin. A serum pool or individual sera from 18 patients with pea allergy were used to detect IgE binding proteins from pea seeds by immunodetection and immunoblot inhibition assays. Protein preparations enriched in pea vicilin were obtained by gel filtration chromatography followed by reverse-phase high-performance liquid chromatography (HPLC). IgE binding components were identified by means of N-terminal amino acid sequencing. Complete cDNAs encoding pea vicilin were isolated by PCR, using primers based on the amino acid sequence of the reactive proteins. IgE immunodetection of crude pea extracts revealed that convicilin (63 kDa), as well as vicilin (44 kDa) and one of its proteolytic fragments (32 kDa), reacted with more than 50% of the individual sera tested. Additional proteolytic subunits of vicilin (36, 16 and 13 kDa) bound IgE from approximately 20% of the sera. The lentil vicilin allergen Len c 1 strongly inhibited the IgE binding to all components mentioned above. The characterization of cDNA clones encoding pea vicilin has allowed the deduction of its complete amino acid sequence (90% of sequence identity to Len c 1), as well as those of its reactive proteolytic processed subunits. Vicilin and convicilin are potential major allergens from pea seeds. Furthermore, proteolytic fragments from vicilin are also relevant IgE binding pea components. All these proteins cross-react with the major lentil allergen Len c 1.

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

PubMed Central

Barac, Miroljub; Cabrilo, Slavica; Pesic, Mirjana; Stanojevic, Sladjana; Zilic, Sladjana; Macej, Ognjen; Ristic, Nikola

2010-01-01

Extractability, extractable protein compositions, technological-functional properties of pea (Pisum sativum) proteins from six genotypes grown in Serbia were investigated. Also, the relationship between these characteristics was presented. Investigated genotypes showed significant differences in storage protein content, composition and extractability. The ratio of vicilin:legumin concentrations, as well as the ratio of vicilin + convicilin: Legumin concentrations were positively correlated with extractability. Our data suggest that the higher level of vicilin and/or a lower level of legumin have a positive influence on protein extractability. The emulsion activity index (EAI) was strongly and positively correlated with the solubility, while no significant correlation was found between emulsion stability (ESI) and solubility, nor between foaming properties and solubility. No association was evident between ESI and EAI. A moderate positive correlation between emulsion stability and foam capacity was observed. Proteins from the investigated genotypes expressed significantly different emulsifying properties and foam capacity at different pH values, whereas low foam stability was detected. It appears that genotype has considerable influence on content, composition and technological-functional properties of pea bean proteins. This fact can be very useful for food scientists in efforts to improve the quality of peas and pea protein products. PMID:21614186

Pre-fractionation strategies to resolve pea (Pisum sativum) sub-proteomes

PubMed Central

Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana; Kukavica, Biljana M.; Lüthje, Sabine

2015-01-01

Legumes are important crop plants and pea (Pisum sativum L.) has been investigated as a model with respect to several physiological aspects. The sequencing of the pea genome has not been completed. Therefore, proteomic approaches are currently limited. Nevertheless, the increasing numbers of available EST-databases as well as the high homology of the pea and medicago genome (Medicago truncatula Gaertner) allow the successful identification of proteins. Due to the un-sequenced pea genome, pre-fractionation approaches have been used in pea proteomic surveys in the past. Aside from a number of selective proteome studies on crude extracts and the chloroplast, few studies have targeted other components such as the pea secretome, an important sub-proteome of interest due to its role in abiotic and biotic stress processes. The secretome itself can be further divided into different sub-proteomes (plasma membrane, apoplast, cell wall proteins). Cell fractionation in combination with different gel-electrophoresis, chromatography methods and protein identification by mass spectrometry are important partners to gain insight into pea sub-proteomes, post-translational modifications and protein functions. Overall, pea proteomics needs to link numerous existing physiological and biochemical data to gain further insight into adaptation processes, which play important roles in field applications. Future developments and directions in pea proteomics are discussed. PMID:26539198

Fibril formation from pea protein and subsequent gel formation.

PubMed

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.

21 CFR 158.170 - Frozen peas.

Code of Federal Regulations, 2014 CFR

2014-04-01

... and designations shall apply: Size designation Round hole sieve size through which peas will pass... paragraph (a)(2) of this section or the applicable sieve size. However, the optional descriptive words... transfer peas to U.S. No. 8 sieve, using (20 cm.) size for container of less than 3 lb. net weight and (30...

21 CFR 158.170 - Frozen peas.

Code of Federal Regulations, 2011 CFR

2011-04-01

... and designations shall apply: Size designation Round hole sieve size through which peas will pass... paragraph (a)(2) of this section or the applicable sieve size. However, the optional descriptive words... transfer peas to U.S. No. 8 sieve, using (20 cm.) size for container of less than 3 lb. net weight and (30...

21 CFR 158.170 - Frozen peas.

Code of Federal Regulations, 2012 CFR

2012-04-01

... and designations shall apply: Size designation Round hole sieve size through which peas will pass... paragraph (a)(2) of this section or the applicable sieve size. However, the optional descriptive words... transfer peas to U.S. No. 8 sieve, using (20 cm.) size for container of less than 3 lb. net weight and (30...

21 CFR 158.170 - Frozen peas.

Code of Federal Regulations, 2013 CFR

2013-04-01

... and designations shall apply: Size designation Round hole sieve size through which peas will pass... paragraph (a)(2) of this section or the applicable sieve size. However, the optional descriptive words... transfer peas to U.S. No. 8 sieve, using (20 cm.) size for container of less than 3 lb. net weight and (30...

PsPMEP, a pollen-specific pectin methylesterase of pea (Pisum sativum L.).

PubMed

Gómez, María Dolores; Renau-Morata, Begoña; Roque, Edelín; Polaina, Julio; Beltrán, José Pío; Cañas, Luis A

2013-09-01

Pectin methylesterases (PMEs) are a family of enzymes involved in plant reproductive processes such as pollen development and pollen tube growth. We have isolated and characterized PsPMEP, a pea (Pisum sativum L.) pollen-specific gene that encodes a protein with homology to PMEs. Sequence analysis showed that PsPMEP belongs to group 2 PMEs, which are characterized by the presence of a processable amino-terminal PME inhibitor domain followed by the catalytic PME domain. Moreover, PsPMEP contains several motifs highly conserved among PMEs with the essential amino acid residues involved in enzyme substrate binding and catalysis. Northern blot and in situ hybridization analyses showed that PsPMEP is expressed in pollen grains from 4 days before anthesis till anther dehiscence and in pollinated carpels. In the PsPMEP promoter region, we have identified several conserved cis-regulatory elements that have been associated with gene pollen-specific expression. Expression analysis of PsPMEP promoter fused to the uidA reporter gene in Arabidopsis thaliana plants showed a similar expression pattern when compared with pea, indicating that this promoter is also functional in a non-leguminous plant. GUS expression was detected in mature pollen grains, during pollen germination, during pollen tube elongation along the transmitting tract, and when the pollen tube reaches the embryo sac in the ovule.

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

PubMed

Rajabi-Memari, H; Jalali-Javaran, M; Rasaee, M J; Rahbarizadeh, F; Forouzandeh-Moghadam, M; Esmaili, A

2006-08-01

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

Potential alternative hosts for a powdery mildew on pea

USDA-ARS?s Scientific Manuscript database

Powdery mildew of pea (Pisum sativum) is an important disease in the field and in the greenhouse. The most widely documented powdery mildew on pea is Erysiphe pisi, but E. trifolii and E. baeumleri have also been reported. From greenhouse-grown peas, we obtained powdery mildew samples with rDNA ITS ...

Heat Stress Response in Pea Involves Interaction of Mitochondrial Nucleoside Diphosphate Kinase with a Novel 86-Kilodalton Protein1

PubMed Central

Escobar Galvis, Martha L.; Marttila, Salla; Håkansson, Gunilla; Forsberg, Jens; Knorpp, Carina

2001-01-01

In this work we have further characterized the first mitochondrial nucleoside diphosphate kinase (mtNDPK) isolated from plants. The mitochondrial isoform was found to be especially abundant in reproductive and young tissues. Expression of the pea (Pisum sativum L. cv Oregon sugarpod) mtNDPK was not affected by different stress conditions. However, the pea mtNDPK was found to interact with a novel 86-kD protein, which is de novo synthesized in pea leaves upon exposure to heat. Thus, we have evidence for the involvement of mtNDPK in mitochondrial heat response in pea in vivo. Studies on oligomerization revealed that mtNDPK was found in complexes of various sizes, corresponding to the sizes of e.g. hexamers, tetramers, and dimers, indicating flexibility in oligomerization. This flexibility, also found for other NDPK isoforms, has been correlated with the ability of this enzyme to interact with other proteins. We believe that the mtNDPK is involved in heat stress response in pea, possibly as a modulator of the 86-kD protein. PMID:11351071

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

PubMed

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.

Characterization of Antibodies to Identify Cellular Expression of Dopamine Receptor 4.

PubMed

Deming, Janise D; Van Craenenbroeck, Kathleen; Eom, Yun Sung; Lee, Eun-Jin; Craft, Cheryl Mae

2016-01-01

The dopamine receptor D4 (DRD4) plays an important role in vision. In order to study the DRD4 expression in vivo, it is important to have antibodies that are specific for DRD4 for both immunoblot and immunohistochemical (IHC) applications. In this study, six antibodies raised against DRD4 peptides were tested in vitro, using transfected mammalian cells, and in vivo, using mouse retinas. Three Santa Cruz (SC) antibodies, D-16, N-20, and R-20, were successful in IHC of transfected DRD4; however, N-20 was the only one effective on immunoblot analysis in DRD4 transfected cells and IHC of mouse retinal sections, while R-20, 2B9, and Antibody Verify AAS63631C were non-specific or below detection.

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

PubMed Central

2011-01-01

Background For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation. Results By vacuum infiltration (0.08 Mpa, 1 min) of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF) was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration. Conclusions Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product. PMID:21548923

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

PubMed

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

2016-11-18

Understanding the molecular mechanisms involved in plant virus-vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus , Luteoviridae ) and Pea enation mosaic virus 2 (PEMV2, Umbravirus , Tombusviridae ) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum , and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum . Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

PubMed Central

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C.; Miller, W. Allen

2016-01-01

Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits. PMID:27869713

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

PubMed

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. © 2014 John Wiley & Sons Ltd.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bujak, Emil; Pretto, Francesca; Ritz, Danilo

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.more » 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

The level of HER2 expression is a predictor of antibody-HER2 trafficking behavior in cancer cells

PubMed Central

Ram, Sripad; Kim, Dongyoung; Ober, Raimund J; Ward, E Sally

2014-01-01

The receptor tyrosine kinase HER2 is known to play a central role in mitogenic signaling, motivating the development of targeted, HER2-specific therapies. However, despite the longstanding use of antibodies to target HER2, controversies remain concerning antibody/HER2 trafficking behavior in cancer cells. Understanding this behavior has direct relevance to the mechanism of action and effective design of such antibodies. In the current study, we analyzed the intracellular dynamics of trastuzumab, a marketed HER2-targeting antibody, in a panel of breast and prostate cancer cell lines that have a wide range of HER2 expression levels. Our results reveal distinct post-endocytic trafficking behavior of antibody-HER2 complexes in cells with different HER2 expression levels. In particular, HER2-overexpressing cells exhibit efficient HER2 recycling and limited reductions in HER2 levels upon antibody treatment, and consequently display a high level of antibody persistence on their plasma membrane. By contrast, in cells with low HER2 expression, trastuzumab treatment results in rapid antibody clearance from the plasma membrane combined with substantial decreases in HER2 levels and undetectable levels of recycling. A cell line with intermediate levels of HER2 expression exhibits both antibody recycling and clearance from the cell surface. Significantly, these analyses demonstrate that HER2 expression levels, rather than cell origin (breast or prostate), is a determinant of subcellular trafficking properties. Such studies have relevance to optimizing the design of antibodies to target HER2. PMID:25517306

PEA: an integrated R toolkit for plant epitranscriptome analysis.

PubMed

Zhai, Jingjing; Song, Jie; Cheng, Qian; Tang, Yunjia; Ma, Chuang

2018-05-29

The epitranscriptome, also known as chemical modifications of RNA (CMRs), is a newly discovered layer of gene regulation, the biological importance of which emerged through analysis of only a small fraction of CMRs detected by high-throughput sequencing technologies. Understanding of the epitranscriptome is hampered by the absence of computational tools for the systematic analysis of epitranscriptome sequencing data. In addition, no tools have yet been designed for accurate prediction of CMRs in plants, or to extend epitranscriptome analysis from a fraction of the transcriptome to its entirety. Here, we introduce PEA, an integrated R toolkit to facilitate the analysis of plant epitranscriptome data. The PEA toolkit contains a comprehensive collection of functions required for read mapping, CMR calling, motif scanning and discovery, and gene functional enrichment analysis. PEA also takes advantage of machine learning technologies for transcriptome-scale CMR prediction, with high prediction accuracy, using the Positive Samples Only Learning algorithm, which addresses the two-class classification problem by using only positive samples (CMRs), in the absence of negative samples (non-CMRs). Hence PEA is a versatile epitranscriptome analysis pipeline covering CMR calling, prediction, and annotation, and we describe its application to predict N6-methyladenosine (m6A) modifications in Arabidopsis thaliana. Experimental results demonstrate that the toolkit achieved 71.6% sensitivity and 73.7% specificity, which is superior to existing m6A predictors. PEA is potentially broadly applicable to the in-depth study of epitranscriptomics. PEA Docker image is available at https://hub.docker.com/r/malab/pea, source codes and user manual are available at https://github.com/cma2015/PEA. chuangma2006@gmail.com. Supplementary data are available at Bioinformatics online.

[Construction of the lentiviral expression vector for anti-p185(erbB2) mouse/human chimeric antibody].

PubMed

Liu, Fang; Li, Li; Zhang, Wei; Wang, Qi

2013-04-01

This research was to construct the lentiviral expression vector for anti- p185(erbB2) mouse/human chimeric antibody and to determine the expression of the chimeric antibody gene in 293T cells transfected with this vector. The genes (vL and vH) coding light and heavy chain of variable regions of anti-p185(erbB2) mAb and the constant regions of human IgG1 (kappa and gamma1) were cloned with PCR method. The target genes were assembled by three-primers PCR method to obtain the chimeric light chain (L) and the chimeric heavy chain (H). Both chains inserted into the down stream and upper stream of IRES gene of the plasmid pVAX1/IRES respectively. We digested the plasmid pVAX1/ H-IRES-L with endoenzyme and subcloned H-IRES-L into the lentiviral vector pWPI. The enzyme digestion and sequence analysis showed that the lentiviral expression vector pWPI/H-IRES-L was constructed correctly. Then, it was transfected into 293T cells and after 48h, GFP protein expression in 293T cells were detected by fluorescent microscope and the chimeric antibody expression was detected by RT-PCR and direct ELISA. The results showed that after 293T cells were transfected with recombination plasmid, both light and heavy chains of the chimeric antibody genes could express together. The chimeric antibody expressed could bind to p185(erbB2) specifically. This research may lay a sound foundation for further study of anti-p185(erbB2) engineered antibody.

Estimating demand for perennial pigeon pea in Malawi using choice experiments.

PubMed

Waldman, Kurt B; Ortega, David L; Richardson, Robert B; Snapp, Sieglinde S

2017-01-01

Perennial crops have numerous ecological and agronomic advantages over their annual counterparts. We estimate discrete choice models to evaluate farmers' preferences for perennial attributes of pigeon pea intercropped with maize in central and southern Malawi. Pigeon pea is a nitrogen-fixing leguminous crop, which has the potential to ameliorate soil fertility problems related to continuous maize cultivation, which are common in Southern Africa. Adoption of annual pigeon pea is relatively low but perennial production of pigeon pea may be more appealing to farmers due to some of the ancillary benefits associated with perenniality. We model perennial production of pigeon pea as a function of the attributes that differ between annual and perennial production: lower labor and seed requirements resulting from a single planting with multiple harvests, enhanced soil fertility and higher levels of biomass production. The primary tradeoff associated with perennial pigeon pea intercropped with maize is competition with maize in subsequent years of production. While maize yield is approximately twice as valuable to farmers as pigeon pea yield, we find positive yet heterogeneous demand for perenniality driven by soil fertility improvements and pigeon pea grain yield.

Growth parameters of vegetable pigeon pea cultivars

USDA-ARS?s Scientific Manuscript database

Pigeon pea is an important crop in the dry regions of eastern Kenya, due to its drought tolerance and high protein content; however, farmer’s yield is limiting. Ojwang et al. (HortTech Vol 26 (1), 2016) evaluated twelve pigeon pea cultivars for flowering, plant height, branches, pod length and yield...

Over expression of anti-MUC1 single-domain antibody fragments in the yeast Pichia pastoris.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad J; Forouzandeh, Mehdi; Allameh, Abdol-Amir

2006-02-01

The methylotrophic yeast Pichia pastoris has become a highly popular expression host system for the recombinant production of a wide variety of proteins, such as antibody fragments. Camelids produce functional antibodies devoid of light chains and constant heavy-chain domain (CH1). The antigen binding fragments of such heavy chain antibodies are therefore comprised in one single domain, the so-called VH of the camelid heavy chain antibody (VHH). To test the feasibility of expressing VHHs in the yeast, which on account of their small size and antigen recognition properties would have a major impact on antibody engineering strategies, we constructed two VHH genes encoding the single-domain antibody fragments with specificity for a cancer associated mucin, MUC1. The recombinant strains of the yeast P. pastoris were developed which secrete single-domain antibody fragment to the culture supernatant as a biologically active protein. Supplementation of medium with sorbitol (in pre-induction phase) and casamino acid or EDTA (in induction phase) provided ideal condition of increasing the yield of VHH production compared to culture condition devoid of above recipe. The secreted protein was purified following a 80% ammonium sulfate precipitation step, followed by a affinity chromatography column. The specific activity in enzyme-linked immunosorbant assay (ELISA) of the purified yeast VHH was higher than that of a bacterial periplasmic counterpart. These results reaffirm that the yeast P. pastoris is a suitable host for high level and correctly folded production of VHH antibody fragments with potential in vivo diagnostic and therapeutic applications. This is the first report of expression of VHH in P. pastoris.

Effect of feeding soybean meal and differently processed peas on intestinal morphology and functional glucose transport in the small intestine of broilers.

PubMed

Röhe, I; Boroojeni, F Goodarzi; Zentek, J

2017-09-01

Peas are locally grown legumes being rich in protein and starch. However, the broad usage of peas as a feed component in poultry nutrition is limited to anti-nutritional factors, which might impair gut morphology and function. This study investigated the effect of feeding raw or differently processed peas compared with feeding a soybean meal-based control diet (C) on intestinal morphology and nutrient transport in broilers. A total of 360 day-old broiler chicks were fed with one of the following diets: The C diet, and 3 diets containing raw peas (RP), fermented peas (FP) and enzymatically pre-digested peas (EP), each supplying 30% of dietary crude protein. After 35 d, jejunal samples of broilers were taken for analyzing histomorphological parameters, active glucose transport in Ussing chambers and the expression of genes related to glucose absorption, intestinal permeability and cell maturation. Villus length (P = 0.017) and crypt depth (P = 0.009) of EP-fed broilers were shorter compared to birds received C. The villus surface area was larger in broilers fed C compared to those fed with the pea-containing feed (P = 0.005). Glucose transport was higher for broilers fed C in comparison to birds fed with the EP diet (P = 0.044). The sodium-dependent glucose co-transporter 1 (SGLT-1) expression was down-regulated in RP (P = 0.028) and FP (P = 0.015) fed broilers. Correlation analyses show that jejunal villus length negatively correlates with the previously published number of jejunal intraepithelial T cells (P = 0.014) and that jejunal glucose transport was negatively correlated with the occurrence of jejunal intraepithelial leukocytes (P = 0.041). To conclude, the feeding of raw and processed pea containing diets compared to a soybean based diet reduced the jejunal mucosal surface area of broilers, which on average was accompanied by lower glucose transport capacities. These morphological and functional alterations were associated with observed mucosal immune

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

Code of Federal Regulations, 2010 CFR

2010-01-01

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

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

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 5 2013-01-01 2013-01-01 false Shelled garden peas from Kenya. 319.56-45 Section 319.56-45 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH... Shelled garden peas from Kenya. Garden peas (Pisum sativum) may be imported into the continental United...

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

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 5 2014-01-01 2014-01-01 false Shelled garden peas from Kenya. 319.56-45 Section 319.56-45 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH... Shelled garden peas from Kenya. Garden peas (Pisum sativum) may be imported into the continental United...

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

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 5 2012-01-01 2012-01-01 false Shelled garden peas from Kenya. 319.56-45 Section 319.56-45 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH... Shelled garden peas from Kenya. Garden peas (Pisum sativum) may be imported into the continental United...

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

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 5 2011-01-01 2011-01-01 false Shelled garden peas from Kenya. 319.56-45 Section 319.56-45 Agriculture Regulations of the Department of Agriculture (Continued) ANIMAL AND PLANT HEALTH... Shelled garden peas from Kenya. Garden peas (Pisum sativum) may be imported into the continental United...

Ly α and UV Sizes of Green Pea Galaxies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Huan; Wang, Junxian; Malhotra, Sangeeta

Green Peas are nearby analogs of high-redshift Ly α -emitting galaxies (LAEs). To probe their Ly α escape, we study the spatial profiles of Ly α and UV continuum emission of 24 Green Pea galaxies using the Cosmic Origins Spectrograph (COS) on the Hubble Space Telescope . We extract the spatial profiles of Ly α emission from their 2D COS spectra, and of the UV continuum from both 2D spectra and NUV images. The Ly α emission shows more extended spatial profiles than the UV continuum, in most Green Peas. The deconvolved full width at half maximum of the Lymore » α spatial profile is about 2–4 times that of the UV continuum, in most cases. Because Green Peas are analogs of high z LAEs, our results suggest that most high- z LAEs probably have larger Ly α sizes than UV sizes. We also compare the spatial profiles of Ly α photons at blueshifted and redshifted velocities in eight Green Peas with sufficient data quality, and find that the blue wing of the Ly α line has a larger spatial extent than the red wing in four Green Peas with comparatively weak blue Ly α line wings. We show that Green Peas and MUSE z = 3–6 LAEs have similar Ly α and UV continuum sizes, which probably suggests that starbursts in both low- z and high- z LAEs drive similar gas outflows illuminated by Ly α light. Five Lyman continuum (LyC) leakers in this sample have similar Ly α to UV continuum size ratios (∼1.4–4.3) to the other Green Peas, indicating that their LyC emissions escape through ionized holes in the interstellar medium.« less

Lyα and UV Sizes of Green Pea Galaxies

NASA Astrophysics Data System (ADS)

Yang, Huan; Malhotra, Sangeeta; Rhoads, James E.; Leitherer, Claus; Wofford, Aida; Jiang, Tianxing; Wang, Junxian

2017-03-01

Green Peas are nearby analogs of high-redshift Lyα-emitting galaxies (LAEs). To probe their Lyα escape, we study the spatial profiles of Lyα and UV continuum emission of 24 Green Pea galaxies using the Cosmic Origins Spectrograph (COS) on the Hubble Space Telescope. We extract the spatial profiles of Lyα emission from their 2D COS spectra, and of the UV continuum from both 2D spectra and NUV images. The Lyα emission shows more extended spatial profiles than the UV continuum, in most Green Peas. The deconvolved full width at half maximum of the Lyα spatial profile is about 2-4 times that of the UV continuum, in most cases. Because Green Peas are analogs of high z LAEs, our results suggest that most high-z LAEs probably have larger Lyα sizes than UV sizes. We also compare the spatial profiles of Lyα photons at blueshifted and redshifted velocities in eight Green Peas with sufficient data quality, and find that the blue wing of the Lyα line has a larger spatial extent than the red wing in four Green Peas with comparatively weak blue Lyα line wings. We show that Green Peas and MUSE z = 3-6 LAEs have similar Lyα and UV continuum sizes, which probably suggests that starbursts in both low-z and high-z LAEs drive similar gas outflows illuminated by Lyα light. Five Lyman continuum (LyC) leakers in this sample have similar Lyα to UV continuum size ratios (˜1.4-4.3) to the other Green Peas, indicating that their LyC emissions escape through ionized holes in the interstellar medium.

Insecticidal components from field pea extracts: sequences of some variants of pea albumin 1b.

PubMed

Taylor, Wesley G; Sutherland, Daniel H; Olson, Douglas J H; Ross, Andrew R S; Fields, Paul G

2004-12-15

Methanol soluble insecticidal peptides with masses of 3752, 3757, and 3805 Da, isolated from crude extracts (C8 extracts) derived from the protein-enriched flour of commercial field peas [Pisum sativum (L.)], were purified by reversed phase chromatography and, after reduction and alkylation, were sequenced by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry with the aid of various peptidases. These major peptides were variants of pea albumin 1b (PA1b) with methionine sulfoxide rather than methionine at position 12. Peptide 3752 showed additional variations at positions 29 (valine for isoleucine) and 34 (histidine for asparagine). A minor, 37 amino acid peptide with a molecular mass of 3788 Da was also sequenced and differed from a known PA1b variant at positions 1, 25, and 31. Sequence variants of PA1b with their molecular masses were compiled, and variants that matched the accurate masses of the experimental peptides were used to narrow the search. MALDI postsource decay experiments on pronase fragments helped to confirm the sequences. Whole and dehulled field peas gave insecticidal C8 extracts in the laboratory that were enriched in peptides with masses of 3736, 3741, and 3789 Da, as determined by high-performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry. It was therefore concluded that oxidation of the methionine residues to methionine sulfoxide occurred primarily during the processing of dehulled peas in a mill.

Expression of enhancing-activity-free neutralizing antibody against dengue type 1 virus in plasmid-inoculated mice.

PubMed

Yamanaka, Atsushi; Pitaksajjakul, Pannamthip; Ramasoota, Pongrama; Konishi, Eiji

2015-11-09

Most candidate dengue vaccines currently under development induce neutralizing antibodies, which are considered important for immunoprotection. However, the concomitant induction of infection-enhancing antibodies is an unavoidable concern. In contrast, a neutralizing antibody developed for passive immunotherapy has been engineered to eliminate its enhancing activity. Therefore, a strategy for the long-term expression of enhancing-activity-free neutralizing antibodies may resolve this concern. A mouse monoclonal antibody, 7F4, of the IgG3 subclass and with no detectable enhancing activity, was selected as the model neutralizing antibody to evaluate the potential of this strategy. Equal amounts of commercial vector (pFUSE)-based plasmids containing 7F4 heavy (H)- or light (L)-chain variable region genes were mixed and used for the cotransfection of 293T cells and co-delivery into ICR and BALB/c mice. The recombinant plasmids were designed to express IgG2b or IgG3 subclass antibodies (p7F4G2b or p7F4G3, respectively). 293T cells transfected with 2 μg of p7F4G2b or p7F4G3 produced approximately 15,000 or 800 ng/ml IgG in the culture fluids, respectively. The dose is expressed as the total amount of H- and L-chain plasmids. Neutralizing antibody was detected dose-dependently in ICR mice inoculated with 50-200 μg of p7F4G2b. A 1:2 dilution of sera from ICR and BALB/c mice inoculated with 100 μg of p7F4G3 showed average plaque reduction levels of >70% on day 3 and >90% on days 5-9. BALB/c mice maintained detectable neutralizing antibody for at least 3 months. The neutralizing antibody expressed by p7F4G3 in mice showed no enhancing activity. Although the expression of neutralizing antibodies from immunoglobulin genes is a type of passive immunization, its durability can be utilized as a dengue vaccine strategy. This "proof-of-concept" study using a mouse model demonstrates that the enhancing-activity-free characteristic of this strategy augurs well for dengue vaccine

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

PubMed

Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

2003-05-01

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

Pea Compound Leaf Architecture Is Regulated by Interactions among the Genes UNIFOLIATA, COCHLEATA, AFILA, and TENDRIL-LESS

PubMed Central

Gourlay, Campbell W.; Hofer, Julie M. I.; Ellis, T. H. Noel

2000-01-01

The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea. PMID:10948249

2-Pentadecyl-2-Oxazoline, the Oxazoline of Pea, Modulates Carrageenan-Induced Acute Inflammation

PubMed Central

Petrosino, Stefania; Campolo, Michela; Impellizzeri, Daniela; Paterniti, Irene; Allarà, Marco; Gugliandolo, Enrico; D’Amico, Ramona; Siracusa, Rosalba; Cordaro, Marika; Esposito, Emanuela; Di Marzo, Vincenzo; Cuzzocrea, Salvatore

2017-01-01

N-acylethanolamines (NAEs) involve a family of lipid molecules existent in animal and plant, with N-palmitoylethanolamide (PEA) that arouses great attention owing to its anti-inflammatory, analgesic and neuroprotective activities. Because PEA is produced on demand and exerts pleiotropic effects, the modulation of specific amidases for NAEs (and in particular NAE-hydrolyzing acid amidase NAAA, which is more selective for PEA) could be a condition to preserve its levels. Here we investigate the effect of 2-Pentadecyl-2-oxazoline (PEA-OXA) the oxazoline of PEA, on human recombinant NAAA in vitro and in an established model of Carrageenan (CAR)-induced rat paw inflammation. PEA-OXA dose-dependently significantly inhibited recombinant NAAA and, orally administered to rats (10 mg/kg), limiting histological damage, thermal hyperalgesia and the increase of infiltrating inflammatory cells after CAR injection in the rat right hindpaw, compared to ultramicronized PEA given orally at the same dose (10 mg/kg). These effects were accompanied by elevation of paw PEA levels. Moreover, PEA-OXA markedly reduced neutrophil infiltration and pro-inflammatory cytokine release and prevented CAR-induced IκB-α degradation, nuclear translocation of NF-κB p65, the increase of inducible nitric oxide synthase, cyclooxygenase-2, intercellular adhesion molecule-1, and mast cell activation. Experiments in PPAR-α knockout mice showed that the anti-inflammatory effects of PEA-OXA were not dependent on the presence of PPAR-α receptors. In conclusion, NAAA modulators as PEA-OXA could help to maximize the tissue availability of PEA by increasing its levels and anti-inflammatory effects. PMID:28611664

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

PubMed

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.

Light regulation of gibberellin biosynthesis in pea is mediated through the COP1/HY5 pathway.

PubMed

Weller, James L; Hecht, Valérie; Vander Schoor, Jacqueline K; Davidson, Sandra E; Ross, John J

2009-03-01

Light regulation of gibberellin (GA) biosynthesis occurs in several species, but the signaling pathway through which this occurs has not been clearly established. We have isolated a new pea (Pisum sativum) mutant, long1, with a light-dependent elongated phenotype that is particularly pronounced in the epicotyl and first internode. The long1 mutation impairs signaling from phytochrome and cryptochrome photoreceptors and interacts genetically with a mutation in LIP1, the pea ortholog of Arabidopsis thaliana COP1. Mutant long1 seedlings show a dramatic impairment in the light regulation of active GA levels and the expression of several GA biosynthetic genes, most notably the GA catabolism gene GA2ox2. The long1 mutant carries a nonsense mutation in a gene orthologous to the ASTRAY gene from Lotus japonicus, a divergent ortholog of the Arabidopsis bZIP transcription factor gene HY5. Our results show that LONG1 has a central role in mediating the effects of light on GA biosynthesis in pea and demonstrate the importance of this regulation for appropriate photomorphogenic development. By contrast, LONG1 has no effect on GA responsiveness, implying that interactions between LONG1 and GA signaling are not a significant component of the molecular framework for light-GA interactions in pea.

Expansion of Genes Encoding piRNA-Associated Argonaute Proteins in the Pea Aphid: Diversification of Expression Profiles in Different Plastic Morphs

PubMed Central

Lu, Hsiao-ling; Tanguy, Sylvie; Rispe, Claude; Gauthier, Jean-Pierre; Walsh, Tom; Gordon, Karl; Edwards, Owain; Tagu, Denis; Chang, Chun-che; Jaubert-Possamai, Stéphanie

2011-01-01

Piwi-interacting RNAs (piRNAs) are known to regulate transposon activity in germ cells of several animal models that propagate sexually. However, the role of piRNAs during asexual reproduction remains almost unknown. Aphids that can alternate sexual and asexual reproduction cycles in response to seasonal changes of photoperiod provide a unique opportunity to study piRNAs and the piRNA pathway in both reproductive modes. Taking advantage of the recently sequenced genome of the pea aphid Acyrthosiphon pisum, we found an unusually large lineage-specific expansion of genes encoding the Piwi sub-clade of Argonaute proteins. In situ hybridisation showed differential expressions between the duplicated piwi copies: while Api-piwi2 and Api-piwi6 are “specialised” in germ cells their most closely related copy, respectively Api-piwi5 and Api-piwi3, are expressed in the somatic cells. The differential expression was also identified in duplicated ago3: Api-ago3a in germ cells and Api-ago3b in somatic cells. Moreover, analyses of expression profiles of the expanded piwi and ago3 genes by semi-quantitative RT-PCR showed that expressions varied according to the reproductive types. These specific expression patterns suggest that expanded aphid piwi and ago3 genes have distinct roles in asexual and sexual reproduction. PMID:22162754

The Pisum Genus: Getting out of Pea Soup!

USDA-ARS?s Scientific Manuscript database

Pea (Pisum sativum L.) has long been a model for plant genetics and is a widely grown pulse crop producing protein-rich seeds in a sustainable manner. However, many questions remain open about (sub)species relationships in the Pisumgenus. The ongoing pea genome sequencing project and the recent geno...

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

PubMed

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. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Naturally occurring anti-glycan antibodies binding to Globo H-expressing cells identify ovarian cancer patients.

PubMed

Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola

2017-02-10

Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P < 0.05). A combination with the clinically used tumour marker CA125 increased the diagnostic performance (AUC 0.8711). We next compared suspension array with standard flow cytometry in plasma samples and found that the level of anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.

A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors

PubMed Central

Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Sysa-Shah, Polina; Azad, Babak Behnam; Pomper, Martin G.; Nimmagadda, Sridhar

2016-01-01

Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings. PMID:26848870

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

PubMed Central

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

Characterization of pea (Pisum sativum) seed protein fractions.

PubMed

Rubio, Luis A; Pérez, Alicia; Ruiz, Raquel; Guzmán, M Ángeles; Aranda-Olmedo, Isabel; Clemente, Alfonso

2014-01-30

Legume seed proteins have to be chemically characterized in order to properly link their nutritional effects with their chemical structure. Vicilin and albumin fractions devoid of cross-contamination, as assessed by mass peptide fingerprinting analysis, were obtained from defatted pea (Pisum sativum cv. Bilbo) meal. The extracted protein fractions contained 56.7-67.7 g non-starch polysaccharides kg⁻¹. The vicilin fraction was higher than legumins in arginine, isoleucine, leucine, phenylalanine and lysine. The most abundant amino acids in the albumin fraction were aspartic acid, glutamic acid, lysine and arginine, and the amounts of methionine were more than double than those in legumins and vicilins. The pea albumin fraction showed a clear enrichment of protease inhibitory activity when compared with the seed meal. In vitro digestibility values for pea proteins were 0.63 ±  0.04, 0.88 ±  0.04 and 0.41 ±  0.23 for legumins, vicilins and albumins respectively. Vicilin and albumin fractions devoid of cross-contamination with other proteins were obtained from pea seed meal. The vicilin fraction also contained low amounts of soluble non-starch polysaccharides and was enriched in isoleucine, leucine, phenylalanine and lysine. In vitro digestibility values for pea proteins were similar or even numerically higher than those for control proteins. © 2013 Society of Chemical Industry.

Potential alternative hosts for the pea powdery mildew pathogen Erysiphe trifolii

USDA-ARS?s Scientific Manuscript database

Powdery mildew of pea (Pisum sativum) is an important disease in the field and in the greenhouse. The most widely documented powdery mildew pathogen on pea is Erysiphe pisi, but E. baeumleri and E. trifolii have also been reported. We recently showed that E. trifolii is frequently found on pea in th...

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

PubMed Central

2011-01-01

Background The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. Results Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. Conclusions Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants. PMID:22018401

Hypolipidemic effect of dietary pea proteins: Impact on genes regulating hepatic lipid metabolism.

PubMed

Rigamonti, Elena; Parolini, Cinzia; Marchesi, Marta; Diani, Erika; Brambilla, Stefano; Sirtori, Cesare R; Chiesa, Giulia

2010-05-01

Controversial data on the lipid-lowering effect of dietary pea proteins have been provided and the mechanisms behind this effect are not completely understood. The aim of the study was to evaluate a possible hypolipidemic activity of a pea protein isolate and to determine whether pea proteins could affect the hepatic lipid metabolism through regulation of genes involved in cholesterol and fatty acid homeostasis. Rats were fed Nath's hypercholesterolemic diets for 28 days, the protein sources being casein or a pea protein isolate from Pisum sativum. After 14 and 28 days of dietary treatment, rats fed pea proteins had markedly lower plasma cholesterol and triglyceride levels than rats fed casein (p<0.05). Pea protein-fed rats displayed higher hepatic mRNA levels of LDL receptor versus those fed casein (p<0.05). Hepatic mRNA concentration of genes involved in fatty acids synthesis, such as fatty acid synthase and stearoyl-CoA desaturase, was lower in pea protein-fed rats than in rats fed casein (p<0.05). In conclusion, the present study demonstrates a marked cholesterol and triglyceride-lowering activity of pea proteins in rats. Moreover, pea proteins appear to affect cellular lipid homeostasis by upregulating genes involved in hepatic cholesterol uptake and by downregulating fatty acid synthesis genes.

A Bivalent, Chimeric Rabies Virus Expressing Simian Immunodeficiency Virus Envelope Induces Multifunctional Antibody Responses.

PubMed

Dunkel, Amber; Shen, Shixue; LaBranche, Celia C; Montefiori, David; McGettigan, James P

2015-11-01

We previously showed that a matrix (M) gene-deleted rabies virus (RABV)-based vaccine (RABV-ΔM) is highly immunogenic and induces potent B cell responses in the context of RABV infection. We speculated that RABV-ΔM expressing HIV proteins would also induce potent B cell responses against HIV antigens. As a prerequisite to future studies in nonhuman primates, we completed immunogenicity studies in mice to confirm the ability of RABV-ΔM to induce polyfunctional B cell responses in the context of HIV. To that end, the envelope protein from the mac239 strain of SIV (SIVmac239Env) was cloned into RABV-ΔM, resulting in RABV-ΔM-Env. Infectious virus was recovered following standard methods and propagated on baby hamster kidney cells stably expressing RABV M [>10(7) focus forming units (ffu)/ml]. Western blot analysis of cell lysates or of purified virions confirmed Env expression on the surface of infected cells and within virus particles, respectively. Positive neutralization activity against a neutralization-sensitive SIV strain and to a lesser extent against a neutralization-resistant SIV strain was detected in mice after a single intramuscular inoculation with RABV-ΔM-Env. The quality, but not quantity, of the antibody response was enhanced via boosting with recombinant gp130 or RABV-ΔM-Env as measured by an increase in antibody avidity and a skewing toward a Th1-type antibody response. We also show that an intradermal inoculation induces higher antibodies than an intramuscular or intranasal inoculation. An intradermal inoculation of RABV-ΔM-Env followed by a boost inoculation with recombinant gp130 produced anti-SIV antibodies with neutralizing and nonneutralizing antibody (nNAb) effector functions. Together, RABV-ΔM-Env induces B cells to secrete antibodies against SIV with the potential to clear both "free" and cell-associated virus. Strategies capable of eliciting both NAbs as well as nNAbs might help to improve the efficacy of HIV-1 vaccines.

Effect of pig bodyweight on ileal amino acid endogenous losses after ingestion of a protein-free diet enriched in pea inner fibre isolates.

PubMed

Leterme, Pascal; Théwis, André

2004-01-01

The present study was conducted to evaluate whether bodyweight and the micronisation of dietary fibre affect the endogenous nitrogen and amino acid losses (ENL and EAAL) in pigs. The effect of the micronising process was tested by providing pigs with 90 g DM x kg(-1) BW0.75 of a N-free diet supplemented with isolated pea inner fibres, presented in native or micronised form and with a water-holding capacity of 12 and 4 g water g(-1) DM, respectively. ENL and EAAL were measured on pigs weighing 24, 62 and 105 kg. In all cases, daily ENL increased linearly (P < 0.05) with BW, for the majority of the AA and total N. As BW increased, daily ENL, total EAAL and the majority of EAAL increased linearly independently of micronisation (P < 0.05). When expressed per kg DMI, total EAAL and the majority of each EAA decreased curvilinearly and reached nadir at around 100 kg BW. For ENL expressed per kg DMI, micronisation resulted in a curvilinear decrease with increasing BW, as compared to a linear decrease for pigs fed the native pea fibre diet (non-micronised). Micronisation of pea inner fibres did not decrease ENL or EAAL daily, except for proline. When the losses were expressed as g x k(-1)g DMI, micronisation did not decrease ENL but decreased (P < 0.05) endogenous losses for a majority of AA as well as for total AA. The results suggest that small pigs excrete more endogenous N per kg DMI than large pigs and that pea fibre micronisation reduces EAAL but not ENL when expressed per kg DMI.

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

PubMed

Maggi, Maristella; Scotti, Claudia

2017-06-01

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

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

NASA Technical Reports Server (NTRS)

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.

THE ORIGIN AND OPTICAL DEPTH OF IONIZING RADIATION IN THE 'GREEN PEA' GALAXIES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaskot, A. E.; Oey, M. S.

2013-04-01

Although Lyman-continuum (LyC) radiation from star-forming galaxies likely drove the reionization of the universe, observations of star-forming galaxies at low redshift generally indicate low LyC escape fractions. However, the extreme [O III]/[O II] ratios of the z = 0.1-0.3 Green Pea galaxies may be due to high escape fractions of ionizing radiation. To analyze the LyC optical depths and ionizing sources of these rare, compact starbursts, we compare nebular photoionization and stellar population models with observed emission lines in the Peas' Sloan Digital Sky Survey (SDSS) spectra. We focus on the six most extreme Green Peas, the galaxies with themore » highest [O III]/[O II] ratios and the best candidates for escaping ionizing radiation. The Balmer line equivalent widths and He I {lambda}3819 emission in the extreme Peas support young ages of 3-5 Myr, and He II {lambda}4686 emission in five extreme Peas signals the presence of hard ionizing sources. Ionization by active galactic nuclei or high-mass X-ray binaries is inconsistent with the Peas' line ratios and ages. Although stacked spectra reveal no Wolf-Rayet (WR) features, we tentatively detect WR features in the SDSS spectra of three extreme Peas. Based on the Peas' ages and line ratios, we find that WR stars, chemically homogeneous O stars, or shocks could produce the observed He II emission. If hot stars are responsible, then the Peas' optical depths are ambiguous. However, accounting for emission from shocks lowers the inferred optical depth and suggests that the Peas may be optically thin. The Peas' ages likely optimize the escape of LyC radiation; they are old enough for supernovae and stellar winds to reshape the interstellar medium, but young enough to possess large numbers of UV-luminous O or WR stars.« less

7 CFR 457.140 - Dry pea crop insurance provisions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 6 2010-01-01 2010-01-01 false Dry pea crop insurance provisions. 457.140 Section 457.140 Agriculture Regulations of the Department of Agriculture (Continued) FEDERAL CROP INSURANCE CORPORATION, DEPARTMENT OF AGRICULTURE COMMON CROP INSURANCE REGULATIONS § 457.140 Dry pea crop insurance...

7 CFR 457.137 - Green pea crop insurance provisions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 6 2010-01-01 2010-01-01 false Green pea crop insurance provisions. 457.137 Section 457.137 Agriculture Regulations of the Department of Agriculture (Continued) FEDERAL CROP INSURANCE CORPORATION, DEPARTMENT OF AGRICULTURE COMMON CROP INSURANCE REGULATIONS § 457.137 Green pea crop insurance...

1. NORTHWEST OBLIQUE AERIAL VIEW OF FORT DELAWARE AND PEA ...

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

1. NORTHWEST OBLIQUE AERIAL VIEW OF FORT DELAWARE AND PEA PATCH ISLAND. REMAINS OF SEA WALL VISIBLE IN FOREGROUND AND RIGHT OF IMAGE. - Fort Delaware, Sea Wall, Pea Patch Island, Delaware City, New Castle County, DE

Studies of thermostability in Camelus bactrianus (Bactrian camel) single-domain antibody specific for the mutant epidermal-growth-factor receptor expressed by Pichia.

PubMed

Omidfar, Kobra; Rasaee, Mohhamad Javad; Kashanian, Soheila; Paknejad, Malieheh; Bathaie, Zahra

2007-01-01

Camelids have a unique immune system capable of producing heavy-chain antibodies lacking the light chains and CH1 (constant heavy-chain domain 1). It has been shown that, in contrast with conventional antibody fragments, the variable domains of these heavy-chain antibodies are functional at or after exposure to high temperatures. In the present study, the VHH (variable domain of heavy-chain antibody) camel antibody was subcloned into vector Ppiczc and expressed in Pichia pastoris. ORB1-83 VHH antibody recognizes the external domain of the mutant EGFR [EGF (epidermal growth factor) receptor], EGFR VIII. This tumour-specific antigen is ligand-independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. We report here that, although expression from P. pastoris resulted in a significantly increased level of expression of the anti-EGFR VIII VHH antibodies compared with Escherichia coli [Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani, Bakhtiari, Paknejad and Kashanian (2004) Tumor Biol. 25, 179-187; Omidfar, Rasaee, Modjtahedi, Forouzandeh, Taghikhani and Golmakany (2004) Tumor Biol. 25, 296-305], this antibody selectively bound to the EGFR VIII peptide and reacted specifically with the immunoaffinity-purified antigen from non-small-cell lung cancer. Furthermore, thermal denaturation stability and CD spectra analysis of the Camelus bactrianus (Bactrian camel) VHH and heavy-chain antibodies at different temperature proved reversibility and binding activity after heat denaturation. Our results indicate that the P. pastoris expression system may be useful for the expression of camel single domain antibody and the ability of the expressed protein to reversibly melt without aggregation, allowing it to regain binding activity after heat denaturation.

Genome Sequence of the Pea Aphid Acyrthosiphon pisum

PubMed Central

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 highlight findings from whole genome analysis that may be related to these unusual biological features. These findings include discovery of extensive gene duplication in more than 2000 gene families as well as loss of evolutionarily conserved genes. Gene family expansions relative to other published genomes 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 from bacteria; thus the reduced gene count of Buchnera does not reflect gene transfer to the host genome. The inventory of metabolic genes in the pea aphid genome suggests that there is extensive metabolite exchange between the aphid and Buchnera, including sharing of amino acid biosynthesis between the aphid and Buchnera. The pea aphid genome provides a foundation for post-genomic studies of fundamental biological questions and applied agricultural problems. PMID:20186266

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

KASP assays for powdery mildew resistance breeding in pea

USDA-ARS?s Scientific Manuscript database

Powdery mildew of pea, caused by Erysiphe pisi DC, is a serious production constraint to pea (Pisum sativum L.) production in the U.S. and elsewhere. Utilization of genetic resistance to powdery mildew using er1 has been an effective strategy to manage this disease. This gene, er1, conferring powde...

CD22 expression on blastic plasmacytoid dendritic cell neoplasms and reactivity of anti-CD22 antibodies to peripheral blood dendritic cells.

PubMed

Reineks, Edmunds Z; Osei, Ebenezer S; Rosenberg, Arlene; Auletta, Jeffrey; Meyerson, Howard J

2009-07-01

We identified CD22 expression on a blastic plasmacytoid dendritic cell (pDC) neoplasm presenting as a leukemia in a child. CD22 expression, as determined by the antibody s-HCL-1, was also noted on the neoplastic cells from three additional patients with blastic pDC tumors identified at our institution. Subsequently we determined that peripheral blood pDCs react with the s-HCL-1 antibody demonstrating that normal pDCs express CD22. Evaluation of five additional anti-CD22 antibodies indicated that staining of pDCs with these reagents was poor except for s-HCL-1. Therefore, the detection of CD22 on pDCs is best demonstrated with the use of this specific antibody clone. All anti-CD22 antibodies stained conventional DCs. We also evaluated the reactivity of the anti-CD22 antibodies with basophils and noted that the pattern of staining was similar to that seen with pDCs. The studies demonstrate that normal DCs and pDC neoplasms express CD22, and highlight clone specific differences in anti-CD22 antibody reactivity patterns on pDCs and basophils. (c) 2009 Clinical Cytometry Society.

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody.

PubMed

Leung, Shui-On; Gao, Kai; Wang, Guang Yu; Cheung, Benny Ka-Wa; Lee, Kwan-Yeung; Zhao, Qi; Cheung, Wing-Tai; Wang, Jun Zhi

2015-01-01

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03. Apart from its being used as a surrogate antigen for monitoring SM03 binding affinities, the anti-idiotype antibody was engineered to express as fusion proteins on cell surfaces in a non-internalizing manner, and the engineered cells were used as novel "surrogate target cells" for SM03. SM03-induced complement-mediated cytotoxicity (CMC) against these "surrogate target cells" proved to be an effective bioassay for monitoring changes in Fc functions, including those resulting from minor structural modifications borne within the Fc-appended carbohydrates. The approach can be generally applied for antibodies that target rapidly internalizing or non-surface bound antigens. The combined use of the anti-idiotype antibody and the surrogate target cells could help evaluate clinical parameters associated with safety and efficacies, and possibly the mechanisms of action of SM03.

Effects of ionizing radiation on sensorial, chemical, and microbiological quality of frozen corn and peas.

PubMed

Fan, Xuetong; Sokorai, Kimberly J B

2007-08-01

The effects of irradiation (0, 1.8, and 4.5 kGy) on the quality of frozen corn and peas were investigated during a 12month period of postirradiation storage at -18 degrees C. Irradiation of frozen corn and peas caused a reduction in ascorbic acid content of both vegetables and a loss of texture in peas but had no significant effects on instrumental color parameters (L*, a*, and b*), carotenoid and chlorophyll content, or antioxidant capacity of corn and peas. Irradiation reduced microbial loads of frozen peas and increased display life at 23 degrees C of thawed peas by preserving the green color, apparently because of slower increases in the population of acid-producing microorganisms in the irradiated samples. Overall, irradiation significantly reduced the microbial load and increased the display life of peas and had minimal detrimental effects on the quality of frozen corn and peas.

Divergent Pseudomonas exotoxin A sensitivity in normal and transformed liver cells is correlated with low-density lipoprotein receptor-related protein expression.

PubMed

Laithwaite, J E; Benn, S J; Marshall, W S; FitzGerald, D J; LaMarre, J

2001-09-01

Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic human pathogen Pseudomonas aerguinosa. PEA intoxification begins when PEA binds to the low-density lipoprotein receptor-related protein (LRP). The liver is the primary target of systemic PEA, due largely to the high levels of functional LRP expressed by liver cells. Using a 3H-leucine incorporation assay to measure inhibition of protein synthesis we have demonstrated that normal (BNL CL.2) and transformed (BNL 1ME A7R.1) liver cells exhibit divergent PEA sensitivity; with BNL 1ME A7R.1 cells demonstrating greater PEA sensitivity than their non-transformed counterparts. The receptor-associated protein, a LRP antagonist, decreased PEA toxicity in BNL 1ME A7R.1 cells, confirming the importance of the LRP in PEA intoxification in this cell type. Increased PEA sensitivity in BNL 1ME A7R.1 cells was associated with increased functional cell surface LRP expression, as measured by alpha2-macroglobulin binding and internalization studies, and increased LRP mRNA levels, as determined by Northern blot analysis. Interestingly, BNL CL.2 cells were more sensitive than BNL 1ME A7R.1 cells to conjugate and mutant PEA toxins that do not utilize the LRP for cellular entry. These data demonstrate that increased LRP expression is an important mechanism by which PEA sensitivity is increased in BNL 1ME A7R.1 transformed liver cells.

Expression, purification and antibody preparation of PCV2 Rep and ORF3 proteins.

PubMed

Peng, Zhiyuan; Ma, Teng; Pang, Daxin; Su, Dan; Chen, Fuwang; Chen, Xinrong; Guo, Ning; Ouyang, Ting; Ouyang, Hongsheng; Ren, Linzhu

2016-05-01

Rep and ORF3 proteins are important functional proteins of porcine circovirus 2 (PCV2). Here, Rep and ORF3 genes were cloned, expressed and used to raise polyclonal antibodies. The result showed the recombinant plasmids of Rep and ORF3 genes constructed in this study were expressed efficiently in the prokaryotic system, and the recombinant proteins had antigenicity and immunogenicity. Furthermore, reactivity and specificity of the antiserums were characterized by western blot and indirect immunofluorescent assays. The results elucidated that polyclonal antiserum prepared with Rep or ORF3 had good reactivity and specificity against PCV2, or the Rep and ORF3 expressed in PK-15 cells, respectively. The Rep protein is promising for PCV2 antibody and vaccine development. These results will be helpful for further studies focusing on pathogenesis of PCV2 and serology diagnostic test or vaccine development against PCV2. Copyright © 2016 Elsevier B.V. All rights reserved.

Stamina pistilloida, the Pea Ortholog of Fim and UFO, Is Required for Normal Development of Flowers, Inflorescences, and Leaves

PubMed Central

Taylor, Scott; Hofer, Julie; Murfet, Ian

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

Ersiphe trifolii-a newly recognized powdery mildew pathogen of pea.

USDA-ARS?s Scientific Manuscript database

Population diversity of powdery mildews infecting pea (Pisum sativum) in the US Pacific Northwest was investigated in order to assess inconsistent resistance performances of pea genotypes in different environments. Phylogenetic analyses based on ITS sequences, in combination with assessment of morph...

76 FR 37136 - Post-Entry Amendment (PEA) Processing Test: Modification, Clarification, and Extension

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-24

.... Customs and Border Protection's (CBP's) Post-Entry Amendment (PEA) Processing test, which allows the...: The Post-Entry Amendment (PEA) Processing test modification set forth in this document is effective...: Background I. Post-Entry Amendment Processing Test Program The Post-Entry Amendment (PEA) Processing test...

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

PubMed

Shukla, Rahul; Rajpoot, Ravi K; Arora, Upasana; Poddar, Ankur; Swaminathan, Sathyamangalam; Khanna, Navin

2017-01-01

Dengue, a significant public health problem in several countries around the world, is caused by four different serotypes of mosquito-borne dengue viruses (DENV-1, -2, -3, and -4). Antibodies to any one DENV serotype which can protect against homotypic re-infection, do not offer heterotypic cross-protection. In fact, cross-reactive antibodies may augment heterotypic DENV infection through antibody-dependent enhancement (ADE). A recently launched live attenuated vaccine (LAV) for dengue, which consists of a mixture of four chimeric yellow-fever/dengue vaccine viruses, may be linked to the induction of disease-enhancing antibodies. This is likely related to viral interference among the replicating viral strains, resulting in an unbalanced immune response, as well as to the fact that the LAV encodes prM, a DENV protein documented to elicit ADE-mediating antibodies. This makes it imperative to explore the feasibility of alternate ADE risk-free vaccine candidates. Our quest for a non-replicating vaccine centered on the DENV envelope (E) protein which mediates virus entry into the host cell and serves as an important target of the immune response. Serotype-specific neutralizing epitopes and the host receptor recognition function map to E domain III (EDIII). Recently, we found that Pichia pastoris -expressed DENV E protein, of all four serotypes, self-assembled into virus-like particles (VLPs) in the absence of prM. Significantly, these VLPs displayed EDIII and elicited EDIII-focused DENV-neutralizing antibodies in mice. We now report the creation and characterization of a novel non-replicating recombinant particulate vaccine candidate, produced by co-expressing the E proteins of DENV-1 and DENV-2 in P. pastoris . The two E proteins co-assembled into bivalent mosaic VLPs (mVLPs) designated as mE1E2 bv VLPs. The mVLP, which preserved the serotype-specific antigenic integrity of its two component proteins, elicited predominantly EDIII-focused homotypic virus

Identification of a cis-Regulatory Element Involved in Phytochrome Down-Regulated Expression of the Pea Small GTPase Gene pra21

PubMed Central

Inaba, Takehito; Nagano, Yukio; Sakakibara, Toshihiro; Sasaki, Yukiko

1999-01-01

The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5′-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5′- and internal deletion analyses revealed that the 93-bp sequence between −734 and −642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression. PMID:10364400

A peptide that binds the pea aphid gut impedes entry of Pea enation mosaic virus into the aphid hemocoel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu Sijun, E-mail: sliu@iastate.ed; Sivakumar, S., E-mail: sivaento@iastate.ed; Sparks, Wendy O., E-mail: wosparks@iastate.ed

2010-05-25

Development of ways to block virus transmission by aphids could lead to novel and broad-spectrum means of controlling plant viruses. Viruses in the Luteoviridae enhanced are obligately transmitted by aphids in a persistent manner that requires virion accumulation in the aphid hemocoel. To enter the hemocoel, the virion must bind and traverse the aphid gut epithelium. By screening a phage display library, we identified a 12-residue gut binding peptide (GBP3.1) that binds to the midgut and hindgut of the pea aphid Acyrthosiphon pisum. Binding was confirmed by labeling the aphid gut with a GBP3.1-green fluorescent protein fusion. GBP3.1 reduced uptakemore » of Pea enation mosaic virus (Luteoviridae) from the pea aphid gut into the hemocoel. GBP3.1 also bound to the gut epithelia of the green peach aphid and the soybean aphid. These results suggest a novel strategy for inhibiting plant virus transmission by at least three major aphid pest species.« less

Prostaglandin E1 reduces the glomerular mRNA expression of monocyte-chemoattractant protein 1 in anti-thymocyte antibody-induced glomerular injury.

PubMed

Jocks, T; Zahner, G; Freudenberg, J; Wolf, G; Thaiss, F; Helmchen, U; Stahl, R A

1996-06-01

To study whether prostaglandins (PG) can regulate the mRNA expression of monocyte-chemoattractant protein 1 (MCP-1) in glomerular immune injury, MCP-1 mRNA levels were evaluated in anti-thymocyte antibody (ATS) -induced glomerular injury by Northern blotting and reverse transcription-polymerase chain reaction. Immune injury was induced in vivo by the intravenous application of ATS to male Wistar rats and in vitro by the perfusion of isolated rat kidneys with ATS and rat serum. In vivo 3 h and 5 days after antibody application, glomerular mRNA expression of MCP-1 was markedly enhanced compared with controls. In the isolated perfused kidney, antibody and complement also induced an increase in MCP-1 expression at 10 min and 60 min after antibody perfusion. When the rats were treated with PGE (250 micrograms, twice daily), the increase in MCP-1 expression was reduced. This was associated with a reduction of intraglomerular recruitment of monocytes/macrophages. In the isolated perfused kidneys, PGE1 (1 mg/L) prevented the antibody- and rat serum-stimulated increase in glomerular MCP-1 mRNA expression. These data demonstrate that PGE1 reduces glomerular MCP-1 mRNA expression in glomerulonephritis and in the isolated perfused rat kidney after induction of immune injury with antibody and complement. The data suggest that prostaglandins might mediate MCP-1 effects in glomerular immune injuries.

Addition of sucralose enhances the release of satiety hormones in combination with pea protein.

PubMed

Geraedts, Maartje C P; Troost, Freddy J; Saris, Wim H M

2012-03-01

Exposing the intestine to proteins or tastants, particularly sweet, affects satiety hormone release. There are indications that each sweetener has different effects on this release, and that combining sweeteners with other nutrients might exert synergistic effects on hormone release. STC-1 cells were incubated with acesulfame-K, aspartame, saccharine, sucralose, sucrose, pea, and pea with each sweetener. After a 2-h incubation period, cholecystokinin(CCK) and glucagon-like peptide 1 (GLP-1) concentrations were measured. Using Ussing chamber technology, the mucosal side of human duodenal biopsies was exposed to sucrose, sucralose, pea, and pea with each sweetener. CCK and GLP-1 levels were measured in basolateral secretions. In STC-1 cells, exposure to aspartame, sucralose, sucrose, pea, and pea with sucralose increased CCK levels, whereas GLP-1 levels increased after addition of all test products. Addition of sucrose and sucralose to human duodenal biopsies did not affect CCK and GLP-1 release; addition of pea stimulated CCK and GLP-1 secretion. Combining pea with sucrose and sucralose induced even higher levels of CCK and GLP-1. Synchronous addition of pea and sucralose to enteroendocrine cells induced higher levels of CCK and GLP-1 than addition of each compound alone. This study shows that combinations of dietary compounds synergize to enhance satiety hormone release. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relating physico-chemical properties of frozen green peas (Pisum sativum L.) to sensory quality.

PubMed

Nleya, Kathleen M; Minnaar, Amanda; de Kock, Henriëtte L

2014-03-30

The acceptability of frozen green peas depends on their sensory quality. There is a need to relate physico-chemical parameters to sensory quality. In this research, six brands of frozen green peas representing product sold for retail and caterer's markets were purchased and subjected to descriptive sensory evaluation and physico-chemical analyses (including dry matter content, alcohol insoluble solids content, starch content, °Brix, residual peroxidase activity, size sorting, hardness using texture analysis and colour measurements) to assess and explain product quality. The sensory quality of frozen green peas, particularly texture properties, were well explained using physico-chemical methods of analysis notably alcohol insoluble solids, starch content, hardness and °Brix. Generally, retail class peas were of superior sensory quality to caterer's class peas although one caterer's brand was comparable to the retail brands. Retail class peas were sweeter, smaller, greener, more moist and more tender than the caterer's peas. Retail class peas also had higher °Brix, a(*) , hue and chroma values; lower starch, alcohol insoluble solids, dry matter content and hardness measured. The sensory quality of frozen green peas can be partially predicted by measuring physico-chemical parameters particularly °Brix and to a lesser extent hardness by texture analyser, alcohol insoluble solids, dry matter and starch content. © 2013 Society of Chemical Industry.

Femtosecond spectroscopy probes the folding quality of antibody fragments expressed as GFP fusions in the cytoplasm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Didier, P.; Weiss, E.; Sibler, A.-P.

2008-02-22

Time-resolved femtosecond spectroscopy can improve the application of green fluorescent proteins (GFPs) as protein-folding reporters. The study of ultrafast excited-state dynamics (ESD) of GFP fused to single chain variable fragment (scFv) antibody fragments, allowed us to define and measure an empirical parameter that only depends on the folding quality (FQ) of the fusion. This method has been applied to the analysis of genetic fusions expressed in the bacterial cytoplasm and allowed us to distinguish folded and thus functional antibody fragments (high FQ) with respect to misfolded antibody fragments. Moreover, these findings were strongly correlated to the behavior of the samemore » scFvs expressed in animal cells. This method is based on the sensitivity of the ESD to the modifications in the tertiary structure of the GFP induced by the aggregation state of the fusion partner. This approach may be applicable to the study of the FQ of polypeptides over-expressed under reducing conditions.« less

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

NASA Astrophysics Data System (ADS)

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

2018-01-01

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

Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (Intrabody).

PubMed

Heng, Boon Chin; Cao, Tong

2005-01-01

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA). Firstly, the high specificity and affinity of intrabodies to target antigens is well-established, whereas iRNA has been frequently shown to exert multiple non-specific effects. Secondly, intrabodies being proteins possess a much longer active half-life compared to iRNA. Thirdly, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA would be slow to yield any effect, whereas the effects of intrabody expression would be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. There is, however, various technical challenges faced with intrabody expression through the application of recombinant DNA technology. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment. Also, there are overwhelming safety concerns surrounding the application of transfected recombinant DNA in human clinical therapy, which is required to achieve intrabody expression within the cell. Of particular concern are the various viral-based vectors that are commonly-used in genetic manipulation. A novel approach around these problems would be to look at the possibility of fusing protein transduction domains (PTD) to scFv antibodies, to create a 'cell-permeable' antibody or 'Transbody'. PTD are short peptide sequences that enable proteins to translocate across the cell membrane and be internalized within the cytosol, through atypical secretory and

Forecasting model for Pea seed-borne mosaic virus epidemics in field pea crops in a Mediterranean-type environment.

PubMed

Congdon, B S; Coutts, B A; Jones, R A C; Renton, M

2017-09-15

An empirical model was developed to forecast Pea seed-borne mosaic virus (PSbMV) incidence at a critical phase of the annual growing season to predict yield loss in field pea crops sown under Mediterranean-type conditions. The model uses pre-growing season rainfall to calculate an index of aphid abundance in early-August which, in combination with PSbMV infection level in seed sown, is used to forecast virus crop incidence. Using predicted PSbMV crop incidence in early-August and day of sowing, PSbMV transmission from harvested seed was also predicted, albeit less accurately. The model was developed so it provides forecasts before sowing to allow sufficient time to implement control recommendations, such as having representative seed samples tested for PSbMV transmission rate to seedlings, obtaining seed with minimal PSbMV infection or of a PSbMV-resistant cultivar, and implementation of cultural management strategies. The model provides a disease forecast risk indication, taking into account predicted percentage yield loss to PSbMV infection and economic factors involved in field pea production. This disease risk forecast delivers location-specific recommendations regarding PSbMV management to end-users. These recommendations will be delivered directly to end-users via SMS alerts with links to web support that provide information on PSbMV management options. This modelling and decision support system approach would likely be suitable for use in other world regions where field pea is grown in similar Mediterranean-type environments. Copyright © 2017 Elsevier B.V. All rights reserved.

Antibody and Immunotoxin Treatments for Mesothelin-expressing Cancers | NCI Technology Transfer Center | TTC

Cancer.gov

The National Cancer Institute Laboratory of Molecular Biology is seeking statements of capability or interest from parties interested in licensing or collaborative research to further develop, evaluate, or commercialize antibody-based treatments of mesothelin-expressing cancers.

Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

PubMed

Yamada, Tadaaki; Bando, Hideaki; Takeuchi, Shinji; Kita, Kenji; Li, Qi; Wang, Wei; Akinaga, Shiro; Nishioka, Yasuhiko; Sone, Saburo; Yano, Seiji

2011-12-01

Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. © 2011 Japanese Cancer Association.

Strains of Pseudomonas syringae pv. syringae from pea are phylogenetically and pathogenically diverse.

PubMed

Martín-Sanz, Alberto; de la Vega, Marcelino Pérez; Murillo, Jesús; Caminero, Constantino

2013-07-01

Pseudomonas syringae pv. syringae causes extensive yield losses in the pea crop worldwide, although there is little information on its host specialization and its interactions with pea. A collection of 88 putative P. syringae pv. syringae strains (including 39 strains isolated from pea) was characterized by repetitive polymerase chain reaction (rep-PCR), multilocus sequence typing (MLST), and syrB amplification and evaluated for pathogenicity and virulence. rep-PCR data grouped the strains from pea into two groups (1B and 1C) together with strains from other hosts; a third group (1A) was formed exclusively with strains isolated from non-legume species. MLST data included all strains from pea in the genomospecies 1 of P. syringae pathovars defined in previous studies; they were distributed in the same three groups defined by rep-PCR. The inoculations performed in two pea cultivars showed that P. syringae pv. syringae strains from groups 1A and 1C were less virulent than strains from group 1B, suggesting a possible pathogenic specialization in this group. This study shows the existence of genetically and pathogenically distinct P. syringae pv. syringae strain groups from pea, which will be useful for the diagnostic and epidemiology of this pathogen and for disease resistance breeding.

A germin-like protein with superoxide dismutase activity in pea nodules with high protein sequence identity to a putative rhicadhesin receptor.

PubMed

Gucciardo, Sébastian; Wisniewski, Jean-Pierre; Brewin, Nicholas J; Bornemann, Stephen

2007-01-01

The cDNAs encoding three germin-like proteins (PsGER1, PsGER2a, and PsGER2b) were isolated from Pisum sativum. The coding sequence of PsGER1 transiently expressed in tobacco leaves gave a protein with superoxide dismutase activity but no detectable oxalate oxidase activity according to in-gel activity stains. The transient expression of wheat germin gf-2.8 oxalate oxidase showed oxalate oxidase but no superoxide dismutase activity under the same conditions. The superoxide dismutase activity of PsGER1 was resistant to high temperature, denaturation by detergent, and high concentrations of hydrogen peroxide. In salt-stressed pea roots, a heat-resistant superoxide dismutase activity was observed with an electrophoretic mobility similar to that of the PsGER1 protein, but this activity was below the detection limit in non-stressed or H(2)O(2)-stressed pea roots. Oxalate oxidase activity was not detected in either pea roots or nodules. Following in situ hybridization in developing pea nodules, PsGER1 transcript was detected in expanding cells just proximal to the meristematic zone and also in the epidermis, but to a lesser extent. PsGER1 is the first known germin-like protein with superoxide dismutase activity to be associated with nodules. It shared protein sequence identity with the N-terminal sequence of a putative plant receptor for rhicadhesin, a bacterial attachment protein. However, its primary location in nodules suggests functional roles other than as a rhicadhesin receptor required for the first stage of bacterial attachment to root hairs.

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

PubMed

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.

Physicochemical properties and in vitro digestibility of flour and starch from pea (Pisum sativum L.) cultivars.

PubMed

Chung, Hyun-Jung; Liu, Qiang

2012-01-01

Flours and isolated starches from three different cultivars (1544-8, 1658-11 and 1760-8) of pea grown under identical environmental conditions were evaluated for their physicochemical properties and in vitro digestibility. The protein content, total starch content and apparent amylose content of pea flour ranged from 24.4 to 26.3%, 48.8 to 50.2%, and 13.9 to 16.7%, respectively. In pea starches, the 1760-8 showed higher apparent amylose content and total starch content than the other cultivars. Pea starch granules were irregularly shaped, ranging from oval to round with a smooth surface. All pea starches showed C-type X-ray diffraction pattern with relative crystallinity ranging between 23.7 and 24.7%. Pea starch had only a single endothermic transition (12.1-14.2 J/g) in the DSC thermogram, whereas pea flour showed two separate endothermic transitions corresponding to starch gelatinization (4.54-4.71 J/g) and disruption of the amylose-lipid complex (0.36-0.78 J/g). In pea cultivars, the 1760-8 had significantly higher setback and final viscosity than the other cultivars in both pea flour (672 and 1170cP, respectively) and isolated starch (2901 and 4811cP). The average branch chain length of pea starches ranged from 20.1 to 20.3. The 1760-8 displayed a larger proportion of short branch chains, DP (degree of polymerization) 6-12 (21.1%), and a smaller proportion of long branch chains, DP≥37 (8.4%). The RDS, SDS and RS contents of pea flour ranged from 23.7 to 24.1%, 11.3 to 12.8%, and 13.2 to 14.8%, respectively. In pea starches, the 1760-8 showed a lower RDS content but higher SDS and RS contents. The expected glycemic index (eGI), based on the hydrolysis index, ranged from 36.9 to 37.7 and 69.8 to 70.7 for pea flour and isolated pea starch, respectively. Copyright © 2011 Elsevier B.V. All rights reserved.

Pea (Pisum sativum L.)

USDA-ARS?s Scientific Manuscript database

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...

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

PubMed Central

Bradbury, Andrew R. M.; Trinklein, Nathan D.; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui-Yan; Lane, David; Lefranc, Marie-Paule; Clark, Mike

2018-01-01

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use. PMID:29485921

Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.)

NASA Technical Reports Server (NTRS)

Slocum, R. D.; Richardson, D. P.

1991-01-01

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

Plant characteristics and growth parameters of vegetable pigeon pea cultivars

USDA-ARS?s Scientific Manuscript database

Pigeon pea is an important crop in dry land and semi-arid regions and is a supplementary source of dietary protein for the resource-constrained farmers. The aim of this research was to evaluate growth parameters of twelve vegetable pigeon pea genotypes at two locations in Eastern Kenya. The number o...

[Possibility of using flour of pigeon pea in products prepared with rice or wheat flour].

PubMed

Mueses, C; de León, L; Bressani, R

1993-03-01

The present study reports on the development of foods containing processed pigeon pea (Cajanus cajan) flour. The pigeon pea flours described in a previous publication were prepared from dehulled pigeon peas by cooking in autoclave, by extrusion-cooking and by cooking/dehydration by drum-drying. Mixtures of cooked pigeon peas and rice were first evaluated biological through a protein complementation design using NPR. The results of this study showed that the two products had high protein quality and were similar when mixed in ratios of 80:20 to 40:60. For the evaluation of the processed pigeon pea flour, mixtures with rice (80:20) were used. All pigeon pea flours gave similar protein quality values. On the basis of these results three products were developed and tested. One was a gruel ("atole"), a second a fruit-flavored thick drink with and without 15% milk. Cookies were also prepared with a series of blends of pigeon pea flour (extrusion-cooked) and wheat. The gruel and the fruit flavored products had high acceptability based on a sensory evaluation test. Cookies with 100% pigeon pea flour were unacceptable, however, mixtures of 75% wheat flour and 25% pigeon pea flour gave cookies of attractive appearance and good taste. The study showed the possibility of preparing and utilizing tropical grain legume flours for food products of relatively high acceptability and nutritive value.

The effects of fermentation and enzymatic treatment of pea on nutrient digestibility and growth performance of broilers.

PubMed

Goodarzi Boroojeni, F; Senz, M; Kozłowski, K; Boros, D; Wisniewska, M; Rose, D; Männer, K; Zentek, J

2017-10-01

The present study examined the impacts of native, fermented or enzymatically treated peas (Pisum sativum L.) inclusion in broiler diets, on growth performance and nutrient digestibility. For the fermentation process, Madonna pea was mixed with water (1/1) containing 2.57×108 Bacillus subtilis (GalliPro®) spores/kg pea and then, incubated for 48 h at 30 °C. For the enzymatic treatment process, the used water for dough production contained three enzymes, AlphaGalTM (α-galactosidase), RONOZYME® ProAct and VP (protease and pectinases respectively - DSM, Switzerland) and the pea dough incubated for 24 h at 30°C. Nine corn-wheat-soybean diets were formulated by supplying 10%, 20% and 30% of the required CP with either native, fermented or enzymatically treated peas. Performance was recorded weekly and at the end of the experiment (day 35), apparent ileal digestibility (AID) of CP, amino acids (AA), crude fat, starch, Ca, P and K were determined. Data were subjected to ANOVA using GLM procedure with a 3×3 factorial arrangement of treatments. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. Increasing levels of pea products up to 300 g/kg diet, reduced BW gain and feed intake (P⩽0.05). Broilers fed diets containing enzymatically treated pea had the best feed conversion ratio at day 35. Different types of pea product and their inclusion levels had no effect on AID of all nutrients. The interaction between type of the pea products and inclusion levels was significant for AID of starch. For native pea diets, 10% group showed similar AID of starch to 20% native pea but it had higher AID than 30% native pea. For fermented and enzymatically treated groups, all three levels displayed similar AID of starch. In conclusion, enzymatic treatment and fermentation could improve the nutritional quality of pea. Inclusion of enzymatically treated pea in broiler diets could improve broiler performance compared with other pea

Germinated Pigeon Pea (Cajanus cajan): a novel diet for lowering oxidative stress and hyperglycemia.

PubMed

Uchegbu, Nneka N; Ishiwu, Charles N

2016-09-01

This work studied the antioxidant activity of extract of germinated pigeon pea (Cajanus cajan) in alloxan-induced diabetic rats. Germination was carried out in a dark chamber under room temperature (28°C). The total phenolic, 1,1,diphenyl-2-picrylhy-drazyl free radical (DPPH) scavenging, the inhibition of α-amylase and α-glucosidase were done in vitro and blood glucose levels of the animal were investigated. Lipid peroxidation (LPO) and reduced glutathione (GSH) were analyzed spectrophotometrically. The total phenolic and DPPH scavenging activity increased by 30% and 63%, respectively, after germinating pigeon pea. Also after germination there was an increase in the inhibitory potential of pigeon pea extract against α-glucosidase compared with the nongerminated pigeon pea extract. There was a significant increase (P < 0.05) in fasting blood glucose level of alloxan-induced rats. Consumption of germinated pigeon pea extract gave rise to a reduced fasting blood glucose level in diabetic rats. On administration of germinated pigeon pea extract, LPO reduced drastically but there was an increase in the level of GSH. This study concluded that intake of germinated pigeon pea is a good dietary supplement for controlling hyperglycemia and LPO.

Influence of the inclusion of cooked cereals and pea starch in diets based on soy or pea protein concentrate on nutrient digestibility and performance of young pigs.

PubMed

Parera, N; Lázaro, R P; Serrano, M P; Valencia, D G; Mateos, G G

2010-02-01

An experiment was conducted to compare different dietary vegetable sources of starch and protein on the coefficient of apparent total tract digestibility (CATTD) of energy and nutrients and performance of piglets from 29 to 60 d of age. The experiment was completely randomized with 6 treatments arranged factorially with 3 sources of starch (cooked-flaked corn, cooked-flaked rice, and pea starch) and 2 sources of protein [soy protein concentrate (SPC) and pea protein concentrate (PPC)]. The pea starch and the PPC used were obtained by dehulling and grinding pea seeds to a mean particle size of 30 microm. Each treatment was replicated 6 times (6 pigs per pen). For the entire experiment, piglets fed cooked rice had greater ADG than piglets fed pea starch with piglets fed cooked corn being intermediate (471, 403, and 430 g/d, respectively; P < 0.05). Protein source did not have any effect on piglet performance. The CATTD of DM, OM, and GE were greater (P < 0.05) for diets based on cooked rice than diets based on cooked corn with diets based on pea starch being intermediate. Crude protein digestibility was not affected by source of starch but was greater for the diets based on SPC than for diets based on PPC (0.836 vs. 0.821; P < 0.01). Protein source did not affect the digestibility of any of the other dietary components. It is concluded that cooked rice is an energy source of choice in diets for young pigs. The inclusion of PPC in the diet reduced protein digestibility but had no effects on energy digestibility or piglet performance. Therefore, the finely ground starch and protein fractions of peas can be used in substitution of cooked corn or SPC, respectively, in diets for young pigs.

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule.

PubMed

Dobosz, Michael; Haupt, Ute; Scheuer, Werner

2017-01-01

Preclinical efficacy studies of antibodies targeting a tumor-associated antigen are only justified when the expression of the relevant antigen has been demonstrated. Conventionally, antigen expression level is examined by immunohistochemistry of formalin-fixed paraffin-embedded tumor tissue section. This method represents the diagnostic "gold standard" for tumor target evaluation, but is affected by a number of factors, such as epitope masking and insufficient antigen retrieval. As a consequence, variances and discrepancies in histological staining results can occur, which may influence decision-making and therapeutic outcome. To overcome these problems, we have used different fluorescence-labeled therapeutic antibodies targeting human epidermal growth factor receptor (HER) family members and insulin-like growth factor-1 receptor (IGF1R) in combination with fluorescence imaging modalities to determine tumor antigen expression, drug-target interaction, and biodistribution and tumor saturation kinetics in non-small cell lung cancer xenografts. For this, whole-body fluorescence intensities of labeled antibodies, applied as a single compound or antibody mixture, were measured in Calu-1 and Calu-3 tumor-bearing mice, then ex vivo multispectral tumor tissue analysis at microscopic resolution was performed. With the aid of this simple and fast imaging method, we were able to analyze the tumor cell receptor status of HER1-3 and IGF1R, monitor the antibody-target interaction and evaluate the receptor binding sites of anti-HER2-targeting antibodies. Based on this, the most suitable tumor model, best therapeutic antibody, and optimal treatment dosage and application schedule was selected. Predictions drawn from obtained imaging data were in excellent concordance with outcome of conducted preclinical efficacy studies. Our results clearly demonstrate the great potential of combined in vivo and ex vivo fluorescence imaging for the preclinical development and characterization of

Protein-enriched pea flour extract protects stored milled rice against the rice weevil, Sitophilus oryzae.

PubMed

Pretheep-Kumar, P; Mohan, S; Ramaraju, K

2004-01-01

Studies were conducted to evaluate the effect of a protein-enriched pea (Pisum sativum var. Bonneville) flour extract against the rice weevil, Sitophilus oryzae in its repellency, toxicity, effect on fecundity, stability and sensory properties. Milled rice admixed with pea flour extract at 1% concentration significantly repelled S. oryzae. Mortality of S. oryzae was found to increase and fecundity was markedly suppressed, in rice treated with 1% pea flour extract. The toxicity and reproductive effects of the pea protein-enriched rice were found to be stable for a period of 5 months. The sensory characteristics of stored rice when eaten were not affected by the treatment with pea flour extract. This study indicates that the protein-enriched flour extract obtained from the Bonneville pea may be feasible to protect stored milled rice from insect attack.

Antibodies among men and children to placental-binding Plasmodium falciparum-infected erythrocytes that express var2csa.

PubMed

Beeson, James G; Ndungu, Francis; Persson, Kristina E M; Chesson, Joanne M; Kelly, Greg L; Uyoga, Sophie; Hallamore, Sandra L; Williams, Thomas N; Reeder, John C; Brown, Graham V; Marsh, Kevin

2007-07-01

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

2004-10-25

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

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

PubMed

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

2004-10-25

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d3) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d3. In addition, both sCD4-gp120 and sCD4-gp120-mC3d3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d3 or sCD4-gp120-mC3d3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d3-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

Characterization of two brassinosteroid C-6 oxidase genes in pea.

PubMed

Jager, Corinne E; Symons, Gregory M; Nomura, Takahito; Yamada, Yumiko; Smith, Jennifer J; Yamaguchi, Shinjiro; Kamiya, Yuji; Weller, James L; Yokota, Takao; Reid, James B

2007-04-01

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.

Enhanced Macrophage Resistance to Pseudomonas Exotoxin A Is Correlated with Decreased Expression of the Low-Density Lipoprotein Receptor-Related Protein

PubMed Central

Laithwaite, James E.; Benn, Sally J.; Yamate, Jyoji; FitzGerald, David J.; LaMarre, Jonathan

1999-01-01

Cellular intoxification by exotoxin A of Pseudomonas aeruginosa (PEA) begins when PEA binds to its cellular receptor, the low-density lipoprotein receptor-related protein (LRP). This receptor is particularly abundant on macrophages. We hypothesize here that inducible changes in cellular expression levels of the LRP represent an important mechanism by which macrophage susceptibility to PEA is regulated by the host. We have examined the effect of lipopolysaccharide (LPS) on LRP expression and PEA sensitivity in the macrophage-like cell line HS-P. Using a [3H]leucine incorporation assay to measure inhibition of protein synthesis, we have demonstrated that HS-P macrophages are highly sensitive to PEA and that PEA toxicity is decreased by the LRP antagonist receptor-associated protein. LPS pretreatment decreases HS-P PEA sensitivity in a time- and dose-dependent manner. The dose of toxin required to inhibit protein synthesis by 50% increased from 11.3 ± 1.2 ng/ml in untreated cells to 25.7 ± 2.0 ng/ml in cells treated with LPS. In pulse experiments, involving brief exposure to saturating concentrations of PEA, [3H]leucine incorporation was more than threefold higher in cells pretreated with LPS than in untreated macrophages. These changes in HS-P PEA sensitivity following LPS treatment were consistently associated with a fivefold decrease in HS-P LRP mRNA expression as measured by Northern blot analysis and a three-and-a-half-fold decrease in HS-P LRP-specific ligand internalization as determined by activated α2-macroglobulin internalization studies. These data demonstrate for the first time that modulation of LRP levels by extracellular signaling molecules can alter cellular PEA sensitivity. PMID:10531236

Protein-enriched pea flour extract protects stored milled rice against the rice weevil, Sitophilus oryzae

PubMed Central

Pretheep-Kumar, P.; Mohan, S.; Ramaraju, K.

2004-01-01

Studies were conducted to evaluate the effect of a protein-enriched pea (Pisum sativum var. Bonneville) flour extract against the rice weevil, Sitophilus oryzae in its repellency, toxicity, effect on fecundity, stability and sensory properties. Milled rice admixed with pea flour extract at 1% concentration significantly repelled S. oryzae. Mortality of S. oryzae was found to increase and fecundity was markedly suppressed, in rice treated with 1% pea flour extract. The toxicity and reproductive effects of the pea protein-enriched rice were found to be stable for a period of 5 months. The sensory characteristics of stored rice when eaten were not affected by the treatment with pea flour extract. This study indicates that the protein-enriched flour extract obtained from the Bonneville pea may be feasible to protect stored milled rice from insect attack. PMID:15861241

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

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 3 2011-01-01 2011-01-01 false Legume or pea family, Fabaceae (Leguminosae). 201.56-6 Section 201.56-6 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL...-6 Legume or pea family, Fabaceae (Leguminosae). Kinds of seed: Alfalfa, alyceclover, asparagusbean...

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

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 3 2010-01-01 2010-01-01 false Legume or pea family, Fabaceae (Leguminosae). 201.56-6 Section 201.56-6 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL...-6 Legume or pea family, Fabaceae (Leguminosae). Kinds of seed: Alfalfa, alyceclover, asparagusbean...

Posterior localization of ApVas1 positions the preformed germ plasm in the sexual oviparous pea aphid Acyrthosiphon pisum

PubMed Central

2014-01-01

Background Germline specification in some animals is driven by the maternally inherited germ plasm during early embryogenesis (inheritance mode), whereas in others it is induced by signals from neighboring cells in mid or late development (induction mode). In the Metazoa, the induction mode appears as a more prevalent and ancestral condition; the inheritance mode is therefore derived. However, regarding germline specification in organisms with asexual and sexual reproduction it has not been clear whether both strategies are used, one for each reproductive phase, or if just one strategy is used for both phases. Previously we have demonstrated that specification of germ cells in the asexual viviparous pea aphid depends on a preformed germ plasm. In this study, we extended this work to investigate how germ cells were specified in the sexual oviparous embryos, aiming to understand whether or not developmental plasticity of germline specification exists in the pea aphid. Results We employed Apvas1, a Drosophila vasa ortholog in the pea aphid, as a germline marker to examine whether germ plasm is preformed during oviparous development, as has already been seen in the viviparous embryos. During oogenesis, Apvas1 mRNA and ApVas1 protein were both evenly distributed. After fertilization, uniform expression of Apvas1 remained in the egg but posterior localization of ApVas1 occurred from the fifth nuclear cycle onward. Posterior co-localization of Apvas1/ApVas1 was first identified in the syncytial blastoderm undergoing cellularization, and later we could detect specific expression of Apvas1/ApVas1 in the morphologically identifiable germ cells of mature embryos. This suggests that Apvas1/ApVas1-positive cells are primordial germ cells and posterior localization of ApVas1 prior to cellularization positions the preformed germ plasm. Conclusions We conclude that both asexual and sexual pea aphids rely on the preformed germ plasm to specify germ cells and that developmental

Isolation and characterization of the pea cytochrome c oxidase Vb gene.

PubMed

Kubo, Nakao; Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Kadowaki, Koh-Ichi; Hirai, Masashi

2006-11-01

Three copies of the gene that encodes cytochrome c oxidase subunit Vb were isolated from the pea (PscoxVb-1, PscoxVb-2, and PscoxVb-3). Northern Blot and reverse transcriptase-PCR analyses suggest that all 3 genes are transcribed in the pea. Each pea coxVb gene has an N-terminal extended sequence that can encode a mitochondrial targeting signal, called a presequence. The localization of green fluorescent proteins fused with the presequence strongly suggests the targeting of pea COXVb proteins to mitochondria. Each pea coxVb gene has 5 intron sites within the coding region. These are similar to Arabidopsis and rice, although the intron lengths vary greatly. A phylogenetic analysis of coxVb suggests the occurrence of gene duplication events during angiosperm evolution. In particular, 2 duplication events might have occurred in legumes, grasses, and Solanaceae. A comparison of amino acid sequences in COXVb or its counterpart shows the conservation of several amino acids within a zinc finger motif. Interestingly, a homology search analysis showed that bacterial protein COG4391 and a mitochondrial complex I 13 kDa subunit also have similar amino acid compositions around this motif. Such similarity might reflect evolutionary relationships among the 3 proteins.

Faba beans and peas in poultry feed: economic assessment.

PubMed

Proskina, Liga; Cerina, Sallija

2017-10-01

Broiler diets mainly consist of cereals and protein-rich feed sources; in the EU-27, poultry farming consumes 24% of the total amount of protein-rich feedstuffs. Since the EU produces only 30% of the total quantity of protein crops used for feed, it is necessary to promote the use of traditional European protein crops (beans, peas) for feed in livestock farming. The research aim is to identify economic gains from the production of broiler chicken meat, replacing soybean meal with domestic faba beans and field peas in broiler chicken diets. Adding field peas and faba beans to the broiler feed ration resulted in a significant live weight increase (5.74-11.95%) at the selling age, a decrease in the feed conversion ratio by 0.61-6.06%, and decrease in the product unit cost (15.34-37.06%) as well as an increase in the production efficiency factor (8.70-48.54), compared with the control group. The optimum kind of legume species used in the broiler diet was peas, which were added in the amount of 200 g kg -1 , resulting in live weight gain, a decrease in the feed conversion ratio and an increase in the production efficiency factor. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Genetic diversity and trait genomic prediction in a pea diversity panel.

PubMed

Burstin, Judith; Salloignon, Pauline; Chabert-Martinello, Marianne; Magnin-Robert, Jean-Bernard; Siol, Mathieu; Jacquin, Françoise; Chauveau, Aurélie; Pont, Caroline; Aubert, Grégoire; Delaitre, Catherine; Truntzer, Caroline; Duc, Gérard

2015-02-21

Pea (Pisum sativum L.), a major pulse crop grown for its protein-rich seeds, is an important component of agroecological cropping systems in diverse regions of the world. New breeding challenges imposed by global climate change and new regulations urge pea breeders to undertake more efficient methods of selection and better take advantage of the large genetic diversity present in the Pisum sativum genepool. Diversity studies conducted so far in pea used Simple Sequence Repeat (SSR) and Retrotransposon Based Insertion Polymorphism (RBIP) markers. Recently, SNP marker panels have been developed that will be useful for genetic diversity assessment and marker-assisted selection. A collection of diverse pea accessions, including landraces and cultivars of garden, field or fodder peas as well as wild peas was characterised at the molecular level using newly developed SNP markers, as well as SSR markers and RBIP markers. The three types of markers were used to describe the structure of the collection and revealed different pictures of the genetic diversity among the collection. SSR showed the fastest rate of evolution and RBIP the slowest rate of evolution, pointing to their contrasted mode of evolution. SNP markers were then used to predict phenotypes -the date of flowering (BegFlo), the number of seeds per plant (Nseed) and thousand seed weight (TSW)- that were recorded for the collection. Different statistical methods were tested including the LASSO (Least Absolute Shrinkage ans Selection Operator), PLS (Partial Least Squares), SPLS (Sparse Partial Least Squares), Bayes A, Bayes B and GBLUP (Genomic Best Linear Unbiased Prediction) methods and the structure of the collection was taken into account in the prediction. Despite a limited number of 331 markers used for prediction, TSW was reliably predicted. The development of marker assisted selection has not reached its full potential in pea until now. This paper shows that the high-throughput SNP arrays that are being

Affinity ranking of antibodies using flow cytometry: application in antibody phage display-based target discovery.

PubMed

Geuijen, Cecilia A W; Clijsters-van der Horst, Marieke; Cox, Freek; Rood, Pauline M L; Throsby, Mark; Jongeneelen, Mandy A C; Backus, Harold H J; van Deventer, Els; Kruisbeek, Ada M; Goudsmit, Jaap; de Kruif, John

2005-07-01

Application of antibody phage display to the identification of cell surface antigens with restricted expression patterns is often complicated by the inability to demonstrate specific binding to a certain cell type. The specificity of an antibody can only be properly assessed when the antibody is of sufficient high affinity to detect low-density antigens on cell surfaces. Therefore, a robust and simple assay for the prediction of relative antibody affinities was developed and compared to data obtained using surface plasmon resonance (SPR) technology. A panel of eight anti-CD46 antibody fragments with different affinities was selected from phage display libraries and reformatted into complete human IgG1 molecules. SPR was used to determine K(D) values for these antibodies. The association and dissociation of the antibodies for binding to CD46 expressed on cell surfaces were analysed using FACS-based assays. We show that ranking of the antibodies based on FACS data correlates well with ranking based on K(D) values as measured by SPR and can therefore be used to discriminate between high- and low-affinity antibodies. Finally, we show that a low-affinity antibody may only detect high expression levels of a surface marker while failing to detect lower expression levels of this molecule, which may lead to a false interpretation of antibody specificity.

Saponin content and trypsin inhibitor activity in processed and cooked pigeon pea cultivars.

PubMed

Duhan, A; Khetarpaul, N; Bishnoi, S

2001-01-01

Four high-yielding varieties of pigeon pea namely UPAS-120, Manak, JCPL-151. ICPL-87 had considerable amounts of antinutrients i.e. saponins and trypsin inhibitors. Saponin content of these unprocessed cultivars ranged from 2164 to 3494 mg/100 g. There were significant varietal variations in trypsin inhibitor activity (1007-1082 TIU/g) of these pigeon pea cultivars. Some simple, inexpensive and easy-to-use domestic processing and cooking methods, namely, soaking (6, 12, 18 h), soaking (12 h)-dehulling, ordinary cooking, pressure cooking and germination (24, 36, 48 h) were found to be quite effective in lowering the level of saponins and trypsin inhibitors in all the pigeon pea cultivars. Pressure cooking of soaked and dehulled seeds lowered the content of saponins to a maximum extent (28 to 38%) followed by ordinary cooking of soaked and dehulled seeds (28 to 35%), soaked dehulled raw seeds (22 to 27%) and 48 h germinated seeds (15 to 19%). Loss of TIA was marginal due to soaking but ordinary as well as pressure cooking of unsoaked and soaked-dehulled pigeon pea seeds reduced the TIA drastically. Pressure cooking of pigeon pea seeds completely destroyed the TIA while it was reduced to the extent of 86-88% against the control in 48 h pigeon pea sprouts.

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

PubMed Central

Ahn, Junsik; Lee, Kyung Jin

2014-01-01

The purpose of this study is to optimize ELISA conditions to quantify the colorectal cancer antigen GA733 linked to the Fc antibody fragment fused to KDEL, an ER retention motif (GA733-FcK) expressed in transgenic plant. Variable conditions of capture antibody, blocking buffer, and detection antibody for ELISA were optimized with application of leaf extracts from transgenic plant expressing GA733-FcK. In detection antibody, anti-EpCAM/CD362 IgG recognizing the GA733 did not detect any GA733-FcK whereas anti-human Fc IgG recognizing the human Fc existed in plant leaf extracts. For blocking buffer conditions, 3% BSA buffer clearly blocked the plate, compared to the 5% skim-milk buffer. For capture antibody, monoclonal antibody (MAb) CO17-1A was applied to coat the plate with different amounts (1, 0.5, and 0.25 μg/well). Among the amounts of the capture antibody, 1 and 0.5 μg/well (capture antibody) showed similar absorbance, whereas 0.25 μg/well of the capture antibody showed significantly less absorbance. Taken together, the optimized conditions to quantify plant-derived GA733-FcK were 0.5 μg/well of MAb CO17-1A per well for the capture antibody, 3% BSA for blocking buffer, and anti-human Fc conjugated HRP. To confirm the optimized ELISA conditions, correlation analysis was conducted between the quantified amount of GA733-FcK in ELISA and its protein density values of different leaf samples in Western blot. The co-efficient value R2 between the ELISA quantified value and protein density was 0.85 (p<0.01), which indicates that the optimized ELISA conditions feasibly provides quantitative information of GA733-FcK expression in transgenic plant. PMID:24555929

Limiting an Insect Infestation of Nitrogen-Fixing Root Nodules of the Pigeon Pea (Cajanus cajan) by Engineering the Expression of an Entomocidal Gene in Its Root Nodules

PubMed Central

Nambiar, P. T. C.; Ma, S.-W.; Iyer, V. N.

1990-01-01

A region of DNA which determined the production of the insecticidal toxin of Bacillus thuringiensis subsp. israelensis was cloned into a derivative of a broad-host-range group IncQ plasmid vector of gram-negative bacteria. The plasmid which we constructed was transferred by conjugative mobilization into a Bradyrhizobium species that nodulates pigeon peas. In this species the construction was maintained stably in the absence of selection and expressed the gene that was installed. Experiments in a greenhouse with the strain which we constructed indicated that this organism provides protection against root nodule damage by the larvae of the insect Rivellia angulata (Diptera). Images PMID:16348294

HYDROXYCHLOROQUINE REDUCES BINDING OF ANTIPHOSPHOLIPID ANTIBODIES TO SYNCYTIOTROPHOBLASTS AND RESTORES ANNEXIN A5 EXPRESSION

PubMed Central

Wu, Xiao-Xuan; Guller, Seth; Rand, Jacob H.

2011-01-01

Objectives Antibody-mediated disruption of the annexin A5 (AnxA5) anticoagulant shield has been posited to be a thrombogenic mechanism in the antiphospholipid syndrome. We recently showed that the antimalarial drug, hydroxychloroquine, dissociates antiphospholipid immune complexes and restores AnxA5 binding to planar phospholipid bilayer. Using quantitative immunoassays, we demonstrated similar effects on BeWo trophoblasts. We therefore investigated the effects of the drug on localization of AnxA5 in primary cultures of human placental syncytiotrophoblasts (SCTs). Study Laser confocal microscopy with computer-based morphometric analysis was used to localize AnxA5 and antiphospholipid antibodies on SCTs exposed to polyclonal and monoclonal antiphospholipid and control IgGs. Results Hydroxychloroquine reversed the effects of the antiphospholipid antibodies on the SCTs by markedly reducing IgG binding and restoring AnxA5 expression. Conclusions These results provide the first morphologic evidence for this effect of hydroxychloroquine on human placental SCTs and support the possibility of novel treatments that target antiphospholipid antibody binding. PMID:21871597

Effects of pea chips on pig performance, carcass quality and composition, and palatability of pork.

PubMed

Newman, D J; Harris, E K; Lepper, A N; Berg, E P; Stein, H H

2011-10-01

Pea chips are produced as a by-product when field peas are processed to produce split peas for human consumption. The objective of this experiment was to test the hypothesis that inclusion of pea chips in diets fed to finishing pigs does not negatively influence pig growth performance, carcass composition, and the palatability of pork. A total of 24 barrows (initial BW: 58.0 ± 6.6 kg) were allotted to 1 of 4 treatments and fed early finishing diets for 35 d and late finishing diets for 35 d. A corn-soybean meal (SBM) control diet and 3 diets containing pea chips were formulated for each phase. Pea chips replaced 33.3, 66.6, or 100% of the SBM in the control diet. Pigs were housed individually, and all pigs were slaughtered at the conclusion of the experiment. Overall, there were no differences (P > 0.11) in final BW, ADFI, and G:F of pigs among treatments, but there was a quadratic response in ADG (P = 0.04), with the smallest value observed in pigs fed the control diet. Dressing percentage linearly decreased (P = 0.04) as pea chips replaced SBM in diets, but there were no differences (P > 0.20) among treatments in HCW, LM area, 10th-rib backfat, lean meat percentage, and marbling. Likewise, pH in loin and ham, drip loss, and purge loss were not influenced (P > 0.13) by treatment. However, there was a quadratic response (P = 0.08) in 24-h pH in the shoulder, with the smallest value present in pigs fed the diet, in which 66.6% of the SBM was replaced by pea chips. Subjective LM color and Japanese color score standard were reduced (quadratic, P = 0.03 and 0.05, respectively) and LM b* values and hue angle were increased (quadratic, P = 0.09 and 0.10, respectively) when pea chips replaced SBM in the diets. Ham L* (quadratic, P = 0.04), a* (linear, P = 0.02), b* (quadratic, P = 0.07), color saturation (linear, P = 0.02), and hue angle (quadratic, P = 0.05) were increased when pea chips replaced SBM. However, there were no differences (P > 0.16) in shoulder and fat

Molecular cloning and characterization of ADP-glucose pyrophosphorylase cDNA clones isolated from pea cotyledons.

PubMed

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.

Insecticidal components from field pea extracts: soyasaponins and lysolecithins.

PubMed

Taylor, Wesley G; Fields, Paul G; Sutherland, Daniel H

2004-12-15

Extracts from field peas (Pisum sativum L.) have previously been shown to have a utility to control insect pests. To identify potentially new bioinsecticides in field crops, we describe the fractionation of impure extracts (C8 extracts) derived from protein-rich fractions of commercial pea flour. The activity of separated fractions was determined by a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)], an insect pest of stored products. Bioassay-guided fractionation showed that the triterpenoid saponin fractions were partly responsible for the antifeedant activity of C8 extracts. Soyasaponin I (soyasaponin Bb), isolated from peas and soybeans, and mixtures of soyasaponins, comprised of soyasaponins I-III and isolated from soybeans, were inactive antifeedants, but dehydrosoyasaponin I (the C-22 ketone derivative of soyasaponin I), a minor component found in C8 extracts, was shown to be an active component. Dehydrosoyasaponin I (soyasaponin Be) and soyasaponin VI (soyasaponin betag) coeluted under conditions of silica gel thin-layer chromatography and C18 high-performance liquid chromatography. However, dehydrosoyasaponin I could be isolated from saponin-enriched fractions with a reversed phase column of styrene/divinylbenzene operated at alkaline pH. Phospholipids of the lysolecithin type were also identified in saponin fractions of C8 extracts from peas. Three of the lysolecithins were inactive alone against rice weevils, but mixtures of these phospholipids enhanced the insecticidal activity of dehydrosoyasaponin I.

Genetic Diversity of Chinese and Global Pea (Pisum sativum L.) Collections.

USDA-ARS?s Scientific Manuscript database

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 ...

Oxidative processes in soybean and pea seeds: effect of light, temperature, and water content

NASA Technical Reports Server (NTRS)

Vertucci, C. W.; Leopold, A. C.

1987-01-01

Oxidative processes are probable determinants of longevity of seeds in storage. Measurements of actual oxygen uptake rates were made for soybean and pea seeds as a comparison of short and long lived seeds when light, temperature, and moisture contents were varied. In both peas and soybeans, the oxygen uptake was depressed at low temperatures (<16 degrees C) and low water contents (< 0.25 gram H2O per gram dry weight). Apparent activation energies under these conditions are very high, while apparent activation energies of seeds at higher water contents and at temperatures greater than 22 degrees C are much less. Light enhances the level of oxygen uptake in pea, but reduces the level of oxygen uptake in soybean. The complexities of the interactions of oxygen uptake with environmental conditions in soybean compared to pea suggest that oxidative processes occur in soybean at low water contents, but are essentially absent in pea. It is suggested that the additional oxidative processes in soybean with moisture contents between 0.10 and 0.24 gram per gram may contribute to the poorer longevity of soybean seed compared to pea seed.

Phenetic, genetic diversity and symbiotic compatibility of rhizobial strains nodulating pigeon pea in Northern India.

PubMed

Arora, Naveen Kumar; Khare, Ekta; Singh, Sachin; Tewari, Sakshi

2018-01-01

Pigeon pea ( Cajanus cajan ) is one of the most important legumes grown in the northern province of Uttar Pradesh, India. However, its productively in Uttar Pradesh is lower than the average yield of adjoining states. During the course of the present study, a survey of pigeon pea growing agricultural fields was carried out and it was found that 80% of plants were inadequately nodulated. The study was aimed to evaluate the pigeon pea symbiotic compatibility and nodulation efficiency of root nodulating bacteria isolated from various legumes, and to explore the phenetic and genetic diversity of rhizobial population nodulating pigeon pea growing in fields of Uttar Pradesh. Amongst all the 96 isolates, 40 isolates showed nodulation in pigeon pea. These 40 isolates were further characterized by phenotypic, biochemical and physiological tests. Intrinsic antibiotic resistance pattern was taken to generate similarity matrix revealing 10 phenons. The study shows that most of the isolates nodulating pigeon pea in this region were rapid growers. The dendrogram generated using the NTSYSpc software grouped RAPD patterns into 19 clusters. The high degree of phenetic and genetic diversity encountered is probably because of a history of mixed cropping of legumes. The assessment of diversity is a very important tool and can be used to improve the nodulation and quality of pigeon pea crop. It is also concluded that difference between phenetic and RAPD clustering pattern is an indication that rhizobial diversity of pigeon pea is not as yet completely understood and settled.

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

PubMed

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.

Nitrous oxide emissions from crop rotations including wheat, rapeseed and dry pea

NASA Astrophysics Data System (ADS)

Jeuffroy, M. H.; Baranger, E.; Carrouée, B.; de Chezelles, E.; Gosme, M.; Hénault, C.; Schneider, A.; Cellier, P.

2012-07-01

Approximately 65% of anthropogenic emissions of N2O, a potent greenhouse gas, originate from soils at global scale, and particularly after N fertilisation of the main crops in Europe. Thanks to their capacity to fix atmospheric N2 through biological fixation, legumes allow to reduce N fertilizer use, and possibly N2O emission. Nevertheless, the decomposition of crop organic matter during the crop cycle and during the residue decomposition, and possibly the N fixation process itself, could lead to N2O emissions. The objective of this study was to quantify N2O emissions from a dry pea crop (Pisum sativum, harvested at maturity) and from the subsequent crops in comparison with N2O emissions from wheat and oilseed-rape crops, fertilized or not, in various rotations. A field experiment was conducted during 4 consecutive years, aiming at comparing the emissions during the pea crop, in comparison with those during the wheat (fertilized or not) or oilseed rape crops, and after the pea crop, in comparison with other preceding crops. N2O fluxes were measured using static chambers. In spite of low N2O fluxes, mainly linked with the site soil characteristics, fluxes during the crop were significantly lower for pea and unfertilized wheat than for fertilized wheat and oilseed rape. The effect of the preceding crop was not significant, while soil mineral N at harvest was higher after pea. These results, combined with the emission reduction allowed by the production and transport of the N fertiliser not applied on the pea crop, should be confirmed in a larger range of soil types. Nevertheless, they demonstrate the absence of N2O emission linked to the symbiotic N fixation process, and allow us to estimate the decrease of N2O emissions to 20-25% by including one pea crop in a three-year rotation. At a larger scale, this reduction of GHG emissions at field level has to be cumulated with the reduction of GHG emissions linked with the lower level of production and transport of the N

PEAS AND PARTICLES, TEACHER'S GUIDE.

ERIC Educational Resources Information Center

1966

THIS TEACHER'S GUIDE IS DESIGNED FOR USE WITH AN ELEMENTARY SCIENCE STUDY UNIT ON "PEAS AND PARTICLES" WHICH DEALS WITH LARGE NUMBERS AND ESTIMATIONS. ITS PURPOSE IS TO GIVE ELEMENTARY SCHOOL CHILDREN AN UNDERSTANDING OF WHAT LARGE NUMBERS MEAN THROUGH INFORMAL ACTIVITIES INVOLVING FAMILIAR OBJECTS. THE MATERIAL HAS BEEN FOUND SUITABLE…

CEI-PEA Alert, Summer 2006

ERIC Educational Resources Information Center

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…

A novel method for detecting neutralizing antibodies against therapeutic proteins by measuring gene expression.

PubMed

Yu, Yanbin; Piddington, Christopher; Fitzpatrick, Dan; Twomey, Brian; Xu, Ren; Swanson, Steven J; Jing, Shuqian

2006-10-20

The presence of neutralizing antibodies against protein therapeutics is a concern in the biomedical field. Such antibodies not only reduce the efficacy of protein therapeutics, but also impose potential dangers to the patients receiving them. To date, a small number of in vitro cell-based bioassays for detecting neutralizing antibodies against therapeutic proteins have been developed. Most of the existing assays, however, either involve the use of radioactive materials or have limited sensitivities and/or poor specificities. With advances in mRNA profiling and detection techniques, we have established a novel and non-radioactive bioassay system using branched DNA (bDNA) technology for detecting protein-therapeutic neutralizing antibodies in patient serum. Our assay measures the variations of target gene expression that reflect the biologic effect of the therapeutic agent and the capability of the antibodies, if present, to neutralize the therapeutics. Compared with most existing assays, the new assay is more sensitive and specific, and completely eliminates the use of radioactive materials. Application of the new assay system can be widely expanded if new target genes and responding cell lines for other therapeutics are identified or engineered.

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli

PubMed Central

Veisi, Kamal; Farajnia, Safar; Zarghami, Nosratollah; Khoram Khorshid, Hamid Reza; Samadi, Nasser; Ahdi Khosroshahi, Shiva; Zarei Jaliani, Hossein

2015-01-01

Purpose: Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. Methods: To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. Results: SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. Conclusion: The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies. PMID:26793607

Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

PubMed Central

Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

2013-01-01

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

Biological control of fusarial wilt of pigeon pea by Bacillus brevis.

PubMed

Bapat, S; Shah, A K

2000-02-01

A virulent strain of pigeon pea wilt pathogen was isolated from wilted pigeon pea plants and was identified as Fusarium oxysporum f. sp. udum. Many bacterial cultures showing antagonism to the pathogen were isolated from various ecological niches. When tested under pot and field conditions, development of fusarial wilt symptoms was prevented in pigeon pea seeds treated with one such antagonist, Bacillus brevis. A formulation of B. brevis with vermiculite as a carrier had a shelf life of at least 6 months. Bacillus brevis produced an extracellular antagonistic substance which induced swelling of the pathogen's hyphal tips, and cells were bulbous and swollen with shrunken and granulated cytoplasm. The antagonistic substance also inhibited germination of conidia, and was fungicidal to the vegetative mycelia of the pathogen. Comparison of the properties of our antagonistic substance with that of known antibiotics produced by B. brevis suggests that our antagonistic substance is a novel compound. The observations reported here indicate that this strain of B. brevis may have potential as a biocontrol agent against fusarial wilt in pigeon pea.

Vegan-mycoprotein concentrate from pea-processing industry byproduct using edible filamentous fungi.

PubMed

Souza Filho, Pedro F; Nair, Ramkumar B; Andersson, Dan; Lennartsson, Patrik R; Taherzadeh, Mohammad J

2018-01-01

Currently around one billion people in the world do not have access to a diet which provides enough protein and energy. However, the production of one of the main sources of protein, animal meat, causes severe impacts on the environment. The present study investigates the production of a vegan-mycoprotein concentrate from pea-industry byproduct (PpB), using edible filamentous fungi, with potential application in human nutrition. Edible fungal strains of Ascomycota ( Aspergillus oryzae , Fusarium venenatum , Monascus purpureus , Neurospora intermedia ) and Zygomycota ( Rhizopus oryzae ) phyla were screened and selected for their protein production yield. A. oryzae had the best performance among the tested fungi, with a protein yield of 0.26 g per g of pea-processing byproduct from the bench scale airlift bioreactor cultivation. It is estimated that by integrating the novel fungal process at an existing pea-processing industry, about 680 kg of fungal biomass attributing to about 38% of extra protein could be produced for each 1 metric ton of pea-processing byproduct. This study is the first of its kind to demonstrate the potential of the pea-processing byproduct to be used by filamentous fungi to produce vegan-mycoprotein for human food applications. The pea-processing byproduct (PpB) was proved to be an efficient medium for the growth of filamentous fungi to produce a vegan-protein concentrate. Moreover, an industrial scenario for the production of vegan-mycoprotein concentrate for human nutrition is proposed as an integrated process to the existing PPI production facilities.

Hypocholesterolaemic effects of lupin protein and pea protein/fibre combinations in moderately hypercholesterolaemic individuals.

PubMed

Sirtori, Cesare R; Triolo, Michela; Bosisio, Raffaella; Bondioli, Alighiero; Calabresi, Laura; De Vergori, Viviana; Gomaraschi, Monica; Mombelli, Giuliana; Pazzucconi, Franco; Zacherl, Christian; Arnoldi, Anna

2012-04-01

The present study was aimed to evaluate the effect of plant proteins (lupin protein or pea protein) and their combinations with soluble fibres (oat fibre or apple pectin) on plasma total and LDL-cholesterol levels. A randomised, double-blind, parallel group design was followed: after a 4-week run-in period, participants were randomised into seven treatment groups, each consisting of twenty-five participants. Each group consumed two bars containing specific protein/fibre combinations: the reference group consumed casein+cellulose; the second and third groups consumed bars containing lupin or pea proteins+cellulose; the fourth and fifth groups consumed bars containing casein and oat fibre or apple pectin; the sixth group and seventh group received bars containing combinations of pea protein and oat fibre or apple pectin, respectively. Bars containing lupin protein+cellulose ( - 116 mg/l, - 4·2%), casein+apple pectin ( - 152 mg/l, - 5·3%), pea protein+oat fibre ( - 135 mg/l, - 4·7%) or pea protein+apple pectin ( - 168 mg/l, - 6·4%) resulted in significant reductions of total cholesterol levels (P<0·05), whereas no cholesterol changes were observed in the subjects consuming the bars containing casein+cellulose, casein+oat fibre or pea protein+cellulose. The present study shows the hypocholesterolaemic activity and potential clinical benefits of consuming lupin protein or combinations of pea protein and a soluble fibre, such as oat fibre or apple pectin.

Cross-reactivity of a new food ingredient, dun pea, with legumes, and risk of anaphylaxis in legume allergic children.

PubMed

Richard, C; Jacquenet, S; Sergeant, P; Moneret-Vautrin, D A

2015-07-01

Legume allergy is the fifth food allergy in Europe. The dun pea (Pisum sativum sativum var. arvense), a pea belonging to the same subspecies as green pea, has been recently introduced as an ingredient in the human food industry. The aims of this study were to evaluate the cross-reactivity between dun pea and other legumes and to search for modification of allergenicity induced by food technologies. A series of 36 patients with legume and/or peanut allergy was studied. They underwent skin tests to peanut and a panel of legumes including dun pea. Specific IgE to dun pea and cross-reactivity to peanut allergens, particularly to Ara h 1, were evaluated by ELISA. Proteins and allergens of different pea extracts were studied by SDS-PAGE and immunoblots. In France and Belgium, 7.7% of severe food anaphylaxis cases were due to legumes. Patients with isolated legume allergy had positive prick tests to dun pea, whereas patients with isolated peanut allergy had negative prick tests. Cross-reactivity between sIgE to peanut and dun pea was observed, and more frequently than expected (96%) peanut-allergic patients with legume sensitization or allergy had sIgE to Ara h 1. Analysis of dun pea allergens suggested that protein epitopes were presented differently in dun pea seeds, isolate and flour. This study identifies, for the first time, a risk of dun pea allergy in legume-allergic patients and in a subset of peanut-allergic patients.

[Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

PubMed

Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

2012-08-01

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

Cockroach GABAB receptor subtypes: molecular characterization, pharmacological properties and tissue distribution.

PubMed

Blankenburg, S; Balfanz, S; Hayashi, Y; Shigenobu, S; Miura, T; Baumann, O; Baumann, A; Blenau, W

2015-01-01

γ-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABAA receptors or metabotropic GABAB receptors. GABAB receptors regulate, via Gi/o G-proteins, ion channels, and adenylyl cyclases. In humans, GABAB receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABAB receptors in the American cockroach, Periplaneta americana. We have cloned cDNAs coding for putative GABAB receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABAB receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABAB receptors act as autoreceptors in this neuron. Copyright © 2014 Elsevier Ltd. All rights reserved.

Carbohydrate binding specificity of pea lectin studied by NMR spectroscopy and molecular dynamics simulations

NASA Astrophysics Data System (ADS)

Cheong, Youngjoo; Shim, Gyuchang; Kang, Dongil; Kim, Yangmee

1999-02-01

The conformational details of Man( α1,6)Man( α)OMe are investigated through NMR spectroscopy in conjunction with molecular modeling. The lowest energy structure (M1) in the adiabatic energy map calculated with a dielectric constant of 50 has glycosidic dihedral angles of φ=-60°, ψ=180° and ω=180°. The other low energy structure (M2) has glycosidic dihedral angles of φ=-60°, ψ=180° and ω=-60°. Molecular dynamics simulations and NMR experiments prove that Man( α1,6)Man( α)OMe in the free form exists with conformational averaging of M1 and M2 conformers predominantly. Molecular dynamics simulations of the pea lectin-carbohydrate complex with explicit water molecules starting from the X-ray crystallographic structure of pea lectin show that the protein-carbohydrate interaction centers mainly on the hydrogen bonds and van der Waals interactions between protein and carbohydrate. From the molecular dynamics simulation, it is found that the M1 structure can bind to pea lectin better than the M2 structure. The origin of this selectivity is the water- mediated hydrogen bond interactions between the remote mannose and the binding site of pea lectin as well as the direct hydrogen bond interaction between the terminal mannose and pea lectin. Extensive networks of interactions in the carbohydrate binding site and the metal binding site are important in maintaining the carbohydrate binding properties of pea lectin. Especially, the predominant factors of mannose binding specificity of pea lectin are the hydrogen bond interactions between the 4th hydroxyl groups of the terminal sugar ring and the side chains of Asp-81 and Asn-125 in the carbohydrate binding site, and the additional interactions between these side chains of Asp-81 and Asn-125 and the calcium ion in the metal binding site of pea lectin.

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

NASA Technical Reports Server (NTRS)

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

1992-01-01

Although the inhibitory effects of high concentrations of mineral N (> 1.0 mM) on nodule development and function have often been studied, the effects of low, static concentrations of NH4+ (< 1.0 mM) on nodulation are unknown. In the present experiments we examine the effects of static concentrations of NH4+ at 0, 0.1 and 0.5 mM in flowing, hydroponic culture on nodule establishment and nitrogenase activity in field peas [Pisum sativum L. cv. Express (Svalof AB)] for the initial 28 days after planting (DAP). Peas grown in the presence of low concentrations of NH4+ had significantly greater nodule numbers (up to 4-fold) than plants grown without NH4+. Nodule dry weight per plant was significantly higher at 14, 21 and 28 DAP in plants grown in the presence of NH4+, but individual nodule mass was lower than in plants grown without NH4+. The nodulation pattern of the plants supplied with NH4+ was similar to that often reported for supernodulating mutants, however the plants did not express other growth habits associated with supernodulation. Estimates of N2 fixation indicate that the plus-NH4+ peas fixed as much or more N2 than the plants supplied with minus-NH4+ nutrient solution. There were no significant differences in nodule numbers, nodule mass or NH4+ uptake between the plants grown at the two concentrations of NH4+. Nodulation appeared to autoregulate by 14 DAP in the minus-NH4+ treatment. Plant growth and N accumulation in the minus-NH4+ plants lagged behind those of the plus-NH4+ treatments prior to N2 fixation becoming well established in the final week of the experiment. The plus-NH4+ treatments appeared not to elicit autoregulation and plants continued to initiate nodules throughout the experiment.

Analysis of Protein Expression in Cell Microarrays: A Tool for Antibody-based Proteomics

PubMed Central

Andersson, Ann-Catrin; Strömberg, Sara; Bäckvall, Helena; Kampf, Caroline; Uhlen, Mathias; Wester, Kenneth; Pontén, Fredrik

2006-01-01

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome. PMID:16957166

Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea.

PubMed

Lee, Bao-Hong; Lai, Yi-Syuan; Wu, She-Ching

2015-12-01

Because of the high incidence of cardiovascular diseases in Asian countries, traditional fermented foods from Asia have been increasingly investigated for antiatherosclerotic effects. This study investigated the production of nattokinase, a serine fibrinolytic enzyme, in pigeon pea by Bacillus subtilis fermentation. B. subtilis 14714, B. subtilis 14715, B. subtilis 14716, and B. subtilis 14718 were employed to produce nattokinase. The highest nattokinase activity in pigeon pea was obtained using B. subtilis 14715 fermentation for 32 hours. In addition, the levels of antioxidants (phenolics and flavonoids) and angiotensin converting enzyme inhibitory activity were increased in B. subtilis 14715-fermented pigeon pea, compared with those in nonfermented pigeon pea. In an animal model, we found that both water extracts of pigeon pea (100 mg/kg body weight) and water extracts of B. subtilis-fermented pigeon pea (100 mg/kg body weight) significantly improved systolic blood pressure (21 mmHg) and diastolic blood pressure (30 mmHg) in spontaneously hypertensive rats. These results suggest that Bacillus-fermented pigeon pea has benefits for cardiovascular health and can be developed as a new dietary supplement or functional food that prevents hypertension. Copyright © 2015. Published by Elsevier B.V.

Expression of a recombinant human sperm-agglutinating mini-antibody in tobacco (Nicotiana tabacum L.).

PubMed

Xu, Bingfang; Copolla, Michael; Herr, John C; Timko, Michael P

2007-01-01

The murine monoclonal antibody (mAB) S19 recognizes an N-linked carbohydrate antigen designated sperm agglutination antigen-1 (SAGA1) located on the membrane protein CD52. This antigen is added to the sperm surface during epididymal maturation. Binding of the S19 mAB to SAGA-1 causes the rapid agglutination of sperm and blocks pre-fertilization events. Previous studies indicated that the S19 mAB may be a potential specific spermicidal agent (termed a spermistatic) capable of replacing current spermicidal products that contain harsh detergents with harmful side effects. The nucleotide sequences encoding the heavy (H) and light (L) chains of the S19 antibody were cloned. A chimeric gene was constructed using the nucleotide sequences encoding the variable regions of both the H and L chains, and this gene (scFv1 9) was expressed in transgenic tobacco (Nicotiana tabacum L.) to produce a recombinant anti-sperm antibody (RASA). Highest levels of RASA expression were observed in BY-2 plant cell suspension cultures and regenerated N. tabacum cv. Xanthi plants transformant in which the RASA coding sequences were expressed under the control of the Cauliflower Mosaic Virus 35S promoter containing a double-enhancer sequence (2X CaMV 35S). Subsequent modifications of the transgene including the addition of a 5'-untranslated sequence from the tobacco etch virus (TEV leader sequence), N-terminal fusion of the coding region with an endoplasmic reticulum targeting signal of patatin (pat) and C-terminal fusion with the endoplasmic reticulum retention signal peptide KDEL showed further enhancement of RASA expression. The plant-expressed RASA formed intrachain disulfide bonds and was primarily soluble in the cytoplasmic fraction of the cells. Introduction of a poly-histidine (6xHIS) tag in the recombinant RASA protein allowed for rapid purification of the recombinant protein using Ni-NTA chromatography. Optimization of scale-up production and purification of this plant

Genome-wide association mapping of partial resistance to Aphanomyces euteiches in pea.

PubMed

Desgroux, Aurore; L'Anthoëne, Virginie; Roux-Duparque, Martine; Rivière, Jean-Philippe; Aubert, Grégoire; Tayeh, Nadim; Moussart, Anne; Mangin, Pierre; Vetel, Pierrick; Piriou, Christophe; McGee, Rebecca J; Coyne, Clarice J; Burstin, Judith; Baranger, Alain; Manzanares-Dauleux, Maria; Bourion, Virginie; Pilet-Nayel, Marie-Laure

2016-02-20

Genome-wide association (GWA) mapping has recently emerged as a valuable approach for refining the genetic basis of polygenic resistance to plant diseases, which are increasingly used in integrated strategies for durable crop protection. Aphanomyces euteiches is a soil-borne pathogen of pea and other legumes worldwide, which causes yield-damaging root rot. Linkage mapping studies reported quantitative trait loci (QTL) controlling resistance to A. euteiches in pea. However the confidence intervals (CIs) of these QTL remained large and were often linked to undesirable alleles, which limited their application in breeding. The aim of this study was to use a GWA approach to validate and refine CIs of the previously reported Aphanomyces resistance QTL, as well as identify new resistance loci. A pea-Aphanomyces collection of 175 pea lines, enriched in germplasm derived from previously studied resistant sources, was evaluated for resistance to A. euteiches in field infested nurseries in nine environments and with two strains in climatic chambers. The collection was genotyped using 13,204 SNPs from the recently developed GenoPea Infinium® BeadChip. GWA analysis detected a total of 52 QTL of small size-intervals associated with resistance to A. euteiches, using the recently developed Multi-Locus Mixed Model. The analysis validated six of the seven previously reported main Aphanomyces resistance QTL and detected novel resistance loci. It also provided marker haplotypes at 14 consistent QTL regions associated with increased resistance and highlighted accumulation of favourable haplotypes in the most resistant lines. Previous linkages between resistance alleles and undesired late-flowering alleles for dry pea breeding were mostly confirmed, but the linkage between loci controlling resistance and coloured flowers was broken due to the high resolution of the analysis. A high proportion of the putative candidate genes underlying resistance loci encoded stress-related proteins and

Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.

PubMed

Robinson, P A; Anderton, B H; Loviny, T L

1988-04-06

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

Estimation of pea (Pisum sativum L.) microsatellite mutation rate based on pedigree and single-seed descent analyses.

PubMed

Cieslarová, Jaroslava; Hanáček, Pavel; Fialová, Eva; Hýbl, Miroslav; Smýkal, Petr

2011-11-01

Microsatellites, or simple sequence repeats (SSRs) are widespread class of repetitive DNA sequences, used in population genetics, genetic diversity and mapping studies. In spite of the SSR utility, the genetic and evolutionary mechanisms are not fully understood. We have investigated three microsatellite loci with different position in the pea (Pisum sativum L.) genome, the A9 locus residing in LTR region of abundant retrotransposon, AD270 as intergenic and AF016458 located in 5'untranslated region of expressed gene. Comparative analysis of a 35 pair samples from seven pea varieties propagated by single-seed descent for ten generations, revealed single 4 bp mutation in 10th generation sample at AD270 locus corresponding to stepwise increase in one additional ATCT repeat unit. The estimated mutation rate was 4.76 × 10(-3) per locus per generation, with a 95% confidence interval of 1.2 × 10(-4) to 2.7 × 10(-2). The comparison of cv. Bohatýr accessions retrieved from different collections, showed intra-, inter-accession variation and differences in flanking and repeat sequences. Fragment size and sequence alternations were also found in long term in vitro organogenic culture, established at 1983, indicative of somatic mutation process. The evidence of homoplasy was detected across of unrelated pea genotypes, which adversaly affects the reliability of diversity estimates not only for diverse germplasm but also highly bred material. The findings of this study have important implications for Pisum phylogeny studies, variety identification and registration process in pea breeding where mutation rate influences the genetic diversity and the effective population size estimates.

VizieR Online Data Catalog: Lyα profile in 43 Green Pea galaxies (Yang+, 2017)

NASA Astrophysics Data System (ADS)

Yang, H.; Malhotra, S.; Gronke, M.; Rhoads, J. E.; Leitherer, C.; Wofford, A.; Jiang, T.; Dijkstra, M.; Tilvi, V.; Wang, J.

2018-03-01

In SDSS DR7, a sample of 251 Green Peas was observed as serendipitous spectroscopic targets (Cardamone+ 2009MNRAS.399.1191C). A subset of 66 Green Peas have sufficient signal-to-noise ratio (S/N) in both continuum and emission lines (Hα, Hβ, and [OIII]λ5007) to study galactic properties. In Paper I (Yang+ 2016ApJ...820..130Y), we matched these 66 Green Peas with the COS archive and studied Lyα escape in a sample of 12 Green Peas with COS UV spectra. To address the bias and expand the sample size, we took the Lyα spectra of 20 additional Green Peas (PI S. Malhotra, GO 14201). We also supplement this sample with 11 additional Green Peas from published literature. In total, we have 43 Green Peas from six HST programs -- 20 galaxies from GO 14201 (PI S. Malhotra), 9 galaxies from GO 12928 (PI A. Henry; Henry+ 2015ApJ...809...19H), 7 galaxies from GO 11727 and GO 13017 (PI T. Heckman; Heckman+ 2011ApJ...730....5H ; Alexandroff+ 2015ApJ...810..104A), 2 galaxies from GO 13293 (PI A. Jaskot; Jaskot & Oey 2014ApJ...791L..19J), and 5 galaxies from GO 13744 (PI T. Thuan; Izotov+ 2016MNRAS.461.3683I). (4 data files).

Fermented pigeon pea (Cajanus cajan) ingredients in pasta products.

PubMed

Torres, Alexia; Frias, J; Granito, M; Vidal-Valverde, C

2006-09-06

Pigeon pea (Cajanus cajan var. aroíto) seeds were fermented in order to remove antinutritional factors and to obtain functional legume flour to be used as pasta ingredients. Fermentation brought about a drastic reduction of alpha-galactosides (82%), phytic acid (48%), and trypsin inhibitor activity (39%). Fermented legume flours presented a notable increase of fat and total soluble available carbohydrates, a slight decrease of protein, dietary fiber, calcium, vitamin B2, vitamin E, and total antioxidant capacity, and a decrease of soluble dietary fiber, Na, K, Mg, and Zn contents. No changes were observed in the level of starch and tannins as a consequence of fermentation. The fermented flour was used as an ingredient to make pasta products in a proportion of 5, 10, and 12%. The supplemented pasta products obtained had longer cooking times, higher cooking water absorptions, higher cooking loss, and higher protein loss in water than control pasta (100% semolina). From sensory evaluations, fortified pasta with 5 and 10% fermented pigeon pea flour had an acceptability score similar to control pasta. Pasta supplemented with 10% fermented pigeon pea flour presented higher levels of protein, fat, dietary fiber, mineral, vitamin E, and Trolox equivalent antioxidant capacity than 100% semolina pasta and similar vitamins B1 and B2 contents. Protein efficiency ratios and true protein digestibility improved (73 and 6%, respectively) after supplementation with 10% fermented pigeon pea flour; therefore, the nutritional value was enhanced.

Nutritional evaluation of low-phytate peas (Pisum sativum L.) for young broiler chicks.

PubMed

Thacker, Philip; Deep, Aman; Petri, Daniel; Warkentin, Thomas

2013-02-01

This experiment determined the effects of including normal and low-phytate peas in diets fed to young broiler chickens on performance, phosphorus availability and bone strength. A total of 180, day-old, male broilers (Ross-308 line) were assigned to six treatments. The control was based on corn and soybean meal while two additional corn-based diets were formulated containing 30% of either normal or low-phytate pea providing 0.45% available phosphorus. For each of these three diets, a similar diet was formulated by reducing the amount of dicalcium phosphate to produce a diet with 0.3% available phosphorus. The total tract apparent availability (TTAA) of phosphorus was higher (p = 0.02) for broilers fed the low-phytate pea than for birds fed the normal pea diets. Birds fed diets containing the lower level of phosphorus had a higher TTAA of phosphorus (50.64 vs. 46.68%) than broilers fed diets adequate in phosphorus. Protein source had no effect on weight gain, feed intake or feed conversion. Broilers fed the low phosphorus diets had lower weight gain (p = 0.04) and feed intake (p < 0.01) than broilers fed the higher phosphorus level. Bone strength was higher (p < 0.01) for broilers fed diets based on low-phytate pea than for those fed diets based on normal pea or soybean meal. Increasing the availability of the phosphorus in peas could mean that less inorganic phosphorus would be required in order to meet the nutritional requirements of broilers. Since inorganic phosphorus sources tend to be expensive, a reduction in their use would lower ration costs. In addition, increased availability of phosphorus would reduce the amount of phosphorus excreted thus reducing the amount of phosphorus that can potentially pollute the environment.

Establishing alighting preferences and species transmission differences for Pea seed-borne mosaic virus aphid vectors.

PubMed

Congdon, B S; Coutts, B A; Renton, M; Flematti, G R; Jones, R A C

2017-09-15

Pea seed-borne mosaic virus (PSbMV) infection causes a serious disease of field pea (Pisum sativum) crops worldwide. The PSbMV transmission efficiencies of five aphid species previously found landing in south-west Australian pea crops in which PSbMV was spreading were studied. With plants of susceptible pea cv. Kaspa, the transmission efficiencies of Aphis craccivora, Myzus persicae, Acyrthosiphon kondoi and Rhopalosiphum padi were 27%, 26%, 6% and 3%, respectively. Lipaphis erysimi did not transmit PSbMV in these experiments. The transmission efficiencies found for M. persicae and A. craccivora resembled earlier findings, but PSbMV vector transmission efficiency data were unavailable for A. kondoi, R. padi and L. erysimi. With plants of partially PSbMV resistant pea cv. PBA Twilight, transmission efficiencies of M. persicae, A. craccivora and R. padi were 16%, 12% and 1%, respectively, reflecting putative partial resistance to aphid inoculation. To examine aphid alighting preferences over time, free-choice assays were conducted with two aphid species representing efficient (M. persicae) and inefficient (R. padi) vector species. For this, alatae were set free on multiple occasions (10-15 repetitions each) amongst PSbMV-infected and mock-inoculated pea or faba bean (Vicia faba) plants. Following release, non-viruliferous R. padi alatae exhibited a general preference for PSbMV-infected pea and faba bean plants after 30min-4h, but preferred mock-inoculated plants after 24h. In contrast, non-viruliferous M. persicae alatae alighted on mock-inoculated pea plants preferentially for up to 48h following their release. With faba bean, M. persicae preferred infected plants at the front of assay cages, but mock-inoculated ones their backs, apparently due to increased levels of natural light there. When preliminary analyses were performed to detect PSbMV-induced changes in the volatile organic compound profiles of pea and faba bean plants, higher numbers of volatiles

Effect of extrusion on the nutritional value of peas for broiler chickens.

PubMed

Hejdysz, Marcin; Kaczmarek, Sebastian Andrzej; Rutkowski, Andrzej

2016-10-01

The study was conducted to investigate the nutritional value of five samples of raw and extruded pea seeds (Pisum sativum L., Tarachalska cv.) from different experimental fields. The study included 150 male 1-day-old Ross 308 chickens, which were randomly assigned to three dietary treatments (50 replications each) and kept in individual cages. From days 1 to 16, all birds received only the basal diets. From days 17 to 21, the control group received still the basal diet, but for the two other groups, 20% of basal diet was replaced by raw or extruded peas. Furthermore, the groups receiving raw or extruded peas were divided into five subgroups of 10 animals each, where the diets contained one of the five pea samples of the same cultivar grown at different locations, respectively. On days 19 and 20, excreta were individually collected, and then all chickens were sacrificed and ileal digesta were sampled for determination of ileal digestibility, which was calculated by the difference method. Extrusion of pea seeds decreased the contents of crude fibre, acid and neutral detergent fibre, trypsin inhibitor activity (TIA), phytic P and resistant starch (RS) (p ≤ 0.05), but increased the contents of apparent metabolisable energy (AMEN) by approximately 2.25 MJ/kg dry matter (DM). Furthermore, extrusion improved the DM and crude protein digestibility significantly by about 21.3% and 11.6%, respectively. Similar results were observed for the digestibility of all analysed amino acids. In conclusion, extrusion markedly influenced the chemical composition of peas, reduced their contents of phytic P, TIA and RS and consequently had a positive impact on nutrient digestibility and AMEN values.

Transcriptome sequencing for high throughput SNP development and genetic mapping in Pea

PubMed Central

2014-01-01

Background Pea has a complex genome of 4.3 Gb for which only limited genomic resources are available to date. Although SNP markers are now highly valuable for research and modern breeding, only a few are described and used in pea for genetic diversity and linkage analysis. Results We developed a large resource by cDNA sequencing of 8 genotypes representative of modern breeding material using the Roche 454 technology, combining both long reads (400 bp) and high coverage (3.8 million reads, reaching a total of 1,369 megabases). Sequencing data were assembled and generated a 68 K unigene set, from which 41 K were annotated from their best blast hit against the model species Medicago truncatula. Annotated contigs showed an even distribution along M. truncatula pseudochromosomes, suggesting a good representation of the pea genome. 10 K pea contigs were found to be polymorphic among the genetic material surveyed, corresponding to 35 K SNPs. We validated a subset of 1538 SNPs through the GoldenGate assay, proving their ability to structure a diversity panel of breeding germplasm. Among them, 1340 were genetically mapped and used to build a new consensus map comprising a total of 2070 markers. Based on blast analysis, we could establish 1252 bridges between our pea consensus map and the pseudochromosomes of M. truncatula, which provides new insight on synteny between the two species. Conclusions Our approach created significant new resources in pea, i.e. the most comprehensive genetic map to date tightly linked to the model species M. truncatula and a large SNP resource for both academic research and breeding. PMID:24521263

High expression and purification of the recombinant camelid anti-MUC1 single domain antibodies in Escherichia coli.

PubMed

Rahbarizadeh, Fatemeh; Rasaee, Mohammad Javad; Forouzandeh-Moghadam, Mehdi; Allameh, Abdol-Amir

2005-11-01

In contrast to the murine and human VHs, camels' single domain antibodies (sdAb) have sufficient solubility. These antigen-specific fragments are expressed well in Escherichia coli. Here, we report high expression and purification of sdAbs against MUC1 mucin. MUC1 is a high molecular weight glycoprotein with an aberrant expression profile in various malignancies. The sdAb genes were sub-cloned into a pET32a(+) vector to overexpress the protein coupled with fusion tags in E. coli BL21(DE3). The expressed single domain antibodies were purified by immobilized metal affinity chromatography and antigen affinity chromatography. Analysis by SDS-PAGE and Western blotting demonstrated the integrity of the sdAbs-tags, while ELISA results confirm that the activity of these molecules compare favorably with that of the parent recombinant antibodies. Enterokinase treated sdAb showed a band at the molecular weight around 12 kDa which demonstrated the naked protein in its natural structure with activities comparable to that of native protein. The high binding activity to MUC1 antigen purified from ascitic fluid (of patients with small-cell lung aggressive carcinoma and metastasis to peritoneum) and the very close similarity of these molecules to human VHs illustrated the potential application of these novel products as an immunodiagnostic and immunotherapeutic reagent.

PsRBR1 encodes a pea retinoblastoma-related protein that is phosphorylated in axillary buds during dormancy-to-growth transition

PubMed Central

Shimizu-Sato, Sae; Ike, Yoko

2007-01-01

In intact plants, cells in axillary buds are arrested at the G1 phase of the cell cycle during dormancy. In mammalian cells, the cell cycle is suppressed at the G1 phase by the activities of retinoblastoma tumor suppressor gene (RB) family proteins, depending on their phosphorylation state. Here, we report the isolation of a pea cDNA clone encoding an RB-related protein (PsRBR1, Accession No. AB012024) with a high degree of amino acid conservation in comparison with RB family proteins. PsRBR1 protein was detected as two polypeptides using an anti-PsRBR1 antibody in dormant axillary buds, whereas it was detected as three polypeptides, which were the same two polypeptides and another larger polypeptide 2 h after terminal decapitation. Both in vitro-synthesized PsPRB1 protein and lambda protein phosphatase-treated PsRBR1 protein corresponded to the smallest polypeptide detected by anti-PsRBR1 antibody, suggesting that the three polypeptides correspond to non-phosphorylated form of PsRBR1 protein, and lower- and higher-molecular mass forms of phosphorylated PsRBR1 protein. Furthermore, in vivo labeling with [32P]-inorganic phosphate indicated that PsRBR1 protein was more phosphorylated before mRNA accumulation of cell cycle regulatory genes such as PCNA. Together these findings suggest that dormancy-to-growth transition in pea axillary buds is regulated by molecular mechanisms of cell cycle control similar to those in mammals, and that the PsRBR1 protein has an important role in suppressing the cell cycle during dormancy in axillary buds. PMID:18034314

Feeding value of field pea as a protein source in forage-based diets fed to beef cattle.

PubMed

Soto-Navarro, S A; Encinias, A M; Bauer, M L; Lardy, G P; Caton, J S

2012-02-01

Three studies were conducted to evaluate the feasibility of field peas as a protein source in diets for beef cattle. In the first study, 4 cultivars of field pea were incubated in situ to determine rate and extent of CP disappearance. Results indicate that field pea cultivars vary in CP content (22.6, 26.1, 22.6, and 19.4%, DM basis for Profi, Arvika, Carneval, and Trapper, respectively). Soluble protein fraction ranged from 34.9% for Trapper to 54.9% for Profi. Degradable CP fraction was greater (P = 0.01) for Trapper compared with the other cultivars, and no differences (P ≥ 0.25) were observed among Profi, Arvika, and Carneval. Rate of CP degradation differed (P ≤ 0.03) for all cultivars, with Profi being the greatest and Trapper the smallest (10.8, 10.0, 8.1, and 6.3 ± 1.4%/h for Profi, Carneval, Arvika, and Trapper, respectively). Estimated RDP was not different (P = 0.21) for all 4 cultivars. In the second study, 30 crossbred beef steers (301 ± 15 kg) were individually fed and used to evaluate effects of field pea processing (whole, rolled, or ground) on steer performance. Diets contained 40% field pea grain. Growing steers consuming whole field pea had greater ADG (P = 0.08) than those consuming processed field pea (1.69, 1.52, and 1.63 ± 0.05 kg/d, for whole, rolled, and ground, respectively). However, DMI (kg/d and as % of BW) and G:F were not different (P ≥ 0.24). In the third study, 35 individually fed gestating beef cows (694 ± 17 kg) were used to evaluate the use of field pea as a protein supplement for medium quality grass hay (9.3% CP). Treatments consisted of whole field peas at 1) 0 g (CON), 2) 680 g (FP680), 3) 1,360 g (FP1360), and 4) 2,040 g (FP2040), and 5) 1,360 g of 74% barley and 26% canola meal (BCM). Total intake (forage + supplement) of gestating beef cows increased with increasing field pea level (linear, P = 0.01; supplemented vs. nonsupplemented, P = 0.01). In summary, protein quantity and rate of ruminal protein degradation

Studying Lyman-alpha escape and reionization in Green Pea galaxies

NASA Astrophysics Data System (ADS)

Yang, Huan; Malhotra, Sangeeta; Rhoads, James E.; Gronke, Max; Leitherer, Claus; Wofford, Aida; Dijkstra, Mark

2017-01-01

Green Pea galaxies are low-redshift galaxies with extreme [OIII]5007 emission line. We built the first statistical sample of Green Peas observed by HST/COS and used them as analogs of high-z Lyman-alpha emitters to study Ly-alpha escape and Ly-alpha sizes. Using the HST/COS 2D spectra, we found that Ly-alpha sizes of Green Peas are larger than the UV continuum sizes. We found many correlations between Ly-alpha escape fraction and galactic properties -- dust extinction, Ly-alpha kinematic features, [OIII]/[OII] ratio, and gas outflow velocities. We fit an empirical relation to predict Ly-alpha escape fraction from dust extinction and Ly-alpha red-peak velocity. In the JWST era, we can use this relation to derive the IGM HI column density along the line of sight of each high-z Ly-alpha emitter and probe the reionization process.

Differential impact of environmental stresses on the pea mitochondrial proteome.

PubMed

Taylor, Nicolas L; Heazlewood, Joshua L; Day, David A; Millar, A Harvey

2005-08-01

Exposure to adverse environmental conditions causes oxidative stress in many organisms, leading either to disease and debilitation or to response and tolerance. Mitochondria are a key site of oxidative stress and of cellular response and play important roles in cell survival. We analyzed the response of mitochondria in pea (Pisum sativum) plants to the common stresses associated with drought, cold, and herbicides. These treatments all altered photosynthetic and respiratory rates of pea leaves to various extents, but only herbicides significantly increased lipid peroxidation product accumulation. Mitochondria isolated from the stressed pea plants maintained their electron transport chain activity, but changes were evident in the abundance of uncoupling proteins, non-phosphorylating respiratory pathways, and oxidative modification of lipoic acid moieties on mitochondrial proteins. These data suggest that herbicide treatment placed a severe oxidative stress on mitochondria, whereas chilling and particularly drought were milder stresses. Detailed analysis of the soluble proteome of mitochondria by gel electrophoresis and mass spectrometry revealed differential degradation of key matrix enzymes during treatments with chilling being significantly more damaging than drought. Differential induction of heat shock proteins and specific losses of other proteins illustrated the diversity of response to these stresses at the protein level. Cross-species matching was required for mass spectrometry identification of nine proteins because only a limited number of pea cDNAs have been sequenced, and the full pea genome is not available. Blue-native separation of intact respiratory chain complexes revealed little if any change in response to environmental stresses. Together these data suggest that although many of the molecular events identified by chemical stresses of mitochondria from a range of model eukaryotes are also apparent during environmental stress of plants, their extent

Insecticidal components from field pea extracts: isolation and separation of peptide mixtures related to pea albumin 1b.

PubMed

Taylor, Wesley G; Fields, Paul G; Elder, James L

2004-12-15

Chromatographic fractionation of crude extracts (C8 extracts) from the protein-enriched flour of commercial field peas (Pisum sativum L.) has been shown here to yield peptide mixtures related to the pea albumin 1b (PA1b) family of cysteine-rich plant peptides. The mixtures were obtained initially by flash chromatography with silica gel. Following elution of soyasaponins and lysolecithins, the end fractions obtained with the use of two flash chromatographic solvent systems displayed activity in a flour disk antifeedant bioassay with the rice weevil [Sitophilus oryzae (L.)]. Chemical properties of these mixtures were compared by thin-layer chromatography, high-performance liquid chromatography (HPLC), IR, MS, and amino acid analyses. The major peptides of C8 extracts, with average masses of 3752, 3757, and 3805 Da, were isolated by anion exchange chromatography. Samples enriched in the peptide of mass 3752 were isolated by cation exchange chromatography. Reduction plus alkylation experiments in combination with electrospray ionization mass spectrometry showed that C8 extracts contained about 10 peptides and, like PA1b, each peptide possessed six cysteine residues (three disulfide bonds). Disulfide bond reduction with 2-mercaptoethanol destroyed the antifeedant activity. The native peptides of C8 extracts were found to be resolved into nine peaks with XTerra HPLC columns operating at alkaline pH. These columns were employed to assess the distribution of pea peptides in the isolated fractions, with photodiode array and electrospray detection.

Pea, Pisum sativum, and Its Anticancer Activity

PubMed Central

Rungruangmaitree, Runchana; Jiraungkoorskul, Wannee

2017-01-01

Pisum sativum (Family: Fabaceae), as known as green pea or garden pea, has long been important in diet due to its content of fiber, protein, starch, trace elements, and many phytochemical substances. It has been shown to possess antibacterial, antidiabetic, antifungal, anti-inflammatory, antihypercholesterolemia, and antioxidant activities and also shown anticancer property. Its nonnutritive biologically active components include alkaloids, flavonoids, glycosides, isoflavones, phenols, phytosterols, phytic acid, protease inhibitors, saponins, and tannins. This plant is rich in apigenin, hydroxybenzoic, hydroxycinnamic, luteolin, and quercetin, all of which have been reported to contribute to its remedial properties including anticarcinogenesis property. Based on established literature on the anticancer property of P. sativum and possible mode of action, this review article has focused to demonstrate that P. sativum could be further explored for the development of anticancer treatment. PMID:28503053

Effective stabilization of CLA by microencapsulation in pea protein.

PubMed

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. Copyright © 2014 Elsevier Ltd. All rights reserved.

NUTRALYS® pea protein: characterization of in vitro gastric digestion and in vivo gastrointestinal peptide responses relevant to satiety

PubMed Central

Overduin, Joost; Guérin-Deremaux, Laetitia; Wils, Daniel; Lambers, Tim T.

2015-01-01

Background Pea protein (from Pisum sativum) is under consideration as a sustainable, satiety-inducing food ingredient. Objective In the current study, pea-protein-induced physiological signals relevant to satiety were characterized in vitro via gastric digestion kinetics and in vivo by monitoring post-meal gastrointestinal hormonal responses in rats. Design Under in vitro simulated gastric conditions, the digestion of NUTRALYS® pea protein was compared to that of two dairy proteins, slow-digestible casein and fast-digestible whey. In vivo, blood glucose and gastrointestinal hormonal (insulin, ghrelin, cholecystokinin [CCK], glucagon-like peptide 1 [GLP-1], and peptide YY [PYY]) responses were monitored in nine male Wistar rats following isocaloric (11 kcal) meals containing 35 energy% of either NUTRALYS® pea protein, whey protein, or carbohydrate (non-protein). Results In vitro, pea protein transiently aggregated into particles, whereas casein formed a more enduring protein network and whey protein remained dissolved. Pea-protein particle size ranged from 50 to 500 µm, well below the 2 mm threshold for gastric retention in humans. In vivo, pea-protein and whey-protein meals induced comparable responses for CCK, GLP-1, and PYY, that is, the anorexigenic hormones. Pea protein induced weaker initial, but equal 3-h integrated ghrelin and insulin responses than whey protein, possibly due to the slower gastric breakdown of pea protein observed in vitro. Two hours after meals, CCK levels were more elevated in the case of protein meals compared to that of non-protein meals. Conclusions These results indicate that 1) pea protein transiently aggregates in the stomach and has an intermediately fast intestinal bioavailability in between that of whey and casein; 2) pea-protein- and dairy-protein-containing meals were comparably efficacious in triggering gastrointestinal satiety signals. PMID:25882536

Assessing the effects of architectural variations on light partitioning within virtual wheat–pea mixtures

PubMed Central

Barillot, Romain; Escobar-Gutiérrez, Abraham J.; Fournier, Christian; Huynh, Pierre; Combes, Didier

2014-01-01

Background and Aims Predicting light partitioning in crop mixtures is a critical step in improving the productivity of such complex systems, and light interception has been shown to be closely linked to plant architecture. The aim of the present work was to analyse the relationships between plant architecture and light partitioning within wheat–pea (Triticum aestivum–Pisum sativum) mixtures. An existing model for wheat was utilized and a new model for pea morphogenesis was developed. Both models were then used to assess the effects of architectural variations in light partitioning. Methods First, a deterministic model (L-Pea) was developed in order to obtain dynamic reconstructions of pea architecture. The L-Pea model is based on L-systems formalism and consists of modules for ‘vegetative development’ and ‘organ extension’. A tripartite simulator was then built up from pea and wheat models interfaced with a radiative transfer model. Architectural parameters from both plant models, selected on the basis of their contribution to leaf area index (LAI), height and leaf geometry, were then modified in order to generate contrasting architectures of wheat and pea. Key results By scaling down the analysis to the organ level, it could be shown that the number of branches/tillers and length of internodes significantly determined the partitioning of light within mixtures. Temporal relationships between light partitioning and the LAI and height of the different species showed that light capture was mainly related to the architectural traits involved in plant LAI during the early stages of development, and in plant height during the onset of interspecific competition. Conclusions In silico experiments enabled the study of the intrinsic effects of architectural parameters on the partitioning of light in crop mixtures of wheat and pea. The findings show that plant architecture is an important criterion for the identification/breeding of plant ideotypes, particularly

Beta-ketoacyl-acyl carrier protein synthase III from pea (Pisum sativum L.): properties, inhibition by a novel thiolactomycin analogue and isolation of a cDNA clone encoding the enzyme.

PubMed

Jones, A Lesley; Gane, Andy M; Herbert, Derek; Willey, David L; Rutter, Andrew J; Kille, Peter; Dancer, Jane E; Harwood, John L

2003-03-01

A beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III; short-chain condensing enzyme) has been partly purified from pea leaves. The enzyme, which had acetyl-CoA:ACP acyltransferase (ACAT) activity, was resolved from a second, specific, ACAT protein. The KAS III enzyme had a derived molecular mass of 42 kDa (from its cDNA sequence) and operated as a dimer. Its enzymological characteristics were similar to those of two other plant KAS III enzymes except for its inhibition by thiolactomycin. A derivative of thiolactomycin containing a longer (C8 saturated) hydrophobic side-chain (compound 332) was a more effective inhibitor of pea KAS III and showed competitive inhibition towards malonyl-ACP whereas thiolactomycin showed uncompetitive characteristics at high concentrations. This difference may be due to the better fit of compound 332 into a hydrophobic pocket at the active site. A full-length cDNA for the pea KAS III was isolated. This was expressed in Escherichia coli as a fusion protein with glutathione S-transferase in order to facilitate subsequent purification. Demonstrated activity in preparations from E. coli confirmed that the cDNA encoded a KAS III enzyme. Furthermore, the expressed KAS III had ACAT activity, showing that the latter was inherent. The derived amino acid sequence of the pea cDNA showed 81-87% similarity to that for other plant dicotyledon KAS IIIs, somewhat less for Allium porrum (leek, 71%) and for Porphyra spp. (62%), Synechocystis spp. (65%) and various bacteria (42-65%). The pea KAS III exhibited four areas of homology, three of which were around the active-site Cys(123), His(323) and Asn(353). In addition, a stretch of 23 amino acids (residues 207-229 in the pea KAS III) was almost completely conserved in the plant KAS IIIs. Modelling this stretch showed they belonged to a peptide fragment that fitted over the active site and contained segments suggested to be involved in substrate binding and in conformational changes during

Studies on antioxidative enzymes induced by cadmium in pea plants (Pisum sativum).

PubMed

Pandey, Nalini; Singh, Gaurav Kumar

2012-03-01

Pea plants (Pisum sativum cv. Swati) exposed to different concentration of cadmium (50,100, 200 microM Cd) under controlled glass house conditions were quantified for different physiological parameters and antioxidative enzymes. In pea plants, Cd produced a significant inhibition of growth and induced chlorosis, marginal yellowing and necrosis in young leaves, the effect being most pronounced at 200 microM Cd supply. An alteration in the activated oxygen metabolism of pea plants were also detected as evidenced by an increase in concentration of H2O2 and TBARS along with decrease in the chlorophyll and carotenoid concentration in leaves. Cadmium toxicity induced an increase in non-protein thiol, ascorbate, proline and cysteine concentration. A significant increment in the activity of SOD, APX and GR, and a decrease in CAT was observed as a result of Cd treatment. The enhanced activity of SOD and inhibition of CAT and POD produces a high build up of H2O2 which appears to be the main cause of oxidative stress due to Cd toxicity in pea plants.

Validation of quantitative method for azoxystrobin residues in green beans and peas.

PubMed

Abdelraheem, Ehab M H; Hassan, Sayed M; Arief, Mohamed M H; Mohammad, Somaia G

2015-09-01

This study presents a method validation for extraction and quantitative analysis of azoxystrobin residues in green beans and peas using HPLC-UV and the results confirmed by GC-MS. The employed method involved initial extraction with acetonitrile after the addition of salts (magnesium sulfate and sodium chloride), followed by a cleanup step by activated neutral carbon. Validation parameters; linearity, matrix effect, LOQ, specificity, trueness and repeatability precision were attained. The spiking levels for the trueness and the precision experiments were (0.1, 0.5, 3 mg/kg). For HPLC-UV analysis, mean recoveries ranged between 83.69% to 91.58% and 81.99% to 107.85% for green beans and peas, respectively. For GC-MS analysis, mean recoveries ranged from 76.29% to 94.56% and 80.77% to 100.91% for green beans and peas, respectively. According to these results, the method has been proven to be efficient for extraction and determination of azoxystrobin residues in green beans and peas. Copyright © 2015 Elsevier Ltd. All rights reserved.

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

NASA Technical Reports Server (NTRS)

Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Characterization of an invertebrate-type dopamine receptor of the American cockroach, Periplaneta americana.

PubMed

Troppmann, Britta; Balfanz, Sabine; Krach, Christian; Baumann, Arnd; Blenau, Wolfgang

2014-01-06

We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology.

Molecular and functional characterization of cry1Ac transgenic pea lines.

PubMed

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-10-01

Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses.

Molecular and functional characterization of cry1Ac transgenic pea lines

PubMed Central

Teressa Negawo, Alemayehu; Baranek, Linda; Jacobsen, Hans-Jörg; Hassan, Fathi

2016-01-01

ABSTRACT Transgenic pea lines transformed with the cry1Ac gene were characterized at molecular (PCR, RT-PCR, qRT-PCR and immunostrip assay) and functional levels (leaf paint and insect feeding bioassays). The results showed the presence, expression, inheritance and functionality of the introduced transgene at different progeny levels. Variation in the expression of the cry1Ac gene was observed among the different transgenic lines. In the insect bioassay studies using the larvae of Heliothis virescens, both larval survival and plant damage were highly affected on the different transgenic plants. Up to 100 % larval mortality was observed on the transgenic plants compared to 17.42 % on control plants. Most of the challenged transgenic plants showed very negligible to substantially reduced feeding damage indicating the insect resistance of the developed transgenic lines. Further analysis under field condition will be required to select promising lines for future uses. PMID:27764552

Strategic deployment of CHO expression platforms to deliver Pfizer's Monoclonal Antibody Portfolio.

PubMed

Scarcelli, John J; Shang, Tanya Q; Iskra, Tim; Allen, Martin J; Zhang, Lin

2017-11-01

Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1463-1467, 2017. © 2017 American Institute of Chemical Engineers.

Changes in the germination process and growth of pea in effect of laser seed irradiation

NASA Astrophysics Data System (ADS)

Podleśna, Anna; Gładyszewska, Bożena; Podleśny, Janusz; Zgrajka, Wojciech

2015-10-01

The aim of this study was to determine the effect of pre-sowing helium-neon (He-Ne) laser irradiation of pea seeds on changes in seed biochemical processes, germination rate, seedling emergence, growth rate, and yield. The first experimental factor was exposure to laser radiation: D0 - no irradiation, D3 - three exposures, D5 - five exposures, and the harvest dates were the second factor. Pre-sowing treatment of pea seeds with He-Ne laser light increased the concentrations of amylolytic enzymes and the content of indole-3-acetic acid (IAA) in pea seeds and seedlings. The exposure of seeds to He-Ne laser light improved the germination rate and uniformity and modified growth stages, which caused acceleration of flowering and ripening of pea plants. Laser light stimulation improved the morphological characteristics of plants by increasing plant height and leaf surface area. Irradiation improved the yield of vegetative and reproductive organs of pea, although the effects varied at the different growth stages. The increase in the seed yield resulted from a higher number of pods and seeds per plant, whereas no significant changes were observed in the number of seeds per pod. Both radiation doses exerted similarly stimulating effects on pea growth, development, and yield.

Pea disease diagnostic series - White mold

USDA-ARS?s Scientific Manuscript database

White mold is a serious disease of pea worldwide, and it is caused by the fungus Sclerotinia sclerotiorum. Water soaked lesions and white mycelial growth may occur on leaves, stems and pods, and are characteristics of the disease. The pathogen may form black fruiting bodies called sclerotia on infec...

Light differentially regulates cell division and the mRNA abundance of pea nucleolin during de-etiolation

NASA Technical Reports Server (NTRS)

Reichler, S. A.; Balk, J.; Brown, M. E.; Woodruff, K.; Clark, G. B.; Roux, S. J.

2001-01-01

The abundance of plant nucleolin mRNA is regulated during de-etiolation by phytochrome. A close correlation between the mRNA abundance of nucleolin and mitosis has also been previously reported. These results raised the question of whether the effects of light on nucleolin mRNA expression were a consequence of light effects on mitosis. To test this we compared the kinetics of light-mediated increases in cell proliferation with that of light-mediated changes in the abundance of nucleolin mRNA using plumules of dark-grown pea (Pisum sativum) seedlings. These experiments show that S-phase increases 9 h after a red light pulse, followed by M-phase increases in the plumule leaves at 12 h post-irradiation, a time course consistent with separately measured kinetics of red light-induced increases in the expression of cell cycle-regulated genes. These increases in cell cycle-regulated genes are photoreversible, implying that the light-induced increases in cell proliferation are, like nucleolin mRNA expression, regulated via phytochrome. Red light stimulates increases in the mRNA for nucleolin at 6 h post-irradiation, prior to any cell proliferation changes and concurrent with the reported timing of phytochrome-mediated increases of rRNA abundance. After a green light pulse, nucleolin mRNA levels increase without increasing S-phase or M-phase. Studies in animals and yeast indicate that nucleolin plays a significant role in ribosome biosynthesis. Consistent with this function, pea nucleolin can rescue nucleolin deletion mutants of yeast that are defective in rRNA synthesis. Our data show that during de-etiolation, the increased expression of nucleolin mRNA is more directly regulated by light than by mitosis.

ALK gene copy number gain and immunohistochemical expression status using three antibodies in neuroblastoma.

PubMed

Kim, Eun Kyung; Kim, Sewha

2016-03-17

Anaplastic lymphoma kinase (ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC positive rate in ALK1 and 5A4 antibodies (p= < 0.001 and 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

ALK Gene Copy Number Gain and Immunohistochemical Expression Status Using Three Antibodies in Neuroblastoma.

PubMed

Kim, Eun Kyung; Kim, Sewha

2017-01-01

Anaplastic lymphoma kinase ( ALK) gene aberrations-such as mutations, amplifications, and copy number gains-represent a major genetic predisposition to neuroblastoma (NB). This study aimed to evaluate the correlation between ALK gene copy number status, ALK protein expression, and clinicopathological parameters. We retrospectively retrieved 30 cases of poorly differentiated NB and constructed tissue microarrays (TMAs). ALK copy number changes were assessed by fluorescence in situ hybridization (FISH) assays, and ALK immunohistochemistry (IHC) testing was performed using three different antibodies (ALK1, D5F3, and 5A4 clones). ALK amplification and copy number gain were observed in 10% (3/30) and 53.3% (16/30) of the cohort, respectively. There were positive correlations between ALK copy number and IHC-positive rate in ALK1 and 5A4 antibodies ( P < 0.001 and P = 0.019, respectively). ALK1, D5F3, and 5A4 antibodies equally showed 100% sensitivity in detecting ALK amplification. However, the sensitivity for detecting copy number gain differed among the three antibodies, with 75% sensitivity in D5F3 and 0% sensitivity in ALK1. ALK-amplified NBs were correlated with synchronous MYCN amplification and chromosome 1p deletion. ALK IHC positivity was frequently observed in INSS stage IV and high-risk group patients. In conclusion, this study identified that an increase in the ALK copy number is a frequent genetic alteration in poorly differentiated NB. ALK-amplified NBs showed consistent ALK IHC positivity with all kinds of antibodies. In contrast, the detection performance of ALK copy number gain was antibody dependent, with the D5F3 antibody showing the best sensitivity.

Effect of Root-Zone Moisture Variations on Growth of Lettuce and Pea Plants

NASA Astrophysics Data System (ADS)

Ilieva, Iliana; Ivanova, Tania

2008-06-01

Variations in substrate moisture lead to changes in water and oxygen availability to plant roots. Ground experiments were carried out in the laboratory prototype of SVET-2 Space Greenhouse to study the effect of variation of root-zone moisture conditions on growth of lettuce and pea plants. The effect of transient increase (for 1 day) and drastic increase (waterlogging for 10 days) of substrate moisture was studied with 16-day old pea and 21-day old lettuce plants respectively. Pea height and fresh biomass accumulation were not affected by transient substrate moisture increase. Net photosynthetic rate (Pn) of pea plants showed fast response to substrate moisture variation, while chlorophyll content did not change. Drastic change of substrate moisture suppressed lettuce Pn, chlorophyll biosynthesis and plant growth. These parameters slowly recovered after termination of waterlogging treatment but lettuce yield was greatly affected. The results showed that the most sensitive physiological parameter to substrate moisture variations is photosynthesis.

Nitrous oxide emissions from crop rotations including wheat, oilseed rape and dry peas

NASA Astrophysics Data System (ADS)

Jeuffroy, M. H.; Baranger, E.; Carrouée, B.; de Chezelles, E.; Gosme, M.; Hénault, C.; Schneider, A.; Cellier, P.

2013-03-01

Approximately 65% of anthropogenic emissions of N2O, a potent greenhouse gas (GHG), originate from soils at a global scale, and particularly after N fertilisation of the main crops in Europe. Thanks to their capacity to fix atmospheric N2 through biological fixation, legumes can reduce N fertilizer use, and possibly N2O emissions. Nevertheless, the decomposition of crop organic matter during the crop cycle and residue decomposition, and possibly the N fixation process itself, could lead to N2O emissions. The objective of this study was to quantify N2O emissions from a dry pea crop (Pisum sativum, harvested at maturity) and from the subsequent crops in comparison with N2O emissions from wheat and oilseed rape crops, fertilized or not, in various rotations. A field experiment was conducted over 4 consecutive years to compare the emissions during the pea crop, in comparison with those during the wheat (fertilized or not) or oilseed rape crops, and after the pea crop, in comparison with other preceding crops. N2O fluxes were measured using static chambers. In spite of low N2O fluxes, mainly due to the site's soil characteristics, fluxes during the crop were significantly lower for pea and unfertilized wheat than for fertilized wheat and oilseed rape. The effect of the preceding crop was not significant, while soil mineral N at harvest was higher after the pea crop. These results should be confirmed over a wider range of soil types. Nevertheless, they demonstrate the absence of N2O emissions linked to the symbiotic N fixation process, and allow us to estimate the decrease in N2O emissions by 20-25% through including one pea crop in a three-year rotation. On a larger scale, this reduction of GHG emissions at field level has to be added to the decrease due to the reduced production and transport of the N fertilizer not applied to the pea crop.

The Pea Photoperiod Response Gene STERILE NODES Is an Ortholog of LUX ARRHYTHMO1[W][OPEN

PubMed Central

Liew, Lim Chee; Hecht, Valérie; Sussmilch, Frances C.; Weller, James L.

2014-01-01

The STERILE NODES (SN) locus in pea (Pisum sativum) was one of the first photoperiod response genes to be described and provided early evidence for the genetic control of long-distance signaling in flowering-time regulation. Lines homozygous for recessive sn mutations are early flowering and photoperiod insensitive, with an increased ability to promote flowering across a graft union in short-day conditions. Here, we show that SN controls developmental regulation of genes in the FT family and rhythmic regulation of genes related to circadian clock function. Using a positional and functional candidate approach, we identify SN as the pea ortholog of LUX ARRHYTHMO, a GARP transcription factor from Arabidopsis (Arabidopsis thaliana) with an important role in circadian clock function. In addition to induced mutants, sequence analysis demonstrates the presence of at least three other independent, naturally occurring loss-of-function mutations among known sn cultivars. Examination of genetic and regulatory interactions between SN and two other circadian clock genes, HIGH RESPONSE TO PHOTOPERIOD (HR) and DIE NEUTRALIS (DNE), suggests a complex relationship in which HR regulates expression of SN and the role of DNE and HR in control of flowering is dependent on SN. These results extend previous work to show that pea orthologs of all three Arabidopsis evening complex genes regulate clock function and photoperiod-responsive flowering and suggest that the function of these genes may be widely conserved. PMID:24706549

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody.

PubMed

Dhungel, Bidur; Ohno, Yoshikazu; Matayoshi, Rie; Otaki, Joji M

2013-03-25

Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

PubMed Central

2013-01-01

Background Candidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo. Results A recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules. Conclusions Our baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer

Improving quality of an innovative pea puree by high hydrostatic pressure.

PubMed

Klug, Tâmmila Venzke; Martínez-Sánchez, Ascensión; Gómez, Perla A; Collado, Elena; Aguayo, Encarna; Artés, Francisco; Artés-Hernández, Francisco

2017-10-01

The food industry is continuously innovating to fulfill consumer demand for new, healthy, ready-to-eat products. Pea purees could satisfy this trend by increasing the intake of legumes, which are an important source of nutrients. Moreover, sensorial properties like viscosity could be improved by high hydrostatic pressure (HHP). In this study the effect of a boiling treatment (10 min) followed by HHP at 550 kPa (0, 5 or 10 min) on the rheological properties, associated with enzymatic activity and particle size, as well as on the microbial and sensory quality of a pea-based puree stored for 36 days at 5 °C, has been assessed. The particle size of pea puree decreased after all processing treatments, but increased during storage in HHP-treated samples. Conversely, boiling treatment showed an increase in polygalacturonase activity at the end of the storage period, with a decrease in particle size, viscosity and stability. However, 5 min of 550 kPa HHP showed the highest mean particle size, mean surface diameter and viscosity regarding the remaining treatments. The microbial load remained low during storage. HHP treatment can be used by the food industry to improve the rheological properties, viscosity and stability of pea purees. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

PubMed

de Hoog, Hans-Peter M; Lin JieRong, Esther M; Banerjee, Sourabh; Décaillot, Fabien M; Nallani, Madhavan

2014-01-01

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4.

Conformational Antibody Binding to a Native, Cell-Free Expressed GPCR in Block Copolymer Membranes

PubMed Central

de Hoog, Hans-Peter M.; Lin JieRong, Esther M.; Banerjee, Sourabh; Décaillot, Fabien M.; Nallani, Madhavan

2014-01-01

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4. PMID:25329156

Patterns of Genetic Structure and Linkage Disequilibrium in a Large Collection of Pea Germplasm

PubMed Central

Siol, Mathieu; Jacquin, Françoise; Chabert-Martinello, Marianne; Smýkal, Petr; Le Paslier, Marie-Christine; Aubert, Grégoire; Burstin, Judith

2017-01-01

Pea (Pisum sativum, L.) is a major pulse crop used both for animal and human alimentation. Owing to its association with nitrogen-fixing bacteria, it is also a valuable component for low-input cropping systems. To evaluate the genetic diversity and the scale of linkage disequilibrium (LD) decay in pea, we genotyped a collection of 917 accessions, gathering elite cultivars, landraces, and wild relatives using an array of ∼13,000 single nucleotide polymorphisms (SNP). Genetic diversity is broadly distributed across three groups corresponding to wild/landraces peas, winter types, and spring types. At a finer subdivision level, genetic groups relate to local breeding programs and type usage. LD decreases steeply as genetic distance increases. When considering subsets of the data, LD values can be higher, even if the steep decay remains. We looked for genomic regions exhibiting high level of differentiation between wild/landraces, winter, and spring pea, respectively. Two regions on linkage groups 5 and 6 containing 33 SNPs exhibit stronger differentiation between winter and spring peas than would be expected under neutrality. Interestingly, QTL for resistance to cold acclimation and frost resistance have been identified previously in the same regions. PMID:28611254

Effect of Processing on the in Vitro and in Vivo Protein Quality of Yellow and Green Split Peas (Pisum sativum).

PubMed

Nosworthy, Matthew G; Franczyk, Adam J; Medina, Gerardo; Neufeld, Jason; Appah, Paulyn; Utioh, Alphonsus; Frohlich, Peter; House, James D

2017-09-06

In order to determine the effect of extrusion, baking, and cooking on the protein quality of yellow and green split peas, a rodent bioassay was conducted and compared to an in vitro method of protein quality determination. The Protein Digestibility-Corrected Amino Acid Score (PDCAAS) of green split peas (71.4%) was higher than that of yellow split peas (67.8%), on average. Similarly, the average Digestible Indispensable Amino Acid Score (DIAAS) of green split peas (69%) was higher than that of yellow split peas (67%). Cooked green pea flour had lower PDCAAS and DIAAS values (69.19% and 67%) than either extruded (73.61%, 70%) or baked (75.22%, 70%). Conversely, cooked yellow split peas had the highest PDCCAS value (69.19%), while extruded yellow split peas had the highest DIAAS value (67%). Interestingly, a strong correlation was found between in vivo and in vitro analysis of protein quality (R 2 = 0.9745). This work highlights the differences between processing methods on pea protein quality and suggests that in vitro measurements of protein digestibility could be used as a surrogate for in vivo analysis.

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

PubMed Central

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

2014-01-01

Schistosomiasis caused by infection with parasitic helminths of Schistosoma spp. is a major global health problem due to inadequate treatment and lack of a vaccine. The immune response to schistosomes includes glycan antigens, which could be valuable diagnostic markers and vaccine targets. However, no precedent exists for how to design vaccines targeting eukaryotic glycoconjugates. The di- and tri-saccharide motifs LacdiNAc (GalNAcβ1,4GlcNAc; LDN) and fucosylated LacdiNAc (GalNAcβ1,4(Fucα1-3)GlcNAc; LDNF) are the basis for several important schistosome glycan antigens. They occur in monomeric form or as repeating units (poly-LDNF) and as part of a variety of different glycoconjugates. Because chemical synthesis and conjugation of such antigens is exceedingly difficult, we sought to develop a recombinant expression system for parasite glycans. We hypothesized that presentation of parasite glycans on the cell surface would induce glycan-specific antibodies. We generated Chinese hamster ovary (CHO) Lec8 cell lines expressing poly-LDN (L8-GT) and poly-LDNF (L8-GTFT) abundantly on their membrane glycoproteins. Sera from Schistosoma mansoni-infected mice were highly cross-reactive with the cells and with cell-surface N-glycans. Immunizing mice with L8-GT and L8-GTFT cells induced glycan-specific antibodies. The L8-GTFT cells induced a sustained booster response, with antibodies that bound to S. mansoni lysates and recapitulated the exquisite specificity of the anti-parasite response for particular presentations of LDNF antigen. In summary, this recombinant expression system promotes successful generation of antibodies to the glycans of S. mansoni, and it can be adapted to study the role of glycan antigens and anti-glycan immune responses in many other infections and pathologies. PMID:24727440

Enzyme-linked immunosorbent assay using a recombinant baculovirus-expressed Bacillus anthracis protective antigen (PA): measurement of human anti-PA antibodies.

PubMed Central

Iacono-Connors, L C; Novak, J; Rossi, C; Mangiafico, J; Ksiazek, T

1994-01-01

We developed an antigen capture enzyme-linked immunosorbent assay (ELISA) which does not require purified protective antigen (PA) for detection of human antibodies to Bacillus anthracis PA. Lysates of Spodoptera frugiperda (Sf-9) cells infected with recombinant baculovirus containing the PA gene were used as the source of PA to develop the ELISA. Recombinant PA from crude Sf-9 cell lysates or PA purified from B. anthracis Sterne strain was captured by an anti-PA monoclonal antibody coated onto microtiter plates. We demonstrated that human serum antibody titers to PA were identical in the ELISA whether we used crude Sf-9 cell lysates containing recombinant baculovirus-expressed PA or purified Sterne PA. Finally, false-positive results observed in a direct ELISA were eliminated with this antigen capture ELISA. Thus, the antigen capture ELISA with crude preparations of baculovirus-expressed PA is reliable, safe, and inexpensive for determining anti-PA antibody levels in human sera. PMID:7496927

Pea disease diagnostic series - Powdery Mildew

USDA-ARS?s Scientific Manuscript database

Powdery mildew is a serious disease of pea worldwide, and it could be caused by two fungal species Erysiphe pisi and E. trifolii. White powdery patches on leaves, stems and pods are characteristics of the disease. The pathogen may form black fruiting bodies called chasmothecia near the end of the gr...

Effects of pigeon pea and plantain starches on the compressional, mechanical, and disintegration properties of paracetamol tablets.

PubMed

Dare, Kunle; Akin-Ajani, Dorothy O; Odeku, Oluwatoyin A; Itiola, Oludele A; Odusote, Omotunde M

2006-03-01

A study has been made of the effects of pigeon pea starch obtained from the plant Cajanus cajan (L) Millisp. (family Fabaceae) and plantain starch obtained from the unripe fruit of Musa paradisiaca L. (family Musaceae) on the compressional, mechanical, and disintegration properties of paracetamol tablets in comparison with official corn starch BP. Analysis of compressional properties was done by using density measurements, and the Heckel and Kawakita equations, whereas the mechanical properties of the tablets were evaluated by using tensile strength (T--a measure of bond strength) and brittle fracture index (BFI--a measure of lamination tendency). The ranking for the mean yield pressure, P(y), for the formulations containing the different starches was generally corn < pigeon pea < plantain starch while the ranking for P(k), an inverse measure of the amount of plasticity, was pigeon pea < plantain < corn starch, which indicated that formulations containing corn starch generally exhibited the fastest onset of plastic deformation, whereas those formulations containing pigeon pea starch exhibited the highest amount of plastic deformation during tableting. The tensile strength of the tablets increased with increase in concentration of the starches while the Brittle Fracture Index decreased. The ranking for T was pigeon pea > plantain > corn starch while the ranking for BFI was corn > plantain > pigeon pea starch. The bonding capacity of the formulations was in general agreement with the tensile strength results. The disintegration time (DT) of the formulation increased with concentration of plantain and corn starches but decreased with concentration of pigeon pea starch. The general ranking of DT values was plantain < pigeon pea < corn starch. Notably, formulations containing pigeon pea starch exhibited the highest bond strength and lowest brittleness, suggesting the usefulness of pigeon pea starch in producing strong tablets with minimal lamination tendency. Plantain

A Single, Plastic Population of Mycosphaerella pinodes Causes Ascochyta Blight on Winter and Spring Peas (Pisum sativum) in France

PubMed Central

Guibert, Michèle; Leclerc, Aurélie; Andrivon, Didier; Tivoli, Bernard

2012-01-01

Plant diseases are caused by pathogen populations continuously subjected to evolutionary forces (genetic flow, selection, and recombination). Ascochyta blight, caused by Mycosphaerella pinodes, is one of the most damaging necrotrophic pathogens of field peas worldwide. In France, both winter and spring peas are cultivated. Although these crops overlap by about 4 months (March to June), primary Ascochyta blight infections are not synchronous on the two crops. This suggests that the disease could be due to two different M. pinodes populations, specialized on either winter or spring pea. To test this hypothesis, 144 pathogen isolates were collected in the field during the winter and spring growing seasons in Rennes (western France), and all the isolates were genotyped using amplified fragment length polymorphism (AFLP) markers. Furthermore, the pathogenicities of 33 isolates randomly chosen within the collection were tested on four pea genotypes (2 winter and 2 spring types) grown under three climatic regimes, simulating winter, late winter, and spring conditions. M. pinodes isolates from winter and spring peas were genetically polymorphic but not differentiated according to the type of cultivars. Isolates from winter pea were more pathogenic than isolates from spring pea on hosts raised under winter conditions, while isolates from spring pea were more pathogenic than those from winter pea on plants raised under spring conditions. These results show that disease developed on winter and spring peas was initiated by a single population of M. pinodes whose pathogenicity is a plastic trait modulated by the physiological status of the host plant. PMID:23023742

A highly infective plant-associated bacterium influences reproductive rates in pea aphids

PubMed Central

Hendry, Tory A.; Clark, Kelley J.; Baltrus, David A.

2016-01-01

Pea aphids, Acyrthosiphon pisum, have the potential to increase reproduction as a defence against pathogens, though how frequently this occurs or how infection with live pathogens influences this response is not well understood. Here we determine the minimum infective dose of an environmentally common bacterium and possible aphid pathogen, Pseudomonas syringae, to determine the likelihood of pathogenic effects to pea aphids. Additionally, we used P. syringae infection to investigate how live pathogens may alter reproductive rates. We found that oral bacterial exposure decreased subsequent survival of aphids in a dose-dependent manner and we estimate that ingestion of less than 10 bacterial cells is sufficient to increase aphid mortality. Pathogen dose was positively related to aphid reproduction. Aphids exposed to low bacterial doses showed decreased, although statistically indistinguishable, fecundity compared to controls. Aphids exposed to high doses reproduced significantly more than low dose treatments and also more, but not significantly so, than controls. These results are consistent with previous studies suggesting that pea aphids may use fecundity compensation as a response to pathogens. Consequently, even low levels of exposure to a common plant-associated bacterium may therefore have significant effects on pea aphid survival and reproduction. PMID:26998321

A highly infective plant-associated bacterium influences reproductive rates in pea aphids.

PubMed

Hendry, Tory A; Clark, Kelley J; Baltrus, David A

2016-02-01

Pea aphids, Acyrthosiphon pisum, have the potential to increase reproduction as a defence against pathogens, though how frequently this occurs or how infection with live pathogens influences this response is not well understood. Here we determine the minimum infective dose of an environmentally common bacterium and possible aphid pathogen, Pseudomonas syringae, to determine the likelihood of pathogenic effects to pea aphids. Additionally, we used P. syringae infection to investigate how live pathogens may alter reproductive rates. We found that oral bacterial exposure decreased subsequent survival of aphids in a dose-dependent manner and we estimate that ingestion of less than 10 bacterial cells is sufficient to increase aphid mortality. Pathogen dose was positively related to aphid reproduction. Aphids exposed to low bacterial doses showed decreased, although statistically indistinguishable, fecundity compared to controls. Aphids exposed to high doses reproduced significantly more than low dose treatments and also more, but not significantly so, than controls. These results are consistent with previous studies suggesting that pea aphids may use fecundity compensation as a response to pathogens. Consequently, even low levels of exposure to a common plant-associated bacterium may therefore have significant effects on pea aphid survival and reproduction.

A novel plasmid pEA68 of Erwinia amylovora and the description of a new family of plasmids.

PubMed

Ismail, Emadeldeen; Blom, Jochen; Bultreys, Alain; Ivanović, Milan; Obradović, Aleksa; van Doorn, Joop; Bergsma-Vlami, Maria; Maes, Martine; Willems, Anne; Duffy, Brion; Stockwell, Virginia O; Smits, Theo H M; Puławska, Joanna

2014-12-01

Recent genome analysis of Erwinia amylovora, the causal agent of fire blight disease on Rosaceae, has shown that the chromosome is highly conserved among strains and that plasmids are the principal source of genomic diversity. A new circular plasmid, pEA68, was found in E. amylovora strain 692 (LMG 28361), isolated in Poland from Sorbus (mountain ash) with fire blight symptoms. Annotation of the 68,763-bp IncFIIa-type plasmid revealed that it contains 79 predicted CDS, among which two operons (tra, pil) are associated with mobility. The plasmid is maintained stably in E. amylovora and does not possess genes associated with antibiotic resistance or known virulence genes. Curing E. amylovora strain 692 of pEA68 did not influence its virulence in apple shoots nor amylovoran synthesis. Of 488 strains of E. amylovora from seventeen countries, pEA68 was only found in two additional strains from Belgium. Although the spread of pEA68 is currently limited to Europe, pEA68 comprises, together with pEA72 and pEA78 both found in North America, a new plasmid family that spans two continents.

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Co-localization of glyceraldehyde-3-phosphate dehydrogenase with ferredoxin-NADP reductase in pea leaf chloroplasts

PubMed Central

Negi, Surendra S.; Carol, Andrew A.; Pandya, Shivangi; Braun, Werner; Anderson, Louise E.

2008-01-01

In immunogold double-labeling of pea leaf thin sections with antibodies raised against ferredoxin-NADP reductase (EC 1.18.1.2, FNR) and antibodies directed against the A or B subunits of the NADP-linked glyceraldehyde-3-P dehydrogenase (GAPD) (EC 1.2.1.13), many small and large gold particles were found together over the chloroplasts. Nearest neighbor analysis of the distribution of the gold particles indicates that FNR and the NADP-linked GAPD are co-localized, in situ. This suggests that FNR might carry FADH2 or NADPH from the thylakoid membrane to GAPD, or that ferredoxin might carry electrons to FNR co-localized with GAPD in the stroma. Crystal structures of the spinach enzymes are available. When they are docked computationally, the proteins appear, as modeled, to be able to form at least two different complexes. One involves a single GAPD monomer and an FNR monomer (or dimer). The amino acid residues located at the putative interface are highly conserved on the chloroplastic forms of both enzymes. The other potential complex involves the GAPD A2B2 tetramer and an FNR monomer (or dimer). The interface residues are conserved in this model as well. Ferredoxin is able to interact with FNR in either complex. PMID:17945509

Effectiveness of rhizobacteria containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought conditions.

PubMed

Zahir, Z A; Munir, A; Asghar, H N; Shaharoona, B; Arshad, M

2008-05-01

A series of experiments were conducted to assess the effectiveness of rhizobacteria containing 1-aminocyclopropane- 1-carboxylate (ACC) deaminase for growth promotion of peas under drought conditions. Ten rhizobacteria isolated from the rhizosphere of different crops (peas, wheat, and maize) were screened for their growth promoting ability in peas under axenic condition. Three rhizobacterial isolates, Pseudomonas fluorescens biotype G (ACC-5), P. fluorescens (ACC-14), and P. putida biotype A (Q-7), were selected for pot trial on the basis of their source, ACC deaminase activity, root colonization, and growth promoting activity under axenic conditions. Inoculated and uninoculated (control) seeds of pea cultivar 2000 were sown in pots (4 seeds/pot) at different soil moisture levels (25, 50, 75, and 100% of field capacity). Results revealed that decreasing the soil moisture levels from 100 to 25% of field capacity significantly decreased the growth of peas. However, inoculation of peas with rhizobacteria containing ACC deaminase significantly decreased the "drought stress imposed effects" on growth of peas, although with variable efficacy at different moisture levels. At the lowest soil moisture level (25% field capacity), rhizobacterial isolate Pseudomonas fluorescens biotype G (ACC-5) was found to be more promising compared with the other isolates, as it caused maximum increases in fresh weight, dry weight, root length, shoot length, number of leaves per plant, and water use efficiency on fresh and dry weight basis (45, 150, 92, 45, 140, 46, and 147%, respectively) compared with respective uninoculated controls. It is highly likely that rhizobacteria containing ACC deaminase might have decreased the drought-stress induced ethylene in inoculated plants, which resulted in better growth of plants even at low moisture levels. Therefore, inoculation with rhizobacteria containing ACC deaminase could be helpful in eliminating the inhibitory effects of drought stress on the

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

PubMed

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

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 vary in the amounts of product produced and in the heterogeneity of the secreted products. The biological mechanisms of this variation are not fully defined. Here we have utilised experimental and modelling strategies to characterise and define the biology underpinning product heterogeneity in cell lines exhibiting varying antibody expression levels, and then experimentally validated these models. In undertaking these studies we applied and validated biochemical (rate-constant based) and engineering (nonlinear) models of antibody expression to experimental data from four NS0 cell lines with different IgG4 secretion rates. The models predict that export of the full antibody and its fragments are intrinsically linked, and cannot therefore be manipulated individually at the level of the secretory machinery. Instead, the models highlight strategies for the manipulation at the precursor species level to increase recombinant protein yields in both high and low producing cell lines. The models also highlight cell line specific limitations in the antibody expression pathway.

Nutritional composition and antinutritional properties of maize ogi cofermented with pigeon pea.

PubMed

Okafor, Uchechukwu I; Omemu, Adebunkola M; Obadina, Adewale O; Bankole, Mobolaji O; Adeyeye, Samuel A O

2018-03-01

Maize was cofermented with pigeon pea for ogi production and evaluated for nutritional (proximate composition, minerals, vitamins, and amino acid profile analyses) and antinutritional (phytate, tannin, and trypsin inhibitor activity analyses) qualities. White maize and pigeon pea were mixed at ratios of 90:10, 80:20, 70:30, 60:40, and 50:50, respectively, with 100:0 serving as the control. Mixtures were cofermented for 96 hr at 27°C ± 2°C and nutritional, mineral, and antinutritional qualities were analyzed using analysis of variance. Results of proximate analysis showed that the values were significantly difference at p  ≤   .05. Maize cofermented with pigeon pea at a ratio of 60:40 had the highest protein (22.79 mg/100 g), fat (19.27 mg/100 g), ash (2.98 mg/100 g), crude fiber (0.73 mg/100 g), and lowest moisture (1.98 mg/100 g) content, and was significantly ( p  ≤   .05) different from the other ratios. Of all the mixtures analyzed, 60:40 was significantly ( p  ≤   .05) different and had the highest Vitamin B 1 , B 2 , and B 3 contents. Amino acid profile results showed that maize cofermented with pigeon pea at a ratio of 60:40 showed the highest contents of lysine (93.95 mg/g), tryptophan (20.38 mg/g), isoleucine (54.78 mg/g), phenylalanine (86.23 mg/g), leucine (109.55 mg/g), and valine (68.29 mg/g), respectively, and was significantly ( p  ≤   .05) different from the other ratios. Results of antinutritional analysis showed low phytate, tannin, and trypsin inhibitor values in maize cofermented with pigeon pea at a ratio of 60:40 when compared with other ratios. The cofermented maize-pigeon pea product 60:40 had high amino acid profile than the others.

21 CFR 155.170 - Canned peas.

Code of Federal Regulations, 2011 CFR

2011-04-01

..., glucose sirup, and fructose. (viii) Spice. (ix) Flavoring (except artificial). (x) Color additives. (xi... additive has been added, the name of the food shall include the term “artificially colored.” (ii) The... 21 Food and Drugs 2 2011-04-01 2011-04-01 false Canned peas. 155.170 Section 155.170 Food and...

Mechanism of gibberellin-dependent stem elongation in peas

NASA Technical Reports Server (NTRS)

Cosgrove, D. J.; Sovonick-Dunford, S. A.

1989-01-01

Stem elongation in peas (Pisum sativum L.) is under partial control by gibberellins, yet the mechanism of such control is uncertain. In this study, we examined the cellular and physical properties that govern stem elongation, to determine how gibberellins influence pea stem growth. Stem elongation of etiolated seedlings was retarded with uniconozol, a gibberellin synthesis inhibitor, and the growth retardation was reversed by exogenous gibberellin. Using the pressure probe and vapor pressure osmometry, we found little effect of uniconozol and gibberellin on cell turgor pressure or osmotic pressure. In contrast, these treatments had major effects on in vivo stress relaxation, measured by turgor relaxation and pressure-block techniques. Uniconozol-treated plants exhibited reduced wall relaxation (both initial rate and total amount). The results show that growth retardation is effected via a reduction in the wall yield coefficient and an increase in the yield threshold. These effects were largely reversed by exogenous gibberellin. When we measured the mechanical characteristics of the wall by stress/strain (Instron) analysis, we found only minor effects of uniconozol and gibberellin on the plastic compliance. This observation indicates that these agents did not alter wall expansion through effects on the mechanical (viscoelastic) properties of the wall. Our results suggest that wall expansion in peas is better viewed as a chemorheological, rather than a viscoelastic, process.

Microbials for the production of monoclonal antibodies and antibody fragments.

PubMed

Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

2014-01-01

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

Auxin Does Not Alter the Permeability of Pea Segments to Tritium-labeled Water.

PubMed

Dowler, M J; Rayle, D L

1974-02-01

The possibility of an auxin effect on the permeability of pea (Pisum sativum L. ev. Alaska) segments to tritium-labeled water has been investigated by three separate laboratories, and the combined results are presented. We were unable to obtain any indication of a rapid effect of indoleacetic acid on the efflux of (3)HHO when pea segments previously "loaded" for 90 minutes with (3)HHO were transferred to unlabeled aqueous medium with indoleacetic acid. We were able to confirm that segments pretreated with (3)HHO plus indoleacetic acid for 60 to 90 minutes can show an enhanced (3)HHO release as compared with minus indoleacetic acid controls. However, this phenomenon appears to be due to an increased uptake of (3)HHO during the prolonged indoleacetic acid pretreatment, and therefore we conclude that auxin does not alter the permeability of pea segments to (3)HHO in either short term or long term tests. We confirm previous reports that the uptake of (3)HHO in pea segments proceeds largely through the cut surfaces, and that the cuticle is a potent barrier to (3)HHO flux.

EDTA a novel inducer of pisatin, a phytoalexin indicator of the non-host resistance in peas.

PubMed

Hadwiger, Lee A; Tanaka, Kiwamu

2014-12-23

Pea pod endocarp suppresses the growth of an inappropriate fungus or non-pathogen by generating a "non-host resistance response" that completely suppresses growth of the challenging fungus within 6 h. Most of the components of this resistance response including pisatin production can be elicited by an extensive number of both biotic and abiotic inducers. Thus this phytoalexin serves as an indicator to be used in evaluating the chemical properties of inducers that can initiate the resistance response. Many of the pisatin inducers are reported to interact with DNA and potentially cause DNA damage. Here we propose that EDTA (ethylenediaminetetraacetic acid) is an elicitor to evoke non-host resistance in plants. EDTA is manufactured as a chelating agent, however at low concentration it is a strong elicitor, inducing the phytoalexin pisatin, cellular DNA damage and defense-responsive genes. It is capable of activating complete resistance in peas against a pea pathogen. Since there is also an accompanying fragmentation of pea DNA and alteration in the size of pea nuclei, the potential biochemical insult as a metal chelator may not be its primary action. The potential effects of EDTA on the structure of DNA within pea chromatin may assist the transcription of plant defense genes.

Redistribution of annexin in gravistimulated pea plumules

NASA Technical Reports Server (NTRS)

Clark, G. B.; Rafati, D. S.; Bolton, R. J.; Dauwalder, M.; Roux, S. J.

2000-01-01

We used immunocytochemistry to investigate the effects of gravistimulation on annexin localization in etiolated pea plumule shoots. In longitudinal sections, an asymmetric annexin immunostaining pattern was observed in a defined group of cells located just basipetal to apical meristems at the main shoot apex and at all of the axillary buds, an area classically referred to as the leaf gap. The pattern was observed using both protein-A-purified anti-annexin and affinity-purified anti-annexin antibodies for the immunostaining. A subset of the cells with the annexin staining also showed an unusually high level of periodic acid Schiff (PAS) staining in their cell walls. Prior to gravistimulation, the highest concentration of annexin was oriented toward the direction of gravity along the apical end of these immunostained cells. In contrast, both at 15 and 30 min after gravistimulation, the annexin immunostain became more evenly distributed all around the cell and more distinctly cell peripheral. The asymmetry along the lower wall of these cells was no longer evident. In accord with current models of annexin action, we interpret the results to indicate that annexin-mediated secretion in the leaf gap area is preferentially toward the apical meristem prior to gravistimulation, and that gravistimulation results in a redirection of this secretion. These data are to our knowledge the first to show a correlation between the vector of gravity and the distribution of annexins in the cells of flowering plants. c 2000 Editions scientifiques et medicales Elsevier SAS.

Cutaneous exposure to clinically-relevant pigeon pea (Cajanus cajan) proteins promote TH2-dependent sensitization and IgE-mediated anaphylaxis in Balb/c mice.

PubMed

Kumar Gupta, Rinkesh; Kumar, Sandeep; Gupta, Kriti; Sharma, Akanksha; Roy, Ruchi; Kumar Verma, Alok; Chaudhari, Bhushan P; Das, Mukul; Ahmad Ansari, Irfan; Dwivedi, Premendra D

2016-11-01

Epicutaneous (EC) sensitization to food allergens may occur when the skin has been lightly damaged. The study here tested whether cutaneous exposure to pigeon pea protein(s) may cause allergic sensitization. BALB/c mice were either orally gavaged or epicutaneously sensitized by repeated application of pigeon pea crude protein extract (CPE) on undamaged areas of skin without any adjuvant; afterwards, both groups were orally challenged with the pigeon pea CPE. Anaphylactic symptoms along with measures of body temperature, MCPT-1, TSLP, pigeon pea-specific IgE and IgG 1 , myeloperoxidase (MPO) activity, T H 2 cytokines, T H 2 transcription factors (TFs) and filaggrin expression were determined. Mast cell staining, eosinophil levels and histopathological analysis of the skin and intestines were also performed. In the epicutaneously-sensitized mice, elevated levels of specific IgE and IgG 1 , as well as of MCPT-1, TSLP, T H 2 cytokines and TFs, higher anaphylactic scores and histological changes in the skin and intestine were indicative of sensitization ability via both routes in the pigeon pea CPE-treated hosts. Elevated levels of mast cells were observed in both the skin and intestine; increased levels of eosinophils and MPO activity were noted only in the skin. Decreased levels of filaggrin in skin may have played a key role in the skin barrier dysfunction, increasing the chances of sensitization. Therefore, the experimental data support the hypothesis that in addition to oral exposure, skin exposure to food allergens can promote T H 2-dependent sensitization, IgE-mediated anaphylaxis and intestinal changes after oral challenge. Based on this, an avoidance of cutaneous exposures to allergens might prevent development of food anaphylaxis.

Dissecting the proteome of pea mature seeds reveals the phenotypic plasticity of seed protein composition.

PubMed

Bourgeois, Michael; Jacquin, Françoise; Savois, Vincent; Sommerer, Nicolas; Labas, Valérie; Henry, Céline; Burstin, Judith

2009-01-01

Pea (Pisum sativum L.) is the most cultivated European pulse crop and the pea seeds mainly serve as a protein source for monogastric animals. Because the seed protein composition impacts on seed nutritional value, we aimed at identifying the determinants of its variability. This paper presents the first pea mature seed proteome reference map, which includes 156 identified proteins (http://www.inra.fr/legumbase/peaseedmap/). This map provides a fine dissection of the pea seed storage protein composition revealing a large diversity of storage proteins resulting both from gene diversity and post-translational processing. It gives new insights into the pea storage protein processing (especially 7S globulins) as a possible adaptation towards progressive mobilization of the proteins during germination. The nonstorage seed proteome revealed the presence of proteins involved in seed defense together with proteins preparing germination. The plasticity of the seed proteome was revealed for seeds produced in three successive years of cultivation, and 30% of the spots were affected by environmental variations. This work pinpoints seed proteins most affected by environment, highlighting new targets to stabilize storage protein composition that should be further analyzed.

Antibody-dependent cellular cytotoxicity and cytokine/chemokine secretion by KHYG-1 cells stably expressing FcγRIIIA.

PubMed

Kobayashi, Eiji; Motoi, Sotaro; Sugiura, Masahito; Kajikawa, Masunori; Kojima, Shuji; Kohroki, Junya; Masuho, Yasuhiko

2014-09-01

Antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells is a major mechanism of tumor therapy with antibodies. NK cells not only manifest cytotoxicity but also secrete a variety of cytokines/chemokines that regulate immune responses. Using a retroviral vector, in this study we established a KHYG-1 cell line that stably expresses FcγRIIIA (CD16A). The KHYG-1/FcγRIIIA cells exerted potent antibody concentration-dependent ADCC, whereas parental KHYG-1 cells did not. In contrast, without antibody, the natural killer activity of KHYG-1/FcγRIIIA cells was less potent than that of parental KHYG-1 cells. During the course of ADCC, KHYG-1/FcγRIIIA cells secreted IFN-γ and MIP-1α dependent upon antibody concentration, but parental KHYG-1 cells did not. These results suggest that KHYG-1/FcγRIIIA cells would be useful in studies to elucidate the function of NK cells and the mechanism of ADCC. Copyright © 2014 Elsevier B.V. All rights reserved.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

NASA Technical Reports Server (NTRS)

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.

Physiological Studies on Pea Tendrils. IV. Flavonoids and Contact Coiling

PubMed Central

Jaffe, M. J.; Galston, A. W.

1967-01-01

Pea tendrils contain high concentrations of flavonoids, mainly quercetin-triglucosyl-p-coumarate (QGC). QGC is most abundant near the highly responsive apex of the tendril, and least abundant at the base. After mechanical stimulation, and during coiling of the tendril, the QGC titer drops to about 30% of its original value. The kinetics of flavonoid disappearance are significantly correlated with the kinetics of coiling. Aqueous extracts of unstimulated pea tendrils or 10 μm QGC inhibit contact coiling of excised tendrils. Extracts of coiled tendrils do not. The evidence indicates a possible regulatory role for flavonoids in contact coiling. PMID:16656581

Human parvovirus B19 antibodies induce altered membrane protein expression and apoptosis of BeWo trophoblasts.

PubMed

Tzang, Bor-Show; Chiang, Szu-Yi; Chan, Hsu-Chin; Liu, Chung-Hsien; Hsu, Tsai-Ching

2016-11-01

Human parvovirus B19 (B19) is harmful during pregnancy since it can be vertically transmitted to the developing fetus. In addition, the anti‑B19 antibodies induced by B19 infection are believed to have a cytopathic role in B19 transmission; however, knowledge regarding the effects of anti‑B19 antibodies during pregnancy is limited. To investigate the possible roles of anti‑B19 antibodies during pregnancy, the present study examined the effects of anti‑B19‑VP1 unique region (VP1u), anti‑B19‑VP2 and anti‑B19‑nonstructural protein 1 (NS1) immunoglobulin G (IgG) antibodies on BeWo trophoblasts. Briefly, BeWo trophoblasts were incubated with purified IgG against B19‑VP1u, B19‑VP2 and B19‑NS1. Subsequently, the expression of surface proteins and apoptotic molecules were assessed in BeWo trophoblasts using flow cytometry, ELISA and western blotting. The expression levels of human leukocyte antigen (HLA)‑G were significantly increased on BeWo trophoblasts treated with rabbit anti‑B19‑VP1u IgG, and were unchanged in those treated with rabbit anti‑B19‑NS1 and anti‑B19‑VP2 IgG, as compared with the control group. Furthermore, the expression levels of globoside (P blood group antigen) and cluster of differentiation (CD)29 (β1 integrin) were significantly increased in BeWo trophoblasts treated with rabbit anti‑B19‑NS1 and anti‑B19‑VP2 IgG, whereas only CD29 was also significantly increased in cells treated with anti‑B19‑VP1u IgG. In addition, the number of cells at sub‑G1 phase; caspase‑3 activity; and the expression of intrinsic and extrinsic apoptotic molecules, including Fas‑associated death domain protein, activated caspase‑8, activated caspase‑3, B‑cell lymphoma 2‑associated X protein, cytochrome c, apoptotic peptidase activating factor 1 and activated caspase‑9, were significantly increased in BeWo trophoblasts treated with anti‑B19‑VP1u and anti‑B19‑NS1 IgG. In conclusion, the present

Ultramicronized palmitoylethanolamide (PEA-um(®)) in the treatment of idiopathic pulmonary fibrosis.

PubMed

Di Paola, Rosanna; Impellizzeri, Daniela; Fusco, Roberta; Cordaro, Marika; Siracusa, Rosalba; Crupi, Rosalia; Esposito, Emanuela; Cuzzocrea, Salvatore

2016-09-01

Pulmonary fibrosis is a chronic condition characterized by progressive scarring of lung parenchyma. The aim of this study was to examine the effects of an ultramicronized preparation of palmitoylethanolamide (PEA-um(®)), an endogenous fatty acid amide, in mice subjected to idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis was induced in male mice by a single intratracheal administration of saline with bleomycin sulphate (1mg/kg body weight) in a volume of 100μL. PEA-um(®) was injected intraperitoneally at 1, 3 or 10mg/kg 1h after bleomycin instillation and daily thereafter. Animals were sacrificed after 7 and 21days by pentobarbitone overdose. One cohort of mice was sacrificed after seven days of bleomycin administration, followed by bronchoalveloar lavage and determination of myeloperoxidase activity, lung edema and histopathology features. In the 21-day cohort, mortality was assessed daily, and surviving mice were sacrificed followed by the above analyses together with immunohistochemical localization of CD8, tumor necrosis factor-α, CD4, interleukin-1β, transforming growth factor-β, inducible nitric oxide synthase and basic fibroblast growth factor. Compared to bleomycin-treated mice, animals that received also PEA-um(®) (3 or 10mg/kg) had significantly decreased weight loss, mortality, inflammation, lung damage at the histological level, and lung fibrosis at 7 and 21days. PEA-um(®) (1mg/kg) did not significantly inhibit the inflammation response and lung fibrosis. This study demonstrates that PEA-um(®) (3 and 10mg/kg) reduces the extent of lung inflammation in a mouse model of idiopathic pulmonary fibrosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

PubMed

Makeyev, E V; Kolb, V A; Spirin, A S

1999-02-12

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.

Auto-antibodies in prostate cancer: humoral immune response to antigenic determinants coded by the differentially expressed transcripts FLJ23438 and VAMP3.

PubMed

Pontes, E R; Matos, L C; da Silva, E A; Xavier, L S; Diaz, B L; Small, I A; Reis, E M; Verjovski-Almeida, S; Barcinski, M A; Gimba, E R P

2006-10-01

Here we evaluate auto-antibody response against two potential antigenic determinants of genes highly expressed in low Gleason Score prostate cancer (PC) tumor samples, namely FLJ23438 and VAMP3. RT-PCR assays were used to analyze mRNA expression profiles of FLJ23438 and VAMP3 transcripts. The auto-antibody response against FLJ23438 and VAMP3 recombinant proteins was tested by immunoblot assays using PC, benign prostate hyperplasia (BPH), healthy donors (HD), and other human cancers plasma samples. Our data showed that 37% (10/27) and 7.4% (2/27) of PC plasma samples presented auto-antibodies against FLJ23438 and VAMP3, respectively. Only 8.3% (1/12) of BPH plasma samples were reactive for both auto-antibodies, while none (0/12) of HD plasma samples tested were reactive. The prevalence of 37% of positive PC plasma samples for anti-FLJ23438 antibodies suggests that humoral immune response against this antigenic determinant could be a potential serum marker for this cancer. (c) 2006 Wiley-Liss, Inc.

AAVrh.10-mediated expression of an anti-cocaine antibody mediates persistent passive immunization that suppresses cocaine-induced behavior.

PubMed

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

2012-05-01

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, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity.

Effect of an extruded pea or rice diet on postprandial insulin and cardiovascular responses in dogs.

PubMed

Adolphe, J L; Drew, M D; Silver, T I; Fouhse, J; Childs, H; Weber, L P

2015-08-01

Peas are increasing in popularity as a source of carbohydrate, protein and fibre in extruded canine diets. The aim of this study was to test the health effects of two canine diets with identical macronutrient profiles, but containing either yellow field peas or white rice as the carbohydrate source on metabolism, cardiovascular outcomes and adiposity. First, the acute glycemic, insulinemic and cardiovascular responses to the pea- or rice-based diets were determined in normal weight beagles (n = 7 dogs). The glycemic index did not differ between the pea diet (56 ± 12) and rice diet (63 ± 9). Next, obese beagles (n = 9) were fed the yellow field pea diet or white rice diet ad libitum for 12 weeks in a crossover study. Adiposity (measured using computed tomography), metabolic (oral glucose tolerance test, plasma leptin, adiponectin, C-reactive protein) and cardiovascular assessments (echocardiography and blood pressure) were performed before and after each crossover study period. After 12 weeks on each diet, peak insulin (p = 0.05) and area under the curve (AUC) for insulin after a 10 g oral glucose tolerance test (p = 0.05) were lower with the pea than the rice diet. Diet did not show a significant effect on body weight, fat distribution, cardiovascular variables, adiponectin or leptin. In conclusion, a diet containing yellow field peas reduced the postprandial insulin response after glucose challenge in dogs despite continued obesity, indicating improved metabolic health. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

Characterization of an Invertebrate-Type Dopamine Receptor of the American Cockroach, Periplaneta americana

PubMed Central

Troppmann, Britta; Balfanz, Sabine; Krach, Christian; Baumann, Arnd; Blenau, Wolfgang

2014-01-01

We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology. PMID:24398985

Application of pigeon pea (Cajanus cajan) stalks as raw material for xylooligosaccharides production.

PubMed

Samanta, A K; Jayapal, Natasha; Kolte, A P; Senani, S; Sridhar, Manpal; Mishra, Sukriti; Prasad, C S; Suresh, K P

2013-04-01

Pigeon pea (Cajanus cajan) is a perennial plant widely cultivated in tropical and subtropical regions of many countries. The present studies aimed to produce xylooligosaccharides (XOS) from pigeon pea stalks in order to do value addition. The chemical analysis of stalks revealed 18.33 ± 1.40 % hemicelluloses in addition to cellulose, protein, and lignin. Sodium hydroxide coupled with steam application enabled almost 96 % recovery of original xylan, present in the pigeon pea stalks. Enzymatic hydrolysis of xylan led to production of XOS namely, xylobiose and xylotriose. Response surface model indicated a maximum yield of xylobiose (0.502 mg/ml) under the hydrolysis conditions of pH 4.91, temperature at 48.11 °C, enzyme dose at 11.01 U, and incubation time at 15.65 h. The ideal conditions for higher xylotriose yield (0.204 mg/ml) were pH 5.44, temperature at 39.29 °C, enzyme dose at 3.23 U, and incubation time at 15.26 h. The present investigation was successful in assessing the prospect of using pigeon pea stalks as a raw material for xylan extraction vis-à-vis XOS production.

A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation.

PubMed

Könitzer, Jennifer D; Müller, Markus M; Leparc, Germán; Pauers, Martin; Bechmann, Jan; Schulz, Patrick; Schaub, Jochen; Enenkel, Barbara; Hildebrandt, Tobias; Hampel, Martin; Tolstrup, Anne B

2015-09-01

Boehringer Ingelheim uses two CHO-DG44 lines for manufacturing biotherapeutics, BI-HEX-1 and BI-HEX-2, which produce distinct cell type-specific antibody glycosylation patterns. A recently established CHO-K1 descended host, BI-HEX-K1, generates antibodies with glycosylation profiles differing from CHO-DG44. Manufacturing process development is significantly influenced by these unique profiles. To investigate the underlying glycosylation related gene expression, we leveraged our CHO host and production cell RNA-seqtranscriptomics and product quality database together with the CHO-K1 genome. We observed that each BI-HEX host and antibody producing cell line has a unique gene expression fingerprint. CHO-DG44 cells only transcribe Fut10, Gfpt2 and ST8Sia6 when expressing antibodies. BI-HEX-K1 cells express ST8Sia6 at host cell level. We detected a link between BI-HEX-1/BI-HEX-2 antibody galactosylation and mannosylation and the gene expression of the B4galt gene family and genes controlling mannose processing. Furthermore, we found major differences between the CHO-DG44 and CHO-K1 lineages in the expression of sialyl transferases and enzymes synthesizing sialic acid precursors, providing a rationale for the lack of immunogenic NeuGc/NGNA synthesis in CHO. Our study highlights the value of systems biotechnology to understand glycoprotein synthesis and product glycoprofiles. Such data improve future production clone selection and process development strategies for better steering of biotherapeutic product quality. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tyrosine pathway regulation is host-mediated in the pea aphid symbiosis during late embryonic and early larval development.

PubMed

Rabatel, Andréane; Febvay, Gérard; Gaget, Karen; Duport, Gabrielle; Baa-Puyoulet, Patrice; Sapountzis, Panagiotis; Bendridi, Nadia; Rey, Marjolaine; Rahbé, Yvan; Charles, Hubert; Calevro, Federica; Colella, Stefano

2013-04-10

Nutritional symbioses play a central role in insects' adaptation to specialized diets and in their evolutionary success. The obligatory symbiosis between the pea aphid, Acyrthosiphon pisum, and the bacterium, Buchnera aphidicola, is no exception as it enables this important agricultural pest insect to develop on a diet exclusively based on plant phloem sap. The symbiotic bacteria provide the host with essential amino acids lacking in its diet but necessary for the rapid embryonic growth seen in the parthenogenetic viviparous reproduction of aphids. The aphid furnishes, in exchange, non-essential amino acids and other important metabolites. Understanding the regulations acting on this integrated metabolic system during the development of this insect is essential in elucidating aphid biology. We used a microarray-based approach to analyse gene expression in the late embryonic and the early larval stages of the pea aphid, characterizing, for the first time, the transcriptional profiles in these developmental phases. Our analyses allowed us to identify key genes in the phenylalanine, tyrosine and dopamine pathways and we identified ACYPI004243, one of the four genes encoding for the aspartate transaminase (E.C. 2.6.1.1), as specifically regulated during development. Indeed, the tyrosine biosynthetic pathway is crucial for the symbiotic metabolism as it is shared between the two partners, all the precursors being produced by B. aphidicola. Our microarray data are supported by HPLC amino acid analyses demonstrating an accumulation of tyrosine at the same developmental stages, with an up-regulation of the tyrosine biosynthetic genes. Tyrosine is also essential for the synthesis of cuticular proteins and it is an important precursor for cuticle maturation: together with the up-regulation of tyrosine biosynthesis, we observed an up-regulation of cuticular genes expression. We were also able to identify some amino acid transporter genes which are essential for the switch

Broadly protective anti-hemagglutinin stalk antibodies induced by live attenuated influenza vaccine expressing chimeric hemagglutinin.

PubMed

Isakova-Sivak, Irina; Korenkov, Daniil; Smolonogina, Tatiana; Kotomina, Tatiana; Donina, Svetlana; Matyushenko, Victoria; Mezhenskaya, Daria; Krammer, Florian; Rudenko, Larisa

2018-05-01

The development of influenza vaccines that can provide broad protection against all drifted seasonal virus variants, zoonotic infections and emerging pandemic strains, has been a priority for two decades. Here we propose a strategy of inducing broadly-reactive anti-stalk antibody by sequential immunizations with live attenuated influenza vaccines (LAIVs) expressing chimeric HAs (cHAs). These vaccines are designed to contain identical hemagglutinin stalk domains from H1N1 virus but antigenically unrelated globular head domains from avian influenza virus subtypes H5, H8 and H9. Mouse experiments demonstrated enhanced cross-protection of cHA-containing LAIVs compared to the relevant vaccine viruses expressing natural HAs, and this enhanced protection was driven by stalk-HA-reactive IgG antibodies. The establishment of fully functional cross-protective immunity after two doses of cHA LAIV vaccination in naïve animals suggests that a similar effect might be expected after a single cHA LAIV dose in primed individuals, or after two to three doses in naïve children. Copyright © 2018 Elsevier Inc. All rights reserved.

Doing the Basics Better in Africa: How School Support, Autonomy, and Accountability Improved Outcomes for Girls in PEAS Schools

ERIC Educational Resources Information Center

Hills, Libby

2017-01-01

Promoting Equality in African Schools (PEAS) seeks to expand access to sustainably delivered, quality secondary education in Africa. PEAS builds and runs chains of not-for-profit, low-cost private schools in public-private partnership with governments. External evaluation data show that PEAS schools in Uganda are delivering higher quality…

Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera)

PubMed Central

Sabater-Muñoz, Beatriz; Legeai, Fabrice; Rispe, Claude; Bonhomme, Joël; Dearden, Peter; Dossat, Carole; Duclert, Aymeric; Gauthier, Jean-Pierre; Ducray, Danièle Giblot; Hunter, Wayne; Dang, Phat; Kambhampati, Srini; Martinez-Torres, David; Cortes, Teresa; Moya, Andrès; Nakabachi, Atsushi; Philippe, Cathy; Prunier-Leterme, Nathalie; Rahbé, Yvan; Simon, Jean-Christophe; Stern, David L; Wincker, Patrick; Tagu, Denis

2006-01-01

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect. PMID:16542494

In vitro digestibility, protein composition and techno-functional properties of Saskatchewan grown yellow field peas (Pisum sativum L.) as affected by processing.

PubMed

Ma, Zhen; Boye, Joyce I; Hu, Xinzhong

2017-02-01

Saskatchewan grown yellow field pea was subjected to different processing conditions including dehulling, micronization, roasting, conventional/microwave cooking, germination, and combined germination and conventional cooking/roasting. Their nutritional and antinutritional compositions, functional properties, microstructure, thermal properties, in vitro protein and starch digestibility, and protein composition were studied. Processed field peas including conventional cooked yellow peas (CCYP), microwave cooked yellow peas (MCYP), germinated-conventional cooked yellow peas (GCCYP), and germinated-roasted yellow peas (GRYP) exhibited the significantly higher in vitro protein digestibility (IVPD), which was in accordance with their significantly lower trypsin inhibitor activity and tannin content. The SDS-PAGE and size exclusion HPLC profiles of untreated pea proteins and their hydrolysates also confirmed the IVPD result that these four treatments facilitated the hydrolysis of pea proteins to a greater extent. The CCYP, MCYP, GCCYP, and GRYP also exhibited significantly higher starch digestibility which was supported by their lower onset (T o ), peak (T p ), and conclusion (T c ) temperatures obtained from DSC thermogram, their lower pasting properties and starch damage results, as well as their distinguished amorphous flakes' configuration observed on the scanning electron microscopic image. LC/ESI-MS/MS analysis following in-gel digests of SDS-PAGE separated proteins allowed detailed compositional characterization of pea proteins. The present study would provide fundamental information to help to better understand the functionality of field peas as ingredients, and particularly in regards to agri-food industry to improve the process efficiency of field peas with enhanced nutritional and techno-functional qualities. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dietary Green Pea Protects against DSS-Induced Colitis in Mice Challenged with High-Fat Diet.

PubMed

Bibi, Shima; de Sousa Moraes, Luís Fernando; Lebow, Noelle; Zhu, Mei-Jun

2017-05-18

Obesity is a risk factor for developing inflammatory bowel disease. Pea is unique with its high content of dietary fiber, polyphenolics, and glycoproteins, all of which are known to be health beneficial. We aimed to investigate the impact of green pea (GP) supplementation on the susceptibility of high-fat diet (HFD)-fed mice to dextran sulfate sodium (DSS)-induced colitis. Six-week-old C57BL/6J female mice were fed a 45% HFD or HFD supplemented with 10% GP. After 7-week dietary supplementation, colitis was induced by adding 2.5% DSS in drinking water for 7 days followed by a 7-day recovery period. GP supplementation ameliorated the disease activity index score in HFD-fed mice during the recovery stage, and reduced neutrophil infiltration, mRNA expression of monocyte chemoattractant protein-1 (MCP-1) and inflammatory markers interleukin (IL)-6, cyclooxygenase-2 (COX-2), IL-17, interferon-γ (IFN-γ), and inducible nitric oxide synthase (iNOS) in HFD-fed mice. Further, GP supplementation increased mucin 2 content and mRNA expression of goblet cell differentiation markers including Trefoil factor 3 (Tff3), Krüppel-like factor 4 (Klf4), and SAM pointed domain ETS factor 1 (Spdef1) in HFD-fed mice. In addition, GP ameliorated endoplasmic reticulum (ER) stress as indicated by the reduced expression of Activating transcription factor-6 (ATF-6) protein and its target genes chaperone protein glucose-regulated protein 78 (Grp78), the CCAAT-enhancer-binding protein homologous protein (CHOP), the ER degradation-enhancing α-mannosidase-like 1 protein (Edem1), and the X-box binding protein 1 (Xbp1) in HFD-fed mice. In conclusion, GP supplementation ameliorated the severity of DSS-induced colitis in HFD-fed mice, which was associated with the suppression of inflammation, mucin depletion, and ER stress in the colon.

Genetic control of floral zygomorphy in pea (Pisum sativum L.).

PubMed

Wang, Zheng; Luo, Yonghai; Li, Xin; Wang, Liping; Xu, Shilei; Yang, Jun; Weng, Lin; Sato, Shusei; Tabata, Satoshi; Ambrose, Mike; Rameau, Catherine; Feng, Xianzhong; Hu, Xiaohe; Luo, Da

2008-07-29

Floral zygomorphy (flowers with bilateral symmetry) has multiple origins and typically manifests two kinds of asymmetries, dorsoventral (DV) and organ internal (IN) asymmetries in floral and organ planes, respectively, revealing the underlying key regulators in plant genomes that generate and superimpose various mechanisms to build up complexity and different floral forms during plant development. In this study, we investigate the loci affecting these asymmetries during the development of floral zygomorphy in pea (Pisum sativum L.). Two genes, LOBED STANDARD 1 (LST1) and KEELED WINGS (K), were cloned that encode TCP transcription factors and have divergent functions to constitute the DV asymmetry. A previously undescribed regulator, SYMMETRIC PETALS 1 (SYP1), has been isolated as controlling IN asymmetry. Genetic analysis demonstrates that DV and IN asymmetries could be controlled independently by the two kinds of regulators in pea, and their interactions help to specify the type of zygomorphy. Based on the genetic analysis in pea, we suggest that variation in both the functions and interactions of these regulators could give rise to the wide spectrum of floral symmetries among legume species and other flowering plants.

An Evaluation of the Pea Pod System for Assessing Body Composition of Moderately Premature Infants.

PubMed

Forsum, Elisabet; Olhager, Elisabeth; Törnqvist, Caroline

2016-04-22

(1) BACKGROUND: Assessing the quality of growth in premature infants is important in order to be able to provide them with optimal nutrition. The Pea Pod device, based on air displacement plethysmography, is able to assess body composition of infants. However, this method has not been sufficiently evaluated in premature infants; (2) METHODS: In 14 infants in an age range of 3-7 days, born after 32-35 completed weeks of gestation, body weight, body volume, fat-free mass density (predicted by the Pea Pod software), and total body water (isotope dilution) were assessed. Reference estimates of fat-free mass density and body composition were obtained using a three-component model; (3) RESULTS: Fat-free mass density values, predicted using Pea Pod, were biased but not significantly (p > 0.05) different from reference estimates. Body fat (%), assessed using Pea Pod, was not significantly different from reference estimates. The biological variability of fat-free mass density was 0.55% of the average value (1.0627 g/mL); (4) CONCLUSION: The results indicate that the Pea Pod system is accurate for groups of newborn, moderately premature infants. However, more studies where this system is used for premature infants are needed, and we provide suggestions regarding how to develop this area.

Marker-trait association analysis of frost tolerance of 672 worldwide pea (Pisum sativum L.) collections.

PubMed

Liu, Rong; Fang, Li; Yang, Tao; Zhang, Xiaoyan; Hu, Jinguo; Zhang, Hongyan; Han, Wenliang; Hua, Zeke; Hao, Junjie; Zong, Xuxiao

2017-07-19

Frost stress is one of the major abiotic stresses causing seedling death and yield reduction in winter pea. To improve the frost tolerance of pea, field evaluation of frost tolerance was conducted on 672 diverse pea accessions at three locations in Northern China in three growing seasons from 2013 to 2016 and marker-trait association analysis of frost tolerance were performed with 267 informative SSR markers in this study. Sixteen accessions were identified as the most winter-hardy for their ability to survive in all nine field experiments with a mean survival rate of 0.57, ranging from 0.41 to 0.75. Population structure analysis revealed a structured population of two sub-populations plus some admixtures in the 672 accessions. Association analysis detected seven markers that repeatedly had associations with frost tolerance in at least two different environments with two different statistical models. One of the markers is the functional marker EST1109 on LG VI which was predicted to co-localize with a gene involved in the metabolism of glycoproteins in response to chilling stress and may provide a novel mechanism of frost tolerance in pea. These winter-hardy germplasms and frost tolerance associated markers will play a vital role in marker-assisted breeding for winter-hardy pea cultivar.

Response of the calf pancreas to differently processed soya bean and pea diets.

PubMed

Le Dréan, G; Le Huërou-Luron, I; Philouze-Romé, V; Toullec, R; Guilloteau, P

1995-01-01

The purpose of this study was to determine the effect of replacing skim-milk powder by differently treated soya bean or pea products on growth, pancreas size and pancreatic enzyme activities in calves. Three separate experiments have been performed. In experiments 1 and 2, 28 and 21 male Holstein calves were divided into 4 or 3 groups, respectively, and fed either dairy products or milk substitutes in which protein was mainly provided by soya bean products differing in their protein concentration due to the technological processing applied. In experiment 3, 45 male Holstein calves were divided into 3 groups and were fed either dairy products, or raw or flaked pea flour as a protein source. After an experimental period of 99 +/- 4 days in experiments 1 and 2, and of 88 days in experiment 3, animal growth rate was significantly lower with raw pea flour (16%) and with the soya bean diet, which was highly concentrated in carbohydrates and allergenic proteins (13-27%). Pancreas weight decreased significantly (16-18%) with pea diets and tended to be lower (NS) with the water extracted, concentrated and heated flour (soya bean). Amylase-specific activity increased significantly (43%) with pea diets but showed opposite tendencies with the most refined soya bean products. Proteolytic enzyme activities were slightly influenced by dietary protein source, but this was not as obvious as in the literature reviewed. Specific messenger RNAs corresponding to amylase, trypsin and chymotrypsin seemed to increase (NS) with the soya bean diets, particularly with the less elaborated one. However, further investigations are required before any conclusions may be drawn concerning regulation levels of pancreatic adaptation to dietary protein. According to this study and the literature, results concerning pancreatic response to diets were different suggesting that the origin of soya bean, pea seeds and technological treatments applied to them were of great importance. Also, the level of

21 CFR 155.172 - Canned dry peas.

Code of Federal Regulations, 2010 CFR

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 dry...

Effect of gamma irradiation on physicochemical properties of stored pigeon pea (Cajanus cajan) flour

PubMed Central

Bamidele, Oluwaseun P; Akanbi, Charles T

2013-01-01

The effect of gamma irradiation at various doses (5, 10, 15, 20 kGy) was observed on pigeon pea flour stored for 3 months on proximate composition, functional properties, and peroxide value. Sensory evaluation was also carried out on bean cake (moinmoin) made from nonirradiated and irradiated pigeon pea flour. The results showed that stored gamma-irradiated samples had significantly lower (P < 0.05) value of protein and little or no effect on moisture content. There were slight decreases in crude fiber and ash content of the irradiated samples compared with the control sample. The result of functional properties of the irradiated flours showed slight increase in water absorption capacity, swelling capacity and bulk density. The peroxide value of crude oil increased significantly with dose increases for the period of storage. The sensory evaluation of moinmoin samples prepared from irradiated pigeon pea flour showed no significant difference from the moinmoin sample prepared from nonirradiated flour. It can be concluded that gamma irradiation can extend the shelf life of pigeon pea flour. PMID:24804044

Effect of gamma irradiation on physicochemical properties of stored pigeon pea (Cajanus cajan) flour.

PubMed

Bamidele, Oluwaseun P; Akanbi, Charles T

2013-09-01

The effect of gamma irradiation at various doses (5, 10, 15, 20 kGy) was observed on pigeon pea flour stored for 3 months on proximate composition, functional properties, and peroxide value. Sensory evaluation was also carried out on bean cake (moinmoin) made from nonirradiated and irradiated pigeon pea flour. The results showed that stored gamma-irradiated samples had significantly lower (P < 0.05) value of protein and little or no effect on moisture content. There were slight decreases in crude fiber and ash content of the irradiated samples compared with the control sample. The result of functional properties of the irradiated flours showed slight increase in water absorption capacity, swelling capacity and bulk density. The peroxide value of crude oil increased significantly with dose increases for the period of storage. The sensory evaluation of moinmoin samples prepared from irradiated pigeon pea flour showed no significant difference from the moinmoin sample prepared from nonirradiated flour. It can be concluded that gamma irradiation can extend the shelf life of pigeon pea flour.

Synergistic enhancement in the co-gelation of salt-soluble pea proteins and whey proteins.

PubMed

Wong, Douglas; Vasanthan, Thava; Ozimek, Lech

2013-12-15

This paper investigated the enhancement of thermal gelation properties when salt-soluble pea proteins were co-gelated with whey proteins in NaCl solutions, using different blend ratios, total protein concentrations, pH, and salt concentrations. Results showed that the thermal co-gelation of pea/whey proteins blended in ratio of 2:8 in NaCl solutions showed synergistic enhancement in storage modulus, gel hardness, paste viscosity and minimum gelation concentrations. The highest synergistic enhancement was observed at pH 6.0 as compared with pH 4.0 and 8.0, and at the lower total protein concentration of 10% as compared with 16% and 22% (w/v), as well as in lower NaCl concentrations of 0.5% and 1.0% as compared with 1.5%, 2.0%, 2.5%, and 3.0% (w/v). The least gelation concentrations were also lower in the different pea/whey protein blend ratios than in pure pea or whey proteins, when dissolved in 1.0% or 2.5% (w/v) NaCl aqueous solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Influence of grinding on the nutritive value of peas for ruminants: comparison between in vitro and in situ approaches

PubMed Central

Giger-Reverdin, Sylvie; Maaroufi, Chiraze; Chapoutot, Patrick; Peyronnet, Corinne; Sauvant, Daniel

2014-01-01

In ruminant nutrition, peas are characterized by high protein solubility and degradability, which impair its protein value estimated by the official in situ method. Grinding can be used as a technological treatment of pea seeds to modify their nutritional value. The aim of this study was to compare the in situ method with an in vitro method on the same pea either in a coarse pea flour form (PCF) or in a ground pea fine flour form (PFF) to understand the effect of grinding. Both forms were also reground (GPCF and GPFF). PCF presented a lower rate of in vitro degradation than PFF, and more stable fermentation parameters (pH, ammonia, soluble carbohydrates) even if gas production was higher for the PCF after 48 h of incubation. In situ dry matter and protein degradation were lower for PCF than those for PFF; these differences were more marked than with the in vitro method. Reground peas were very similar to PFF. The values for pea protein digestible in the intestine (PDI) were higher for PCF than those for PFF. This study points out the high sensitivity of the in situ method to grinding. The study needs to be validated by in vivo measurements. PMID:25473488

Quality evaluation of stiff porridges prepared from Irish potato (Solanum tuberosum) and pigeon pea (Cajanus cajan) starch blends.

PubMed

Abu, Joseph Oneh; Enyinnaya, Chinma Chiemela; James, Samaila; Okeleke, Ezinne

2012-06-01

Quality attributes of stiff porridges prepared from Irish potato and pigeon pea starch blends were studied. Starches were extracted from Irish potato and pigeon pea using a wet extraction method. Various ratios of the starches were mixed and analyzed for chemical, functional and pasting properties. The starch blends were then prepared into stiff porridges for sensory evaluation using a 20-man sensory panel. Substitution of Irish potato starch with pigeon pea starch led to increases in protein (0.15 to 1.2%), fat (0.26 to 0.56%) and ash (0.30 to 0.69%) while the amylose content of the starch blends decreased (from 23.8 to 18.4%) respectively. Functional properties such as bulk density (0.75 to 0.60 g/cm(3)), water absorption capacity (3.1 to 2.6 g water/ g sample) and dispersibility (58.6 to 42.7%) decreased significantly (P < 0.05) at the highest concentration (50%) of pigeon pea starch respectively. Pasting properties such as peak, breakdown, final and setback viscosities increased with increasing levels of pigeon pea starch while peak time and pasting temperature decreased. The sensory attributes of stiff porridges were not adversely affected by pigeon pea starch inclusion. Therefore it should be possible to incorporate up to 50% of low digestible pigeon pea starch into Irish potato starch from legumes such as pigeon pea as alternatives to cassava starch in the preparation of stiff porridges. Such porridges made from Irish potato and legume starches could provide additional incentive for individuals requiring decreased and or slow starch digestibility such as diabetics.

Thermochemical characterization of pigeon pea stalk for its efficient utilization as an energy source

DOE Office of Scientific and Technical Information (OSTI.GOV)

Katyal, S.K.; Iyer, P.V.R.

2000-05-01

Pigeon pea stalk is a widely available biomass species in India. In this article the potential use of pigeon pea stalk as a fuel source through thermochemical conversion methods such as combustion, gasification, and pyrolysis has been investigated through experimentation using a thermogravimetric analyzer and pilot-plant-scale equipment. It has been proposed that pigeon pea stalks can be effectively utilized in two ways. The first is to pyrolyze the material to produce value-added products such as char, tar, and fuel gas. The second alternative is to partially pyrolyze the material to remove tar-forming volatiles, followed by gasification of reactive char tomore » generate producer gas.« less

Quantitative and Qualitative Involvement of P3N-PIPO in Overcoming Recessive Resistance against Clover Yellow Vein Virus in Pea Carrying the cyv1 Gene

PubMed Central

Choi, Sun Hee; Hagiwara-Komoda, Yuka; Atsumi, Go; Shimada, Ryoko; Hisa, Yusuke; Naito, Satoshi

2013-01-01

In pea carrying cyv1, a recessive gene for resistance to Clover yellow vein virus (ClYVV), ClYVV isolate Cl-no30 was restricted to the initially infected cells, whereas isolate 90-1 Br2 overcame this resistance. We mapped the region responsible for breaking of cyv1-mediated resistance by examining infection of cyv1 pea with chimeric viruses constructed from parts of Cl-no30 and 90-1 Br2. The breaking of resistance was attributed to the P3 cistron, which is known to produce two proteins: P3, from the main open reading frame (ORF), and P3N-PIPO, which has the N-terminal part of P3 fused to amino acids encoded by a small open reading frame (ORF) called PIPO in the +2 reading frame. We introduced point mutations that were synonymous with respect to the P3 protein but nonsynonymous with respect to the P3N-PIPO protein, and vice versa, into the chimeric viruses. Infection of plants with these mutant viruses revealed that both P3 and P3N-PIPO were involved in overcoming cyv1-mediated resistance. Moreover, P3N-PIPO quantitatively affected the virulence of Cl-no30 in cyv1 pea. Additional expression in trans of the P3N-PIPO derived from Cl-no30, using White clover mosaic virus as a vector, enabled Cl-no30 to move to systemic leaves in cyv1 pea. Susceptible pea plants infected with chimeric ClYVV possessing the P3 cistron of 90-1 Br2, and which were therefore virulent toward cyv1 pea, accumulated more P3N-PIPO than did those infected with Cl-no30, suggesting that the higher level of P3N-PIPO in infected cells contributed to the breaking of resistance by 90-1 Br2. This is the first report showing that P3N-PIPO is a virulence determinant in plants resistant to a potyvirus. PMID:23616656

Pea Chaperones under Centrifugation

NASA Astrophysics Data System (ADS)

Talalaiev, Oleksandr

2008-06-01

Etiolated Pisum sativum seedlings were subjected to altered g-forces by centrifugation (3-14g). By using semiquantitative RT-PCR, we studied transcripts of pea genes coding for chaperones that are representatives of small heat shock proteins (sHsps) family. Four members from the different classes of sHsps: cytosolic Hsp17.7 and Hsp18.1 (class I and class II accordingly), chloroplast Hsp21 (class III) and endoplasmic reticulum Hsp22.7 (class IV) were investigated. We conclude that exposure to 3, 7, 10 and 14g for 1h did not affect the level of sHsp transcripts.

Broad bean and pea by-products as sources of fibre-rich ingredients: potential antioxidant activity measured in vitro.

PubMed

Mateos-Aparicio, Inmaculada; Redondo-Cuenca, Araceli; Villanueva-Suárez, María-José

2012-02-01

By-products generated during the processing of plant food can be considered a promising source of dietary fibre as a functional compound. The dietary fibre composition, soluble sugars and antioxidant activity of the extractable polyphenols of pea and broad bean by-products have been analysed in this study. Total dietary fibre using AOAC methods plus hydrolysis (broad bean pod: 337.3 g kg⁻¹; pea pod: 472.6 g kg⁻¹) is higher (P < 0.05) in both by-products than with the Englyst method (broad bean pod: 309.7 g kg⁻¹; pea pod: 434.6 g kg⁻¹). The main monomers are uronic acids, glucose, arabinose and galactose in broad bean pods. However, pea pods are very rich in glucose and xylose. The soluble sugars analysed by high-performance liquid chromatography in both by-products have glucose as the most important component, followed by sucrose and fructose. The ferric reducing antioxidant power (broad bean pod: 406.4 µmol Trolox equivalents g⁻¹; pea pod: 25.9 µmol Trolox equivalents g⁻¹) and scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radical (EC₅₀ of broad bean pod: 0.4 mg mL⁻¹; EC₅₀ of pea pod: 16.0 mg mL⁻¹) were also measured. Broad bean and pea by-products are very rich in dietary fibre, particularly insoluble dietary fibre and their extractable polyphenols demonstrate antioxidant activity. Therefore they might be regarded as functional ingredients. Copyright © 2011 Society of Chemical Industry.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

NASA Technical Reports Server (NTRS)

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.

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

PubMed Central

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

2012-01-01

Abstract 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, an AAVrh.10 gene transfer vector expressing the heavy and light chains of the high affinity anti-cocaine monoclonal antibody GNC92H2. Intravenous administration of AAVrh.10antiCoc.Mab to mice mediated high, persistent serum levels of high-affinity, cocaine-specific antibodies that sequestered intravenously administered cocaine in the blood. With repeated intravenous cocaine challenge, naive mice exhibited hyperactivity, while the AAVrh.10antiCoc.Mab-vaccinated mice were completely resistant to the cocaine. These observations demonstrate a novel strategy for cocaine addiction by requiring only a single administration of an AAV vector mediating persistent, systemic anti-cocaine passive immunity. PMID:22486244

Large-scale evaluation of pea (Pisum sativum L.) germplasm for cold tolerance in the open field during winter in Qingdao.

USDA-ARS?s Scientific Manuscript database

As a cool season crop, pea (Pisum sativum L.) can tolerate frost at the vegetative stage but has yield loss when freezing stress occurs at reproductive stage. Cold tolerance improvement of pea varieties is important for the stable yield and the expansion of winter pea planting area. Under the natura...

Microbiological quality of frozen cauliflower, corn, and peas obtained at retail markets.

PubMed Central

Barnard, R J; Duran, A P; Swartzentruber, A; Schwab, A H; Wentz, B A; Read, R B

1982-01-01

The microbiological quality of blanched frozen cauliflower, cut corn, and peas at the retail level was determined. At 35 degrees C, mean aerobic plate count (APC) values for cauliflower, corn, and peas, respectively, were 30,000, 6,100, and 4,700 per g; at 30 degrees C, the mean APC values were 45,000, 8,500, and 6,800 per g, respectively. Geometric means for coliform, Escherichia coli, and Staphylococcus aureus counts for all three vegetables were less than 10 per g. PMID:6751226

Isoform specificity of progesterone receptor antibodies

PubMed Central

Fabris, Victoria; Abascal, María F; Giulianelli, Sebastián; May, María; Sequeira, Gonzalo R; Jacobsen, Britta; Lombès, Marc; Han, Julie; Tran, Luan; Molinolo, Alfredo

2017-01-01

Abstract Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone‐dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N‐terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin‐fixed paraffin‐embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D‐YA and ‐YB cells expressing PRA or PRB, respectively, MDA‐MB‐231 cells modified to synthesize PRB, and MDA‐MB‐231/iPRAB cells which can bi‐inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H‐190, clone 636, clone 16, and Ab‐6 anti‐PR antibodies, the latter exclusively recognizing PRB. Except for Ab‐6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H‐190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA‐specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer. PMID:29085663

Isoform specificity of progesterone receptor antibodies.

PubMed

Fabris, Victoria; Abascal, María F; Giulianelli, Sebastián; May, María; Sequeira, Gonzalo R; Jacobsen, Britta; Lombès, Marc; Han, Julie; Tran, Luan; Molinolo, Alfredo; Lanari, Claudia

2017-10-01

Progesterone receptors (PR) are prognostic and predictive biomarkers in hormone-dependent cancers. Two main PR isoforms have been described, PRB and PRA, that differ only in that PRB has 164 extra N-terminal amino acids. It has been reported that several antibodies empirically exclusively recognize PRA in formalin-fixed paraffin-embedded (FFPE) tissues. To confirm these findings, we used human breast cancer xenograft models, T47D-YA and -YB cells expressing PRA or PRB, respectively, MDA-MB-231 cells modified to synthesize PRB, and MDA-MB-231/iPRAB cells which can bi-inducibly express either PRA or PRB. Cells were injected into immunocompromised mice to generate tumours exclusively expressing PRA or PRB. PR isoform expression was verified using immunoblots. FFPE samples from the same tumours were studied by immunohistochemistry using H-190, clone 636, clone 16, and Ab-6 anti-PR antibodies, the latter exclusively recognizing PRB. Except for Ab-6, all antibodies displayed a similar staining pattern. Our results indicate that clones 16, 636, and the H-190 antibody recognize both PR isoforms. They point to the need for more stringency in evaluating the true specificity of purported PRA-specific antibodies as the PRA/PRB ratio may have prognostic and predictive value in breast cancer.

Analysis of the state of posttranslational calmodulin methylation in developing pea plants. [Pisum sativum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oh, Sukheung; Roberts, D.M.

1990-07-01

A specific calmodulin-N-methyltransferase was used in a radiometric assay to analyze the degree of methylation of lysine-115 in pea (Pisum sativum) plants. Calmodulin was isolated from dissected segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by incubation with the calmodulin methyltransferase in the presence of ({sup 3}H)methyl-S-adenosylmethionine. By this approach, the presence of unmethylated calmodulins were demonstrated in pea tissues, and the levels of methylation varied depending on the developmental state of the tissue tested. Calmodulin methylation levels were lower in apical root segments of both etiolated andmore » green plants, and in the young lateral roots compared with the mature, differentiated root tissues. The incorporation of methyl groups into these calmodulin samples appears to be specific for position 115 since site-directed mutants of calmodulin with substitutions at this position competitively inhibited methyl group incorporation. The present findings, combined with previous data showing differences in the ability of methylated and unmethylated calmodulins to activate pea NAD kinase raise the possibility that posttranslational methylation of calmodulin could be another mechanism for regulating calmodulin activity.« less

Structure of allelic variants of subtype 5 of histone H1 in pea Pisum sativum L.

PubMed

Bogdanova, V S; Lester, D R; Berdnikov, V A; Andersson, I

2005-06-01

The pea genome contains seven histone H1 genes encoding different subtypes. Previously, the DNA sequence of only one gene, His1, coding for the subtype H1-1, had been identified. We isolated a histone H1 allele from a pea genomic DNA library. Data from the electrophoretic mobility of the pea H1 subtypes and their N-bromosuccinimide cleavage products indicated that the newly isolated gene corresponded to the H1-5 subtype encoded by His5. We confirmed this result by sequencing the gene from three pea lines with H1-5 allelic variants of altered electrophoretic mobility. The allele of the slow H1-5 variant differed from the standard allele by a nucleotide substitution that caused the replacement of the positively charged lysine with asparagine in the DNA-interacting domain of the histone molecule. A temperature-related occurrence had previously been demonstrated for this H1-5 variant in a study on a worldwide collection of pea germplasm. The variant tended to occur at higher frequencies in geographic regions with a cold climate. The fast allelic variant of H1-5 displayed a deletion resulting in the loss of a duplicated pentapeptide in the C-terminal domain.

Enzymatic breakdown of raffinose oligosaccharides in pea seeds.

PubMed

Blöchl, Andreas; Peterbauer, Thomas; Hofmann, Julia; Richter, Andreas

2008-06-01

Both alkaline and acidic alpha-galactosidases (alpha-D: -galactoside galactohydrolases, E.C.3.2.1.22) isolated from various plant species have been described, although little is known about their co-occurrence and functions in germinating seeds. Here, we report on the isolation of two cDNAs, encoding for alpha-galactosidases from maturing and germinating seeds of Pisum sativum. One was identified as a member of the acidic alpha-galactosidase of the family 27 glycosyl hydrolase cluster and the other as a member of the family of alkaline alpha-galactosidases, which are highly homologous to seed imbibition proteins (SIPs). PsGAL1 transcripts, encoding for the ACIDIC alpha-GALACTOSIDASE, were predominately expressed during seed maturation and acidic enzyme activities were already present in dry seeds, showing little changes during seed germination. Compartmentation studies revealed that acidic alpha-galactosidases were located in protein storage vacuoles (PSVs). PsAGA1, encoding for the ALKALINE alpha-GALACTOSIDASE, was only expressed after radicle protrusion, when about 50% of RFOs have already been broken down. RFO breakdown was markedly decreased when the translation of the alkaline enzyme was inhibited, providing evidence that PsAGA1 indeed functioned in RFO degradation. Based on these data, we present an integrated model of RFO breakdown by two sequentially active alpha-galactosidases in pea seeds.

Expression of cytokeratins in odontogenic jaw cysts: monoclonal antibodies reveal distinct variation between different cyst types.

PubMed

Hormia, M; Ylipaavalniemi, P; Nagle, R B; Virtanen, I

1987-08-01

Immunostaining with monoclonal antibodies was used to study and compare the cytokeratin content of odontogenic cysts and normal gingival epithelium. Two monoclonal antibodies, PKK2 and KA1, stained the whole epithelium in all cyst samples. In gingiva, PKK2 gave a suprabasal staining and KA1 reacted with all epithelial cell layers. Antibodies PKK1, KM 4.62 and KS 8.12 gave a heterogeneous staining in follicular and radicular cysts. In keratocysts and in gingiva PKK1 and KM 4.62 reacted mainly with basal cells and KS 8.12 gave a suprabasal staining. Antibodies reacting with the simple epithelial cytokeratin polypeptide No. 18 (PKK3, KS 18.18) recognized in gingiva only solitary cells compatible with Merkel cells. In a case of follicular ameloblastoma a distinct staining of tumor epithelium was revealed with these antibodies. In 2 follicular cysts, but not in other cyst types, a layer of cytokeratin 18-positive cells was revealed. KA5 and KK 8.60 antibodies, reacting exclusively with keratinizing epithelia, including normal gingiva, gave no reaction in radicular cysts, keratocysts and ameloblastoma. Two of the follicular cysts, were negative for PKK3 and KS 18.18, but reacted strongly with KA5 and KK 8.60. The present results show that odontogenic jaw cysts have distinct differences in their cytokeratin content. With the exception of some follicular cysts, they lack signs of keratinizing epithelial differentiation. Only follicular cysts appear to share with some types of ameloblastoma the expression of cytokeratin polypeptide No. 18.

Inheritance patterns of secondary symbionts during sexual reproduction of pea aphid biotypes.

PubMed

Peccoud, Jean; Bonhomme, Joël; Mahéo, Frédérique; de la Huerta, Manon; Cosson, Olivier; Simon, Jean-Christophe

2014-06-01

Herbivorous insects frequently harbor bacterial symbionts that affect their ecology and evolution. Aphids host the obligatory endosymbiont Buchnera, which is required for reproduction, together with facultative symbionts whose frequencies vary across aphid populations. These maternally transmitted secondary symbionts have been particularly studied in the pea aphid, Acyrthosiphon pisum, which harbors at least 8 distinct bacterial species (not counting Buchnera) having environmentally dependent effects on host fitness. In particular, these symbiont species are associated with pea aphid populations feeding on specific plants. Although they are maternally inherited, these bacteria are occasionally transferred across insect lineages. One mechanism of such nonmaternal transfer is paternal transmission to the progeny during sexual reproduction. To date, transmission of secondary symbionts during sexual reproduction of aphids has been investigated in only a handful of aphid lineages and 3 symbiont species. To better characterize this process, we investigated inheritance patterns of 7 symbiont species during sexual reproduction of pea aphids through a crossing experiment involving 49 clones belonging to 9 host-specialized biotypes, and 117 crosses. Symbiont species in the progeny were detected with diagnostic qualitative PCR at the fundatrix stage hatching from eggs and in later parthenogenetic generations. We found no confirmed case of paternal transmission of symbionts to the progeny, and we observed that maternal transmission of a particular symbiont species (Serratia symbiotica) was quite inefficient. We discuss these observations in respect to the ecology of the pea aphid. © 2013 Institute of Zoology, Chinese Academy of Sciences.

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

PubMed Central

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

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 SDS-polyacrylamide gel electrophoresis were applied to purify the antigen for sequence analysis. The antigen coming from Mycoplasma hyorhinis (M. hyorhinis) was further confirmed with Western blot analysis by infecting M. hyorhinis -free HeLa cells and eliminating the M. hyorhinis from MGC803 cells. The full p37 gene was cloned by PCR and expressed successfully in Escherichia coli after site-directed mutations. Immunofluorescence assay was used to demonstrate if p37 protein 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 Mycoplasma 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 p37 protein 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. PMID:14562370

Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

NASA Astrophysics Data System (ADS)

Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

1987-05-01

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

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

PubMed

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

2003-04-02

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.

Dynamic effects of adrenaline (epinephrine) in out-of-hospital cardiac arrest with initial pulseless electrical activity (PEA).

PubMed

Nordseth, Trond; Olasveengen, Theresa Mariero; Kvaløy, Jan Terje; Wik, Lars; Steen, Petter Andreas; Skogvoll, Eirik

2012-08-01

In cardiac arrest, pulseless electrical activity (PEA) is a challenging clinical syndrome. In a randomized study comparing intravenous (i.v.) access and drugs versus no i.v. access or drugs during advanced life support (ALS), adrenaline (epinephrine) improved return of spontaneous circulation (ROSC) in patients with PEA. Originating from this study, we investigated the time-dependent effects of adrenaline on clinical state transitions in patients with initial PEA, using a non-parametric multi-state statistical model. Patients with available defibrillator recordings were included, of whom 101 received adrenaline and 73 did not. There were significantly more state transitions in the adrenaline group than in the no-adrenaline group (rate ratio = 1.6, p<0.001). Adrenaline markedly increased the rate of transition from PEA to ROSC during ALS and slowed the rate of being declared dead; e.g. by 20 min 20% of patients in the adrenaline group had been declared dead and 25% had obtained ROSC, whereas 50% in the no-adrenaline group have been declared dead and 15% had obtained ROSC. The differential effect of adrenaline could be seen after approx. 10 min of ALS for most transitions. For both groups the probability of deteriorating from PEA to asystole was highest during the first 15 min. Adrenaline increased the rate of transition from PEA to ventricular fibrillation or -tachycardia (VF/VT), and from ROSC to VF/VT. Adrenaline has notable clinical effects during ALS in patients with initial PEA. The drug extends the time window for ROSC to develop, but also renders the patient more unstable. Further research should investigate the optimal dose, timing and mode of adrenaline administration during ALS. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The Diversity of Pea Microsymbionts in Various Types of Soils and Their Effects on Plant Host Productivity

PubMed Central

Wielbo, Jerzy; Podleśna, Anna; Kidaj, Dominika; Podleśny, Janusz; Skorupska, Anna

2015-01-01

The growth and yield of peas cultivated on eight different soils, as well as the diversity of pea microsymbionts derived from these soils were investigated in the present study. The experimental plot was composed of soils that were transferred from different parts of Poland more than a century ago. The soils were located in direct vicinity of each other in the experimental plot. All soils examined contained pea microsymbionts, which were suggested to belong to Rhizobium leguminosarum sv. viciae based on the nucleotide sequence of the partial 16S rRNA gene. PCR-RFLP analyses of the 16S-23S rRNA gene ITS region and nodD alleles revealed the presence of numerous and diversified groups of pea microsymbionts and some similarities between the tested populations, which may have been the result of the spread or displacement of strains. However, most populations retained their own genetic distinction, which may have been related to the type of soil. Most of the tested populations comprised low-effective strains for the promotion of pea growth. No relationships were found between the characteristics of soil and symbiotic effectiveness of rhizobial populations; however, better seed yield was obtained for soil with medium biological productivity inhabited by high-effective rhizobial populations than for soil with high agricultural quality containing medium-quality pea microsymbionts, and these results showed the importance of symbiosis for plant hosts. PMID:26370165

Ectopic expression of phloem motor protein pea forisome PsSEO-F1 enhances salinity stress tolerance in tobacco.

PubMed

Srivastava, Vineet Kumar; Raikwar, Shailendra; Tuteja, Renu; Tuteja, Narendra

2016-05-01

PsSEOF-1 binds to calcium and its expression is upregulated by salinity treatment. PsSEOF - 1 -overexpressing transgenic tobacco showed enhanced salinity stress tolerance by maintaining cellular ion homeostasis and modulating ROS-scavenging pathway. Calcium (Ca(2+)) plays important role in growth, development and stress tolerance in plants. Cellular Ca(2+) homeostasis is achieved by the collective action of channels, pumps, antiporters and by Ca(2+) chelators present in the cell like calcium-binding proteins. Forisomes are ATP-independent mechanically active motor proteins known to function in wound sealing of injured sieve elements of phloem tissue. The Ca(2+)-binding activity of forisome and its role in abiotic stress signaling were largely unknown. Here we report the Ca(2+)-binding activity of pea forisome (PsSEO-F1) and its novel function in promoting salinity tolerance in transgenic tobacco. Native PsSEO-F1 promoter positively responded in salinity stress as confirmed using GUS reporter. Overexpression of PsSEO-F1 tobacco plants confers salinity tolerance by alleviating ionic toxicity and increased ROS scavenging activity which probably results in reduced membrane damage and improved yield under salinity stress. Evaluation of several physiological indices shows an increase in relative water content, electrolyte leakage, proline accumulation and chlorophyll content in transgenic lines as compared with null-segregant control. Expression of several genes involved in cellular homeostasis is perturbed by PsSEO-F1 overexpression. These findings suggest that PsSEO-F1 provides salinity tolerance through cellular Ca(2+) homeostasis which in turn modulates ROS machinery providing indirect link between Ca(2+) and ROS signaling under salinity-induced perturbation. PsSEO-F1 most likely functions in salinity stress tolerance by improving antioxidant machinery and mitigating ion toxicity in transgenic lines. This finding should make an important contribution in our better

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets*

PubMed Central

Rhoden, John J.; Dyas, Gregory L.

2016-01-01

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. PMID:27022022

A Modeling and Experimental Investigation of the Effects of Antigen Density, Binding Affinity, and Antigen Expression Ratio on Bispecific Antibody Binding to Cell Surface Targets.

PubMed

Rhoden, John J; Dyas, Gregory L; Wroblewski, Victor J

2016-05-20

Despite the increasing number of multivalent antibodies, bispecific antibodies, fusion proteins, and targeted nanoparticles that have been generated and studied, the mechanism of multivalent binding to cell surface targets is not well understood. Here, we describe a conceptual and mathematical model of multivalent antibody binding to cell surface antigens. Our model predicts that properties beyond 1:1 antibody:antigen affinity to target antigens have a strong influence on multivalent binding. Predicted crucial properties include the structure and flexibility of the antibody construct, the target antigen(s) and binding epitope(s), and the density of antigens on the cell surface. For bispecific antibodies, the ratio of the expression levels of the two target antigens is predicted to be critical to target binding, particularly for the lower expressed of the antigens. Using bispecific antibodies of different valencies to cell surface antigens including MET and EGF receptor, we have experimentally validated our modeling approach and its predictions and observed several nonintuitive effects of avidity related to antigen density, target ratio, and antibody affinity. In some biological circumstances, the effect we have predicted and measured varied from the monovalent binding interaction by several orders of magnitude. Moreover, our mathematical framework affords us a mechanistic interpretation of our observations and suggests strategies to achieve the desired antibody-antigen binding goals. These mechanistic insights have implications in antibody engineering and structure/activity relationship determination in a variety of biological contexts. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Symbiotic activity of pea (Pisum sativum) after application of Nod factors under field conditions.

PubMed

Siczek, Anna; Lipiec, Jerzy; Wielbo, Jerzy; Kidaj, Dominika; Szarlip, Paweł

2014-04-29

Growth and symbiotic activity of legumes are mediated by Nod factors (LCO, lipo-chitooligosaccharides). To assess the effects of application of Nod factors on symbiotic activity and yield of pea, a two-year field experiment was conducted on a Haplic Luvisol developed from loess. Nod factors were isolated from Rhizobium leguminosarum bv. viciae strain GR09. Pea seeds were treated with the Nod factors (10⁻¹¹ M) or water (control) before planting. Symbiotic activity was evaluated by measurements of nitrogenase activity (acetylene reduction assay), nodule number and mass, and top growth by shoot mass, leaf area, and seed and protein yield. Nod factors generally improved pea yield and nitrogenase activity in the relatively dry growing season 2012, but not in the wet growing season in 2013 due to different weather conditions.

Theranostics Using Antibodies and Antibody-Related Therapeutics.

PubMed

Moek, Kirsten L; Giesen, Danique; Kok, Iris C; de Groot, Derk Jan A; Jalving, Mathilde; Fehrmann, Rudolf S N; Lub-de Hooge, Marjolijn N; Brouwers, Adrienne H; de Vries, Elisabeth G E

2017-09-01

In theranostics, radiolabeled compounds are used to determine a treatment strategy by combining therapeutics and diagnostics in the same agent. Monoclonal antibodies (mAbs) and antibody-related therapeutics represent a rapidly expanding group of cancer medicines. Theranostic approaches using these drugs in oncology are particularly interesting because antibodies are designed against specific targets on the tumor cell membrane and immune cells as well as targets in the tumor microenvironment. In addition, these drugs are relatively easy to radiolabel. Noninvasive molecular imaging techniques, such as SPECT and PET, provide information on the whole-body distribution of radiolabeled mAbs and antibody-related therapeutics. Molecular antibody imaging can potentially elucidate drug target expression, tracer uptake in the tumor, tumor saturation, and heterogeneity for these parameters within the tumor. These data can support drug development and may aid in patient stratification and monitoring of the treatment response. Selecting a radionuclide for theranostic purposes generally starts by matching the serum half-life of the mAb or antibody-related therapeutic and the physical half-life of the radionuclide. Furthermore, PET imaging allows better quantification than the SPECT technique. This information has increased interest in theranostics using PET radionuclides with a relatively long physical half-life, such as 89 Zr. In this review, we provide an overview of ongoing research on mAbs and antibody-related theranostics in preclinical and clinical oncologic settings. We identified 24 antibodies or antibody-related therapeutics labeled with PET radionuclides for theranostic purposes in patients. For this approach to become integrated in standard care, further standardization with respect to the procedures involved is required. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

PubMed

Hassaïne, Ghérici; Deluz, Cédric; Grasso, Luigino; Wyss, Romain; Hovius, Ruud; Stahlberg, Henning; Tomizaki, Takashi; Desmyter, Aline; Moreau, Christophe; Peclinovska, Lucie; Minniberger, Sonja; Mebarki, Lamia; Li, Xiao-Dan; Vogel, Horst; Nury, Hugues

2017-01-01

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

GREEN PEA GALAXIES REVEAL SECRETS OF Lyα ESCAPE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Huan; Wang, Junxian; Malhotra, Sangeeta

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 fractionmore » 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.« less

Biological changes of green pea (Pisum sativum L.) by selenium enrichment.

PubMed

Garousi, Farzaneh; Kovács, Béla; Domokos-Szabolcsy, Éva; Veres, Szilvia

2017-03-01

Supplement of common fertilizers with selenium (Se) for crop production will be an effective way to produce selenium-rich food and feed. The value of green pea seeds and forages as alternative protein source can be improved by using agronomic biofortification. Therefore, biological changes of green pea (Pisum sativum L.) and influences of inorganic forms of Se (sodium selenite and sodium selenate) at different concentrations on the accumulation of magnesium (Mg) and phosphorus (P) were investigated in greenhouse experiment. 3 mg kg -1 of selenite had positive effects to enhance photosynthetic attributes and decrease lipid peroxidation significantly. At the same time, Se accumulation increased in all parts of plant by increasing Se supply. Moreover, Mg and P accumulations were significantly increased at 3 mg kg -1 selenite and 1 mg kg -1 selenate treatments, respectively. By contrast higher selenite concentrations (≥30 mg kg -1 ) exerted toxic effects on plants. Relative chlorophyll content, actual photochemical efficiency of PSII (Ф PSII ) and Mg accumulation showed significant decrease while membrane lipid peroxidation increased. Thus, the present findings prove Se biofortification has positive effects on biological traits of green pea to provide it as a proper functional product.

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

PubMed Central

Rahimi, Roghayeh; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Mostafaie, Ali; Mahdavi, Mehdi

2015-01-01

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. Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. The final concentration of purified protein was 500 µg/ml. PMID:25810888

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate

PubMed Central

Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito; Kabatova, Barbora; Mackow, Natalie; Lingemann, Matthias; Yang, Lijuan; McLellan, Jason S.; Graham, Barney S.; Kwong, Peter D.; Schaap-Nutt, Anne; Collins, Peter L.

2015-01-01

ABSTRACT Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. IMPORTANCE Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F

Emulsifying and foaming properties of commercial yellow pea (Pisum sativum L.) seed flours.

PubMed

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 < 0.05) oil droplet sizes, 16-30 and 23-54 microm, respectively, than flours that primarily contained fiber such as Centara III and IV, or those that consisted mainly of starch: Centu-tex, Uptake 80 and Accu-gel. PPI was a better emulsifier than SPI at pH 7.0, and a better foaming agent at pH 3.0 and pH 7.0, although foaming capacity varied with sample concentration. Centu-tex and Uptake 80 have exactly the same chemical composition, but the latter has a much smaller flour particle size range, and had significantly smaller (p < 0.05) emulsion oil droplets. Incorporation of pea starch into SPI emulsions produced a synergistic effect that led to significant increases (p < 0.05) in emulsification capacity (reduced emulsion oil droplet size) when compared to SPI or starch alone. These results showed that PPI had generally significantly higher (p < 0.05) emulsion and foam forming properties than SPI, and that pea starch could be used to improve the quality of SPI-stabilized food emulsions.

Thioredoxin Redox Regulates ATPase Activity of Magnesium Chelatase CHLI Subunit and Modulates Redox-Mediated Signaling in Tetrapyrrole Biosynthesis and Homeostasis of Reactive Oxygen Species in Pea Plants1[C][W][OA

PubMed Central

Luo, Tao; Fan, Tingting; Liu, Yinan; Rothbart, Maxi; Yu, Jing; Zhou, Shuaixiang; Grimm, Bernhard; Luo, Meizhong

2012-01-01

The chloroplast thioredoxins (TRXs) function as messengers of redox signals from ferredoxin to target enzymes. In this work, we studied the regulatory impact of pea (Pisum sativum) TRX-F on the magnesium (Mg) chelatase CHLI subunit and the enzymatic activation of Mg chelatase in vitro and in vivo. In vitro, reduced TRX-F activated the ATPase activity of pea CHLI and enhanced the activity of Mg chelatase reconstituted from the three recombinant subunits CHLI, CHLD, and CHLH in combination with the regulator protein GENOMES UNCOUPLED4 (GUN4). Yeast two-hybrid and bimolecular fluorescence complementation assays demonstrated that TRX-F physically interacts with CHLI but not with either of the other two subunits or GUN4. In vivo, virus-induced TRX-F gene silencing (VIGS-TRX-F) in pea plants did not result in an altered redox state of CHLI. However, simultaneous silencing of the pea TRX-F and TRX-M genes (VIGS-TRX-F/TRX-M) resulted in partially and fully oxidized CHLI in vivo. VIGS-TRX-F/TRX-M plants demonstrated a significant reduction in Mg chelatase activity and 5-aminolevulinic acid synthesizing capacity as well as reduced pigment content and lower photosynthetic capacity. These results suggest that, in vivo, TRX-M can compensate for a lack of TRX-F and that both TRXs act as important redox regulators of Mg chelatase. Furthermore, the silencing of TRX-F and TRX-M expression also affects gene expression in the tetrapyrrole biosynthesis pathway and leads to the accumulation of reactive oxygen species, which may also serve as an additional signal for the transcriptional regulation of photosynthesis-associated nuclear genes. PMID:22452855

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.

PubMed

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.

Potential of roselle and blue pea in the dye-sensitized solar cell

NASA Astrophysics Data System (ADS)

Dayang, S.; Irwanto, M.; Gomesh, N.; Ismail, B.

2017-09-01

This paper discovers the use of natural dyes from Roselle flower and Blue Pea flower which act as a sensitizer in DSSC and in addition has a potential in absorbing visible light spectrum. The dyes were extracted using distilled water (DI) and ethanol (E) extract solvent in an ultrasonic cleaner for 30 minutes with a frequency of 37 Hz by using `degas' mode at the temperature of 30°C. Absorption spectra of roselle dye and blue pea dye with different extract solvent were tested using Evolution 201 UV-Vis Spectrophotometer. It was found that Roselle dye absorbs at a range of 400 nm - 620 nm and Blue Pea absorbs at the range of wavelength 500 nm - 680 nm. Fourier-Transform Infrared (FTIR) was used to identify the functional active group in extract dye. The concept of Dye-Sensitized Solar Cell (DSSC) similar to photosynthesis process has attracted much attention since it demonstrates a great potential due to the use of low-cost materials and environmentally friendly sources of technology.

Dryland pea production and water use in responses to tillage, crop rotation, and weed management practice

USDA-ARS?s Scientific Manuscript database

Pea has been used to replace fallow and sustain dryland crop yields in arid and semiarid regions, but information to optimize its management is required. We evaluated pea growth, yield, and water use in response to tillage, crop rotation, and weed management practice from 2005 to 2010 in the norther...

The pea TCP transcription factor PsBRC1 acts downstream of Strigolactones to control shoot branching.

PubMed

Braun, Nils; de Saint Germain, Alexandre; Pillot, Jean-Paul; Boutet-Mercey, Stéphanie; Dalmais, Marion; Antoniadi, Ioanna; Li, Xin; Maia-Grondard, Alessandra; Le Signor, Christine; Bouteiller, Nathalie; Luo, Da; Bendahmane, Abdelhafid; Turnbull, Colin; Rameau, Catherine

2012-01-01

The function of PsBRC1, the pea (Pisum sativum) homolog of the maize (Zea mays) TEOSINTE BRANCHED1 and the Arabidopsis (Arabidopsis thaliana) BRANCHED1 (AtBRC1) genes, was investigated. The pea Psbrc1 mutant displays an increased shoot-branching phenotype, is able to synthesize strigolactone (SL), and does not respond to SL application. The level of pleiotropy of the SL-deficient ramosus1 (rms1) mutant is higher than in the Psbrc1 mutant, rms1 exhibiting a relatively dwarf phenotype and more extensive branching at upper nodes. The PsBRC1 gene is mostly expressed in the axillary bud and is transcriptionally up-regulated by direct application of the synthetic SL GR24 and down-regulated by the cytokinin (CK) 6-benzylaminopurine. The results suggest that PsBRC1 may have a role in integrating SL and CK signals and that SLs act directly within the bud to regulate its outgrowth. However, the Psbrc1 mutant responds to 6-benzylaminopurine application and decapitation by increasing axillary bud length, implicating a PsBRC1-independent component of the CK response in sustained bud growth. In contrast to other SL-related mutants, the Psbrc1 mutation does not cause a decrease in the CK zeatin riboside in the xylem sap or a strong increase in RMS1 transcript levels, suggesting that the RMS2-dependent feedback is not activated in this mutant. Surprisingly, the double rms1 Psbrc1 mutant displays a strong increase in numbers of branches at cotyledonary nodes, whereas branching at upper nodes is not significantly higher than the branching in rms1. This phenotype indicates a localized regulation of branching at these nodes specific to pea.

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

[Expression of epidermal growth factor receptor mutation specific antibodies in lung adenocarcinoma: evaluation of sensitivity, specificity and relationship to histologic subtypes].

PubMed

Lai, Y M; Feng, Q; Sun, Y; Wang, P; Shi, Y F; Zhao, M; Wu, Q; Li, X H

2016-09-08

To evaluate the expression of epidermal growth factor receptor (EGFR) mutation specific antibodies in invasive lung adenocarcinomas, and their sensitivity, specificity, as well as relationship to histological subtypes. Immunostaining with EGFR mutation-specific antibodies, del E746-A750 in exon 19 and L858R in exon 21, was performed in tissue microarrays of 884 cases of resection specimens to study the relationship between the immunophenotypes and morphologic subtypes. The sensitivity and specificity of the stains were compared with gene mutations detected by amplified refractory mutation system-polymerase chain reaction (ARMS-PCR). Of the 884 cases, the expression of del E746-A750 in exon 19 was 3+ , 2+ , 1+ and 0 in 7 cases (0.79%), 38 cases (4.30%), 129 cases (14.59%) and 710 cases (80.32%), respectively. For L858R in exon 21, 3+ , 2+ , 1+ and 0 staining were seen in 82 cases (9.28%), 93 cases (10.52%), 82 cases (9.28%) and 627 cases (70.93%), respectively. For both antibodies, positive expression (1+ or more) was mainly observed in lepidic, acinar and papillary predominant subtypes, and rarely seen in solid subtype or invasive mucinous adenocarcinoma (P=0.014 and 0.016). If 1+ to 3+ expression was set as positive, the specificity of exon 19/exon 21 reached 98.59%/92.98%, while the sensitivity was relatively lower (62.86%/88.89%). If 2+ to 3+ expression was read as positive, the specificity and sensitivity were 99.30%/97.37% and 25.71%/74.60% for exon 19/exon 21. If only 3+ expression was considered positive, the specificity was 100.0% for both antibodies, with a low sensitivity (8.57% for exon 19 and 34.92% for exon 21). Of the 18 cases with E746-A750 del in exon 19 based on molecular detection, the sensitivity of immunohistochemistry for exon 19 was 88.89% if a positive cutoff value ≥1+ was used; in contrast, of the 8 cases harboring other deletions in exon 19, only two cases were positive as 1+ . Both the EGFR mutation specific antibodies del E746-A750 in

Purification, crystallization and preliminary X-ray analysis of urease from pigeon pea (Cajanus cajan)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balasubramanian, Anuradha; Ponnuraj, Karthe, E-mail: pkarthe@hotmail.com

Urease from pigeon pea was purified and crystallized and X-ray diffraction data were collected at 2.5 Å resolution. Urease is a seed protein that is common to most Leguminosae. It also occurs in many bacteria, fungi and several species of yeast. Urease catalyzes the hydrolysis of urea to ammonia and carbon dioxide, thus allowing organisms to use exogenous and internally generated urea as a nitrogen source. Urease from pigeon pea seeds has been purified to electrophoretic homogeneity using a series of steps involving ammonium sulfate fractionation, acid precipitation, ion-exchange and size-exclusion chromatography techniques. The pigeon pea urease was crystallized andmore » the resulting crystals diffracted to 2.5 Å resolution. The crystals belong to the rhombohedral space group R32, with unit-cell parameters a = b = 176.29, c = 346.44 Å.« less

The composition of pigeon peas (Cajanus cajan (L.) Millsp.) grown in Botswana.

PubMed

Amarteifio, J O; Munthali, D C; Karikari, S K; Morake, T K

2002-01-01

This study investigated the composition of pigeon peas (Cajanus cajan), grown at Sebele, Botswana. The raw seeds of six varieties were analyzed for dry matter, crude fat, protein, fiber, and ash, using Association of Official Analytical Chemists procedures. Major minerals, Ca, K, P, Mg, Na and trace minerals, Cu, Fe and Zn were also assessed. The range of nutrient contents obtained were: dry matter 86.6-88.0%, crude protein 19.0-21.7%, crude fat 1.2-1.3%, crude fiber 9.8-13.0%, and ash 3.9-4.3%. Minerals ranges (mg/100 g dry matter) were: K 1845-1941, P 163-293, Ca 120-167, Mg 113-127, Na 11.3-12.0, Zn 7.2-8.2, Fe 2.5-4.7 and Cu 1.6-1.8. There were no significant differences in Na among the six varieties (p > 0.05). For the other components, varietal differences (p < 0.05) were observed. The values obtained for the dry matter, crude protein, fat, ash, Ca, Cu, Fe, and Mg were similar to those in pigeon peas grown elsewhere, while those for crude fiber and Zn were higher. In general, the composition of pigeon peas compared favorably with those of other legumes such as Bambara groundnut (Vigna subterranea). The levels of crude protein, crude fiber, K, Ca, P and Mg indicated that pigeon peas could be valuable in the diet of the people of Botswana. This crop would positively contribute protein in the diet and the diversification of agricultural produce.

Anti-Ma2 antibody related paraneoplastic limbic/brain stem encephalitis associated with breast cancer expressing Ma1, Ma2, and Ma3 mRNAs.

PubMed

Sahashi, K; Sakai, K; Mano, K; Hirose, G

2003-09-01

A 69 year old woman presented with cognitive impairment and supranuclear gaze palsy caused by paraneoplastic limbic/brain stem encephalitis associated with atypical medullary breast carcinoma. The cerebrospinal fluid from the patient harboured an anti-neuronal cell antibody against Ma2 antigen, but not against Ma1 or Ma3 antigen. Despite the antibody being restricted to the Ma2 antigen, the patient's cancer tissue expressed Ma1, Ma2, and Ma3 mRNAs. These results, and the expression of Ma2 mRNA in an atypical medullar breast carcinoma in another patient without paraneoplastic encephalitis, indicate that the induction of anti-Ma2 antibody depends on host immunoreponsiveness and not on the presence of the antigen itself in the cancer.

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.

PubMed

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.

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization.

PubMed Central

Short, T. W.; Reymond, P.; Briggs, W. R.

1993-01-01

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase. PMID:12231721

Plant-produced anti-dengue virus monoclonal antibodies exhibit reduced antibody-dependent enhancement of infection activity.

PubMed

Dent, Matthew; Hurtado, Jonathan; Paul, Amber M; Sun, Haiyan; Lai, Huafang; Yang, Ming; Esqueda, Adrian; Bai, Fengwei; Steinkellner, Herta; Chen, Qiang

2016-12-01

The mAb E60 has the potential to be a desirable therapeutic molecule since it efficiently neutralizes all four serotypes of dengue virus (DENV). However, mammalian-cell-produced E60 exhibits antibody-dependent enhancement of infection (ADE) activity, rendering it inefficacious in vivo, and treated animals more susceptible to developing more severe diseases during secondary infection. In this study, we evaluated a plant-based expression system for the production of therapeutically suitable E60. The mAb was transiently expressed in Nicotiana benthamianaWT and a ∆XFT line, a glycosylation mutant lacking plant-specific N-glycan residues. The mAb was efficiently expressed and assembled in leaves and exhibited highly homogenous N-glycosylation profiles, i.e. GnGnXF3 or GnGn structures, depending on the expression host. Both E60 glycovariants demonstrated equivalent antigen-binding specificity and in vitro neutralization potency against DENV serotypes 2 and 4 compared with their mammalian-cell-produced counterpart. By contrast, plant-produced E60 exhibited reduced ADE activity in Fc gamma receptor expressing human cells. Our results suggest the ability of plant-produced antibodies to minimize ADE, which may lead to the development of safe and highly efficacious antibody-based therapeutics against DENV and other ADE-prone viral diseases. Our study provides so far unknown insight into the relationship between mAb N-glycosylation and ADE, which contributes to our understanding of how sugar moieties of antibodies modulate Fc-mediated functions and viral pathogenesis.

Acute effects of pea protein and hull fibre alone and combined on blood glucose, appetite, and food intake in healthy young men--a randomized crossover trial.

PubMed

Mollard, Rebecca C; Luhovyy, Bohdan L; Smith, Christopher; Anderson, G Harvey

2014-12-01

Whether pulse components can be used as value-added ingredients in foods formulated for blood glucose (BG) and food intake (FI) control requires investigation. The objective of this study was to examine of the effects of pea components on FI at an ad libitum meal, as well as appetite and BG responses before and after the meal. In a repeated-measures crossover trial, men (n = 15) randomly consumed (i) pea hull fibre (7 g), (ii) pea protein (10 g), (iii) pea protein (10 g) plus hull fibre (7 g), (iv) yellow peas (406 g), and (v) control. Pea hull fibre and protein were served with tomato sauce and noodles, while yellow peas were served with tomato sauce. Control was noodles and tomato sauce. FI was measured at a pizza meal (135 min). Appetite and BG were measured pre-pizza (0-135 min) and post-pizza (155-215 min). Protein plus fibre and yellow peas led to lower pre-pizza BG area under the curve compared with fibre and control. At 30 min, BG was lower after protein plus fibre and yellow peas compared with fibre and control, whereas at 45 and 75 min, protein plus fibre and yellow peas led to lower BG compared with fibre (p < 0.05). Following the pizza meal (155 min), yellow peas led to lower BG compared with fibre (p < 0.05). No differences were observed in FI or appetite. This trial supports the use of pea components as value-added ingredients in foods designed to improve glycemic control.

An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2

PubMed Central

Boswell, C Andrew; Mundo, Eduardo E; Firestein, Ron; Zhang, Crystal; Mao, Weiguang; Gill, Herman; Young, Cynthia; Ljumanovic, Nina; Stainton, Shannon; Ulufatu, Sheila; Fourie, Aimee; Kozak, Katherine R; Fuji, Reina; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

2013-01-01

Background and Purpose The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. Experimental Approach A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography – X-ray computed tomography imaging and immunohistochemistry. Key Results The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Conclusions and Implications Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. PMID:22889168

An integrated approach to identify normal tissue expression of targets for antibody-drug conjugates: case study of TENB2.

PubMed

Boswell, C Andrew; Mundo, Eduardo E; Firestein, Ron; Zhang, Crystal; Mao, Weiguang; Gill, Herman; Young, Cynthia; Ljumanovic, Nina; Stainton, Shannon; Ulufatu, Sheila; Fourie, Aimee; Kozak, Katherine R; Fuji, Reina; Polakis, Paul; Khawli, Leslie A; Lin, Kedan

2013-01-01

The success of antibody-drug conjugates (ADCs) depends on the therapeutic window rendered by the differential expression between normal and pathological tissues. The ability to identify and visualize target expression in normal tissues could reveal causes for target-mediated clearance observed in pharmacokinetic characterization. TENB2 is a prostate cancer target associated with the progression of poorly differentiated and androgen-independent tumour types, and ADCs specific for TENB2 are candidate therapeutics. The objective of this study was to locate antigen expression of TENB2 in normal tissues, thereby elucidating the underlying causes of target-mediated clearance. A series of pharmacokinetics, tissue distribution and mass balance studies were conducted in mice using a radiolabelled anti-TENB2 ADC. These data were complemented by non-invasive single photon emission computed tomography - X-ray computed tomography imaging and immunohistochemistry. The intestines were identified as a saturable and specific antigen sink that contributes, at least in part, to the rapid target-mediated clearance of the anti-TENB2 antibody and its drug conjugate in rodents. As a proof of concept, we also demonstrated the selective disposition of the ADC in a tumoural environment in vivo using the LuCaP 77 transplant mouse model. High tumour uptake was observed despite the presence of the antigen sink, and antigen specificity was confirmed by antigen blockade. Our findings provide the anatomical location and biological interpretation of target-mediated clearance of anti-TENB2 antibodies and corresponding drug conjugates. Further investigations may be beneficial in addressing the relative contributions to ADC disposition from antigen expression in both normal and pathological tissues. © 2012 Genentech, Inc.. British Journal of Pharmacology © 2012 The British Pharmacological Society.

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

PubMed

Kitabatake, Masahiro; Inoue, Shingo; Yasui, Fumihiko; Yokochi, Shoji; Arai, Masaaki; Morita, Kouichi; Shida, Hisatoshi; Kidokoro, Minoru; Murai, Fukashi; Le, Mai Quynh; Mizuno, Kyosuke; Matsushima, Kouji; Kohara, Michinori

2007-01-08

A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses

PubMed Central

Wykes, Michelle N; Beattie, Lynette; MacPherson, Gordon G; Hart, Derek N

2004-01-01

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b− DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs. PMID:15500618

Dendritic cells and follicular dendritic cells express a novel ligand for CD38 which influences their maturation and antibody responses.

PubMed

Wykes, Michelle N; Beattie, Lynette; Macpherson, Gordon G; Hart, Derek N

2004-11-01

CD38 is a cell surface molecule with ADP-ribosyl cyclase activity, which is predominantly expressed on lymphoid and myeloid cells. CD38 has a significant role in B-cell function as some anti-CD38 antibodies can deliver potent growth and differentiation signals, but the ligand that delivers this signal in mice is unknown. We used a chimeric protein of mouse CD38 and human immunogobulin G (IgG) (CD38-Ig) to identify a novel ligand for murine CD38 (CD38L) on networks of follicular dendritic cells (FDCs) as well as dendritic cells (DCs) in the spleen. Flow-cytometry found that all DC subsets expressed cytoplasmic CD38L but only fresh ex vivo CD11c+ CD11b- DCs had cell surface CD38L. Anti-CD38 antibody blocked the binding of CD38-Ig to CD38L, confirming the specificity of detection. CD38-Ig immuno-precipitated ligands of 66 and 130 kDa. Functional studies found that CD38-Ig along with anti-CD40 and anti-major histocompatibility complex (MHC) class II antibody provided maturation signals to DCs in vitro. When CD38-Ig was administered in vivo with antigen, IgG2a responses were significantly reduced, suggesting that B and T cells expressing CD38 may modulate the isotype of antibodies produced through interaction with CD38L on DCs. CD38-Ig also expanded FDC networks when administered in vivo. In conclusion, this study has identified a novel ligand for CD38 which has a role in functional interactions between lymphocytes and DCs or FDCs.

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.

PubMed

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.

Use of the 22C3 anti-PD-L1 antibody to determine PD-L1 expression in multiple automated immunohistochemistry platforms.

PubMed

Ilie, Marius; Khambata-Ford, Shirin; Copie-Bergman, Christiane; Huang, Lingkang; Juco, Jonathan; Hofman, Veronique; Hofman, Paul

2017-01-01

For non-small cell lung cancer (NSCLC), treatment with pembrolizumab is limited to patients with tumours expressing PD-L1 assessed by immunohistochemistry (IHC) using the PD-L1 IHC 22C3 pharmDx (Dako, Inc.) companion diagnostic test, on the Dako Autostainer Link 48 (ASL48) platform. Optimised protocols are urgently needed for use of the 22C3 antibody concentrate to test PD-L1 expression on more widely available IHC autostainers. We evaluated PD-L1 expression using the 22C3 antibody concentrate in the three main commercially available autostainers Dako ASL48, BenchMark ULTRA (Ventana Medical Systems, Inc.), and Bond-III (Leica Biosystems) and compared the staining results with the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Several technical conditions for laboratory-developed tests (LDTs) were evaluated in tonsil specimens and a training set of three NSCLC samples. Optimised protocols were then validated in 120 NSCLC specimens. Optimised protocols were obtained on both the VENTANA BenchMark ULTRA and Dako ASL48 platforms. Significant expression of PD-L1 was obtained on tissue controls with the Leica Bond-III autostainer when high concentrations of the 22C3 antibody were used. It therefore was not tested on the 120 NSCLC specimens. An almost 100% concordance rate for dichotomized tumour proportion score (TPS) results was observed between TPS ratings using the 22C3 antibody concentrate on the Dako ASL48 and VENTANA BenchMark ULTRA platforms relative to the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Interpathologist agreement was high on both LDTs and the PD-L1 IHC 22C3 pharmDx kit on the Dako ASL48 platform. Availability of standardized protocols for determining PD-L1 expression using the 22C3 antibody concentrate on the widely available Dako ASL48 and VENTANA BenchMark ULTRA IHC platforms will expand the number of laboratories able to determine eligibility of patients with NSCLC for treatment with pembrolizumab in a reliable and concordant

Cloning and expression of N22 region of Torque Teno virus (TTV) genome and use of peptide in developing immunoassay for TTV antibodies

PubMed Central

2014-01-01

Background Torque Teno Virus (TTV) is a DNA virus with high rate of prevalence globally. Since its discovery in 1997, several studies have questioned the role of this virus in causing disease. However, it still remains an enigma. Although methods are available for detection of TTV infection, there is still a need for simple, rapid and reliable method for screening of this virus in human population. Present investigation describes the cloning and expression of N22 region of TTV-genome and the use of expressed peptide in development of immunoassay to detect anti-TTV antibodies in serum. Since TTV genotype-1 is more common in India, the serum positive for genotype-1 was used as source of N22 for expression purpose. Methods Full length N22 region of ORF1 from TTV genotype-1 was amplified and cloned in pGEM®-T Easy vector. After cloning, the amplicon was transformed and expressed as a fusion protein containing hexa-histidine tag in pET-28a(+) vector using BL21 E. coli cells as host. Expression was conducted both in LB medium as well as ZYP-5052 auto-induction medium. The expressed peptide was purified using metal-chelate affinity chromatography and used as antigen in developing a blot immunoassay. Results Analysis of translated product by SDS-PAGE and western blotting demonstrated the presence of 25 kDa polypeptide produced after expression. Solubility studies showed the polypeptide to be associated with insoluble fraction. The use of this peptide as antigen in blot assay produced prominent spot on membrane treated with sera from TTV-infected patients. Analysis of sera from 75 patients with liver and renal diseases demonstrated a successful implication of N22 polypeptide based immunoassay in screening sera for anti-TTV antibodies. Comparison of the immunoassay developed using expressed N22 peptide with established PCR method for TTV-DNA detection showed good coherence between TTV-DNA and presence of anti-TTV antibodies in the sera analysed. Conclusions This concludes

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments.

PubMed

Greunke, Kerstin; Spillner, Edzard; Braren, Ingke; Seismann, Henning; Kainz, Sabine; Hahn, Ulrich; Grunwald, Thomas; Bredehorst, Reinhard

2006-07-13

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.

Characterization of five fungal endophytes producing Cajaninstilbene acid isolated from pigeon pea [Cajanus cajan (L.) Millsp].

PubMed

Gao, Yuan; Zhao, Jin Tong; Zu, Yuan Gang; Fu, Yu Jie; Wang, Wei; Luo, Meng; Efferth, Thomas

2011-01-01

Five fungal endophytes (K4, K5, K6, K9, K14) producing Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) were isolated from the roots of pigeon pea [Cajanus cajan (L.) Millsp.]. CSA is responsible for the prominent pharmacological activities in pigeon pea. The amount of CSA in culture solution varied among the five fungal endophytes. K4 produced the highest levels of CSA (1037.13 µg/L) among the endophytes tested after incubation for five days. Both morphological characteristics and molecular methods were used for species identification of fungal endophytes. The five endophytic isolates were characterized by analyzing the internal transcribed spacer (ITS) rRNA and β-tubulin genes. The K4, K5, K9 and K14 strains isolated from pigeon pea roots were found to be closely related to the species Fusarium oxysporum. K6 was identified as Neonectria macrodidym. The present study is the first report on the isolation and identification of fungal endophytes producing CSA in pigeon pea. The study also provides a scientific base for large scale production of CSA.

Effect of Xyloglucan Oligosaccharides on Growth, Viscoelastic Properties, and Long-Term Extension of Pea Shoots.

PubMed Central

Cutillas-Iturralde, A.; Lorences, E. P.

1997-01-01

The growth-promoting effect of xyloglucan-derived oligosaccharides was investigated using a bioassay with entire pea (Pisum sativum L., var Alaska) shoots. After a 24-h incubation period at 25[deg]C, xyloglucan oligosaccharide (XGO) solutions with concentrations of 10-6 M notably increased the growth rate of pea shoots, whereas the same oligosaccharides at 10-7 M were less effective. To investigate the possible correlation between growth rate changes in the XGO-treated shoots and changes in the wall mechanical properties of their growing regions (third internodes), we used a short-term creep assay. The promotion of elongation by XGOs was reflected in an enhancement of the viscoelasticity of the growing regions of the shoots. To show whether this effect on wall viscoelastic properties was the cause or a consequence of their growth promotion, we tested the effect of XGOs on the long-term extension of isolated cell walls. We characterized an acid-induced extension in isolated cell walls from pea shoots that was not inhibited by preincubation in neutral buffers. Exogenously added XGOs did not alter the pattern of pea segment extension at any pH tested, indicating that XGOs have no direct effect on cell wall viscoelasticity. Finally, preincubation of pea segments in neutral buffers with XGOs enhanced their capacity to extend under acidic conditions. This finding suggests that XGOs at a neutral pH can act via transglycosylation, weakening the wall matrix and making the wall more responsive to other mechanisms of acid-induced extension as an expansin-mediated extension. PMID:12223593

Development of SCAR markers linked to sin-2, the stringless pod locus in pea (Pisum sativum L.)

USDA-ARS?s Scientific Manuscript database

With increasing consumer demand for vegetables, edible-podded peas have become more popular. Stringlessness is one of most important traits for snap peas. A single recessive gene, sin-2, controls this trait. Because pollen carrying the stringless gene is less competitive than pollen carrying the str...

A community resource for exploring and utilizing genetic diversity in the USDA Pea Single Plant Plus Collection

USDA-ARS?s Scientific Manuscript database

Globally, pea (Pisum sativum L.) is an important temperate legume crop for food, feed, and fodder, and many breeding programs exist to develop cultivars adapted to these end uses. In order to conserve genetic diversity useful to researchers, large pea collections have been constructed by numerous na...

Growth performance and carcass characteristics of guinea fowl broilers fed micronized-dehulled pea (Pisum sativum L.) as a substitute for soybean meal.

PubMed

Laudadio, V; Nahashon, S N; Tufarelli, V

2012-11-01

This study was conducted to evaluate the effect of substitution of soybean meal (SBM) with dehulled-micronized peas (Pisum sativum) in diets of guinea fowl broilers on their growth performance, carcass yields, and fatty acid composition of meat. One hundred forty 1-d-old guinea fowl keets were randomly assigned to 2 dietary treatments, which were fed from hatch to 12 wk. The birds were fed 2 wheat middling-based diets comprising a control diet, which contained SBM (78 g/kg) and a test diet containing dehulled-micronized peas (180 g/kg) as the main protein source. The substitution of SBM with peas had no adverse effect on growth performance, dressing percentage, or breast and thigh muscle relative weights of the guinea broilers. However, a reduction of abdominal fat content (P < 0.05) was observed in birds fed the pea diet compared with the control. Breast and thigh meat of birds fed the pea diet had higher lightness scores (P < 0.05) and water-holding capacity (P < 0.01) than the control. Meat from guinea fowls fed the pea diet had less cholesterol (P < 0.01) and lipids (P < 0.05), and higher concentrations of phospholipids (P < 0.05). Feeding peas increased polyunsaturated fatty acid concentration in breast and thigh muscles, and decreased the saturated fatty acid concentration. Feeding the pea diet also lowered the n-6/n-3 polyunsaturated fatty acid ratio of the guinea broiler muscles. Our results suggest that replacing the conventional SBM as the protein source with dehulled-micronized pea meal in diets of guinea fowls broilers can improve carcass quality and favorable lipid profile without adversely affecting growth performance traits.

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

PubMed

Maggi, Maristella; Scotti, Claudia

2017-08-01

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

Induction of host defences by Rhizobium during ineffective nodulation of pea (Pisum sativum L.) carrying symbiotically defective mutations sym40 (PsEFD), sym33 (PsIPD3/PsCYCLOPS) and sym42.

PubMed

Ivanova, Kira A; Tsyganova, Anna V; Brewin, Nicholas J; Tikhonovich, Igor A; Tsyganov, Viktor E

2015-11-01

Rhizobia are able to establish a beneficial interaction with legumes by forming a new organ, called the symbiotic root nodule, which is a unique ecological niche for rhizobial nitrogen fixation. Rhizobial infection has many similarities with pathogenic infection and induction of defence responses accompanies both interactions, but defence responses are induced to a lesser extent during rhizobial infection. However, strong defence responses may result from incompatible interactions between legumes and rhizobia due to a mutation in either macro- or microsymbiont. The aim of this research was to analyse different plant defence reactions in response to Rhizobium infection for several pea (Pisum sativum) mutants that result in ineffective symbiosis. Pea mutants were examined by histochemical and immunocytochemical analyses, light, fluorescence and transmission electron microscopy and quantitative real-time PCR gene expression analysis. It was observed that mutations in pea symbiotic genes sym33 (PsIPD3/PsCYCLOPS encoding a transcriptional factor) and sym40 (PsEFD encoding a putative negative regulator of the cytokinin response) led to suberin depositions in ineffective nodules, and in the sym42 there were callose depositions in infection thread (IT) and host cell walls. The increase in deposition of unesterified pectin in IT walls was observed for mutants in the sym33 and sym42; for mutant in the sym42, unesterified pectin was also found around degrading bacteroids. In mutants in the genes sym33 and sym40, an increase in the expression level of a gene encoding peroxidase was observed. In the genes sym40 and sym42, an increase in the expression levels of genes encoding a marker of hypersensitive reaction and PR10 protein was demonstrated. Thus, a range of plant defence responses like suberisation, callose and unesterified pectin deposition as well as activation of defence genes can be triggered by different pea single mutations that cause perception of an otherwise

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

PubMed

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

2017-09-01

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

Cloning and characterization of TPE4A, a thiol-protease gene induced during ovary senescence and seed germination in pea.

PubMed

Cercós, M; Santamaría, S; Carbonell, J

1999-04-01

A cDNA clone encoding a thiol-protease (TPE4A) was isolated from senescent ovaries of pea (Pisum sativum) by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of TPE4A has the conserved catalytic amino acids of papain. It is very similar to VSCYSPROA, a thiol-protease induced during seed germination in common vetch. TPE4A mRNA levels increase during the senescence of unpollinated pea ovaries and are totally suppressed by treatment with gibberellic acid. In situ hybridization indicated that TPE4A mRNA distribution in senescent pea ovaries is different from that of previously reported thiol-proteases induced during senescence, suggesting the involvement of different proteases in the mobilization of proteins from senescent pea ovaries. TPE4A is also induced during the germination of pea seeds, indicating that a single protease gene can be induced during two different physiological processes, senescence and germination, both of which require protein mobilization.

Effect of isolation techniques on the characteristics of pigeon pea (Cajanus cajan) protein isolates.

PubMed

Adenekan, Monilola K; Fadimu, Gbemisola J; Odunmbaku, Lukumon A; Oke, Emmanuel K

2018-01-01

In this study, the effect of different isolation techniques on the isolated proteins from pigeon pea was investigated. Water, methanol, ammonium sulfate, and acetone were used for the precipitation of proteins from pigeon pea. Proximate composition, and antinutritional and functional properties of the pigeon pea flour and the isolated proteins were measured. Data generated were statistically analyzed. The proximate composition of the water-extracted protein isolate was moisture 8.30%, protein 91.83%, fat 0.25%, ash 0.05%, and crude fiber 0.05%. The methanol-extracted protein isolate composition was moisture 7.87%, protein 91.83%, fat 0.17%, and ash 0.13%, while crude fiber and carbohydrates were not detected. The composition of the ammonium sulfate-extracted protein isolate was moisture 7.73%, protein 91.73%, fat 0.36, ash 0.13%, and crude fiber 0.67%. The acetone-extracted protein isolate composition was moisture 8.03%, protein 91.50%, ash 0.67%, and fat 0.30%, but crude fiber and carbohydrates were not detected. The isolate precipitated with ammonium sulfate displayed the highest foaming capacity (37.63%) and foaming stability (55.75%). Isolates precipitated with methanol and acetone had the highest water absorption capacity (160%). Pigeon pea protein isolates extracted with methanol and ammonium sulfate had the highest oil absorption capacity of 145%. Protein isolates recovered through acetone and methanol had the highest emulsifying capacity of 2.23% and emulsifying stability of 91.47%, respectively. The proximate composition of the recovered protein isolates were of high purity. This shows the efficiency of the extraction techniques. The isolates had desirable solubility index. All the isolation techniques brought significant impact on the characteristics of the isolated pigeon pea protein.

Systemic Induction of the Defensin and Phytoalexin Pisatin Pathways in Pea (Pisum sativum) against Aphanomyces euteiches by Acetylated and Nonacetylated Oligogalacturonides.

PubMed

Selim, Sameh; Sanssené, Jean; Rossard, Stéphanie; Courtois, Josiane

2017-06-19

Oligogalacturonides (OGs) are known for their powerful ability to stimulate the plant immune system but little is known about their mode of action in pea ( Pisum sativum ). In the present study, we investigated the elicitor activity of two fractions of OGs, with polymerization degrees (DPs) of 2-25, in pea against Aphanomyces euteiches . One fraction was nonacetylated (OGs - Ac) whereas the second one was 30% acetylated (OGs + Ac). OGs were applied by injecting the upper two rachises of the plants at three- and/or four-weeks-old. Five-week-old roots were inoculated with 10⁵ zoospores of A. euteiches . The root infection level was determined at 7, 10 and 14 days after inoculation using the quantitative real-time polymerase chain reaction (qPCR). Results showed significant root infection reductions namely 58, 45 and 48% in the plants treated with 80 µg OGs + Ac and 59, 56 and 65% with 200 µg of OGs - Ac. Gene expression results showed the upregulation of genes involved in the antifungal defensins, lignans and the phytoalexin pisatin pathways and a priming effect in the basal defense, SA and ROS gene markers as a response to OGs. The reduction of the efficient dose in OGs + Ac is suggesting that acetylation is necessary for some specific responses. Our work provides the first evidence for the potential of OGs in the defense induction in pea against Aphanomyces root rot.

Analysis of monoclonal antibodies reactive with molecules upregulated or expressed only on activated lymphocytes.

PubMed

Davis, W C; Naessens, J; Brown, W C; Ellis, J A; Hamilton, M J; Cantor, G H; Barbosa, J I; Ferens, W; Bohach, G A

1996-08-01

Monoclonal antibodies potentially specific for antigens expressed or upregulated on activated leukocytes were selected for further analysis from the panel submitted to the third international workshop on ruminant leukocyte antigens. The kinetics of expression of these activation antigens on resting peripheral mononuclear cells (PBMC) and PBMC stimulated with concanavalin A or staphylococcal superantigen SECI for 4, 24 or 96 h were compared, as well as their appearance on various subsets of cells. For some of them, a molecular mass could be determined after immunoprecipitation from radio-labeled, lectin-stimulated cells. Based on the results from the clustering, kinetic studies and biochemical data, evidence was gathered for assigning two additional mAbs to cluster BoCD25 (IL-2 receptor) and two mAbs to cluster BoCD71 (transferrin receptor). Four mAbs recognized an early activation antigen predominantly expressed on gamma delta T cells in short-term cultures. A number of other activation antigens were further characterized.

Small heat shock protein message in etiolated Pea seedlings under altered gravity

NASA Astrophysics Data System (ADS)

Talalaiev, O.

Plants are subjected to various environmental changes during their life cycle To protect themselves against unfavorable influences plant cells synthesize several classes of small heat shock proteins sHsp ranging in size from 15 to 30 kDa This proteins are able to enhance the refolding of chemically denatured proteins in an ATP-independent manner in other words they can function as molecular chaperones The potential contribution of effects of space flight at the plant cellular and gene regulation level has not been characterized yet The object of our study is sHsp gene expression in etiolated Pisum sativum seedlings exposed to altered gravity and environmental conditions We designed primers to detect message for two inducible forms of the cytosolic small heat shock proteins sHsp 17 7 and sHsp 18 1 Applying the RT- PCR we explore sHsps mRNA in pea seedling cells subjected to two types of altered gravity achieved by centrifugation from 3 to 8g by clinorotation 2 rpm and temperature elevation 42oC Temperature elevation as the positive control significantly increased PsHspl7 7 PsHspl8 1 expression We investigate the expression of actin it was constant and comparable for unstressed controls for all variants Results are under discussion

Assessment of flubendiamide residues in pigeon pea in different agro-climatic zones of India.

PubMed

Kale, V D; Walunj, A R; Battu, R S; Sahoo, Sanjay K; Singh, Balwinder; Paramasivam, M; Roy, Sankhajit; Banerjee, Tirthankar; Banerjee, Hemanta; Rao, Cherukuri Sreenivasa; Reddy, D Jagdishwar; Reddy, K Narasimha; Reddy, C Narendra; Tripathy, Vandana; Jaya, Maisnam; Pant, Shashi; Gupta, Monika; Singh, Geeta; Sharma, K K

2012-07-01

Supervised field trials were conducted at the research farms of four agricultural universities located at different agro-climatic zones of India to find out the harvest time residues of flubendiamide and its des-iodo metabolite on pigeon pea (Cajanus cajan) during the year 2006-2007. Two spray applications of flubendiamide 20 WDG at 50 g (T(1)) and 100 g (T(2)) a.i./ha were given to the crop at 15-days interval. The foliage samples at different time intervals were drawn at only one location, however, the harvest time samples of pigeon pea grain, shell, and straw were drawn at all the four locations. The residues were estimated by HPLC coupled with UV-VIS variable detector. No residues of flubendiamide and its des-iodo metabolite were found at harvest of the crop at or above the LOQ level of 0.05 μg/g. On the basis of the data generated, a pre-harvest interval (PHI) of 28 days has been recommended and the flubendiamide 20 WDG has been registered for use on pigeon pea by Central Insecticide Board and Registration Committee, Ministry of Agriculture, Government of India and the MRL has been fixed by Ministry of Health and Family Welfare, Government of India under Prevention of Food and Adulteration as 0.05 μg/g on pigeon pea grains.

Enhanced Neutralizing Antibody Response Induced by Respiratory Syncytial Virus Prefusion F Protein Expressed by a Vaccine Candidate.

PubMed

Liang, Bo; Surman, Sonja; Amaro-Carambot, Emerito; Kabatova, Barbora; Mackow, Natalie; Lingemann, Matthias; Yang, Lijuan; McLellan, Jason S; Graham, Barney S; Kwong, Peter D; Schaap-Nutt, Anne; Collins, Peter L; Munir, Shirin

2015-09-01

Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are the first and second leading viral agents of severe respiratory tract disease in infants and young children worldwide. Vaccines are not available, and an RSV vaccine is particularly needed. A live attenuated chimeric recombinant bovine/human PIV3 (rB/HPIV3) vector expressing the RSV fusion (F) glycoprotein from an added gene has been under development as a bivalent vaccine against RSV and HPIV3. Previous clinical evaluation of this vaccine candidate suggested that increased genetic stability and immunogenicity of the RSV F insert were needed. This was investigated in the present study. RSV F expression was enhanced 5-fold by codon optimization and by modifying the amino acid sequence to be identical to that of an early passage of the original clinical isolate. This conferred a hypofusogenic phenotype that presumably reflects the original isolate. We then compared vectors expressing stabilized prefusion and postfusion versions of RSV F. In a hamster model, prefusion F induced increased quantity and quality of RSV-neutralizing serum antibodies and increased protection against wild-type (wt) RSV challenge. In contrast, a vector expressing the postfusion F was less immunogenic and protective. The genetic stability of the RSV F insert was high and was not affected by enhanced expression or the prefusion or postfusion conformation of RSV F. These studies provide an improved version of the previously well-tolerated rB/HPIV3-RSV F vaccine candidate that induces a superior RSV-neutralizing serum antibody response. Respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are two major causes of pediatric pneumonia and bronchiolitis. The rB/HPIV3 vector expressing RSV F protein is a candidate bivalent live vaccine against HPIV3 and RSV. Previous clinical evaluation indicated the need to increase the immunogenicity and genetic stability of the RSV F insert. Here, we

RADIO DETECTION OF GREEN PEAS: IMPLICATIONS FOR MAGNETIC FIELDS IN YOUNG GALAXIES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chakraborti, Sayan; Yadav, Naveen; Ray, Alak

Green Peas are a new class of young, emission line galaxies that were discovered by citizen volunteers in the Galaxy Zoo project. Their low stellar mass, low metallicity, and very high star formation rates make Green Peas the nearby (z {approx} 0.2) analogs of the Lyman break galaxies which account for the bulk of the star formation in the early universe (z {approx} 2-5). They thus provide accessible laboratories in the nearby universe for understanding star formation, supernova feedback, particle acceleration, and magnetic field amplification in early galaxies. We report the first direct radio detection of Green Peas with lowmore » frequency Giant Metrewave Radio Telescope observations and our stacking detection with archival Very Large Array FIRST data. We show that the radio emission implies that these extremely young galaxies already have magnetic fields ({approx}> 30 {mu}G) even larger than that of the Milky Way. This is at odds with the present understanding of magnetic field growth based on amplification of seed fields by dynamo action over a galaxy's lifetime. Our observations strongly favor models with pregalactic magnetic fields at {mu}G levels.« less

7 CFR 457.140 - Dry pea crop insurance provisions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... business enterprise regularly engaged in the processing of contract seed peas, that possesses all licenses... your production guarantee. Base contract price. The price per pound stipulated in the processor/seed... producer for at least 50 percent of the total production under contract with the processor/seed company...

7 CFR 457.140 - Dry pea crop insurance provisions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... business enterprise regularly engaged in the processing of contract seed peas, that possesses all licenses... your production guarantee. Base contract price. The price per pound stipulated in the processor/seed... producer for at least 50 percent of the total production under contract with the processor/seed company...

7 CFR 457.140 - Dry pea crop insurance provisions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... business enterprise regularly engaged in the processing of contract seed peas, that possesses all licenses... your production guarantee. Base contract price. The price per pound stipulated in the processor/seed... producer for at least 50 percent of the total production under contract with the processor/seed company...

Pea (Pisum sp.) genetic resources, its analysis and exploration

USDA-ARS?s Scientific Manuscript database

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...

Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

PubMed

Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

2009-09-01

The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

The effect of pea albumin 1F on glucose metabolism in mice.

PubMed

Dun, Xin-Peng; Li, Fa-Fang; Wang, Jian-He; Chen, Zheng-Wang

2008-06-01

Pea albumin 1F (PA1F), a plant peptide isolated from pea seeds, can dramatically increase blood glucose concentration by subcutaneous injection with a dosage of 5 or 10 microg/g (body weight) in normal and type II diabetic mice (KK/upj-Ay). The voltage-dependent anion channel 1 (VDAC-1) has been identified as the PA1F binding protein from mice pancreatic cell membrane, which may be involved in the regulation of enhancing blood glucose in response to PA1F binding. The results clearly show that peptide-signaling molecules from plants can affect mammalian physiological functions, especially, in association with glucose metabolism.

Differential uptake of photosynthetic and non-photosynthetic proteins by pea root plastids.

PubMed

Yan, Xianxi; Khan, Sultan; Hase, Toshiharu; Emes, Michael J; Bowsher, Caroline G

2006-11-27

The photosynthetic proteins RuBiSCO, ferredoxin I and ferredoxin NADP(+)-oxidoreductase (pFNR) were efficiently imported into isolated pea chloroplasts but not into pea root plastids. By contrast non-photosynthetic ferredoxin III and heterotrophic FNR (hFNR) were efficiently imported into both isolated chloroplasts and root plastids. Chimeric ferredoxin I/III (transit peptide of ferredoxin I attached to the mature region of ferredoxin III) only imported into chloroplasts. Ferredoxin III/I (transit peptide of ferredoxin III attached to the mature region of ferredoxin I) imported into both chloroplasts and root plastids. This suggests that import depends on specific interactions between the transit peptide and the translocon apparatus.

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

PubMed

Barberà, M; Martínez-Torres, D

2017-10-01

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

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells.

PubMed

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible.

Effective combination treatment of GD2-expressing neuroblastoma and Ewing's sarcoma using anti-GD2 ch14.18/CHO antibody with Vγ9Vδ2+ γδT cells

PubMed Central

Fisher, Jonathan P H; Flutter, Barry; Wesemann, Florian; Frosch, Jennifer; Rossig, Claudia; Gustafsson, Kenth; Anderson, John

2016-01-01

Gamma delta T lymphocytes (γδT cells) have pleiotropic properties including innate cytotoxicity, which make them attractive effectors for cancer immunotherapy. Combination treatment with zoledronic acid and IL-2 can activate and expand the most common subset of blood γδT, which express the Vγ9Vδ2 T cell receptor (TCR) (Vδ2 T cells). Vγ9Vδ2 T cells are equipped for antibody-dependent cell-mediated cytotoxicity (ADCC) through expression of the low-affinity FcγR CD16. GD2 is a highly ranked tumor associated antigen for immunotherapy due to bright expression on the cell surface, absent expression on normal tissues and availability of therapeutic antibodies with known efficacy in neuroblastoma. To explore the hypothesis that zoledronic acid, IL-2 and anti-GD2 antibodies will synergize in a therapeutic combination, we evaluated in vitro cytotoxicity and tumor growth inhibition in the GD2 expressing cancers neuroblastoma and Ewing's sarcoma. Vδ2 T cells exert ADCC against GD2-expressing Ewing's sarcoma and neuroblastoma cell lines, an effect which correlates with the brightness of GD2 expression. In an immunodeficient mouse model of small established GD2-expressing Ewing's sarcoma or neuroblastoma tumors, the combination of adoptively transferred Vδ2+ T cells, expanded in vitro with zoledronic acid and IL-2, with anti-GD2 antibody ch14.18/CHO, and with systemic zoledronic acid, significantly suppressed tumor growth compared to antibody or γδT cell-free controls. Combination treatment using ch14.18/CHO, zoledronic acid and IL-2 is more effective than their use in isolation. The already-established safety profiles of these agents make testing of the combination in GD2 positive cancers such as neuroblastoma or Ewing's sarcoma both rational and feasible. PMID:26942051

Osmolytic Effect of Sucrose on Thermal Denaturation of Pea Seedling Copper Amine Oxidase.

PubMed

Amani, Mojtaba; Barzegar, Aboozar; Mazani, Mohammad

2017-04-01

Protein stability is a subject of interest by many researchers. One of the common methods to increase the protein stability is using the osmolytes. Many studies and theories analyzed and explained osmolytic effect by equilibrium thermodynamic while most proteins undergo an irreversible denaturation. In current study we investigated the effect of sucrose as an osmolyte on the thermal denaturation of pea seedlings amine oxidase by the enzyme activity, fluorescence spectroscopy, circular dichroism, and differential scanning calorimetry. All experiments are in agreement that pea seedlings amine oxidase denaturation is controlled kinetically and its kinetic stability is increased in presence of sucrose. Differential scanning calorimetry experiments at different scanning rates showed that pea seedlings amine oxidase unfolding obeys two-state irreversible model. Fitting the differential scanning calorimetry data to two-state irreversible model showed that unfolding enthalpy and T * , temperature at which rate constant equals unit per minute, are increased while activation energy is not affected by increase in sucrose concentration. We concluded that osmolytes decrease the molecular oscillation of irreversible proteins which leads to decline in unfolding rate constant.

Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

PubMed

Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

2017-08-01

Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

Evaluation of Sorption Mechanism of Pb (II) and Ni (II) onto Pea (Pisum sativum) Peels.

PubMed

Haq, Atta Ul; Saeed, Muhammad; Anjum, Salma; Bokhari, Tanveer Hussain; Usman, Muhammad; Tubbsum, Saiqa

2017-07-01

The present study was carried out to know the sorption mechanism of Pb (II) and Ni (II) in aqueous solution using pea peels under the influence of sorbent dose, pH, temperature, initial metal ion concentration and contact time. SEM and FTIR were used for characterization of pea peels. The study showed that solution pH affects sorption process and the optimum pH for Pb (II) was 6.0 while for that of Ni (II) was 7.0. Pseudo-second order kinetic model was found to be the most suitable one to explain the kinetic data not only due to high value of R 2 (>0.99) but also due to the closeness of the experimental sorption capacity values to that of calculated sorption capacity values of pseudo second order kinetic model. It can be seen from the results that Freundlich isotherm explains well the equilibrium data (R 2 >0.99). Sorption capacity of pea peels was 140.84 and 32.36 for Pb (II) and Ni (II) mg g -1 respectively. The positive value of ΔH° and negative values of ΔG° suggest that sorption of Pb (II) and Ni (II) onto pea peels is an endothermic and spontaneous process respectively.

Co-inoculation of a Pea Core-Collection with Diverse Rhizobial Strains Shows Competitiveness for Nodulation and Efficiency of Nitrogen Fixation Are Distinct traits in the Interaction

PubMed Central

Bourion, Virginie; Heulin-Gotty, Karine; Aubert, Véronique; Tisseyre, Pierre; Chabert-Martinello, Marianne; Pervent, Marjorie; Delaitre, Catherine; Vile, Denis; Siol, Mathieu; Duc, Gérard; Brunel, Brigitte; Burstin, Judith; Lepetit, Marc

2018-01-01

Pea forms symbiotic nodules with Rhizobium leguminosarum sv. viciae (Rlv). In the field, pea roots can be exposed to multiple compatible Rlv strains. Little is known about the mechanisms underlying the competitiveness for nodulation of Rlv strains and the ability of pea to choose between diverse compatible Rlv strains. The variability of pea-Rlv partner choice was investigated by co-inoculation with a mixture of five diverse Rlv strains of a 104-pea collection representative of the variability encountered in the genus Pisum. The nitrogen fixation efficiency conferred by each strain was determined in additional mono-inoculation experiments on a subset of 18 pea lines displaying contrasted Rlv choice. Differences in Rlv choice were observed within the pea collection according to their genetic or geographical diversities. The competitiveness for nodulation of a given pea-Rlv association evaluated in the multi-inoculated experiment was poorly correlated with its nitrogen fixation efficiency determined in mono-inoculation. Both plant and bacterial genetic determinants contribute to pea-Rlv partner choice. No evidence was found for co-selection of competitiveness for nodulation and nitrogen fixation efficiency. Plant and inoculant for an improved symbiotic association in the field must be selected not only on nitrogen fixation efficiency but also for competitiveness for nodulation. PMID:29367857

Co-inoculation of a Pea Core-Collection with Diverse Rhizobial Strains Shows Competitiveness for Nodulation and Efficiency of Nitrogen Fixation Are Distinct traits in the Interaction.

PubMed

Bourion, Virginie; Heulin-Gotty, Karine; Aubert, Véronique; Tisseyre, Pierre; Chabert-Martinello, Marianne; Pervent, Marjorie; Delaitre, Catherine; Vile, Denis; Siol, Mathieu; Duc, Gérard; Brunel, Brigitte; Burstin, Judith; Lepetit, Marc

2017-01-01

Pea forms symbiotic nodules with Rhizobium leguminosarum sv. viciae (Rlv). In the field, pea roots can be exposed to multiple compatible Rlv strains. Little is known about the mechanisms underlying the competitiveness for nodulation of Rlv strains and the ability of pea to choose between diverse compatible Rlv strains. The variability of pea-Rlv partner choice was investigated by co-inoculation with a mixture of five diverse Rlv strains of a 104-pea collection representative of the variability encountered in the genus Pisum . The nitrogen fixation efficiency conferred by each strain was determined in additional mono-inoculation experiments on a subset of 18 pea lines displaying contrasted Rlv choice. Differences in Rlv choice were observed within the pea collection according to their genetic or geographical diversities. The competitiveness for nodulation of a given pea-Rlv association evaluated in the multi-inoculated experiment was poorly correlated with its nitrogen fixation efficiency determined in mono-inoculation. Both plant and bacterial genetic determinants contribute to pea-Rlv partner choice. No evidence was found for co-selection of competitiveness for nodulation and nitrogen fixation efficiency. Plant and inoculant for an improved symbiotic association in the field must be selected not only on nitrogen fixation efficiency but also for competitiveness for nodulation.

Strigolactones suppress adventitious rooting in Arabidopsis and pea.

PubMed

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.

A diagnostic guide for Fusarium Root Rot of pea

USDA-ARS?s Scientific Manuscript database

Fusarium root rot, caused by Fusarium solani f. sp. pisi, is a major root rot pathogen in pea production areas worldwide. Here we provide a diagnostic guide that describes: the taxonomy of the pathogen, signs and symptoms of the pathogen, host range, geographic distribution, methods used to isolate ...

A shark antibody heavy chain encoded by a nonsomatically rearranged VDJ is preferentially expressed in early development and is convergent with mammalian IgG.

PubMed

Rumfelt, L L; Avila, D; Diaz, M; Bartl, S; McKinney, E C; Flajnik, M F

2001-02-13

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM(1gj), from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells ("germline-joined"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H(1gj) in primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H(1gj) chain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM(1gj). Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.