Academic literature on the topic 'Excretory/secretory (E/S) proteins'
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Journal articles on the topic "Excretory/secretory (E/S) proteins"
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MAK, C. H., and R. C. KO. "DNA-binding activity in the excretory–secretory products ofTrichinella pseudospiralis(Nematoda: Trichinelloidea)." Parasitology 123, no. 3 (March 2001): 301–8. http://dx.doi.org/10.1017/s0031182001008459.
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A novel DNA-binding peptide ofMr∼30 kDa was documented for the first time in the excretory–secretory (E–S) products of the infective-stage larvae ofTrichinella pseudospiralis.Larvae recovered from muscles of infected mice were maintained for 48 h in DMEM medium. E–S products of worms extracted from the medium were analysed for DNA-binding activity by the electrophoretic mobility shift assay (EMSA). Multiple DNA-protein complexes were detected. A comparison of theMrof proteins in the complexes indicated that they could bind to the target DNA as a dimer, tetramer or multiples of tetramers. Site selection and competition analysis showed that the binding has a low specificity. A (G/C-rich)-gap-(G/T-rich)-DNA sequence pattern was extracted from a pool of degenerate PCR fragments binding to the E–S products. Results of immunoprecipitation and electrophoretic mobility supershift assay confirmed the authenticity of the DNA-binding protein as an E–S product.
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CERVI, L., G. ROSSI, and D. T. MASIH. "Potential role for excretory–secretory forms of glutathione-S-transferase (GST) in Fasciola hepatica." Parasitology 119, no. 6 (December 1999): 627–33. http://dx.doi.org/10.1017/s003118209900517x.
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The excretory–secretory antigen of Fasciola hepatica (ESA) is involved in the suppressive phenomena of cellular immune responses in rats. The ESA can depress the proliferative response of spleen mononuclear cells and inhibit nitric oxide (NO) production by peritoneal cells. In the present study we identified ESA proteins of ca 24 kDa, which shared significant sequence homology to glutathione-S-transferase (GST) obtained from homogenates of F. hepatica adults, other helminths and different mammals. When the dimeric form of these proteins ca 48 kDa was cultured with rat spleen cells, a significant decrease of proliferative response to Con A was detected, starting from 20 μg/ml of ESA protein (P<0·03). We also observed a significant inhibition of nitrite production by incubation with the dimeric form in normal peritoneal macrophages (P<0·04). These results indicated that the GST secreted by the parasite could be involved in evasion of the parasite from the host immune response.
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Cantacessi, Cinzia, Jason Mulvenna, Neil D. Young, Martin Kasny, Petr Horak, Ammar Aziz, Andreas Hofmann, Alex Loukas, and Robin B. Gasser. "A Deep Exploration of the Transcriptome and “Excretory/Secretory” Proteome of Adult Fascioloides magna." Molecular & Cellular Proteomics 11, no. 11 (August 16, 2012): 1340–53. http://dx.doi.org/10.1074/mcp.m112.019844.
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Parasitic liver flukes of the family Fasciolidae are responsible for major socioeconomic losses worldwide. However, at present, knowledge of the fundamental molecular biology of these organisms is scant. Here, we characterize, for the first time, the transcriptome and secreted proteome of the adult stage of the “giant liver fluke,” Fascioloides magna, using Illumina sequencing technology and one-dimensional SDS-PAGE and OFFGEL protein electrophoresis, respectively. A total of ∼54,000,000 reads were generated and assembled into ∼39,000 contiguous sequences (contigs); ∼20,000 peptides were predicted and classified based on homology searches, protein motifs, gene ontology, and biological pathway mapping. From the predicted proteome, 48.1% of proteins could be assigned to 384 biological pathway terms, including “spliceosome,” “RNA transport,” and “endocytosis.” Putative proteins involved in amino acid degradation were most abundant. Of the 835 secreted proteins predicted from the transcriptome of F. magna, 80 were identified in the excretory/secretory products from this parasite. Highly represented were antioxidant proteins, followed by peptidases (particularly cathepsins) and proteins involved in carbohydrate metabolism. The integration of transcriptomic and proteomic datasets generated herein sets the scene for future studies aimed at exploring the potential role(s) that molecules might play at the host–parasite interface and for establishing novel strategies for the treatment or control of parasitic fluke infections.
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Becerro-Recio, David, Javier González-Miguel, Alberto Ucero, Javier Sotillo, Álvaro Martínez-Moreno, José Pérez-Arévalo, Krystyna Cwiklinski, John P. Dalton, and Mar Siles-Lucas. "Recognition Pattern of the Fasciola hepatica Excretome/Secretome during the Course of an Experimental Infection in Sheep by 2D Immunoproteomics." Pathogens 10, no. 6 (June 9, 2021): 725. http://dx.doi.org/10.3390/pathogens10060725.
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Excretory/secretory products released by helminth parasites have been widely studied for their diagnostic utility, immunomodulatory properties, as well as for their use as vaccines. Due to their location at the host/parasite interface, the characterization of parasite secretions is important to unravel the molecular interactions governing the relationships between helminth parasites and their hosts. In this study, the excretory/secretory products from adult worms of the trematode Fasciola hepatica (FhES) were employed in a combination of two-dimensional electrophoresis, immunoblot and mass spectrometry, to analyze the immune response elicited in sheep during the course of an experimental infection. Ten different immunogenic proteins from FhES recognized by serum samples from infected sheep at 4, 8, and/or 12 weeks post-infection were identified. Among these, different isoforms of cathepsin L and B, peroxiredoxin, calmodulin, or glutathione S-transferase were recognized from the beginning to the end of the experimental infection, suggesting their potential role as immunomodulatory antigens. Furthermore, four FhES proteins (C2H2-type domain-containing protein, ferritin, superoxide dismutase, and globin-3) were identified for the first time as non-immunogenic proteins. These results may help to further understand host/parasite relationships in fasciolosis, and to identify potential diagnostic molecules and drug target candidates of F. hepatica.
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Kochanowski, Maciej, Joanna Dąbrowska, Mirosław Różycki, Jacek Sroka, Jacek Karamon, Aneta Bełcik, Weronika Korpysa-Dzirba, and Tomasz Cencek. "Proteomic Profiling and In Silico Characterization of the Secretome of Anisakis simplex Sensu Stricto L3 Larvae." Pathogens 11, no. 2 (February 14, 2022): 246. http://dx.doi.org/10.3390/pathogens11020246.
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Anisakis simplex sensu stricto (s.s.) L3 larvae are one of the major etiological factors of human anisakiasis, which is one of the most important foodborne parasitic diseases. Nevertheless, to date, Anisakis secretome proteins, with important functions in nematode pathogenicity and host-parasite interactions, have not been extensively explored. Therefore, the aim of this study was to identify and characterize the excretory-secretory (ES) proteins of A. simplex L3 larvae. ES proteins of A. simplex were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the identified proteins were then analyzed using bioinformatics tools. A total of 158 proteins were detected. Detailed bioinformatic characterization of ES proteins was performed, including Gene Ontology (GO) analysis, identification of enzymes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, protein family classification, secretory pathway prediction, and detection of essential proteins. Furthermore, of all detected ES proteins, 1 was identified as an allergen, which was Ani s 4, and 18 were potential allergens, most of which were homologs of nematode and arthropod allergens. Nine potential pathogenicity-related proteins were predicted, which were predominantly homologs of chaperones. In addition, predicted host-parasite interactions between the Anisakis ES proteins and both human and fish proteins were identified. In conclusion, this study represents the first global analysis of Anisakis ES proteins. The findings provide a better understanding of survival and invasion strategies of A. simplex L3 larvae.
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MAK, C. H., Y. Y. Y. CHUNG, and R. C. KO. "Single-stranded endonuclease activity in the excretory–secretory products of Trichinella spiralis and Trichinella pseudospiralis." Parasitology 120, no. 5 (May 2000): 527–33. http://dx.doi.org/10.1017/s0031182099005879.
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A novel acidic extracellular single-stranded endonuclease was demonstrated for the first time in the excretory–secretory (E–S) products of 2 species of Trichinella. Unlike the double-stranded endonuclease reported earlier, the single-stranded molecule is divalent cation independent and is detected in both T. spiralis and T. pseudospiralis E–S products. It hydrolysed single-stranded DNA and RNA at comparable rates. The single-stranded endonuclease was sensitive to inhibition by Zn2+ and to high concentrations of NaCl. Zymographic analysis indicated that it was encoded by at least 3 peptides of Mr ∼ 50–60 kDa. The rate of hydrolysis of single-stranded targets by the E–S products was substantially higher than that of the double-stranded molecule. Due to the differences in peptide profile, divalent cation dependence, and species-specific expression, the single and double-stranded endonucleases are likely to be encoded by different proteins and may have different functions.
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Siles-Lucas, M., and C. Cuesta-Bandera. "Echinococcus granulosus in Spain: strain differentiation by SDS-PAGE of somatic and excretory/secretory proteins." Journal of Helminthology 70, no. 3 (September 1996): 253–57. http://dx.doi.org/10.1017/s0022149x00015492.
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AbstractA comparison was made, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), of excretory/secretory (ES) – crude and immunopurified (with the corresponding anti-host serum) hydatid fluids – and somatic (S) – protoscoleces – proteins, from several ovine, equine, swine, bovine and human Echinococcus granulosus Spanish isolates. Likewise, the host influence on parasitic ES protein expression was studied, comparing purified hydatid fluids from ovine and equine cysts obtained from natural hosts and in RNMI mice. Purified hydatid fluids patterns, under reducing conditions, yielded the most precise differentiation of Spanish strains of E. granulosus into three groups (ovine—bovine-human, equine and swine), the finding of a characteristic 82 kDa band in equine isolates, and an unusual arrangement of bands between 50 and 6 kDa in swine samples. In addition, differences were found amongst crude and purified hydatid fluids, especially in bovine and swine isolates. The total protein patterns of protoscoleces were most complex, and therefore could not be used for strain differentiation. Finally, the purified hydatid fluids from cysts developed in natural and experimental hosts showed similar protein patterns, suggesting the lack of host influence, under our experimental conditions, on the expression of parasitic ES proteins.
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Nagano, Isao, Zhiliang Wu, and Yuzo Takahashi. "Species-Specific Antibody Responses to the Recombinant 53-Kilodalton Excretory and Secretory Proteins in Mice Infected with Trichinella spp." Clinical and Vaccine Immunology 15, no. 3 (January 9, 2008): 468–73. http://dx.doi.org/10.1128/cvi.00467-07.
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ABSTRACT The 53-kDa proteins in larval excretory and secretory (E-S) products were expressed from five Trichinella species (T. spiralis, T. britovi, T. nativa, T. pseudospiralis, and T. papuae), using the Escherichia coli expression system, and the antibody responses to the 53-kDa recombinant proteins in mice infected with Trichinella spp. were analyzed by Western blotting. The 53-kDa protein is conserved among the five Trichinella species, with >60% similarity in amino acid sequences. The 53-kDa recombinant proteins of T. spiralis and T. pseudospiralis reacted to sera from mice infected with T. spiralis and T. pseudospiralis at 8 days postinfection (p.i.), respectively. An antibody against the 53-kDa recombinant protein of T. spiralis recognized the 53-kDa protein in the crude extracts from adult worms and 30-day p.i. muscle larvae and E-S products from muscle larvae of T. spiralis but did not recognize any proteins from T. pseudospiralis. The sera from the mice infected with T. spiralis strongly reacted with the 53-kDa recombinant protein of T. spiralis but did not react with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, and T. papuae. Similarly, the sera from mice infected with T. britovi, T. nativa, T. pseudospiralis, or T. papuae strongly reacted with the 53-kDa recombinant proteins of T. britovi, T. nativa, T. pseudospiralis, or T. papuae, respectively. These results showed that the 53-kDa recombinant proteins provide early and species-specific antibody responses in mice infected with Trichinella spp.
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SPINNER, W. G., F. J. THOMPSON, D. C. EMERY, and M. E. VINEY. "Characterization of genes with a putative key role in the parasitic lifestyle of the nematode Strongyloides ratti." Parasitology 139, no. 10 (May 2, 2012): 1317–28. http://dx.doi.org/10.1017/s0031182012000637.
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SUMMARYParasitic nematodes are significant pathogens of humans and other animals. The molecular and genetic basis of animal parasitism is not yet fully understood. Strongyloides spp. are a genus of gastrointestinal nematodes of which species infect approximately 100–200 million people worldwide. S. ratti is a natural parasite of the rat, and a useful and amenable laboratory model. Previous EST and microarray analyses of the S. ratti life cycle have identified genes whose expression was specific, or biased, to the parasitic adult stage, suggesting that they may play a key role in parasitism in this species. Here we have further investigated the expression of these genes (by RT-PCR) throughout the S. ratti life-cycle. We produced recombinant proteins in vitro for a subset of these genes, which were used in Western blot analyses to investigate the distribution of the gene products among different stages of the S. ratti life cycle. We tested the efficacy of these recombinant proteins as anti-S. ratti vaccines. One of the proteins was detected in the excretory/secretory products of the parasitic stages.
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Grzelak, Sylwia, Anna Stachyra, Jerzy Stefaniak, Karolina Mrówka, Bożena Moskwa, and Justyna Bień-Kalinowska. "Immunoproteomic analysis of Trichinella spiralis and Trichinella britovi excretory-secretory muscle larvae proteins recognized by sera from humans infected with Trichinella." PLOS ONE 15, no. 11 (November 5, 2020): e0241918. http://dx.doi.org/10.1371/journal.pone.0241918.
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The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.
Dissertations / Theses on the topic "Excretory/secretory (E/S) proteins"
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Kemter, Andrea Maria. "Immunomodulatory proteins in Heligmosomoides polygyrus excretory/secretory products." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/23656.
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Infections with parasitic helminths are counted as neglected tropical diseases; they infect millions of people worldwide, causing high morbidity and economic loss. Many parasites establish long lasting infections in the host by blocking immune recognition, activation and effector pathways. To allow in depth research on their modes of immune evasion, several mouse models for parasitic helminth infections have been established. Heligmosomoides polygyrus for example is a gastrointestinal nematode of rodents exhibiting a wide spectrum of immunomodulatory effects, mediated in part by soluble molecules released by adult worms in vitro, the excretory/secretory products (HES). HES is a potent inhibitor of dendritic cell (DC) activation by Toll-like receptor (TLR) ligands, completely abolishing LPS induced IL-12 production and reducing the upregulation of cell surface activation markers. As of now, neither the modulatory molecule nor its mechanism of action are known. Here, the effect of HES on TLR ligand induced DC maturation was characterized in considerably more detail compared to previous publications. It could be shown to inhibit DC maturation induced by various TLR ligands, on both protein and mRNA levels. These effects were comparable in both C57BL/6 and BALB/c derived cells; in contrast to this HES differentially affected alternative activation of BMDC from these two mouse strains. Although for most of the experiments GM-CSF differentiated BMDC were used, HES also inhibited LPS induced activation of splenic CD11c+ cells as well as the activation of all three populations described in Flt3-L differentiated BMDC - pDCs, CD11b+ cDCs and CD24+ cDCs. Furthermore, it could be shown here that HES also inhibits LPS induced maturation in human monocyte derived DCs. In the search for the component in HES responsible for its inhibition of TLR ligand induced DC maturation, exosome depleted HES rather than exosomes was inhibitory, and the effect was heat labile. This lead to the conclusion that the modulatory molecule has a protein component which is indispensable for its effect; following this reasoning HES was subjected to fractionation, with subsequent analysis of the fraction protein contents by mass spectrometry. The top nine candidate proteins were expressed recombinantly; however, the recombinants were not able to inhibit LPS induced DC activation. In parallel, experiments to elucidate the mechanism by which HES inhibits TLR ligand induced DC maturation were performed. This led to the conclusion that HES induces changes in the cells that, while not affecting the induction of signalling downstream of TLRs, do impair its maintenance. As a complement to these experiments, the transcriptomes of LPS and LPS+HES treated cells eight hours after LPS stimulation were compared. This revealed that transcripts encoding a number of transcription factors inducing the expression of activation markers after TLR ligation were reduced upon treatment of cells with HES, as were the transcript levels of IRAK2, a kinase necessary for persistent signalling. In addition, HES increased the transcript levels for several factors known to negatively regulate DC maturation, including ATF3. Furthermore, this analysis revealed changes in transcript levels of factors like HIF-1a, indicating an even greater reliance on aerobic glycolysis if cells were treated with HES, in addition to hints at increased ER and oxidative stress. In conclusion, this work narrows down the list of potential DC modulators in HES, gives a first insight into changes in DC metabolism induced by HES and sheds light on the role of a number of signalling pathways with important roles in DC activation as targets of DC inhibition by HES.
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Ackroyd, Mark. "Development of model systems to study the function of excretory-secretory proteins from the parasitic nematode 'Trichinella spiralis'." Thesis, University of Aberdeen, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430389.
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Invasion of host muscle by the larvae of the nematode parasite Trichinella spiralis is associated with a series of ultrastructural, biochemical and molecular changes to the host muscle fibre that amalgamates in the formation of a unique host-parasite complex. It is hypothesised that proteins that are secreted or excreted (ES proteins) by the muscle larva are responsible for triggering some or all of the changes to the host cell. No such effector molecules have yet been identified although a number of candidate molecules are now emerging. There is therefore a need for an experimental system (or systems) that will enable the study of function of candidate ES proteins as they arise. In this investigation I describe the development of three model systems that allow the characterisation of different aspects of putative protein function with the aim of determining whether a candidate protein could contribute to the formation of the muscle host parasite complex. These models included: firstly, a cell culture model to investigate the effect of T. spiralis ES proteins of the differentiation of the myogenic cell line C2C 12; secondly, two in vivo heterologous expression systems to investigate the secretion and localisation of T. spiralis ES proteins; and thirdly, a cell-free in vitro translation system to investigate the secretion and potential processing and post-translation modifications of candidate T. spiralis ES proteins. The model systems described have enabled conclusions to be drawn on the potential role that a candidate ES protein, TsJ5, may play in both the biology of the muscle larvae and within the parasitised host cell.
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Moustafa, Amr [Verfasser], and Christian [Akademischer Betreuer] Betzel. "Structural and Functional Analyses of Secretory and Excretory Proteins from Onchocerca volvulus as Basis for Rational Drug Design / Amr Moustafa. Betreuer: Christian Betzel." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://d-nb.info/1098428927/34.
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Moustafa, Amr Verfasser], and Christian [Akademischer Betreuer] [Betzel. "Structural and Functional Analyses of Secretory and Excretory Proteins from Onchocerca volvulus as Basis for Rational Drug Design / Amr Moustafa. Betreuer: Christian Betzel." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2016. http://nbn-resolving.de/urn:nbn:de:gbv:18-78577.
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Shukeir, Nicholas. "Molecular mechanism(s) of prostate cancer progression : potential of therapeutic modalities." Thesis, McGill University, 2009. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115853.
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Prostate cancer remains one of the most commonly diagnosed cancers in men and is a leading cause of cancer death. While great success has been achieved at curing early stage prostate cancer, limited success has been obtained when treating late-stage hormone independent prostate cancer. This is due to the increased propensity for skeletal and non-skeletal metastases. Thus development of novel effective therapeutic modalities against late stage prostate cancer is of critical importance.Towards these objectives, I have focused my attention on the role of prostate secretory protein (PSP-94) which is expressed in normal individuals and in patients with early stage prostate cancer. Using our well established in vivo models of prostate cancer, I have evaluated the ability of PSP-94 and its amino acids 31-45 required (PCK3145) to decrease tumor growth and skeletal metastases in vivo and evaluated the potential mechanism(s) associated with PCK3145 anti-cancer actions.
Prostatic cancer can also develop as a result of epigenetic activation of tumor promoting genes. To evaluate the role of methylation in prostate cancer, late stage prostate cancer cells were treated with the universal methylating agent S-adenosylmethionine (SAM) and an anti-sense oligonucleotide directed against MBD2 (AS). Scrambled oligonucleotide was included as a control (S). Both SAM and MBD2-AS resulted in inhibition in uPA, MMP-2 and VEGF production leading to decreased tumor cell invasive capacity. However, SAM and MBD2-AS were not able to either further repress partially methylated genes (GSTP1) or reactivate already methylated genes (AR). Furthermore, SAM and MBD2-AS treatment resulted in significant reduction in tumor growth in vivo . Immunohistochemical and RT-PCR analyses carried out on SAM and MBD2-AS tumors revealed decreased protein and mRNA expression of uPA and MMP-2 which was partially due to increased methylation of the respective promoters even after 10 weeks post in vitro treatment as analyzed by bisulfate sequencing. In addition decreased levels of angiogenesis and tumor survival markers were observed.
Collectively, these studies are aimed at the development of novel reliable approached to diagnose and treat advanced, hormone refractory prostate cancer to reduce tumor associated morbidity and mortality.
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Soblik, Hanns [Verfasser]. "Proteomic analysis of excretory, secretory proteins from parasitic and free living stages of Strongyloides ratti / vorgelegt von Hanns Soblik." 2009. http://d-nb.info/995983267/34.
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Hsu, I.-Jou, and 許怡柔. "Construction of Byblis to express secretory proteins." Thesis, 2019. http://ndltd.ncl.edu.tw/cgi-bin/gs32/gsweb.cgi/login?o=dnclcdr&s=id=%22107NCHU5111021%22.&searchmode=basic.
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碩士國立中興大學
生物科技學研究所
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Carnivorous plants usually grow in a nutrient-poor environment. They have evolved various traps to capture and digest other organisms for nutritional purpose. Byblis guehoi, also known as rainbow grass, can grow fast and is easy to culture. Moreover, the secretory glandular trichomes that distributed all over the leaf can secrete viscous dewdrops which contain abundant enzymes including protease, phosphatase, galactosidase, esterase/lipase etc. Owing to the powerful secretion characteristics of Byblis, we aim to construct it as a bioreactor to produce secretory proteins. By RNA-seq analysis and de novo sequence assembly, we obtain 123,944 sequence contigs. We look for genes having high RPKM (Reads Per Kilobase per Million mapped reads) and encoding proteins with typical signal sequences at their N-terminus. Moreover, to confirm that which genes are for the secretome, the surface dewdrops were collected and the proteins were identified by mass spectrometry (LC/MS/MS). By cross comparison of the RNA-seq and LC/MS/MS data, nine genes were chosen as the targets for promoter hunting. qPCR experiment revealed the genome size of Byblis to be about 8.8 Gb, 23 times that of the rice genome. By nanopore sequencing, 3.1 GB genomic sequences were obtained and the promoter regions for the first three candidate genes were found for primer design. The promoter fragments, each with 2~3 kb, were PCR amplified, subcloned and sequencing confirmed. Together with the signal peptide, the vector systems will be constructed for future insertion of foreign genes. On the other hand, in order to generate transgenic Byblis, we established its Agrobacterium-mediated transformation protocol using GUS-intron vector as a reporter. About 30% callus did form green callus 2 weeks after transformation. Some shoot regenerated to normal looking transgenic Byblis, with GUS-staining confirmed. However, the shoot formation process was extremely long that took several months to go with unstable frequency. Therefore direct regeneration from tissue together with vacuum-aided Agrobacterium-mediated transformation are underway, hope to further improve the transformation efficiency of Byblis.
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Beránková, Kateřina. "Vlastnosti exkrečně-sekrečních proteinů motolice Fascioloides magna." Master's thesis, 2011. http://www.nusl.cz/ntk/nusl-312726.
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Fascioloides magna (the giant liver fluke) originated from North America, is known in the Czech Republic since 1930s. This pathogenic fluke invades mostly cervids, but livestock too. Excretory-secretory products (ES products) contain number of esential biomolecules which are produced by excretory and secretory system of the fluke. These molecules play key role in many biological process during the life cycle not only of fascioloid flukes (e.g. migration in the host tissues, immune evasion and digestion). Due to their antigenic properties they could be also used in immunodiagnostics. Excretory-secretory proteins from adult Fascioloides magna and comparative related species Fasciola hepatica were purified and separated by the basic biochemical methods (1D, 2D electrophoresis, ion-exchange chromatography) and their activity was confirmed by specific (fluorogenic peptide) and nonspecific (gelatine) substrates. By using the mass spectrometry methods (MALDI TOF/TOF), the most abundant peptidolytically active proteins from ES products of F. magna were identified as cathepsin L (FmCL). Recombinant analog of FmCL was expressed in Pichia pastoris expression system. The peptidolytic activity was again confirmed using the synthetic fluorogenic substrates; the specifity of recombinant FmCL active site was...
Conference papers on the topic "Excretory/secretory (E/S) proteins"
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Gronewegen, W. A., S. Heptinstall, W. Loesche, and P. Spangenberg. "EFFECTS OF FEVERFEW EXTRACT AND PARTHENOLIDE ON PLATELET SECRETION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643441.
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Feverfew (Tanacetum parthenium) is used for prophylaxis of migraine and it had been suggested that the plant may have antithrombotic potential. We have prepared extracts from the leaves of feverfew and have demonstrated inhibition of 14C-5HT secretion in platelet-rich plasma induced by the phorbol ester PMA, l-oleoyl-2-acetyl-sn-glycerol (OAG), arachidonic acid, the thromboxane analogue U46619, adrenaline, collagen and ADP. The effects of a solution of parthenolide (an∝-methylenebutyro-lactone isolated from feverfew) were determined in parallel. Both feverfew extract (FE) and parthenolide inhibited 14C-5HT release in a concentration-dependent manner and the effectiveness depended on the nature of the aggregating agent used. Both FE and parthenolide were most effective as inhibitors of the secretion induced by PMA and OAG. When we compared the concentrations of FE and parthenolide which gave 50% inhibition of secretion for all the agents tested, a good correlation was found (r= 0.936). Further studies showed that feverfew extract and parthenolide inhiMt release of β-thromboglobulin from platelets as well as 14C-5HT. FE did not cause liberation of LDH. Inhibition of secretion by FE appears to be irreversible since washing platelets after treatment did not restore secretory activity.The structure of parthenolide suggests that it can alkylate sulphydryl (SH) groups. When agents containing SH groups (e.g. cysteine) were added to FE, anti-secretory activity was reduced. We also obtained a considerable decrease in the number of acid-soluble SH groups in platelets treated with feverfew extract or parthenolide at concentrations which inhibit secretion. However there was a less marked decrease in the number of acid-insoluble SH groups. FE itself does not induce formation of disulphide-linked proteins but such proteins were formed when platelets were activated in the presence of FE, probably as a result of decreased glutathione levels.We conclude that parthenolide or parthenolide-like compounds are responsible for the anti-secretory effects of FE, and that alkylation of sulphydryl groups in platelets may be involved.
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Hunter, N. R., I. R. MacGregor, J. Dawes, and D. S. Pepper. "MICROCARRIER CULTURE OF HUMAN ENDOTHELIAL CELL TYPES - A SOURCE OF METABOLITES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643348.
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The production of human endothelial cell secretory products in amounts sufficient for biochemical studies is largely restricted by the culture growth area. Conventional flat bed systems yield at best 20-30 x 106 cells per 180cm2 culture flask. To overcome this problem, cells may be grown on Cytodex 3 microcarriers allowing large numbers of cells to be grown and conditioned in small culture volumes. A typical microcarrier unit will contain 200-300 x 106 cells and may be expanded in excess of 1000 x 106 cells at confluence. High viability (95%) and recovery (70-80%) in sub-culturing of microcarrier to microcarrier culture can be achieved with careful management of culture conditions and brief exposure to enzymes.Human umbilical artery and vein, and saphenous vein endothelial cells were prepared and grogn on microcarrier cultures to cell populations of 200-450 x 106 cells and conditioned for 14 day periods in serum-free media.The production profiles of several endothelial cell proteins including thrombospondin (TSP), von Willebrand Factor (vWF) and issue plasminogen activator (t-PA) were measured by radioimmunoassay under these conditions, and demonstrate the use of microcarrier cultures in producing milligram quantities of engothelial cell protein. For example, a HUVEC culture of 200 x 106 cells conditioned with serum-free media for 14 days yielded a total of 6.9mg TSP, 0.7mg vWF and 48.9ug t-PA. In this laboratory one such application of the system was the purification of endothelial proteins in amounts sufficient for immunisation of mice prior to the production of monoclonal antibodies and for subsequent characterisation.
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Walker, Isobel D., J. F. Davidson, D. J. Wheatley, K. MacArthur, and T. J. Spyt. "EFFECTS OF CONSTANT INFUSION OF ILOPROST, A STABLE PROSTACYCLIN DERIVATIVE DURING CARDIOPULMONARY BYPASS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643585.
Full textAbstract:
Prostacyclin (PGI2) is a potent inhibitor of platelet aggregation but it is highly unstable. Iloprost (Schering A.G.) is a stable carbacyclin derivative of PGI2 which is also a potent inhibitor of platelet aggregation. The effect of constant Iloprost infusion during cardiopulmonary bypass (CPB) on blood loss, platelet numbers, secretory proteins and platelet sequestration was studied in 50 adult males undergoing CPB for elective coronary vein bypass graft surgery.In a double-blind, randomised, placebo-controlled study (25 patients in each group) intravenous infusion of Iloprost was commenced at 5ng/kg/min immediately after the induction of anaesthesia and increased to 10ng/kg/min when the patient was established on CPB. The infusion was discontinued on completion of CPB. Blood loss and units of blood transfused were not significantly different in the two treatment groups. During CPB, mean platelet numbers fell significantly in both groups but were significantly higher (P<.001) in the Iloprost group than in the placebo group at the end of CPB and until 18-24 hours post CPB (P<.05). Mean plasma thromboxane B2 and β thromboglobulin levels were marginally lower (P<.05 and P<.02) in the Iloprost group but the distribution of platelet factor 4 results were similar in both groups. Post operative spontaneous platelet aggregation was similar in both groups. Platelet sequestration in oxygenators and arterial line filters was assessed using Indium labelled patients’ platelets reinjected immediately after commencing Iloprost infusion. Platelet sequestration was significantly greater in the placebo group than in the Iloprost group both in the oxygenators (placebo 9.2%, Iloprost 3.4% : P<.001) and in the arterial line filters (placebo 4.8%, Iloprost 0.6% : PC.05).Blood pressure was significantly lower in the Iloprost group than in the placebo group throughout the infusion period.Constant infusion of Iloprost during CPB is associated with significantly less platelet sequestration. The importance of this is not only in the conservation of platelet numbers but also the potential reduction in risk of platelet aggregate embolisation.
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Levine, P. H. "ACQUIRED IMMUNODEFICIENCY SYNDROME, HUMAN IMMUNODEFICIENCY VIRUS AND HEMOPHILIA." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644752.
Full textAbstract:
Less than 15 years ago the National Heart, Lung and Blood Institute surveyed physicians in the United States in order to characterize the demographics of hemophilia. The average age of persons with hemophilia in the United States was found to be 11.5 years old. By 10 years later, the life expectancy was predicted to be normal, and indeed the average age of persons with hemophilia in the U.S. is now in the early twenties. Early, intensive and predictably efficacious control of hemorrhage has made this result possible, and the therapeutic product which has allowed such control is commercial clotting factor concentrate.We now know that starting in 1978, and with great frquency during 1982 and 1983, the majority of U.S. hemophiliacs were infected with human immunodeficiency virus (HIV). It is estimated that as of January, 1987, approximately two thirds of the 20,000' persons with hemophilia in the United States have been infected with HIV. Among those with severe factor VIII deficiency, more than 9056 are seropositive. As of 1/5/87, there were 288 cases of AIDS among U.S. hemophiliacs, for an AIDS rate of approximately 2.256 of those with HIV infection. This number included 185 with severe, 32 with moderate and 28 with mild hemophilia A; 12 with severe, 6 with moderate and 1 with mild hemophilia B; 9 with vWD, and 4 others. A disproportionate number were older patients: 55 were ages 1-19; 62 ages 20-29; 85 ages 30-39, and 86 age 40 or older. Although the AIDS attack rate is no longer climbing logarhythmically, new cases are certainly still occurring.A variety of other HIV-related syndromes have emerged. Of great concern is immune thrombocytopenia, which is now relatively common; among a group of 209 carefully followed HIV-positive patients at our center, 31 (1556) are or have been thrombocytopenic. Progressive failure to normally gain height and weight in children with hemophilia has recently been shown by our group to correlate with HIV antibody positivity, and also with decreased T4/T8 ratio, decreased T4 cell count, decreased skin test reactivity, and subsequent development of ARC or AIDS in some such children. Finally, a picture of progressive fall in T4 count associated with recurrent non-specific infections and increased likelihood of positive viral culture, may predict an increased risk of developing AIDS.We know that the immune dysfunction in hemophilia is complex, and not wholly explained by HIV infection. One important factor may be the many foreign proteins contained in commercial clotting factor concentrates, and their ability to stimulate T cells. It is known that latent HIV infection in cultured T4 lymphocytes can be induced to enter the proliferative, viral secretory phase by the addition of soluble foreign antigens to the cell culture. Recent data of Brettler and colleagues, to be presented at this meeting, suggest that the use of highly purified VI!I:C (specific activity >3000 u/mg) in place of the present extremely impure products, may improve the immune dysfunction in hemophilia. This observation offers a new hypothetical approach to the prevention of progressive T4 cell depletion in HIV infected hemophiliacs, and requires immediate and extensive further study.The psychosocial burden of HIV infection is immense. The need for extensive, formal education and support programs is largely unmet in most parts of the world. Such programs are best run out of hemophilia treatment centers in most cases, and must include an active program on prevention of sexual transmission, provision of HIV testing before and during pregnancies, provision for maintenance of confidentiality, etc. Education concerning HIV is like all other forms of education. It requires formal organization, a curriculum, active rather than passive learning in which there is interaction between the teacher and the pupil, time for planned repetition, reinforcement with written materials, and assessment of goals achieved. For all of these reasons it is inappropriate to assume that the physician at the hemophilia center will be able to provide an adequate education program. Adquate paramedical personnel will need to undertake this effort, under the directjon of the physician.